AU2018319349B2 - Enzyme assisted crude palm oil extraction - Google Patents

Abstract

The present invention concerns a process for extraction or separation of crude palm oil (CPO), comprising the steps of: contacting a substrate comprising palm oil with an enzyme composition comprising a GH10 xylanase and extracting or separating the crude palm oil.

Description

W O 2019/038418 II A81 II1 1111|||||||||||||||||||||||11111 |||||||||||||||||||||||||||||||| Published: - with international search report (Art. 21(3)) - before the expiration of the time limit for amending the claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) - with sequence listingpart of description (Rule 5.2(a))

Enzyme assisted crude palm oil extraction

Reference to a Sequence Listing

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

Background of the Invention

Field of the Invention The present invention relates to a process for extraction or separation of crude palm oil, the crude palm oil obtainable by the process and an enzyme composition for use in the process.

Description of the Related Art Palm oil is an edible vegetable oil which is obtained from the mesocarp of palm fruits. Palm fruits or fruitlets grow in large bunches. The palm fruitlets are stripped from the fruit bunches after being sterilized. The high temperature causes the enzymes naturally occurring enzymes in the palm fruits to denature and facilitates stripping of the fruits from the bunch stalks. The palm fruitlets are discharged into vessels commonly referred to as digesters, whereby a digested mash of palm fruits are produced under controlled temperature. The digested mash is then pressed, e.g. by using a screw press for subsequent recovery of palm oil. The crude palm oil may be subjected to screening, e.g. to remove coarse fibers, and then to a clarification process to separate oil from water, cell debris and any remaining fibrous material. Palm fruit mesocarp contains large amounts of oil present as oil droplets within the mesocarp cells. Generally, the oil extraction rate (OER), which is a measure of the amount of extracted oil relative to the weight of the palm fruits is within the range of 20-24%, depending e.g. on fruit quality, and is subject to seasonal variation. In general, the palm oil milling process has been carefully optimized at each mill in order to minimize oil losses to the extent possible but there is still a strong incentive to improve the OER. International patent application WO 2012/011130 (Advanced enzyme technologies Ltd.) concerns an enzyme composition (with exocellulolytic, pectinolytic, mammanolytic and glucanoloytic activity) used in a process for palm oil extraction.

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field. Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

Summary of the Invention

In an aspect, the present invention provides a process for extraction or separation of crude palm oil (CPO), comprising the steps of: i) contacting a substrate comprising palm oil with an enzyme composition, ii) extracting or separating the crude palm oil wherein the enzyme composition comprises a GH10 xylanase and a GH62 arabinofuranosidase wherein the GH10 xylanase has at least at least 90% sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:8 and wherein the GH62 arabinofuranosidase has at least 99% sequence identity to the polypeptide of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. The present invention relates to a process for extraction or separation of crude palm oil (CPO), comprising the steps of: i) contacting a substrate comprising palm oil with an enzyme composition, ii) extracting or separating the crude palm oil (CPO) wherein the enzyme composition comprises a GH10 xylanase. The invention further relates to a crude palm oil obtainable by the above process. The invention further concerns a composition comprising a GH10 xylanase, wherein the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:8; or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 8. The invention further concerns the use of a GH10 xylanase in the process according to the invention, wherein the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 1, SEQ ID NO: 2 SEQ ID NO: 3 or SEQ ID NO:8; or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3 or SEQ ID NO: 8.

Definitions

Arabinofuranosidase: The term "arabinofuranosidase" or "GH62 arabinofuranosidase" means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)- and/or (1,2)- and/or (1,5)-linkages, arabinoxylans, and arabinogalactans. Alpha-L-arabinofuranosidase is also known as arabinosidase, alpha-arabinosidase, alpha-L-arabinosidase, alpha arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase, L-arabinosidase, or alpha-L-arabinanase. Arabinofuranosidase activity can be determined using 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland,

2a

Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5 in a total volume of 200 pl for 30 minutes at 40°C followed by arabinose analysis by AMINEX@ HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Crude oil: The term "crude oil" (also called a non-degummed oil) refers to a pressed or extracted oil or a mixture thereof. In the present context, it is to be understood that the oil is palm oil, in particular un-refined palm oil. In particular, the term "crude oil" refers to the effluent from the screw press of a palm oil mill; i.e. to the mixture of oil and water pressed out of the palm fruit mash, before it has been subject to clarification and separation of oil from water. The crude palm oil is also designated CPO. Crude palm oil comprises water. Digestion: The term "digestion" refers to a process where the substrate comprising palm oil is kept at a temperature in the range of 65-85°C for disintegrating the substrate and releasing palm oil from the mesocarp. The digestion can be carried out in a digestion tank and/or a precooker tank equipped with baffles. During the digestion, the substrate comprising palm oil e.g. the palm fruitlets are disintegrated and oil released from the mesocarp. According to the invention the substrate comprising palm oil can be contacted with the enzyme composition before or during the digestion. Oil extraction rate (OER): For the purpose of the present invention, "Oil extraction rate (OER)" may be defined as by Chang et al., oil palm Industry economic journal, volume 3, 2003[9]. Chang et al. defines the Oil extract rate as ratio of oil recovered and Fresh fruit branch (FFB) times 100. According to this definition, the mathematical formula is: OER = (weight of oil recovered/weight of FFB processed) x 100 Palm oil mill effluent (POME): Palm oil mill effluent (POME) is the waste water discharged e.g. from the sterilization process, crude oil clarification process. Palm press liquid: The term "palm press liquid" refers to the liquid discharged from the pressing of the substrate comprising palm oil. Palm press liquid is not a crude palm oil and water has not been separated from the palm press liquid. Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment) For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment) Mature polypeptide: Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form following translation and any post-translational modifications, such as N terminal processing, C terminal truncation, glycosylation, phosphorylation, etc. Xylanase: The term "xylanase" means a 1,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans. For purposes of the present invention, xylan degrading activity is determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, MO, USA) by xylan-degrading enzyme(s) under the following typical conditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50°C, 24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47: 273-279. In one aspect, the polypeptide of the present invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the xylanase activity of the mature polypeptide of SEQ ID NO: 1, SEQ ID NO: 2 SEQ ID NO: 3 or SEQ ID NO: 8. In another aspect the polypeptide of the present invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the xylanase activity of the mature polypeptide of SEQ ID NO: 1, SEQ ID NO: 2 , SEQ ID NO: 3 or SEQ ID NO: 8. In still another aspect the polypeptide of the present invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the xylanase activity of the mature polypeptide of SEQ ID NO:1,SEQ ID NO:2,SEQ ID NO:3orSEQ ID NO:8. Xylan degrading activity or xylanolytic activity: The term "xylan degrading activity" or "xylanolytic activity" means a biological activity that hydrolyzes xylan-containing material. The two basic approaches for measuring xylanolytic activity include: (1) measuring the total xylanolytic activity, and (2) measuring the individual xylanolytic activities (e.g., endoxylanases, beta xylosidases, arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, and alpha-glucuronyl esterases). Recent progress in assays of xylanolytic enzymes was summarized in several publications including Biely and Puchart, 2006, Recent progress in the assays of xylanolytic enzymes, Journal of the Science ofFood and Agriculture 86(11): 1636 1647; Spanikova and Biely, 2006, Glucuronoyl esterase - Novel carbohydrate esterase produced by Schizophyllum commune, FEBS Letters 580(19): 4597-4601; Herrmann etal., 1997, The beta D-xylosidase of Trichoderma reese is a multifunctional beta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381. Total xylan degrading activity can be measured by determining the reducing sugars formed from various types of xylan, including, for example, oat spelt, beechwood, and larchwood xylans, or by photometric determination of dyed xylan fragments released from various covalently dyed xylans. The most common total xylanolytic activity assay is based on production of reducing sugars from polymeric 4-0-methyl glucuronoxylan as described in Bailey et al., 1992, Interlaboratory testing of methods for assay of xylanase activity, Journal ofBiotechnology 23(3): 257-270. Xylanase activity can also be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON@ X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200 mM sodium phosphate buffer pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 mole of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 buffer. For purposes of the present invention, xylan degrading activity is determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, MO, USA) by xylan-degrading enzyme(s) under the following typical conditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50°C, 24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47: 273-279. The abbreviation TL GH10 is used herein to refer to GH10 xylanase derived from Talaromycesleycettanus. Theabbreviation RB GH10 is used herein to referto GH10 xylanase derived from Rasamsonia byssochlamydoides. The abbreviation AF GH10 is used herein to refer to GH10 xylanase derived from Aspergillus fumigatus.

Overview of sequences SEQ ID NO: 1 is a GH10 xylanase derived from Taaromyces leycettanus SEQ ID NO: 2 is a GH10 xylanase derived from Talaromyces leycettanus, mature polypeptide SEQ ID NO: 3 is a GH10 xylanase derived from Rasamsonia byssochamydoides

SEQ ID NO: 4 is a GH62 arabinofuranosidase derived from Talaromyces pinophilus SEQ ID NO: 5 is a GH62 arabinofuranosidase derived from Penicilium capsulatum SEQ ID NO: 6 is a GH62 arabinofuranosidase derived from Penicilium oxaicum SEQ ID NO: 7 is a GH62 arabinofuranosidase derived from Aspergilius niger SEQ ID NO: 8 is a GH10 xylanase derived from Aspergilus niger SEQ ID NO: 9 is a GH10 xylanase derived from Aspergi/us fumigatus. SEQ ID NO 10 is a GH3 beta-xylosidase derived from Talaromyces emersonii

Brief description of figures

Figure 1 shows oil yield (see Example 9) Figure 2 shows Residual activity of enzyme (see Example 10) Figure 3 shows comparison of enzyme activity (% xylose conversion) at pH 4.0 at varying temperatures. E Aspergilius fumigatus (SEQ ID NO. 9), X- Rasamsonia byssochlamydoides GH10 (SEQ ID NO. 3); I- Talaromyces Leycettanus GH10 (SEQ ID NO. 2). See also Example 11. Figure 4 shows comparison of enzyme activity (% xylose conversion) at temperature 65 °C at varying pH. E Aspergillus fumigatus (SEQ ID NO. 9), X- Rasamsonia byssochlamydoides GH10(SEQIDNO.3); TalaromycesLeycettanus GH10 (SEQ ID NO. 2). See also Example 11.

Detailed Description of the Invention

The present invention concerns an enzyme composition and a process for enzyme assisted extraction of crude palm oil from a substrate comprising palm oil. The substrate comprising palm oil can be selected from the group consisting of palm fruitlets, pressed palm fruit liquid, mashed or partly mashed palm fruitlets. The inventors have found that by using a GH10 xylanase on the substrate comprising palm oil, the oil extraction rate (OER) can be increased. The invention concerns a process for extraction or separation of crude palm oil (CPO), comprising the steps of: i) contacting a substrate comprising palm oil with an enzyme composition, ii) extracting or separating the crude palm oil (CPO) wherein the enzyme composition comprises a GH10 xylanase. In one embodiment of the invention, the substrate comprising palm oil is palm fruitlets, which comprise oil in the mesocarp of the fruit. The palm fruitlets are contacted with the enzyme composition. In one embodiment, the substrate is palm fruitlets, which are mashed or partly mashed and contacted with the enzyme composition. This increases availability of mesocarp cells and thereby enhances enzyme activity on the mesocarp cells. In one embodiment, the substrate comprising palm oil is crude palm oil which is contacted with the enzyme composition. In the various aspects and embodiments of the invention the substrate, which comprises palm oil may be a substrate which also comprises fiber, in particular fiber from the mesocarp of palm fruitlets. In one embodiment of the invention the substrate comprising palm oil is sterilized before being contacted with the enzyme composition. Palm fruits grow in large bunches and needs to be stripped from the bunch stalks before being contacted with the enzyme composition. Steam sterilization of the fresh fruit bunches facilitates fruits being stripped from bunches to give the palm fruitlet. The sterilization step has several advantages one being that it softens the fruit mesocarp for subsequent digestion. A further advantage is that the quality of the final palm oil product is better if the palm fruits are stripped from the bunch stalks. The sterilization can be a batch sterilization or a continuous sterilization. The sterilization process can be carried out at a temperature of 100°C-1500C. In one embodiment of the invention, the pressure is reduced during the sterilization procedure. After the sterilization, the palm fruitlets are stripped from the bunch stalks. Stripping or threshing can be carried out in a mechanized system having a rotating drum or fixed drum equipped with rotary beater bars which detach the fruit from the bunch and leaves the spikelets on the stem. The stripped palm fruitlets can be contacted with the enzyme composition according to the invention. In one embodiment of the invention, the substrate comprising palm oil is subjected to digestion before extracting the crude palm oil. The stripped palm fruitlets can be transported into a digester by one or more transportation means, e.g. a conveyor belt. In the digester, the fruitlets are further heated in order to loosen the pericarp. The digester is typically a steam heated vessel, which has rotating shafts to which stirring arms are attached or is equipped with baffles. The fruitlets are rotated, causing the loosening of the pericarps from the nuts and degradation of the mesocarp. The digester is a continuous process where the digester is kept full and as the digested fruit is drawn out, freshly stripped fruits are brought in. In one embodiment of the invention, the first part of the digestion is carried out in a pre cooker. The substrate may be held at a temperature within the range of 65-85°C for some time and then transferred to the digester tank. In one embodiment of the invention, the substrate comprising palm oil is contacted with the enzyme composition at a temperature of above 50°C and the crude palm oil is then extracted. In one embodiment of the invention, the substrate comprising palm oil is contacted with the enzyme composition at a temperature within the range of 55-90°C, such as a temperature within the range of 55-85°C, 55-80°C, 60-90°C, 60-85°C, 60-80°C, 66-90°C, 67-90°C, 68-90°C, 69-90°C,70-90°C, 66-85°C,66-80°C, 67-80°C, 66-79°C, 66-78°C,66-77°C, 66-76°C,66-75°C,

66-74°C, 66-73°C, 66-72°C, 66-71°C, 67-80°C, 67-79°C, 67-78°C, 67-77°C, 67-76°C, 67-75°C, 67-74°C, 67-73°C, 67-72°C, 67-71°C, 68-79°C, 68-78°C, 68-77°C, 68-76°C, 68-75°C, 68-74°C, 68-73°C, 68-72°C, 68-71°C, 69-79°C, 69-78°C, 69-77°C, 69-76°C, 69-75°C, 69-74°C, 69-73°C, 69-72°C, 69-71°C, 70-90°C, 70-89°C, 70-88°C, 70-87°C, 70-86°C, or 70-85°C. In particular embodiments, the substrate comprising palm oil is contacted with the enzyme composition at a temperature within the range of 60-90°C, such as 60 - 85 90°C. The substrate comprising palm oil is contacted with the enzyme composition for a period of 5-120 minutes, such as a period of 20-120 minutes, 25-120 minutes 5-60 minutes, 20-60 minutes, 25-60 minutes, 30-60 minutes, 15-50 minutes, 20-50 minutes, 25-50 minutes, 30-50 minutes, 15-40 minutes, 20-40 minutes, 25-40 minutes, 30-40 minutes, 15-30 minutes, 20-30 minutes, 25-28 minutes, 25-30 minutes, 25-35 minutes, 15-25 minutes, 20-25 minutes, 20-28 minutes, 15-20 minutes, 10-15 minutes or 5-10 minutes. In one embodiment of the invention, the substrate comprising palm oil is contacted with the enzyme composition at a temperature of above 50°C, such as a temperature within the range of 55-90°C for a period of 5-120 minutes and the crude palm oil is then extracted. The digested substrate is passed into a press, e.g. a screw press, from which the palm press liquid is discharged. The palm press liquid is a mixture of oil, water, press cake/fibre and nuts. The palm press liquid is delivered from the press to a clarification tank forfurther processing. In one embodiment of the invention, the digestion comprises retaining the substrate comprising palm oil at temperatures above 65°C and up to 85°C for 10-30 minutes, such as for 10-28 minutes, 15-28 minutes, 12-30 minutes,12-28 minutes or 12-25 minutes.

It is to be understood that the enzyme composition used according to the invention may be applied at any point in the crude palm oil extraction and/separation process, after the palm fruit bunches have been sterilized and until the oil is separated from water the water and from cell debris and fibrous material, which is also present in the palm press liquid. In particular, the substrate may be selected from the group consisting palm fruitlets, palm press liquid, mashed or partly mashed palm fruitlets.

It will be well within the capacity of the skilled person to optimize the dosing of the enzyme composition in view of other process parameters. In particular embodiments of the invention, the enzyme composition is dosed in amounts corresponding to 10-500 mg enzyme protein/kg FFB (fresh fruit bunch) comprising palm oil, such as 10-450 mg enzyme protein/kg FFB comprising palm oil, 10-400 mg enzyme protein/kg FFB comprising palm oil, 10-350 mg enzyme protein/kg FFB comprising palm oil, 10-300 mg enzyme protein/kg FFB comprising palm oil, 10-250 mg enzyme protein/kg FFB comprising palm oil, 10-200 mg enzyme protein/kg FFB comprising palm oil, 10-150 mg enzyme protein/kg FFB comprising palm oil, 10-100 mg enzyme protein/kg FFB comprising palm oil, 10-75 mg enzyme protein/kg FFB comprising palm oil, 10 50 mg enzyme protein/kg FFB comprising palm oil, 10-500 mg enzyme protein/kg FFB comprising palm oil, 10-500 mg enzyme protein/kg FFB comprising palm oil, 10-500 mg enzyme protein/kg FFB comprising palm oil, 75-500 mg enzyme protein/kg FFB comprising palm oil, 100 500 mg enzyme protein/kg FFB comprising palm oil, 150-500 mg enzyme protein/kg FFB comprising palm oil, 200-500 mg enzyme protein/kg FFB comprising palm oil, 250-500 mg enzyme protein/kg FFB comprising palm oil, 300-500 mg enzyme protein/kg FFB comprising palm oil, 350-500 mg enzyme protein/kg FFB comprising palm oil, 400-500 mg enzyme protein/kg FFB comprising palm oil, 30-400 mg enzyme protein/kg FFB comprising palm oil, 30-300 mg enzyme protein/kg FFB comprising palm oil, 30-200 mg enzyme protein/kg FFB comprising palm oil, 30-150 mg enzyme protein/kg FFB comprising palm oil, 30-100 mg enzyme protein/kg FFB comprising palm oil, 30-75 mg enzyme protein/kg FFB comprising palm oil, or such as 30-50 mg enzyme protein/kg FFB comprising palm oil. According to some embodiments of the invention, the enzyme(s) are dosed at amounts corresponding to 10-1000 ppm, such as 20-1000 ppm, 30-1000 ppm, 40-1000ppm, 50-1000ppm, 100-1000 ppm, 200-1000 ppm, 100-500 ppm, such as 200-500 ppm, 250-400 ppm or 350-1000 ppm relative to the amount of substrate comprising palm oil. In one aspect, the contacting of the substrate comprising palm oil with an enzyme composition is done when the substrate is conveyed towards the digester. In one aspect, the substrate comprising palm oil is contacted with the enzyme during conveyance from threshing to the digester, such as during transport of the fruitlets on a conveyer belt, in a screw conveyor or augerconveyor. In the process according to the invention, the enzyme may alternatively be dosed directly into the digester, such that it is first contacted with the palm fruitlets in the upper one third of the digester. In one aspect, the process comprises steps of: sterilizing and threshing fresh palm fruit bunches to provide palm fruitlets; and conveying the palm fruitlets into a digester. In one aspect, the palm fruitlets are threshed and conveyed from threshing to a digester without being subject to disintegration other than the disintegration, which occurs during threshing and conveyance, such as without being subject to maceration/pre-cooking/mashing. In one aspect, the substrate comprising palm oil is contacted with the enzyme composition by distributing the enzyme composition onto the surface of the palm fruitlets, such as by sprinkling or spraying the enzyme onto the fruitlets, during conveyance. In a particular embodiment of the present invention, enzymes are sprinkled or sprayed onto the palm fruitlets during conveyance to the digester, which leads to improved exposure of the palm fruitlets to enzyme and a more homogenous mixture as compared to mixing within the digester. The skilled person would by default add the enzyme composition in the digester, without having realized that more even distribution of enzyme composition on the fruitlet surface could be obtained by sprinkling or spraying enzyme onto the fruitlet prior to entry into the digester. A further advantage of applying the enzyme composition during conveyance of substrate comprising palm oil towards the digester is early penetration of the enzyme composition into the mesocarp through scratches or bruises on the exocarp. This helps in "positioning" the enzyme in the mesocarp and further reduces the reaction time needed when appropriate temperatures are reached in the digester. As the skilled person will realize, the palm fruitlets must be retained within the digester for sufficient time to allow the enzymes to act e.g. on the cellulosic material of the palm fruitlets. The exact retention time needed in the digester will depend on the exact conditions, and whether the enzyme composition is dosed directly in the digester or onto the palm fruitlets while being conveyed to the digester. Dosing the enzymes upstream of the digester onto the palm fruitlets while they are transported to the digester will generally lower the minimum retention time needed in the digester. In a preferred embodiment, the palm fruitlets are contacted with the enzyme composition in the digester for 15-60 minutes, such as for 20-60 minutes, 25-60 minutes, 30-60 minutes, 40-60 minutes, 50-60 minutes, 15-50 minutes, 20-50 minutes, 25-50 minutes, 30-50 minutes, 40-50 minutes, 15-40 minutes, 20-40 minutes, 25-40 minutes, 30-40 minutes, 15-30 minutes, 20-30 minutes, 25-30 minutes, 15-25 minutes or 15-20 minutes. In addition to slightly lowering the temperature in the digester as compared to the temperature used in conventional palm oil milling processes, it may also be desirable to slightly increase the retention time in the digester. This may be advantageous even when applying the enzyme composition onto the palm fruitlets while being conveyed to the digester. The temperature in the upper one third of digester may in particular be in the range of 45-55°C, e.g. in the range of 55-65°C; in the middle one third of the digester it may be about 65°C, such as in the range of 55-65°C, in the range of 60-70°C or in the range of 65-70°C; and in the lower one third of the digester it may be about 85°C, such as in the range of 70-85°C, typically about 80°C. According to these and other embodiments contacting or incubation of the palm fruitlet with an enzyme composition is done for a period of 5-60 minutes, such as for a period of 20-60 minutes, 25-60 minutes, 30-60 minutes, 15-50 minutes, 20-50 minutes, 25-50 minutes, 30-50 minutes, 15-40 minutes, 20-40 minutes, 25-40 minutes, 30-40 minutes, 15-30 minutes, 20 30 minutes, 25-30 minutes, 25-35 minutes, 15-25 minutes, 20-25 minutes, 15-20 minutes, 10-15 minutes or 5-10 minutes; the time period being calculated as the time from which the enzyme composition is applied onto the palm fruitlets and until the digested fruit is discharged into the press. The contacting or incubation of the palm fruitlet with an enzyme composition may in particular be done for a period of 25-35 minutes, more preferably a period of 25-30 minutes, most preferably a period of 25-28 minutes.

It is further to be understood that the enzymes used in the process according to the Invention, may be inactivated, or at least substantially inactivated, when the digested fruit is pressed, due to the high temperatures reached in the screw press. Preferably, the incubation time or retention time of the palm fruit mash at temperatures above 65°C and up to 85°C is from 10-30 minutes, such as from 10-28 minutes, 15-28 minutes, 12-30 minutes,12-28 minutes or 12-25 minutes. Retention time at temperatures above 65°C and up to 85°C may be controlled according to need; e.g. by increasing the digester volume, or by slowing down the screw press. Also, throughout the milling process retention time at temperatures close to 65°C may be increased by the use of a predigester, and/or by use of a slow conveyor method. In one aspect, contacting with an enzyme composition is done at one or more contact points. In the present invention, the palm fruitlets are conveyed into the digester by means such as but not limited to screw-conveyer or auger conveyor or belt conveyor or roller conveyor or skate-wheel conveyor or chain conveyor or bucket elevator. In one aspect, the palm fruitlets are subject to temperatures during passage through the digester, which increase from 45-85°C. such as from 45-900C, from 50-85°C or such as from 50 900C.

In one aspect, the invention concerns a crude palm oil, which is obtainable by the process according to any of the process of the invention.

In one embodiment of the invention, the substrate comprising palm oil is contacted with the enzyme composition comprising a GH10 xylanase.

A polypeptide having xylanase activity of the present invention (GH10 xylanase) may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly. The polypeptide may be a Talaromyces polypeptide, an Aspergilius polypeptide, or for example a Rasamsonia polypeptide. In another aspect, the polypeptide is a Talaromyces leycettanus polypeptide, e.g., a polypeptide obtained from Talaromyces leycettanus Strain CBS398.68. In another aspect, the polypeptide is a Rasamsonia byssochlamydoides polypeptide, e.g., a polypeptide obtained from Rasamsonia byssochiamydoides.

In another aspect, the polypeptide is an Aspergillus niger polypeptide, e.g., a polypeptide obtained from Aspergillus niger. It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents. Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL). The polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et a., 1989, supra). Preferred embodiments of the aspect of the invention relating to the GH10 polypeptide having xyalanse activity are disclosed herein below. Additional details of preferred GH10 polypeptides having xyalanse activity are found in PCT/CN2016/107281 filed 25 November2016, hereby incorporated by reference. In one embodiment of the invention, the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 1, orthe GH10 xylanase comprises orconsists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 1. In one embodiment of the invention, the GH10 xylanase comprises or consists of the mature polypeptideof SEQ ID NO: 2, or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least

92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 2. In one embodiment of the invention, the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 3, or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 3. In one embodiment of the invention, the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 8, or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 8. According to the invention, the enzyme composition may further comprise one or more enzymes obtained from Trichoderma reesei. Said enzymes may in some embodiments be substantially inactive at a temperature of about 70°C. Said enzymes can be glycoside hydrolases, such as cellulases. In one embodiment of the invention, the enzymes which are obtained from Trichoderma reesei comprise all enzyme activities necessary for complete degradation of cellulose. In one embodiment of the invention, the enzyme composition comprises a GH62 arabinofuranosidase. Preferred embodiments of the aspect of the invention relating to the GH62 polypeptide having arabinofuranosidase activity are disclosed herein below. Additional details of preferred GH62 polypeptides having arabinofuranosidase activity are found in PCT/CN2015/071015 filed 19 January 2015., which is hereby incorporated by reference. In an embodiment, the GH62 polypeptide having arabinofuranosidase activity has a sequence identity to the mature polypeptide of SEQ ID NO: 4 of at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In an embodiment, the GH62 polypeptide having arabinofuranosidase activity has a sequence identity to the mature polypeptide of SEQ ID NO: 5 of at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In an embodiment, the GH62 polypeptide having arabinofuranosidase activity has a sequence identity to SEQ ID NO: 6 of at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In another embodiment, the GH62 polypeptide having arabinofuranosidase activity has a sequence identity to the mature polypeptide of SEQ ID NO: 7 of at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. A polypeptide having arabinofuranosidase activity may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly. The polypeptide may be a fungal polypeptide. In one embodiment, the polypeptide is from a fungus of the order Eurotiales, or from the family Aspergillaceae, or from the genus Penicilium or from the species Penicillium aurantiogriseum, Penicillium oxalicum or Penicillium capsulatum. In one embodiment, the polypeptide is from a fungus of the order Eurotiales, or from the family Aspergillaceae, or from the genus Aspergillus or from the species Aspergillus clavatus or Aspergillus wentii or Aspergillus niger. In one embodiment, the polypeptide is from a fungus of the order Eurotiales, or from the family Aspergillaceae, or from the genus Neosartorya or from the species Neosartoryafischeri. In one embodiment, the polypeptide is from a fungus of the order Eurotiales, or from the family Trichocomaceae, or from the genus Talaromyces or from the species Talaromyces pinophilus. In one embodiment, the polypeptide is from a fungus of the order Ustilaginales, or from the family Ustilaginaceae, or from the genus Ustilago or from the species Ustilago maydis. In one embodiment, the polypeptide is from a fungus of the phylum Ascomycota, or from the genus Acrophialophora or from the species Acrophialophorafusispora. The polypeptide may be a bacterial polypeptide. In one embodiment, the polypeptide is from a bacterium of the order Actinomycetales, or from the family Streptomycetaceae, or from the genus Streptomyces or from the species Streptomyces nitrosporeus or Streptomyces beijiangensis.

In one embodiment, the polypeptide is from a bacterium of the order Actinomycetales, or from the family Streptosporangiaceae, or from the genus Streptosporangium or from the species Streptosporangium sp-60756. It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents. Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL). The polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et a., 1989, supra). The invention further concerns an enzyme composition comprising a GH10 xylanase, wherein the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 1, SEQ ID NO: 2 ,SEQ ID NO: 3 or SEQ ID NO:8; or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO:3orSEQ ID NO:8. The enzyme composition can further comprise a GH62 arabinofuranosidase, wherein the GH62 arabinofuranosidase comprises or consists of the mature polypeptide of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6or SEQ ID NO: 7; or the GH62 arabinofuranosidase comprises or consists comprising or consisting of an amino acid sequence having at least 80%, such as at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the polypeptide of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. In one embodiment of the invention, the enzyme composition further comprises one or more enzymes obtained from Trichoderma reesei. These enzymes are substantially inactive at a temperature of about 70°C.

Examples

Example 1 Preparation of sterilized palm fruit mesocarp

Step Action 1 In palm oil mill, oil palm FFBs (fresh fruit bunches) are received directly from the field, subjected to sterilization in industrial autoclave (1200C for 120 minutes) and then threshed to obtain oil palm fruitlets along with calyx leaves. Collect the sterilized palm fruitlets. 2 Separate the oil palm fruitlets and discard the rest of the biomass [calyx leaves and small pieces of fruit bunch stalk (called as empty fruit bunch or EFB)]. 3 Pack the fruitlets in an autoclavable plastic cover and cook it in a kitchen pressure cooker (10 L Capacity; Aluminum) for below induction cooktop program: Program Name: Pressure Cooker Time: 30 minutes Watt/Temperature: 1300W/1800C 4 Spread the cooked fruitlets in a tray and allow it to cool down to approximately 500C. 5 Peel off the mesocarp from the nut. Collect the mesocarp in a pre-weighed plastic storage container and record the weight. 6 Record the weight of nuts and discard it. 7 The peeled mesocarp is stored at 40C until use.

Example 2 Digesting and pressing of palm fruitlets The sterilized palm fruit mesocarp is pressed

Step Action 1 Mash required amount of mesocarp in mash bath at -200 r.p.m. for: a. 3 minutes, if mesocarp quantity is more than 2 Kilograms. b. 2 minutes, if mesocarp quantity is 2-1 Kilograms. 2 Manually mix the mashed mesocarp until it is uniformly mixed 3 Aliquot 50g (±0.2g) of mashed mesocarp into the steel container.

4 Pre-incubate the mashed mesocarp by placing the containers in a water bath at 90°C for 10 minutes followed by 70°C for 5 minutes. [For control, pre-incubate only at 90°C for 10 minutes] 5 Inoculate the required enzyme composition into the substrate. For control, add 1ml of reverse osmosis water 6 Mix the substrate and inoculum with a spatula in clockwise followed anti-clockwise direction, 5 rounds in each direction. 7 Close the lid and incubate the substrate in a water bath at 70°C for 30 minutes. Keep mixing the substrate as mentioned in step 6 mixing after every 10 minutes. [For control, skip this step and go directly to step 8] 8 Add 30ml of boiling water to the substrate, mix it with a spatula and transfer it into a water bath at 900C for 15 minutes for first conditioning and to stop the enzyme reaction. 9 Press the substrate using a para-press at 4 Kg/cm2 for 25 seconds. (Take 25 seconds to increase the pressure to 4 Kg/cm 2 , and maintain it for another 25 seconds before releasing the pressure). 10 Manually transfer the pressed extract into a pre-weighed 250ml conical bottom centrifuge tubes. 11 Put the pressed fiber cake in the substrate vessel and add 50ml of boiling water to it. 12 Using a spatula, try to open the compact fiber cake such that it is completely soaked in the water. 13 Put the container back at 900C for 15 minutes for second conditioning. 14 Press the fiber cake using a para-press at 4 Kg/cm 2 for 25 seconds. (Take 25 seconds to increase the pressure to 4 Kg/cm 2 , and maintain it for another 25 seconds before releasing the pressure). 15 Add the collected extract to the respective centrifuge tube (containing the extract collected from step 9). 16 Note the weight of tube containing the extract. 17 Put the tube in a water bath at 90'C for 30 minutes for clarification. 18 Centrifuge the clarified extract at 5000 r.p.m. for 10 minutes in a swinging bucket type rotor. 19 Using a glass pipette and pipette controller, pipette out the complete top oil layer into a pre-weighed petri dish. Make sure that the sludge layer is not disturbed during the process and no sludge particles come with the oil. 20 Aspirate some boiling water in the pipette and slowly dispense it into a waste beaker. A layer of oil will form on the water while dispensing. Add this oil layer to the rest of the oil collected in the petri dish. 21 Note the weight of centrifuge tube containing the sludge. 22 Note the weight of petri plate containing the oil.

The oil yield can by calculated by: Oil yield = Weight of petri dish containing oil extracted - Weight of empty petri dish

Example 3

Oil yield in gram from a 50 gram mashed substrate was compared on a batch sterilized substrate obtained from Ribubonus mill, Malaysia. The substrate comprising palm oil was prepared from palm fruitlets following the method described in example 1. The substrate was then digested and pressed as described in example 2. The experiment was carried out three times for each sample and the result is given as the average of the three oil yields. For the samples, where enzyme composition was added, the enzyme addition was done in step 5 as described in example 2. Control sample was without enzyme addition. Samples Dosage (ppm) Oil yield in gram

Control 0 18.6 ±0.1 Palmora© OER 1.0 300 19.3± 0.3 Palmora© OER 1.0 750 20.1± 0.2 Enzyme composition comprising SEQ ID NO: 1 and SEQ ID NO: 300 20.5± 0.2 4 Palmora© OER 1.0 is a commercial product sold by Novozymes A/S

Oil yield in gram from a 50 gram mashed substrate was compared on a continuous mill sterilized substrate obtained from AIP mill, Bengkulu, Indonesia. The mashed substrate was obtained from a continuous sterilizer at the mentioned mill and kept at 4°C until the substrate wash then digested and pressed as described in example 2. The experiment was carried out three times for each sample and the result is given as the average of the three oil yields. For the samples, where enzyme composition was added, the enzyme addition was done in step 5 as described in example 2. Control sample was without enzyme addition.

Samples Dosage (ppm) Oil yield in gram Control 0 12.6 ±0.2 Palmora© OER 1.0 300 13.5 ±0.3 Palmora© OER 1.0 750 15.0 ±0.2 Enzyme composition comprising SEQ ID NO: 1 and SEQ ID NO: 300 14.8 ±0.1 4 Palmora© OER 1.0 is a commercial product sold by Novozymes A/S

As seen from the above two tables, the enzyme composition comprising SEQ ID NO: 1 and SEQ ID NO: 4 performed equally well on substrate from both batch and continuous sterilizer. And the enzyme composition was comparable to Palmora© OER 1.0 at a dosage of 750 ppm and also performed better than current dosage of Palmora© OER 1.0 at 300ppm.

Example 4 Oil yield in gram from a 50 gram mashed substrate was compared on a batch sterilized substrate obtained from Ribubonus mill, Malaysia.

This experiment was done to demonstrate robustness of the enzyme composition comprising SEQ ID NO: 1 and SEQ ID NO: 4 by incubating for 15 and 30 minutes. The objective was to simulate digester residence time with enzyme for lowered time vs. higher time. The mashed substrate was prepared following method listed in example 1. The experiment was carried out three times for each sample and the result is given as the average of the three oil yields. For the samples, where enzyme composition was added, the enzyme addition was done in step 5 as described in example 2. Control sample was without enzyme addition. For experiments with an incubation time of 15 minutes, incubation was carried out for 15 minutes instead of the 30 minutes stated in step 7 of example 2.

A comparison of oil yield of the mashed substrate was done as against Palmora© OER 1.0 for 15 and 30 minutes respectively.

Samples Dosage Oil yield in Oil yield in (ppm) gram gram (15 min) (30 min) Control 18.6 ±0.1 18.7 ±0.1 Palmora© OER 1.0 300 19.1 ±0.2 19.6 ±0.2 Palmora© OER 1.0 750 19.9 ±0.3 20.1 ±0.3 Enzyme composition comprising SEQ ID NO: 1 and 300 20.0 ±0.2 20.5 ±0.2 SEQ ID NO: 4

Example 5 Contributing enzyme activities enzyme composition

Oil yield in gram from a 50 gram mashed substrate was compared on a batch sterilized substrate obtained from Ribubonus mill, Malaysia. The substrate comprising palm oil was prepared from palm fruitlets following the method described in example 1. The substrate was then digested and pressed as described in example 2. The experiment was carried out three times for each sample and the result is given as the average of the three oil yields. For the samples, where enzyme composition was added, the enzyme addition was done in step 5 as described in example 2. Control sample was without enzyme addition.

Samples Dosage Oil yield in (ppm) gram Control 19.4 ±0.2

Enzyme composition comprising SEQ ID NO: 1 and SEQ ID NO: 300 20.1 ±0.1 4 Enzyme composition comprising SEQ ID NO: 1 11.52 20.2 ±0.3 Enzyme composition comprising SEQ ID NO: 4 3.77 19.2 ±0.1 Enzyme composition comprising SEQ ID NO: 1 and SEQ ID NO: 11.52+3.77 20.2 ±0.1 4

Example 6 Determination of Td by Differential Scanning Calorimetry. The thermostability of SEQ ID NO: 1 was determined by Differential Scanning Calorimetry (DSC) using a VP-Capillary Differential Scanning Calorimeter (MicroCal Inc., Piscataway, NJ, USA). The thermal denaturation temperature, Td (°C), was taken as the top of denaturation peak (major endothermic peak) in thermograms (Cp vs. T) obtained after heating enzyme solutions (approx. 0.5 mg/ml) in buffer (50 mM acetate buffer pH 5.0) at a constant programmed heating rate of 200 K/hr. Sample- and reference-solutions (approx. 0.2 ml) were loaded into the calorimeter (reference: bufferwithout enzyme)from storage conditions at 10°C and thermally pre-equilibrated for 20 minutes at 20°C prior to DSC scan from 20°C to 105°C. Denaturation temperatures were determined at an accuracy of approximately +/- 1C. Td obtained under these conditions for SEQ ID NO: 1 was approx. 89°C.

Example 7 Preparation of substrate. The sterilized palm fruit mesocarp is pressed. Step Action 1 Mash required amount of mesocarp in mash bath at -200 r.p.m. for: a. 3 minutes, if mesocarp quantity is more than 2 Kilograms. b. 2 minutes, if mesocarp quantity is less than 2 Kilograms.

2 Manually mix the mashed mesocarp until it is uniformly mixed

Example 8: Enzymes: GH62 Arabinofuranosidase A: GH62 arabinofuranosidase derived from Aspergillus niger (SEQ ID NO: 7). GH10 Xylanase A: GH10 xylanase derived from Aspergillus niger (SEQ ID NO: 8).

10 qms assay protocol for palm substrate 1. 10g of prepared mash is aliquoted into 50ml Falcon tubes with intermittent mixing to ensure substrate homogeneity. Note down the exact weight of substrate weighed; 2. Also, note down the empty weight of plastic pertriplates that are to be used for collecting extracted oil; 3. Pre-condition the tubes with substrate, keeping them at 90°C for 5 minutes; 4. Transfer the tubes to respective incubation temperature (55°C) water bath and pre condition them for 10 minutes; 5. Inoculate the tubes with 500pL of water in case of Control and 500pL of enzyme solution in case of other enzyme treatments; 6. After adding enzyme/ water, mix the contents with a microspatula 5 times in clock-wise and 5 times in anti-clockwise direction to ensure proper mixing; 7. Incubate for specified time (15 mins / 30mins) with intermittent mixing at every 15th minute of incubation with spatula, as specified in Step 6; 8. At the end of incubation, add 20ml of water into each tube and mix well; 9.For clarification, transfer the tubes to 90°C water bath and allow it to clarify for 30 mins; 10.Centrifuge the tubes in table top centrifuge at 7000 rpm, 30°C for 10 min to get oil layer at the top; 11.Pipette out the oil layer into pre-weighed petriplates. Use hot water to completely extract free oil from each tube; 12. Note down the weight of petriplates with extracted oil; 13. The oil yield can by calculated by: Oil yield = Weight of petri dish containing oil extracted - Weight of empty petri dish. As shown in table below, the addition of GH10 Xylanase A ( a GH10 Xylanase from Aspergillus niger, SEQ ID NO: 8) and GH62 Arabinofuranosidase A (GH62 Arabinofuranosidase from Aspergillus niger, SEQ ID NO 7), lead to an increase the oil yield from 10 gms of palm mesocarp.

Samples Mg Enzyme for 10 g substrate Oil yield in grams Control No enzyme 2.88 ±0.2 GH10 Xylanase A+ 0.12+0.04 3.18 ±0.1 GH62 Arabinofuranosidase A

Example 9: Oil yield Experimental procedure: 50g mesocarp tissue is used as substrate. The mesocarp is generated after separating the nuts from the pressure-cooked oil palm fruits. This separated mesocarp part is then mashed for 2-3min to make a uniform mash. The mash substrate is warmed to 7011C in the pre-set water bath followed by addition of required enzyme dilution. For control, equivalent amount of water is added. The enzyme is manually mixed with the substrate and kept for incubation at 70C for 30min to digest the substrate. Intermittent manual mixing is carried out every 10min interval during incubation period. Then 30ml of boiling water is added to the enzyme substrate mix and transferred it into a water bath at 9011C for 15 minutes to stop the enzyme reaction. The digested mash is then pressed using a mechanical press at 4 Kg/cm2 for 25 seconds and collected the extract in a pre-weighed vessel. The pressed-fiber is washed with 50ml of hot water and further pressed after conditioning the material at 9011C for 15 minutes under same condition and mixed the extract with the respective previous extract. The entire amount of extract was clarified at 901C for 60 min in a water bath under static condition. The clarified material is then cen-trifuged at 5000 rpm at ~ 30DC for 10min in a swing bucket centrifuge. The free oil is pipetted out and weighed in a fine balance. This is assessed for the oil yield.

Table 1. Oil yield

Level Least Sq Mean

Talaromyces Lecyttanus A* 19.54 GH10 (SEQ ID NO. 2)

Aspergillus fumigatus GH10 B 18.92 (SEQ ID NO. 9)

Control B 18.66

*Levels not connected by same letter are significantly different. See also figure 1. The data shows that Talaromyces Leycettanus GH10 (SEQ ID NO. 2) leads to significantly higher oil yields, than both control and Aspergillus fumigatus GH10 (SEQ ID NO. 9).

Example 10: Residual activity of enzyme Set up:

Sample stock: 10 mg/ml enzyme> 0.25mg/ml (5ml) in 50mM Na acetate pH 5 buffer Stress temperature: 50, 60, 70, 80, 90 0C Unstress temperature: 4°C Stress duration: 30 min Sample volume for stress : 100pl ( 0.25 mg/ml) in a PCR Plate Assay

Substrate: 0.2% Arabinoxylan in diluted buffer. 2 4X dilution after stress (1 0pl buffer +40pl sample) dilution buffer: 50Mm Na acetate +0.01% TRITON X Assay:20min 251C @ 800 RPM (2 0pl sample+ 180pl substrate) settling time: 10 min Absorbance read at 590nm using 50ul sample in 384 well plate (50pl) Note: Sample diluted on volume basis. Results:

Table 2- Residual activity (see also Figure 1)

%Residual activity SD

Temperature Aspergillusfumigatus Talaromyces Aspergillus Talaromyces Tpt GH10 (SEQ ID 9) leycettanus fumigatus leycettanus GH10 (SEQ GH10 (SEQ GH10 (SEQ ID NO. 2) ID 9) ID NO. 2) 50 94.6 106.9 6.8 8.8 60 93.9 93.3 16.4 14.6 70 41.1 75.2 3.6 5.6 80 -0.2 29.0 0.8 3.0 90 -0.9 -0.1 0.4 0.2

The results are also shown in Figure 2. The results show that the Talaromyces leycettanus GH10 (SEQ ID NO. 2) displays a significantly higher residual activity at higher temperatures as compared to Aspergillus fumigatus GH 10 (SEQ ID NO. 9).

Example 11: Xylose conversion The enzymes Talaromyces leycettanus GH10 and Rasamsonia byssochamydoides GH10 behave similarly in temperature and pH sensitivity testing, while differing from Aspergillus fumigatus GH10. This is shown below Materials and method: Pretreated corn cobs hydrolysis assay Corn cobs were pretreated with NaOH (0.08 g/g dry weight cobs) at 120[]Cfor60 minutes at 15% total dry weight solids (TS). The resulting material was washed with water until it was pH 8.2, resulting in washed alkaline pretreated corn cobs (APCC). Ground Sieved Alkaline Pretreated Corn Cobs (GS-APCC) was prepared by adjusting the pH of APCC to 5.0 by addition of 6 M HCI and water with extensive mixing, milling APCC in a Cosmos ICMG 40 wet multi-utility grinder (EssEmm Corporation, Tamil Nadu, India), and autoclaving for 45 minutes at 121D C, with a final TS of 3.33%. The hydrolysis of GS-APCC was conducted using 2.2 ml deep-well plates (Axygen, Union City, CA, USA) in a total reaction volume of 1.0 ml. The hydrolysis was performed with 10 mg of GS-APCC total solids per ml of 50 mM sodium acetate (pH 4.0 to 5.5) or 50 mM Tris (pH 6.0 to 7.0) buffer containing 1 mM manganese sulfate and various protein loadings of various enzyme compositions (expressed as mg protein per gram of cellulose). Enzyme compositions were prepared and then added simultaneously to all wells in a volume ranging from 50 pl to 200 pl, for a final volume of 1 ml in each reaction. The plate was then sealed using an ALPS-300 TM plate heat sealer (Abgene, Epsom, United Kingdom), mixed thoroughly, and incubated at a specific temperature for 72 hours. All experiments reported were performed in triplicate. Following hydrolysis, samples were filtered using a 0.45 pm MULTISCREEN@ 96-well filter plate (Millipore, Bedford, MA, USA) and filtrates were analyzed for sugar content as described below. When not used immediately, filtered aliquots were frozen at -20°C. The sugar concentrations of samples diluted in 0.005 M H2SO4 were measured using a 4.6 x 250 mm AMINEX@ HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA) by elution with 0.05% w/w benzoic acid-0.005 M H2SO4 at 65°C at a flow rate of 0.6 ml per minute, and quantitation by integration of the glucose, cellobiose, and xylose signals from refractive index detection (CHEMSTATION@, AGILENT@ 1100 HPLC, Agilent Technologies, Santa Clara, CA, USA) calibrated by pure sugar samples. The resultant glucose equivalents were used to calculate the percentage of cellulose conversion for each reaction. The resultant xylose equivalents were used to calculate the percentage of xylo-oligosaccharide conversion for each reaction. Glucose, cellobiose, and xylose were measured individually. Measured sugar concentrations were adjusted for the appropriate dilution factor. All HPLC data processing was performed using MICROSOFT EXCEL TM software (Microsoft, Richland, WA, USA). The degree of xylo-oligosaccharide conversion to xylose was calculated using the following equation: % xylose conversion = xylose concentration/xylose concentration in a limit digest. In order to calculate % conversion, a 100% conversion point was set based on a cellulase control (100 mg of Trichoderma reesei cellulase supplemented with P. emersonii GH61A polypeptide (WO 2011/041397), A. fumigatus GH10 xylanase (xyn3) (WO 2006/078256), and T. emersonii GH3 beta-xylosidase (WO 2003/070956) per gram cellulose), and all values were divided by this number and then multiplied by 100. The % relative activity for each temperature was calculated using the following equation: % relative activity = (% xylose conversion of a xylanase at a certain pH and temperature - % xylose conversion of beta-xylosidase at that certain pH and temperature) / (% xylose conversion of the xylanase for the pH and temperature containing the highest % xylose conversion - % xylose conversion of beta-xylosidase for the pH and temperature containing the highest % xylose conversion) X 100 Results Table 3: % xylose conversion at ph 4.0 and varied temperatures (see also fig 3)

Enzyme 50°C 55°C 60°C 65°C (% relative xylose conversion)

Aspergillus 55 49 42 41 fumigatus GH10 (SEQ ID NO 9) Talaromyces 76 76 76 78 Lecyttanus GH10 (SEQ ID NO. 2) Rasamsonia 70 72 78 83 byssochlamydoides GH10 (SEQ ID NO. 3) It can be seen that while both Talaromyces Leycettanus GH10 (SEQ ID NO. 1) and Rasamsonia byssochlamydoides GH10 (SEQ ID NO. 3) display maintained higher levels of xylose conversion, the xylose conversion by Aspergillus fumigatus GH10 (SEQ ID NO 9) decreases as the temperature increases.

Table 4: % xylose conversion at Temp and varied pH (see also figure 4)

Enzyme pH 4 pH 4.5 0C pH 5.0 pH 5.5 pH 6.0 pH 7.0 (% relative xylose conversion) Aspergillus 41 53 66 64 63 14 fumigatus GH10 (SEQ ID NO 9) Talaromyces 78 82 81 70 77 17 Lecyttanus GH10 (SEQ ID NO. 2) Rasamsonia 83 81 77 65 70 17 byssochlamydoides GH10 (SEQ ID NO. 3)

These experiments demonstrate the similar activity profile of Talaromyces Leycettanus GH10 (SEQ ID NO. 2) and Rasamsonia byssochlamydoides GH10 (SEQ ID NO. 3); and that this activity profile is different from that of Aspergillus fumigatus (SEQ ID NO. 9).

Example 12: Preparation of Talaromyces leycettanus GH10 xylanase (SEQ ID NO 1) The Talaromyces leycettanus GH10 xylanase (SEQ ID NO 1) was prepared recombinantly and purified according to WO 2013/019827 using Aspergillus oryzae as a host.

Example 13: Preparation of Rasamsonia byssochamydoidesGH10 xylanase (SEQ ID NO 3) The Rasamsonia byssochlamydoides GH10 xylanase (SEQ ID NO. 3) was prepared recombinantly and purified according to WO 2014/182990 using Aspergillus oryzae as a host.

Example 14: Preparation of Aspergillusfumigatus GH10 xylanase (SEQ ID NO. 9) Aspergillus fumigatus NN055679 GH10 xylanase (xyn3) (GENESEQP:AEC74753) was prepared recombinantly according to WO 2006/078256 using Aspergillus oryzae BECh2 (WO 2000/39322) as a host. The filtered broth was desalted and buffer-exchanged into 50 mM sodium acetate pH 5.0 using a HIPREP@26/10 Desalting Column (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's instructions. Protein concentration was determined using a Microplate BCATM Protein Assay Kit with bovine serum albumin as a protein standard.

Example 15: Preparation of Talaromyces emersonii CBS 393.64 GH3 beta-xylosidase (SEDQ ID NO. 10) A Talaromyces emersonii CBS 393.64 beta-xylosidase (GENESEQP:AZI104896) was prepared recombinantly according to Rasmussen et al., 2006, Biotechnology and Bioengineering 94: 869-876 using Aspergillus oryzae JaL355 as a host (WO 2003/070956). The filtered broth was concentrated and desalted with 50 mM sodium acetate pH 5.0 using a tangential flow concentrator equipped with a 10 kDa polyethersulfone membrane. Protein concentration was determined using a Microplate BCA TM Protein Assay Kit (Thermo Fischer Scientific, Waltham, MA, USA) in which bovine serum albumin was used as a protein standard eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt SEQUENCE LISTING SEQUENCE LISTING

<110> Novozymes A/S <110> Novozymes A/S <120> Enzyme assisted crude palm oil extraction <120> Enzyme assisted crude palm oil extraction

<130> 14637‐WO‐PCT <130> 14637-WO-PCT

<140> XX <140> XX <141> 2018‐08‐24 <141> 2018-08-24

<160> 10 <160> 10

<170> PatentIn version 3.5 <170> PatentIn version 3.5

<210> 1 <210> 1 <211> 385 <211> 385 <212> PRT <212> PRT <213> Talaromyces leycettanus <213> Talaromyces leycettanus

<400> 1 <400> 1

Ala Gly Leu Asn Thr Ala Ala Lys Ala Ile Gly Lys Leu Tyr Phe Gly Ala Gly Leu Asn Thr Ala Ala Lys Ala Ile Gly Lys Leu Tyr Phe Gly 1 5 10 15 1 5 10 15

Thr Ala Thr Asp Asn Pro Glu Leu Ser Asp Ser Thr Tyr Met Gln Glu Thr Ala Thr Asp Asn Pro Glu Leu Ser Asp Ser Thr Tyr Met Gln Glu 20 25 30 20 25 30

Thr Asp Asn Thr Asp Asp Phe Gly Gln Leu Thr Pro Ala Asn Ser Met Thr Asp Asn Thr Asp Asp Phe Gly Gln Leu Thr Pro Ala Asn Ser Met 35 40 45 35 40 45

Lys Trp Asp Ala Thr Glu Pro Ser Gln Asn Thr Phe Thr Phe Thr Asn Lys Trp Asp Ala Thr Glu Pro Ser Gln Asn Thr Phe Thr Phe Thr Asn 50 55 60 50 55 60

Gly Asp Gln Ile Ala Asn Leu Ala Lys Ser Asn Gly Gln Met Leu Arg Gly Asp Gln Ile Ala Asn Leu Ala Lys Ser Asn Gly Gln Met Leu Arg 65 70 75 80 70 75 80

Cys His Asn Leu Val Trp Tyr Asn Gln Leu Pro Ser Trp Val Thr Ser Cys His Asn Leu Val Trp Tyr Asn Gln Leu Pro Ser Trp Val Thr Ser 85 90 95 85 90 95

Gly Ser Trp Thr Asn Ala Thr Leu Leu Ala Ala Met Lys Asn His Ile Gly Ser Trp Thr Asn Ala Thr Leu Leu Ala Ala Met Lys Asn His Ile 100 105 110 100 105 110

Thr Asn Val Val Thr His Tyr Lys Gly Gln Cys Tyr Ala Trp Asp Val Thr Asn Val Val Thr His Tyr Lys Gly Gln Cys Tyr Ala Trp Asp Val 115 120 125 115 120 125

Page 1 Page 1 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt

Val Asn Glu Ala Leu Asn Asp Asp Gly Thr Tyr Arg Ser Asn Val Phe Val Asn Glu Ala Leu Asn Asp Asp Gly Thr Tyr Arg Ser Asn Val Phe 130 135 140 130 135 140

Tyr Gln Tyr Ile Gly Glu Ala Tyr Ile Pro Ile Ala Phe Ala Thr Ala Tyr Gln Tyr Ile Gly Glu Ala Tyr Ile Pro Ile Ala Phe Ala Thr Ala 145 150 155 160 145 150 155 160

Ala Ala Ala Asp Pro Asn Ala Lys Leu Tyr Tyr Asn Asp Tyr Asn Ile Ala Ala Ala Asp Pro Asn Ala Lys Leu Tyr Tyr Asn Asp Tyr Asn Ile 165 170 175 165 170 175

Glu Tyr Pro Gly Ala Lys Ala Thr Ala Ala Gln Asn Ile Val Lys Met Glu Tyr Pro Gly Ala Lys Ala Thr Ala Ala Gln Asn Ile Val Lys Met 180 185 190 180 185 190

Val Lys Ala Tyr Gly Ala Lys Ile Asp Gly Val Gly Leu Gln Ser His Val Lys Ala Tyr Gly Ala Lys Ile Asp Gly Val Gly Leu Gln Ser His 195 200 205 195 200 205

Phe Ile Val Gly Ser Thr Pro Ser Gln Ser Ser Gln Gln Ser Asn Met Phe Ile Val Gly Ser Thr Pro Ser Gln Ser Ser Gln Gln Ser Asn Met 210 215 220 210 215 220

Ala Ala Phe Thr Ala Leu Gly Val Glu Val Ala Ile Thr Glu Leu Asp Ala Ala Phe Thr Ala Leu Gly Val Glu Val Ala Ile Thr Glu Leu Asp 225 230 235 240 225 230 235 240

Ile Arg Met Thr Leu Pro Ser Thr Ser Ala Leu Leu Ala Gln Gln Ser Ile Arg Met Thr Leu Pro Ser Thr Ser Ala Leu Leu Ala Gln Gln Ser 245 250 255 245 250 255

Thr Asp Tyr Gln Ser Thr Val Ser Ala Cys Val Asn Thr Pro Lys Cys Thr Asp Tyr Gln Ser Thr Val Ser Ala Cys Val Asn Thr Pro Lys Cys 260 265 270 260 265 270

Ile Gly Ile Thr Leu Trp Asp Trp Thr Asp Lys Tyr Ser Trp Val Pro Ile Gly Ile Thr Leu Trp Asp Trp Thr Asp Lys Tyr Ser Trp Val Pro 275 280 285 275 280 285

Asn Thr Phe Ser Gly Gln Gly Asp Ala Cys Pro Trp Asp Ser Asn Tyr Asn Thr Phe Ser Gly Gln Gly Asp Ala Cys Pro Trp Asp Ser Asn Tyr 290 295 300 290 295 300

Gln Lys Lys Pro Ala Tyr Tyr Gly Ile Leu Thr Ala Leu Gly Gly Ser Gln Lys Lys Pro Ala Tyr Tyr Gly Ile Leu Thr Ala Leu Gly Gly Ser 305 310 315 320 305 310 315 320

Ala Ser Thr Ser Thr Thr Thr Thr Leu Val Thr Ser Thr Arg Thr Ser Ala Ser Thr Ser Thr Thr Thr Thr Leu Val Thr Ser Thr Arg Thr Ser 325 330 335 325 330 335 Page 2 Page 2 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt

Thr Thr Thr Ser Thr Ser Ala Thr Ser Thr Ser Thr Gly Val Ala Gln Thr Thr Thr Ser Thr Ser Ala Thr Ser Thr Ser Thr Gly Val Ala Gln 340 345 350 340 345 350

His Trp Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro Thr Thr Cys His Trp Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro Thr Thr Cys 355 360 365 355 360 365

Ala Ser Pro Tyr Thr Cys Gln Glu Leu Asn Pro Tyr Tyr Tyr Gln Cys Ala Ser Pro Tyr Thr Cys Gln Glu Leu Asn Pro Tyr Tyr Tyr Gln Cys 370 375 380 370 375 380

Leu Leu 385 385

<210> 2 <210> 2 <211> 373 <211> 373 <212> PRT <212> PRT <213> Talaromyces leycettanus <213> Talaromyces leycettanus

<400> 2 <400> 2

Ala Ala Val Asp Leu Gln Ser Arg Gln Ala Ala Gln Ser Ile Asn Thr Ala Ala Val Asp Leu Gln Ser Arg Gln Ala Ala Gln Ser Ile Asn Thr 1 5 10 15 1 5 10 15

Leu Ile Gln Ala Lys Gly Lys Lys Tyr Trp Gly Thr Cys Ala Asp Glu Leu Ile Gln Ala Lys Gly Lys Lys Tyr Trp Gly Thr Cys Ala Asp Glu 20 25 30 20 25 30

Gly Arg Leu Thr Glu Asn Ser Gln Asn Pro Ala Ile Ala Lys Ala Asp Gly Arg Leu Thr Glu Asn Ser Gln Asn Pro Ala Ile Ala Lys Ala Asp 35 40 45 35 40 45

Phe Gly Gln Val Thr Pro Glu Asn Ser Met Lys Trp Asp Ala Thr Glu Phe Gly Gln Val Thr Pro Glu Asn Ser Met Lys Trp Asp Ala Thr Glu 50 55 60 50 55 60

Pro Ser Gln Gly Gln Phe Asn Phe Ala Gln Ala Asp Trp Leu Val Asn Pro Ser Gln Gly Gln Phe Asn Phe Ala Gln Ala Asp Trp Leu Val Asn 65 70 75 80 70 75 80

Trp Ala Gln Gln Asn Gly Lys Leu Ile Arg Gly His Asn Leu Val Trp Trp Ala Gln Gln Asn Gly Lys Leu Ile Arg Gly His Asn Leu Val Trp 85 90 95 85 90 95

His Ser Gln Leu Pro Ser Trp Val Cys Gly Ile Thr Asp Lys Thr Ala His Ser Gln Leu Pro Ser Trp Val Cys Gly Ile Thr Asp Lys Thr Ala 100 105 110 100 105 110

Page 3 Page 3 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt

Leu Thr Asn Ala Met Thr Asn His Ile Thr Thr Leu Val Ser Arg Tyr Leu Thr Asn Ala Met Thr Asn His Ile Thr Thr Leu Val Ser Arg Tyr 115 120 125 115 120 125

Lys Gly Lys Ile Tyr Ala Trp Asp Val Val Asn Glu Pro Phe Asn Glu Lys Gly Lys Ile Tyr Ala Trp Asp Val Val Asn Glu Pro Phe Asn Glu 130 135 140 130 135 140

Asp Gly Ser Leu Arg Gln Thr Cys Phe Tyr Asn Val Ile Gly Pro Asp Asp Gly Ser Leu Arg Gln Thr Cys Phe Tyr Asn Val Ile Gly Pro Asp 145 150 155 160 145 150 155 160

Tyr Ile Lys Ile Ala Phe Gln Thr Ala Arg Ala Ala Asp Pro Asn Ala Tyr Ile Lys Ile Ala Phe Gln Thr Ala Arg Ala Ala Asp Pro Asn Ala 165 170 175 165 170 175

Lys Leu Tyr Val Asn Asp Tyr Asn Leu Asp Ser Ala Ser Tyr Ala Lys Lys Leu Tyr Val Asn Asp Tyr Asn Leu Asp Ser Ala Ser Tyr Ala Lys 180 185 190 180 185 190

Thr Thr Gly Val Ala Asn Gln Val Lys Gln Trp Ile Ala Gln Gly Val Thr Thr Gly Val Ala Asn Gln Val Lys Gln Trp Ile Ala Gln Gly Val 195 200 205 195 200 205

Pro Ile Asp Gly Ile Gly Ser Glu Ser His Leu Ser Ala Gly Ala Gly Pro Ile Asp Gly Ile Gly Ser Glu Ser His Leu Ser Ala Gly Ala Gly 210 215 220 210 215 220

Ala Gly Val Pro Ala Ala Leu Gln Val Leu Ala Asn Ser Gly Val Ser Ala Gly Val Pro Ala Ala Leu Gln Val Leu Ala Asn Ser Gly Val Ser 225 230 235 240 225 230 235 240

Glu Val Ala Ile Thr Glu Leu Asp Ile Ala Gln Ala Ser Ser Thr Asp Glu Val Ala Ile Thr Glu Leu Asp Ile Ala Gln Ala Ser Ser Thr Asp 245 250 255 245 250 255

Tyr Asp Asn Val Ala Gln Ala Cys Leu Asn Val Ala Lys Cys Val Gly Tyr Asp Asn Val Ala Gln Ala Cys Leu Asn Val Ala Lys Cys Val Gly 260 265 270 260 265 270

Ile Thr Ser Trp Gly Ile Ser Asp Lys Asp Ser Trp Arg Ser Ser Glu Ile Thr Ser Trp Gly Ile Ser Asp Lys Asp Ser Trp Arg Ser Ser Glu 275 280 285 275 280 285

Asn Pro Asp Leu Phe Asp Ser Asn Tyr Gln Pro Lys Ala Ala Tyr Asn Asn Pro Asp Leu Phe Asp Ser Asn Tyr Gln Pro Lys Ala Ala Tyr Asn 290 295 300 290 295 300

Ala Leu Val Thr Leu Leu Gly Gly Ser Ser Gly Ser Gly Ser Gly Ser Ala Leu Val Thr Leu Leu Gly Gly Ser Ser Gly Ser Gly Ser Gly Ser 305 310 315 320 305 310 315 320

Page 4 Page 4 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 325 330 335 325 330 335

Gly Gln Ala Gln His Trp Gly Gln Cys Gly Gly Glu Gly Trp Thr Gly Gly Gln Ala Gln His Trp Gly Gln Cys Gly Gly Glu Gly Trp Thr Gly 340 345 350 340 345 350

Pro Thr Ser Cys Val Ser Pro Tyr Thr Cys Gln Tyr Gln Asn Gln Trp Pro Thr Ser Cys Val Ser Pro Tyr Thr Cys Gln Tyr Gln Asn Gln Trp 355 360 365 355 360 365

Tyr Ser Gln Cys Leu Tyr Ser Gln Cys Leu 370 370

<210> 3 <210> 3 <211> 383 <211> 383 <212> PRT <212> PRT <213> Rasamsonia byssochlamydoides <213> Rasamsonia byssochlamydoides

<400> 3 <400> 3

Asp Gly Leu Asn Thr Ala Ala Lys Ala Ile Gly Lys Leu Tyr Phe Gly Asp Gly Leu Asn Thr Ala Ala Lys Ala Ile Gly Lys Leu Tyr Phe Gly 1 5 10 15 1 5 10 15

Thr Ala Thr Asp Asn Pro Glu Leu Ser Asp Val Ala Tyr Glu Thr Gln Thr Ala Thr Asp Asn Pro Glu Leu Ser Asp Val Ala Tyr Glu Thr Gln 20 25 30 20 25 30

Leu Asn Asn Thr Gln Asp Phe Gly Gln Ile Thr Pro Ala Asn Ser Met Leu Asn Asn Thr Gln Asp Phe Gly Gln Ile Thr Pro Ala Asn Ser Met 35 40 45 35 40 45

Lys Trp Asp Ala Thr Glu Pro Glu Gln Asn Thr Phe Thr Phe Ala Ala Lys Trp Asp Ala Thr Glu Pro Glu Gln Asn Thr Phe Thr Phe Ala Ala 50 55 60 50 55 60

Gly Asp Gln Ile Ala Asp Leu Ala Glu Ala Asn Gly Gln Ile Leu Arg Gly Asp Gln Ile Ala Asp Leu Ala Glu Ala Asn Gly Gln Ile Leu Arg 65 70 75 80 70 75 80

Cys His Asn Leu Val Trp Tyr Asn Gln Leu Pro Ser Trp Val Thr Ser Cys His Asn Leu Val Trp Tyr Asn Gln Leu Pro Ser Trp Val Thr Ser 85 90 95 85 90 95

Gly Ser Trp Thr Asn Glu Thr Leu Leu Ala Ala Met Lys Asn His Ile Gly Ser Trp Thr Asn Glu Thr Leu Leu Ala Ala Met Lys Asn His Ile 100 105 110 100 105 110

Page 5 Page 5 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) . txt Thr Asn Val Val Thr His Tyr Lys Gly Arg Cys Tyr Ala Trp Asp Val Thr Asn Val Val Thr His Tyr Lys Gly Arg Cys Tyr Ala Trp Asp Val 115 120 125 115 120 125

Val Asn Glu Ala Leu Asn Asp Asp Gly Thr Tyr Arg Asp Asn Val Phe Val Asn Glu Ala Leu Asn Asp Asp Gly Thr Tyr Arg Asp Asn Val Phe 130 135 140 130 135 140

Tyr Gln Tyr Ile Gly Glu Ala Tyr Ile Pro Ile Ala Phe Glu Thr Ala Tyr Gln Tyr Ile Gly Glu Ala Tyr Ile Pro Ile Ala Phe Glu Thr Ala 145 150 155 160 145 150 155 160

Ala Ala Ala Asp Pro Asn Val Lys Leu Tyr Tyr Asn Asp Tyr Asn Ile Ala Ala Ala Asp Pro Asn Val Lys Leu Tyr Tyr Asn Asp Tyr Asn Ile 165 170 175 165 170 175

Glu Tyr Ala Gly Val Lys Ala Thr Ala Ala Gln Asn Ile Val Lys Leu Glu Tyr Ala Gly Val Lys Ala Thr Ala Ala Gln Asn Ile Val Lys Leu 180 185 190 180 185 190

Val Gln Ser Tyr Gly Ala Arg Ile Asp Gly Val Gly Leu Gln Ser His Val Gln Ser Tyr Gly Ala Arg Ile Asp Gly Val Gly Leu Gln Ser His 195 200 205 195 200 205

Phe Ile Val Gly Glu Thr Pro Ser Thr Ser Thr Gln Ala Ser Asn Met Phe Ile Val Gly Glu Thr Pro Ser Thr Ser Thr Gln Ala Ser Asn Met 210 215 220 210 215 220

Ala Ser Phe Thr Ala Leu Gly Val Glu Val Ala Ile Thr Glu Leu Asp Ala Ser Phe Thr Ala Leu Gly Val Glu Val Ala Ile Thr Glu Leu Asp 225 230 235 240 225 230 235 240

Ile Arg Met Gln Leu Pro Glu Thr Thr Ala Leu Leu Thr Gln Gln Ser Ile Arg Met Gln Leu Pro Glu Thr Thr Ala Leu Leu Thr Gln Gln Ser 245 250 255 245 250 255

Thr Asp Tyr Gln Ser Thr Val Gln Ala Cys Val Asn Thr Pro Gly Cys Thr Asp Tyr Gln Ser Thr Val Gln Ala Cys Val Asn Thr Pro Gly Cys 260 265 270 260 265 270

Val Gly Ile Thr Leu Trp Asp Trp Thr Asp Lys Tyr Ser Trp Val Pro Val Gly Ile Thr Leu Trp Asp Trp Thr Asp Lys Tyr Ser Trp Val Pro 275 280 285 275 280 285

Ser Thr Phe Ser Gly Tyr Gly Asp Ala Cys Pro Trp Asp Asp Asn Tyr Ser Thr Phe Ser Gly Tyr Gly Asp Ala Cys Pro Trp Asp Asp Asn Tyr 290 295 300 290 295 300

Gln Lys Lys Pro Ala Tyr Tyr Gly Ile Leu Thr Ala Leu Gly Gly Ser Gln Lys Lys Pro Ala Tyr Tyr Gly Ile Leu Thr Ala Leu Gly Gly Ser 305 310 315 320 305 310 315 320

Page 6 Page 6 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) . txt Ala Ser Thr Thr Thr Val Gly Thr Gly Thr Thr Thr Thr Ser Thr Ala Ala Ser Thr Thr Thr Val Gly Thr Gly Thr Thr Thr Thr Ser Thr Ala 325 330 335 325 330 335

Thr Thr Ser Ser Thr Gly Ser Ser Gly Thr Gly Val Ala Gln His Trp Thr Thr Ser Ser Thr Gly Ser Ser Gly Thr Gly Val Ala Gln His Trp 340 345 350 340 345 350

Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro Thr Val Cys Ala Ser Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro Thr Val Cys Ala Ser 355 360 365 355 360 365

Gly Tyr Thr Cys Thr Val Val Asn Pro Tyr Tyr Ser Gln Cys Leu Gly Tyr Thr Cys Thr Val Val Asn Pro Tyr Tyr Ser Gln Cys Leu 370 375 380 370 375 380

<210> 4 <210> 4 <211> 302 <211> 302 <212> PRT <212> PRT <213> Talaromyces pinophilus <213> Talaromyces pinophilus

<400> 4 <400> 4

Ser Gly Cys Ala Leu Pro Ser Thr Tyr Lys Trp Thr Ser Thr Gly Pro Ser Gly Cys Ala Leu Pro Ser Thr Tyr Lys Trp Thr Ser Thr Gly Pro 1 5 10 15 1 5 10 15

Leu Ala Ser Pro Lys Ser Gly Leu Val Ala Leu Arg Asp Tyr Ser His Leu Ala Ser Pro Lys Ser Gly Leu Val Ala Leu Arg Asp Tyr Ser His 20 25 30 20 25 30

Val Ile Tyr Asn Gly Gln His Leu Val Tyr Gly Ser Thr Ala Asn Thr Val Ile Tyr Asn Gly Gln His Leu Val Tyr Gly Ser Thr Ala Asn Thr 35 40 45 35 40 45

Ala Gly Ser Tyr Gly Ser Met Asn Phe Gly Leu Phe Ser Asp Trp Ser Ala Gly Ser Tyr Gly Ser Met Asn Phe Gly Leu Phe Ser Asp Trp Ser 50 55 60 50 55 60

Glu Met Ser Ser Ala Ser Gln Asn Thr Met Ser Thr Gly Ala Val Ala Glu Met Ser Ser Ala Ser Gln Asn Thr Met Ser Thr Gly Ala Val Ala 65 70 75 80 70 75 80

Pro Thr Ile Phe Tyr Phe Ala Pro Lys Ser Val Trp Ile Leu Ala Tyr Pro Thr Ile Phe Tyr Phe Ala Pro Lys Ser Val Trp Ile Leu Ala Tyr 85 90 95 85 90 95

Gln Trp Gly Pro Tyr Ala Phe Ser Tyr Arg Thr Ser Thr Asp Pro Ser Gln Trp Gly Pro Tyr Ala Phe Ser Tyr Arg Thr Ser Thr Asp Pro Ser 100 105 110 100 105 110

Asn Ala Asn Gly Trp Ser Ser Pro Gln Pro Leu Phe Thr Gly Thr Ile Asn Ala Asn Gly Trp Ser Ser Pro Gln Pro Leu Phe Thr Gly Thr Ile Page 7 Page 7 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt 115 120 125 115 120 125

Ser Gly Ser Ser Thr Gly Val Ile Asp Gln Thr Val Ile Gly Asp Ser Ser Gly Ser Ser Thr Gly Val Ile Asp Gln Thr Val Ile Gly Asp Ser 130 135 140 130 135 140

Glu Asn Met Tyr Leu Phe Phe Ala Gly Asp Asn Gly His Ile Tyr Arg Glu Asn Met Tyr Leu Phe Phe Ala Gly Asp Asn Gly His Ile Tyr Arg 145 150 155 160 145 150 155 160

Ala Ser Met Pro Ile Gly Asp Phe Pro Gly Ser Phe Gly Ser Ala Ser Ala Ser Met Pro Ile Gly Asp Phe Pro Gly Ser Phe Gly Ser Ala Ser 165 170 175 165 170 175

Thr Ile Val Leu Ser Asp Ser Thr Asn Asn Leu Phe Glu Ala Val Glu Thr Ile Val Leu Ser Asp Ser Thr Asn Asn Leu Phe Glu Ala Val Glu 180 185 190 180 185 190

Val Tyr Thr Val Glu Gly Gln Asn Gln Tyr Leu Met Ile Val Glu Ala Val Tyr Thr Val Glu Gly Gln Asn Gln Tyr Leu Met Ile Val Glu Ala 195 200 205 195 200 205

Ile Gly Ala Asn Gly Arg Tyr Phe Arg Ser Phe Thr Ala Ser Ser Leu Ile Gly Ala Asn Gly Arg Tyr Phe Arg Ser Phe Thr Ala Ser Ser Leu 210 215 220 210 215 220

Gly Gly Thr Trp Thr Ala Gln Ala Ser Thr Glu Ser Asn Pro Phe Ala Gly Gly Thr Trp Thr Ala Gln Ala Ser Thr Glu Ser Asn Pro Phe Ala 225 230 235 240 225 230 235 240

Gly Lys Ala Asn Ser Gly Ala Thr Trp Thr Asn Asp Ile Ser Ser Gly Gly Lys Ala Asn Ser Gly Ala Thr Trp Thr Asn Asp Ile Ser Ser Gly 245 250 255 245 250 255

Asp Leu Val Arg Thr Asn Pro Asp Gln Thr Gln Thr Ile Asp Ala Cys Asp Leu Val Arg Thr Asn Pro Asp Gln Thr Gln Thr Ile Asp Ala Cys 260 265 270 260 265 270

Asn Leu Gln Phe Leu Tyr Gln Gly Arg Ser Thr Ser Ser Gly Gly Asp Asn Leu Gln Phe Leu Tyr Gln Gly Arg Ser Thr Ser Ser Gly Gly Asp 275 280 285 275 280 285

Tyr Asn Leu Leu Pro Tyr Gln Pro Gly Leu Leu Thr Leu Ala Tyr Asn Leu Leu Pro Tyr Gln Pro Gly Leu Leu Thr Leu Ala 290 295 300 290 295 300

<210> 5 <210> 5 <211> 302 <211> 302 <212> PRT <212> PRT <213> Penicillium capsulatum <213> Penicillium capsulatum

Page 8 Page 8 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt <400> 5 <400> 5

Asn Cys Ala Leu Pro Ser Thr Tyr Ser Trp Thr Ser Thr Ser Ala Leu Asn Cys Ala Leu Pro Ser Thr Tyr Ser Trp Thr Ser Thr Ser Ala Leu 1 5 10 15 1 5 10 15

Ala Asn Pro Lys Pro Gly Trp Thr Ala Ile Lys Asp Phe Thr Asn Val Ala Asn Pro Lys Pro Gly Trp Thr Ala Ile Lys Asp Phe Thr Asn Val 20 25 30 20 25 30

Val Phe Asn Asn Arg His Val Val Tyr Ala Ser Thr Thr Asp Thr Ser Val Phe Asn Asn Arg His Val Val Tyr Ala Ser Thr Thr Asp Thr Ser 35 40 45 35 40 45

Gly Asn Tyr Gly Ala Met Ser Phe Gly Val Phe Ser Asp Trp Pro Gly Gly Asn Tyr Gly Ala Met Ser Phe Gly Val Phe Ser Asp Trp Pro Gly 50 55 60 50 55 60

Met Ala Ser Ala Ser Gln Asn Ala Leu Ser Phe Ala Ala Val Ala Pro Met Ala Ser Ala Ser Gln Asn Ala Leu Ser Phe Ala Ala Val Ala Pro 65 70 75 80 70 75 80

Thr Leu Phe Tyr Phe Gln Pro Lys Ser Ile Trp Val Leu Ala Tyr Gln Thr Leu Phe Tyr Phe Gln Pro Lys Ser Ile Trp Val Leu Ala Tyr Gln 85 90 95 85 90 95

Trp Gly Ser Ser Thr Phe Thr Tyr Arg Thr Ser Ser Asp Pro Thr Asn Trp Gly Ser Ser Thr Phe Thr Tyr Arg Thr Ser Ser Asp Pro Thr Asn 100 105 110 100 105 110

Ala Tyr Gly Trp Ser Ser Glu Gln Ala Leu Phe Ser Gly Lys Val Thr Ala Tyr Gly Trp Ser Ser Glu Gln Ala Leu Phe Ser Gly Lys Val Thr 115 120 125 115 120 125

Gly Ser Ser Thr Gly Ala Ile Asp Gln Thr Leu Ile Gly Asp Ala Thr Gly Ser Ser Thr Gly Ala Ile Asp Gln Thr Leu Ile Gly Asp Ala Thr 130 135 140 130 135 140

His Met Tyr Leu Phe Phe Ala Gly Asp Asn Gly Lys Ile Tyr Arg Ser His Met Tyr Leu Phe Phe Ala Gly Asp Asn Gly Lys Ile Tyr Arg Ser 145 150 155 160 145 150 155 160

Ser Met Pro Ile Ser Asn Phe Pro Gly Asn Phe Gly Thr Val Ser Glu Ser Met Pro Ile Ser Asn Phe Pro Gly Asn Phe Gly Thr Val Ser Glu 165 170 175 165 170 175

Val Val Leu Ser Asp Thr Gln Asn Asn Leu Phe Glu Ala Val Gln Val Val Val Leu Ser Asp Thr Gln Asn Asn Leu Phe Glu Ala Val Gln Val 180 185 190 180 185 190

Tyr Thr Val Lys Gly Gln Asn Gln Tyr Leu Met Ile Val Glu Ala Ile Tyr Thr Val Lys Gly Gln Asn Gln Tyr Leu Met Ile Val Glu Ala Ile 195 200 205 195 200 205 Page 9 Page 9 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) . txt

Gly Ser Glu Gly Arg Tyr Phe Arg Ser Phe Thr Ala Ser Ser Leu Gly Gly Ser Glu Gly Arg Tyr Phe Arg Ser Phe Thr Ala Ser Ser Leu Gly 210 215 220 210 215 220

Gly Leu Trp Thr Ala Gln Ala Ala Ser Glu Thr Lys Pro Phe Ala Gly Gly Leu Trp Thr Ala Gln Ala Ala Ser Glu Thr Lys Pro Phe Ala Gly 225 230 235 240 225 230 235 240

Lys Ala Asn Ser Gly Ala Thr Trp Thr Asn Asp Ile Ser His Gly Asp Lys Ala Asn Ser Gly Ala Thr Trp Thr Asn Asp Ile Ser His Gly Asp 245 250 255 245 250 255

Leu Val Arg Ser Asn Pro Asp Gln Thr Met Thr Ile Asp Pro Cys Asn Leu Val Arg Ser Asn Pro Asp Gln Thr Met Thr Ile Asp Pro Cys Asn 260 265 270 260 265 270

Leu Gln Phe Leu Tyr Gln Gly Arg Asn Pro Gly Ala Ser Gly Asn Tyr Leu Gln Phe Leu Tyr Gln Gly Arg Asn Pro Gly Ala Ser Gly Asn Tyr 275 280 285 275 280 285

Asn Thr Leu Pro Trp Arg Pro Gly Val Leu Thr Leu Asn Asn Asn Thr Leu Pro Trp Arg Pro Gly Val Leu Thr Leu Asn Asn 290 295 300 290 295 300

<210> 6 <210> 6 <211> 302 <211> 302 <212> PRT <212> PRT <213> Penicillium oxalicum <213> Penicillium oxalicum

<400> 6 <400> 6

Pro Val Pro Ser Gln Gly Gln Tyr Arg Trp Ser Ser Thr Gly Ala Leu Pro Val Pro Ser Gln Gly Gln Tyr Arg Trp Ser Ser Thr Gly Ala Leu 1 5 10 15 1 5 10 15

Ala Gln Pro Gln His Gly Trp Thr Ser Ile Lys Asp Phe Thr Asn Val Ala Gln Pro Gln His Gly Trp Thr Ser Ile Lys Asp Phe Thr Asn Val 20 25 30 20 25 30

Val Tyr Asn Gly Lys His Leu Val Tyr Ala Ser Val Ala Asp Ser Lys Val Tyr Asn Gly Lys His Leu Val Tyr Ala Ser Val Ala Asp Ser Lys 35 40 45 35 40 45

Gly Asn Tyr His Ser Met Asn Phe Gly Leu Phe Ser Asp Trp Ser Gln Gly Asn Tyr His Ser Met Asn Phe Gly Leu Phe Ser Asp Trp Ser Gln 50 55 60 50 55 60

Met Ala Ser Ala Ser Gln Asn Pro Met Asn Phe Asn Ala Val Ala Pro Met Ala Ser Ala Ser Gln Asn Pro Met Asn Phe Asn Ala Val Ala Pro 65 70 75 80 70 75 80

Page 10 Page 10 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt

Thr Leu Phe Phe Phe Ala Pro Lys Asn Val Trp Val Leu Ala Tyr Gln Thr Leu Phe Phe Phe Ala Pro Lys Asn Val Trp Val Leu Ala Tyr Gln 85 90 95 85 90 95

Trp Gly Ala Asn Ala Phe Ser Tyr Arg Thr Ser Asn Asp Pro Ala Asn Trp Gly Ala Asn Ala Phe Ser Tyr Arg Thr Ser Asn Asp Pro Ala Asn 100 105 110 100 105 110

Ala Asn Gly Trp Ser Ser Glu His Pro Leu Phe Thr Gly Lys Ile Ala Ala Asn Gly Trp Ser Ser Glu His Pro Leu Phe Thr Gly Lys Ile Ala 115 120 125 115 120 125

Asn Ser Gly Thr Gly Pro Ile Asp Gln Thr Leu Ile Gly Asp Asn Gln Asn Ser Gly Thr Gly Pro Ile Asp Gln Thr Leu Ile Gly Asp Asn Gln 130 135 140 130 135 140

Asn Met Tyr Leu Phe Phe Ala Gly Asp Asn Gly Lys Ile Tyr Arg Ser Asn Met Tyr Leu Phe Phe Ala Gly Asp Asn Gly Lys Ile Tyr Arg Ser 145 150 155 160 145 150 155 160

Ser Met Pro Leu Asn Asn Phe Pro Gly Ser Phe Gly Gly Ala Ser Glu Ser Met Pro Leu Asn Asn Phe Pro Gly Ser Phe Gly Gly Ala Ser Glu 165 170 175 165 170 175

Val Ile Leu Ser Asp Thr Thr Ala Asn Leu Phe Glu Ala Val Gln Val Val Ile Leu Ser Asp Thr Thr Ala Asn Leu Phe Glu Ala Val Gln Val 180 185 190 180 185 190

Tyr Lys Val Ala Gly Glu Asn Lys Tyr Leu Met Ile Val Glu Ala Met Tyr Lys Val Ala Gly Glu Asn Lys Tyr Leu Met Ile Val Glu Ala Met 195 200 205 195 200 205

Gly Ala His Gly Arg Tyr Phe Arg Ser Phe Thr Ala Thr Ser Leu Asn Gly Ala His Gly Arg Tyr Phe Arg Ser Phe Thr Ala Thr Ser Leu Asn 210 215 220 210 215 220

Gly Lys Trp Thr Leu Asn Ala Gly Ser Glu Gly Ala Pro Phe Ala Gly Gly Lys Trp Thr Leu Asn Ala Gly Ser Glu Gly Ala Pro Phe Ala Gly 225 230 235 240 225 230 235 240

Lys Ala Asn Ser Gly Ala Gly Trp Thr Asn Asp Ile Ser His Gly Asp Lys Ala Asn Ser Gly Ala Gly Trp Thr Asn Asp Ile Ser His Gly Asp 245 250 255 245 250 255

Leu Val Arg Thr Asn Pro Asp Gln Thr Met Thr Val Asp Met Cys Asn Leu Val Arg Thr Asn Pro Asp Gln Thr Met Thr Val Asp Met Cys Asn 260 265 270 260 265 270

Leu Gln Phe Leu Tyr Gln Gly Arg Asp Pro Asn Ala Asn Pro Thr Tyr Leu Gln Phe Leu Tyr Gln Gly Arg Asp Pro Asn Ala Asn Pro Thr Tyr 275 280 285 275 280 285

Page 11 Page 11 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt

Asn Ala Leu Pro Tyr Arg Pro Gly Val Leu Thr Leu Lys His Asn Ala Leu Pro Tyr Arg Pro Gly Val Leu Thr Leu Lys His 290 295 300 290 295 300

<210> 7 <210> 7 <211> 306 <211> 306 <212> PRT <212> PRT <213> Aspergillus Niger <213> Aspergillus Niger

<400> 7 <400> 7

Lys Cys Ser Leu Pro Ser Ser Tyr Ser Trp Ser Ser Thr Asp Ala Leu Lys Cys Ser Leu Pro Ser Ser Tyr Ser Trp Ser Ser Thr Asp Ala Leu 1 5 10 15 1 5 10 15

Ala Thr Pro Lys Ser Gly Trp Thr Ala Leu Lys Asp Phe Thr Asp Val Ala Thr Pro Lys Ser Gly Trp Thr Ala Leu Lys Asp Phe Thr Asp Val 20 25 30 20 25 30

Val Ser Asp Gly Lys His Ile Val Tyr Ala Ser Thr Thr Asp Glu Ala Val Ser Asp Gly Lys His Ile Val Tyr Ala Ser Thr Thr Asp Glu Ala 35 40 45 35 40 45

Gly Asn Tyr Gly Ser Met Thr Phe Gly Ala Phe Ser Glu Trp Ser Asn Gly Asn Tyr Gly Ser Met Thr Phe Gly Ala Phe Ser Glu Trp Ser Asn 50 55 60 50 55 60

Met Ala Ser Ala Ser Gln Thr Ala Thr Pro Phe Asn Ala Val Ala Pro Met Ala Ser Ala Ser Gln Thr Ala Thr Pro Phe Asn Ala Val Ala Pro 65 70 75 80 70 75 80

Thr Leu Phe Tyr Phe Lys Pro Lys Ser Ile Trp Val Leu Ala Tyr Gln Thr Leu Phe Tyr Phe Lys Pro Lys Ser Ile Trp Val Leu Ala Tyr Gln 85 90 95 85 90 95

Trp Gly Ser Ser Thr Phe Thr Tyr Arg Thr Ser Gln Asp Pro Thr Asn Trp Gly Ser Ser Thr Phe Thr Tyr Arg Thr Ser Gln Asp Pro Thr Asn 100 105 110 100 105 110

Val Asn Gly Trp Ser Ser Glu Gln Ala Leu Phe Thr Gly Lys Leu Ser Val Asn Gly Trp Ser Ser Glu Gln Ala Leu Phe Thr Gly Lys Leu Ser 115 120 125 115 120 125

Asp Ser Ser Thr Gly Ala Ile Asp Gln Thr Val Ile Gly Asp Asp Thr Asp Ser Ser Thr Gly Ala Ile Asp Gln Thr Val Ile Gly Asp Asp Thr 130 135 140 130 135 140

Asn Met Tyr Leu Phe Phe Ala Gly Asp Asn Gly Lys Ile Tyr Arg Ser Asn Met Tyr Leu Phe Phe Ala Gly Asp Asn Gly Lys Ile Tyr Arg Ser 145 150 155 160 145 150 155 160

Page 12 Page 12 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt Ser Met Ser Ile Asp Glu Phe Pro Gly Ser Phe Gly Ser Gln Tyr Glu Ser Met Ser Ile Asp Glu Phe Pro Gly Ser Phe Gly Ser Gln Tyr Glu 165 170 175 165 170 175

Glu Ile Leu Ser Gly Ala Thr Asn Asp Leu Phe Glu Ala Val Gln Val Glu Ile Leu Ser Gly Ala Thr Asn Asp Leu Phe Glu Ala Val Gln Val 180 185 190 180 185 190

Tyr Thr Val Asp Gly Gly Glu Gly Asn Ser Lys Tyr Leu Met Ile Val Tyr Thr Val Asp Gly Gly Glu Gly Asn Ser Lys Tyr Leu Met Ile Val 195 200 205 195 200 205

Glu Ala Ile Gly Ser Thr Gly His Arg Tyr Phe Arg Ser Phe Thr Ala Glu Ala Ile Gly Ser Thr Gly His Arg Tyr Phe Arg Ser Phe Thr Ala 210 215 220 210 215 220

Ser Ser Leu Gly Gly Glu Trp Thr Ala Gln Ala Ala Ser Glu Asp Lys Ser Ser Leu Gly Gly Glu Trp Thr Ala Gln Ala Ala Ser Glu Asp Lys 225 230 235 240 225 230 235 240

Pro Phe Ala Gly Lys Ala Asn Ser Gly Ala Thr Trp Thr Glu Asp Ile Pro Phe Ala Gly Lys Ala Asn Ser Gly Ala Thr Trp Thr Glu Asp Ile 245 250 255 245 250 255

Ser His Gly Asp Leu Val Arg Asn Asn Pro Asp Gln Thr Met Thr Val Ser His Gly Asp Leu Val Arg Asn Asn Pro Asp Gln Thr Met Thr Val 260 265 270 260 265 270

Asp Pro Cys Asn Leu Gln Leu Leu Tyr Gln Gly His Asp Pro Asn Ser Asp Pro Cys Asn Leu Gln Leu Leu Tyr Gln Gly His Asp Pro Asn Ser 275 280 285 275 280 285

Ser Gly Asp Tyr Asn Leu Leu Pro Trp Lys Pro Gly Val Leu Thr Leu Ser Gly Asp Tyr Asn Leu Leu Pro Trp Lys Pro Gly Val Leu Thr Leu 290 295 300 290 295 300

Lys Gln Lys Gln 305 305

<210> 8 <210> 8 <211> 319 <211> 319 <212> PRT <212> PRT <213> Aspergillus Niger <213> Aspergillus Niger

<400> 8 <400> 8

Met Val Gln Ile Lys Val Ala Ala Leu Ala Met Leu Phe Ala Ser Gln Met Val Gln Ile Lys Val Ala Ala Leu Ala Met Leu Phe Ala Ser Gln 1 5 10 15 1 5 10 15

Val Leu Ser Glu Pro Ile Glu Pro Arg Gln Ala Ser Val Ser Ile Asp Val Leu Ser Glu Pro Ile Glu Pro Arg Gln Ala Ser Val Ser Ile Asp Page 13 Page 13 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt 20 25 30 20 25 30

Thr Lys Phe Lys Ala His Gly Lys Lys Tyr Leu Gly Asn Ile Gly Asp Thr Lys Phe Lys Ala His Gly Lys Lys Tyr Leu Gly Asn Ile Gly Asp 35 40 45 35 40 45

Gln Tyr Thr Leu Thr Lys Asn Ser Lys Thr Pro Ala Ile Ile Lys Ala Gln Tyr Thr Leu Thr Lys Asn Ser Lys Thr Pro Ala Ile Ile Lys Ala 50 55 60 50 55 60

Asp Phe Gly Ala Leu Thr Pro Glu Asn Ser Met Lys Trp Asp Ala Thr Asp Phe Gly Ala Leu Thr Pro Glu Asn Ser Met Lys Trp Asp Ala Thr 65 70 75 80 70 75 80

Glu Pro Ser Arg Gly Gln Phe Ser Phe Ser Gly Ser Asp Tyr Leu Val Glu Pro Ser Arg Gly Gln Phe Ser Phe Ser Gly Ser Asp Tyr Leu Val 85 90 95 85 90 95

Asn Phe Ala Gln Ser Asn Asn Lys Leu Ile Arg Gly His Thr Leu Val Asn Phe Ala Gln Ser Asn Asn Lys Leu Ile Arg Gly His Thr Leu Val 100 105 110 100 105 110

Trp His Ser Gln Leu Pro Ser Trp Val Gln Ser Ile Thr Asp Lys Asn Trp His Ser Gln Leu Pro Ser Trp Val Gln Ser Ile Thr Asp Lys Asn 115 120 125 115 120 125

Thr Leu Ile Glu Val Met Glu Asn His Ile Thr Thr Val Met Gln His Thr Leu Ile Glu Val Met Glu Asn His Ile Thr Thr Val Met Gln His 130 135 140 130 135 140

Tyr Lys Gly Lys Ile Tyr Ala Trp Asp Val Val Asn Glu Ile Phe Asn Tyr Lys Gly Lys Ile Tyr Ala Trp Asp Val Val Asn Glu Ile Phe Asn 145 150 155 160 145 150 155 160

Glu Asp Gly Ser Leu Arg Asp Ser Val Phe Tyr Lys Val Ile Gly Glu Glu Asp Gly Ser Leu Arg Asp Ser Val Phe Tyr Lys Val Ile Gly Glu 165 170 175 165 170 175

Asp Tyr Val Arg Ile Ala Phe Glu Thr Ala Arg Ala Ala Asp Pro Asn Asp Tyr Val Arg Ile Ala Phe Glu Thr Ala Arg Ala Ala Asp Pro Asn 180 185 190 180 185 190

Ala Lys Leu Tyr Ile Asn Asp Tyr Asn Leu Asp Ser Ala Ser Tyr Pro Ala Lys Leu Tyr Ile Asn Asp Tyr Asn Leu Asp Ser Ala Ser Tyr Pro 195 200 205 195 200 205

Lys Leu Thr Gly Met Val Ser His Val Lys Lys Trp Ile Ala Ala Gly Lys Leu Thr Gly Met Val Ser His Val Lys Lys Trp Ile Ala Ala Gly 210 215 220 210 215 220

Ile Pro Ile Asp Gly Ile Gly Ser Gln Thr His Leu Ser Ala Ala Leu Ile Pro Ile Asp Gly Ile Gly Ser Gln Thr His Leu Ser Ala Ala Leu Page 14 Page 14 eolf‐othd‐000002 (33).txt F-othd-000002 (33) txt 225 230 235 240 225 230 235 240

Asn Ala Leu Ala Gly Ala Gly Thr Lys Glu Ile Ala Val Thr Glu Leu Asn Ala Leu Ala Gly Ala Gly Thr Lys Glu Ile Ala Val Thr Glu Leu 245 250 255 245 250 255

Asp Ile Ala Gly Ala Ser Ser Thr Asp Tyr Val Glu Val Val Glu Ala Asp Ile Ala Gly Ala Ser Ser Thr Asp Tyr Val Glu Val Val Glu Ala 260 265 270 260 265 270

Cys Leu Asn Gln Pro Lys Cys Ile Gly Ile Thr Val Trp Gly Val Ala Cys Leu Asn Gln Pro Lys Cys Ile Gly Ile Thr Val Trp Gly Val Ala 275 280 285 275 280 285

Asp Pro Asp Ser Trp Arg Ser Ser Ser Thr Pro Leu Leu Phe Asp Ser Asp Pro Asp Ser Trp Arg Ser Ser Ser Thr Pro Leu Leu Phe Asp Ser 290 295 300 290 295 300

Asn Tyr Asn Pro Lys Pro Ala Tyr Thr Ala Ile Ala Asn Ala Leu Asn Tyr Asn Pro Lys Pro Ala Tyr Thr Ala Ile Ala Asn Ala Leu 305 310 315 305 310 315

<210> 9 <210> 9 <211> 364 <211> 364 <212> PRT <212> PRT <213> Aspergillus fumigatus <213> Aspergillus fumigatus

<400> 9 <400> 9

Met Arg Phe Ser Leu Ala Ala Thr Ala Leu Leu Ala Gly Leu Ala Thr Met Arg Phe Ser Leu Ala Ala Thr Ala Leu Leu Ala Gly Leu Ala Thr 1 5 10 15 1 5 10 15

Ala Ala Pro Ser Ser Asn Lys Asn Asn Val Asn Leu Asp Lys Leu Ala Ala Ala Pro Ser Ser Asn Lys Asn Asn Val Asn Leu Asp Lys Leu Ala 20 25 30 20 25 30

Arg Arg Asn Gly Met Leu Trp Phe Gly Thr Ala Ala Asp Ile Pro Gly Arg Arg Asn Gly Met Leu Trp Phe Gly Thr Ala Ala Asp Ile Pro Gly 35 40 45 35 40 45

Thr Ser Glu Thr Thr Asp Lys Pro Tyr Leu Ser Ile Leu Arg Lys Gln Thr Ser Glu Thr Thr Asp Lys Pro Tyr Leu Ser Ile Leu Arg Lys Gln 50 55 60 50 55 60

Phe Gly Glu Met Thr Pro Ala Asn Ala Leu Lys Val Ser Gln Ser Asp Phe Gly Glu Met Thr Pro Ala Asn Ala Leu Lys Val Ser Gln Ser Asp 65 70 75 80 70 75 80

Phe Met Tyr Thr Glu Pro Glu Gln Asn Val Phe Asn Phe Thr Gln Gly Phe Met Tyr Thr Glu Pro Glu Gln Asn Val Phe Asn Phe Thr Gln Gly 85 90 95 85 90 95 Page 15 Page 15 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt

Asp Tyr Phe Met Asp Leu Ala Asp His Tyr Gly His Ala Val Arg Cys Asp Tyr Phe Met Asp Leu Ala Asp His Tyr Gly His Ala Val Arg Cys 100 105 110 100 105 110

His Asn Leu Val Trp Ala Ser Gln Val Ser Asp Trp Val Thr Ser Arg His Asn Leu Val Trp Ala Ser Gln Val Ser Asp Trp Val Thr Ser Arg 115 120 125 115 120 125

Asn Trp Thr Ala Thr Glu Leu Lys Glu Val Met Lys Asn His Ile Phe Asn Trp Thr Ala Thr Glu Leu Lys Glu Val Met Lys Asn His Ile Phe 130 135 140 130 135 140

Lys Thr Val Gln His Phe Gly Lys Arg Cys Tyr Ala Trp Asp Val Val Lys Thr Val Gln His Phe Gly Lys Arg Cys Tyr Ala Trp Asp Val Val 145 150 155 160 145 150 155 160

Asn Glu Ala Ile Asn Gly Asp Gly Thr Phe Ser Ser Ser Val Trp Tyr Asn Glu Ala Ile Asn Gly Asp Gly Thr Phe Ser Ser Ser Val Trp Tyr 165 170 175 165 170 175

Asp Thr Ile Gly Glu Glu Tyr Phe Tyr Leu Ala Phe Gln Tyr Ala Gln Asp Thr Ile Gly Glu Glu Tyr Phe Tyr Leu Ala Phe Gln Tyr Ala Gln 180 185 190 180 185 190

Glu Ala Leu Ala Gln Ile His Ala Asn Gln Val Lys Leu Tyr Tyr Asn Glu Ala Leu Ala Gln Ile His Ala Asn Gln Val Lys Leu Tyr Tyr Asn 195 200 205 195 200 205

Asp Tyr Gly Ile Glu Asn Pro Gly Pro Lys Ala Asp Ala Val Leu Lys Asp Tyr Gly Ile Glu Asn Pro Gly Pro Lys Ala Asp Ala Val Leu Lys 210 215 220 210 215 220

Leu Val Ala Glu Leu Arg Lys Arg Gly Ile Arg Ile Asp Gly Val Gly Leu Val Ala Glu Leu Arg Lys Arg Gly Ile Arg Ile Asp Gly Val Gly 225 230 235 240 225 230 235 240

Leu Glu Ser His Phe Ile Val Gly Glu Thr Pro Ser Leu Ala Asp Gln Leu Glu Ser His Phe Ile Val Gly Glu Thr Pro Ser Leu Ala Asp Gln 245 250 255 245 250 255

Leu Ala Thr Lys Lys Ala Tyr Ile Glu Ala Gly Leu Glu Val Ala Ile Leu Ala Thr Lys Lys Ala Tyr Ile Glu Ala Gly Leu Glu Val Ala Ile 260 265 270 260 265 270

Thr Glu Leu Asp Val Arg Phe Ser Gln Ala Pro Phe Tyr Thr Ala Glu Thr Glu Leu Asp Val Arg Phe Ser Gln Ala Pro Phe Tyr Thr Ala Glu 275 280 285 275 280 285

Ala Gln Lys Gln Gln Ala Ala Asp Tyr Tyr Ala Ser Val Ala Ser Cys Ala Gln Lys Gln Gln Ala Ala Asp Tyr Tyr Ala Ser Val Ala Ser Cys 290 295 300 290 295 300 Page 16 Page 16 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt

Lys His Ala Gly Pro Arg Cys Val Gly Val Val Val Trp Asp Phe Asp Lys His Ala Gly Pro Arg Cys Val Gly Val Val Val Trp Asp Phe Asp 305 310 315 320 305 310 315 320

Asp Ala Tyr Ser Trp Ile Pro Gly Thr Phe Glu Gly Gln Gly Gly Ala Asp Ala Tyr Ser Trp Ile Pro Gly Thr Phe Glu Gly Gln Gly Gly Ala 325 330 335 325 330 335

Cys Leu Tyr Asn Glu Thr Leu Glu Val Lys Pro Ala Phe Tyr Ala Ala Cys Leu Tyr Asn Glu Thr Leu Glu Val Lys Pro Ala Phe Tyr Ala Ala 340 345 350 340 345 350

Ala Glu Ala Leu Glu Asn Lys Pro Cys Thr Val Cys Ala Glu Ala Leu Glu Asn Lys Pro Cys Thr Val Cys 355 360 355 360

<210> 10 <210> 10 <211> 796 <211> 796 <212> PRT <212> PRT <213> Talaromyces emersonii <213> Talaromyces emersonii

<400> 10 <400> 10

Met Met Thr Pro Thr Ala Ile Leu Thr Ala Val Ala Ala Leu Leu Pro Met Met Thr Pro Thr Ala Ile Leu Thr Ala Val Ala Ala Leu Leu Pro 1 5 10 15 1 5 10 15

Thr Ala Thr Trp Ala Gln Asp Asn Gln Thr Tyr Ala Asn Tyr Ser Ser Thr Ala Thr Trp Ala Gln Asp Asn Gln Thr Tyr Ala Asn Tyr Ser Ser 20 25 30 20 25 30

Gln Ser Gln Pro Asp Leu Phe Pro Arg Thr Val Ala Thr Ile Asp Leu Gln Ser Gln Pro Asp Leu Phe Pro Arg Thr Val Ala Thr Ile Asp Leu 35 40 45 35 40 45

Ser Phe Pro Asp Cys Glu Asn Gly Pro Leu Ser Thr Asn Leu Val Cys Ser Phe Pro Asp Cys Glu Asn Gly Pro Leu Ser Thr Asn Leu Val Cys 50 55 60 50 55 60

Asn Lys Ser Ala Asp Pro Trp Ala Arg Ala Glu Ala Leu Ile Ser Leu Asn Lys Ser Ala Asp Pro Trp Ala Arg Ala Glu Ala Leu Ile Ser Leu 65 70 75 80 70 75 80

Phe Thr Leu Glu Glu Leu Ile Asn Asn Thr Gln Asn Thr Ala Pro Gly Phe Thr Leu Glu Glu Leu Ile Asn Asn Thr Gln Asn Thr Ala Pro Gly 85 90 95 85 90 95

Val Pro Arg Leu Gly Leu Pro Gln Tyr Gln Val Trp Asn Glu Ala Leu Val Pro Arg Leu Gly Leu Pro Gln Tyr Gln Val Trp Asn Glu Ala Leu 100 105 110 100 105 110

Page 17 Page 17 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt

His Gly Leu Asp Arg Ala Asn Phe Ser His Ser Gly Glu Tyr Ser Trp His Gly Leu Asp Arg Ala Asn Phe Ser His Ser Gly Glu Tyr Ser Trp 115 120 125 115 120 125

Ala Thr Ser Phe Pro Met Pro Ile Leu Ser Met Ala Ser Phe Asn Arg Ala Thr Ser Phe Pro Met Pro Ile Leu Ser Met Ala Ser Phe Asn Arg 130 135 140 130 135 140

Thr Leu Ile Asn Gln Ile Ala Ser Ile Ile Ala Thr Gln Ala Arg Ala Thr Leu Ile Asn Gln Ile Ala Ser Ile Ile Ala Thr Gln Ala Arg Ala 145 150 155 160 145 150 155 160

Phe Asn Asn Ala Gly Arg Tyr Gly Leu Asp Ser Tyr Ala Pro Asn Ile Phe Asn Asn Ala Gly Arg Tyr Gly Leu Asp Ser Tyr Ala Pro Asn Ile 165 170 175 165 170 175

Asn Gly Phe Arg Ser Pro Leu Trp Gly Arg Gly Gln Glu Thr Pro Gly Asn Gly Phe Arg Ser Pro Leu Trp Gly Arg Gly Gln Glu Thr Pro Gly 180 185 190 180 185 190

Glu Asp Ala Phe Phe Leu Ser Ser Thr Tyr Ala Tyr Glu Tyr Ile Thr Glu Asp Ala Phe Phe Leu Ser Ser Thr Tyr Ala Tyr Glu Tyr Ile Thr 195 200 205 195 200 205

Gly Leu Gln Gly Gly Val Asp Pro Glu His Val Lys Ile Val Ala Thr Gly Leu Gln Gly Gly Val Asp Pro Glu His Val Lys Ile Val Ala Thr 210 215 220 210 215 220

Ala Lys His Phe Ala Gly Tyr Asp Leu Glu Asn Trp Gly Asn Val Ser Ala Lys His Phe Ala Gly Tyr Asp Leu Glu Asn Trp Gly Asn Val Ser 225 230 235 240 225 230 235 240

Arg Leu Gly Phe Asn Ala Ile Ile Thr Gln Gln Asp Leu Ser Glu Tyr Arg Leu Gly Phe Asn Ala Ile Ile Thr Gln Gln Asp Leu Ser Glu Tyr 245 250 255 245 250 255

Tyr Thr Pro Gln Phe Leu Ala Ser Ala Arg Tyr Ala Lys Thr Arg Ser Tyr Thr Pro Gln Phe Leu Ala Ser Ala Arg Tyr Ala Lys Thr Arg Ser 260 265 270 260 265 270

Ile Met Cys Ser Tyr Asn Ala Val Asn Gly Val Pro Ser Cys Ala Asn Ile Met Cys Ser Tyr Asn Ala Val Asn Gly Val Pro Ser Cys Ala Asn 275 280 285 275 280 285

Ser Phe Phe Leu Gln Thr Leu Leu Arg Glu Asn Phe Asp Phe Val Asp Ser Phe Phe Leu Gln Thr Leu Leu Arg Glu Asn Phe Asp Phe Val Asp 290 295 300 290 295 300

Asp Gly Tyr Val Ser Ser Asp Cys Asp Ala Val Tyr Asn Val Phe Asn Asp Gly Tyr Val Ser Ser Asp Cys Asp Ala Val Tyr Asn Val Phe Asn 305 310 315 320 305 310 315 320

Page 18 Page 18 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt

Pro His Gly Tyr Ala Leu Asn Gln Ser Gly Ala Ala Ala Asp Ser Leu Pro His Gly Tyr Ala Leu Asn Gln Ser Gly Ala Ala Ala Asp Ser Leu 325 330 335 325 330 335

Leu Ala Gly Thr Asp Ile Asp Cys Gly Gln Thr Leu Pro Trp His Leu Leu Ala Gly Thr Asp Ile Asp Cys Gly Gln Thr Leu Pro Trp His Leu 340 345 350 340 345 350

Asn Glu Ser Phe Val Glu Gly Tyr Val Ser Arg Gly Asp Ile Glu Lys Asn Glu Ser Phe Val Glu Gly Tyr Val Ser Arg Gly Asp Ile Glu Lys 355 360 365 355 360 365

Ser Leu Thr Arg Leu Tyr Ser Asn Leu Val Arg Leu Gly Tyr Phe Asp Ser Leu Thr Arg Leu Tyr Ser Asn Leu Val Arg Leu Gly Tyr Phe Asp 370 375 380 370 375 380

Gly Asn Asn Ser Glu Tyr Arg Asn Leu Asn Trp Asn Asp Val Val Thr Gly Asn Asn Ser Glu Tyr Arg Asn Leu Asn Trp Asn Asp Val Val Thr 385 390 395 400 385 390 395 400

Thr Asp Ala Trp Asn Ile Ser Tyr Glu Ala Ala Val Glu Gly Ile Thr Thr Asp Ala Trp Asn Ile Ser Tyr Glu Ala Ala Val Glu Gly Ile Thr 405 410 415 405 410 415

Leu Leu Lys Asn Asp Gly Thr Leu Pro Leu Ser Lys Lys Val Arg Ser Leu Leu Lys Asn Asp Gly Thr Leu Pro Leu Ser Lys Lys Val Arg Ser 420 425 430 420 425 430

Ile Ala Leu Ile Gly Pro Trp Ala Asn Ala Thr Val Gln Met Gln Gly Ile Ala Leu Ile Gly Pro Trp Ala Asn Ala Thr Val Gln Met Gln Gly 435 440 445 435 440 445

Asn Tyr Tyr Gly Thr Pro Pro Tyr Leu Ile Ser Pro Leu Glu Ala Ala Asn Tyr Tyr Gly Thr Pro Pro Tyr Leu Ile Ser Pro Leu Glu Ala Ala 450 455 460 450 455 460

Lys Ala Ser Gly Phe Thr Val Asn Tyr Ala Phe Gly Thr Asn Ile Ser Lys Ala Ser Gly Phe Thr Val Asn Tyr Ala Phe Gly Thr Asn Ile Ser 465 470 475 480 465 470 475 480

Thr Asp Ser Thr Gln Trp Phe Ala Glu Ala Ile Ala Ala Ala Lys Lys Thr Asp Ser Thr Gln Trp Phe Ala Glu Ala Ile Ala Ala Ala Lys Lys 485 490 495 485 490 495

Ser Asp Val Ile Ile Tyr Ala Gly Gly Ile Asp Asn Thr Ile Glu Ala Ser Asp Val Ile Ile Tyr Ala Gly Gly Ile Asp Asn Thr Ile Glu Ala 500 505 510 500 505 510

Glu Gly Gln Asp Arg Thr Asp Leu Lys Trp Pro Gly Asn Gln Leu Asp Glu Gly Gln Asp Arg Thr Asp Leu Lys Trp Pro Gly Asn Gln Leu Asp 515 520 525 515 520 525

Page 19 Page 19 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt

Leu Ile Glu Gln Leu Ser Gln Val Gly Lys Pro Leu Val Val Leu Gln Leu Ile Glu Gln Leu Ser Gln Val Gly Lys Pro Leu Val Val Leu Gln 530 535 540 530 535 540

Met Gly Gly Gly Gln Val Asp Ser Ser Ser Leu Lys Ala Asn Lys Asn Met Gly Gly Gly Gln Val Asp Ser Ser Ser Leu Lys Ala Asn Lys Asn 545 550 555 560 545 550 555 560

Val Asn Ala Leu Val Trp Gly Gly Tyr Pro Gly Gln Ser Gly Gly Ala Val Asn Ala Leu Val Trp Gly Gly Tyr Pro Gly Gln Ser Gly Gly Ala 565 570 575 565 570 575

Ala Leu Phe Asp Ile Leu Thr Gly Lys Arg Ala Pro Ala Gly Arg Leu Ala Leu Phe Asp Ile Leu Thr Gly Lys Arg Ala Pro Ala Gly Arg Leu 580 585 590 580 585 590

Val Ser Thr Gln Tyr Pro Ala Glu Tyr Ala Thr Gln Phe Pro Ala Asn Val Ser Thr Gln Tyr Pro Ala Glu Tyr Ala Thr Gln Phe Pro Ala Asn 595 600 605 595 600 605

Asp Met Asn Leu Arg Pro Asn Gly Ser Asn Pro Gly Gln Thr Tyr Ile Asp Met Asn Leu Arg Pro Asn Gly Ser Asn Pro Gly Gln Thr Tyr Ile 610 615 620 610 615 620

Trp Tyr Thr Gly Thr Pro Val Tyr Glu Phe Gly His Gly Leu Phe Tyr Trp Tyr Thr Gly Thr Pro Val Tyr Glu Phe Gly His Gly Leu Phe Tyr 625 630 635 640 625 630 635 640

Thr Glu Phe Gln Glu Ser Ala Ala Ala Gly Thr Asn Lys Thr Ser Thr Thr Glu Phe Gln Glu Ser Ala Ala Ala Gly Thr Asn Lys Thr Ser Thr 645 650 655 645 650 655

Phe Asp Ile Leu Asp Leu Phe Ser Thr Pro His Pro Gly Tyr Glu Tyr Phe Asp Ile Leu Asp Leu Phe Ser Thr Pro His Pro Gly Tyr Glu Tyr 660 665 670 660 665 670

Ile Glu Gln Val Pro Phe Ile Asn Val Thr Val Asp Val Lys Asn Val Ile Glu Gln Val Pro Phe Ile Asn Val Thr Val Asp Val Lys Asn Val 675 680 685 675 680 685

Gly His Thr Pro Ser Pro Tyr Thr Gly Leu Leu Phe Ala Asn Thr Thr Gly His Thr Pro Ser Pro Tyr Thr Gly Leu Leu Phe Ala Asn Thr Thr 690 695 700 690 695 700

Ala Gly Pro Lys Pro Tyr Pro Asn Lys Trp Leu Val Gly Phe Asp Trp Ala Gly Pro Lys Pro Tyr Pro Asn Lys Trp Leu Val Gly Phe Asp Trp 705 710 715 720 705 710 715 720

Leu Pro Thr Ile Gln Pro Gly Glu Thr Ala Lys Leu Thr Ile Pro Val Leu Pro Thr Ile Gln Pro Gly Glu Thr Ala Lys Leu Thr Ile Pro Val 725 730 735 725 730 735

Page 20 Page 20 eolf‐othd‐000002 (33).txt eolf-othd-000002 (33) txt

Pro Leu Gly Ala Ile Ala Trp Ala Asp Glu Asn Gly Asn Lys Val Val Pro Leu Gly Ala Ile Ala Trp Ala Asp Glu Asn Gly Asn Lys Val Val 740 745 750 740 745 750

Phe Pro Gly Asn Tyr Glu Leu Ala Leu Asn Asn Glu Arg Ser Val Val Phe Pro Gly Asn Tyr Glu Leu Ala Leu Asn Asn Glu Arg Ser Val Val 755 760 765 755 760 765

Val Ser Phe Thr Leu Thr Gly Asp Ala Ala Thr Leu Glu Lys Trp Pro Val Ser Phe Thr Leu Thr Gly Asp Ala Ala Thr Leu Glu Lys Trp Pro 770 775 780 770 775 780

Leu Trp Glu Gln Ala Val Pro Gly Val Leu Gln Gln Leu Trp Glu Gln Ala Val Pro Gly Val Leu Gln Gln 785 790 795 785 790 795

Page 21 Page 21

Claims (1)

1. Claims

   1. A process for extraction or separation of crude palm oil (CPO), comprising the steps of: i) contacting a substrate comprising palm oil with an enzyme composition, ii) extracting or separating the crude palm oil wherein the enzyme composition comprises a GH10 xylanase and a GH62 arabinofuranosidase wherein the GH10 xylanase has at least at least 90% sequence identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:8 and wherein the GH62 arabinofuranosidase has at least 99% sequence identity to the polypeptide of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

   2. The process according to claim 1, wherein the GH10 xylanase is obtained from Talaromyces leycettanus.

   3. The process according to claim 1, wherein the GH10 xylanase is obtained from Rasamsoniabyssochlamydoides.

   4. The process according to claim 1, wherein the GH10 xylanase is obtained from Aspergillus niger. )

   5. The process according to claim 1 wherein the GH10 xylanase and a GH62 arabinofuranosidase are inactive at a temperature of 70°C.

   6. The process according to any one of the preceding claims, wherein the substrate comprising palm oil is selected from the group consisting of palm fruitlets, palm press liquid, mashed or partly mashed palm fruitlets.

   7. The process according to any one of the preceding claims, wherein the substrate comprising palm oil is subjected to sterilization before being contacted with the enzyme composition.

   8. The process according to claim 7, wherein the substrate is sterilization is batch sterilization or continuous sterilization.

   9. The process according to claim 7 or claim 8, wherein the sterilization temperature is in the range of100-150°C.

   10. The process according to any one of claims 7-9, wherein the palm fruitlets are stripped from fresh fruit bunches (FFB) after being sterilized.

   11. The process according to any one of the preceding claims, wherein the process comprises contacting the substrate with the enzyme composition at a temperature of above 50C before extracting the crude palm oil.

   12. The process according to any one of the preceding claims, wherein the substrate contacted with the enzyme composition is subjected to digestion, before extracting the crude palm oil.

   13. The process according to claim 11 or claim 12, wherein the temperature is within the range of 55-85°C.

   14. The process according to any one of the preceding claims, wherein the process comprises contacting the substrate with the enzyme composition for a period of 5-120 minutes.

   15. The process according to any one of the preceding claims, wherein the substrate comprising palm oil is retained at a temperature above 65°C and below 85°C for 10-30 minutes, 10-28 minutes, 15-28 minutes, 12-30 minutes,12-28 minutes or 12-25 minutes.

   16. The process according to any one of the preceding claims, wherein the enzyme composition is dosed in amounts corresponding to 10-500 mg enzyme protein/kg FFB (fresh fruit bunch) comprising palm oil, or 10-450 mg enzyme protein/kg FFB comprising palm oil.

   17. The process according to any one of the preceding claims, wherein the enzyme composition is dosed such that the amount of enzyme protein corresponds to 10-1000 ppm relative to the amount of substrate comprising palm oil.

   Fig. 1 Mean( Oil Yield (g)) vs. Treatment 22

   21

   19.5

   19 18.9

   18.7

   18

   17

   16

   Control T. Leycettanus A. Fumigatus GH 10 GH10

   2 / 4

   Fig. 2

   140

   120

   100

   80 Verificable

   60

   40 T 20

   o 50 60 70 80 90 Temperature (°C)

   I Aspergillus fumigatus GH10

   Talaromyces leycettanus GH10 pH 4.0 F 00

   90% 80% 70% 60% 50% GH10 fumigatus A. 40% GH10 byssochlamydoides R. 30% GH10 leycettanus T. 20% 10% 0% 70

   60 65

   55

   50

   45 °C Temperature,

   GH10 byssochlamydoides R. GH10 leycettanus T. GH10 fumigatus A. 7,5

   7

   6,5

   65 oC

   6

   5,5 pH

   5

   4,5

   I 4

   3,5

   90% 80% 70% 60% 50% 40% 30% 20% 10% 0%