ES2909902B2 - BETA-GALACTOSIDASE AND USES OF THE SAME - Google Patents

Description

DESCRIPTION

P-galactosidase and uses of it

The present invention is related to the fields of enzymology and the agri-food industry. In particular, the present invention relates to a new pgalactosidase enzyme and its use in the hydrolysis of lactose, as well as in the production of galactooligosaccharides (GOS) from lactose. The unique properties of the enzyme of the invention make it especially useful for carrying out the production of GOS both under pasteurization conditions and at low temperature, which represents an important industrial advantage over other enzymes.

STATE OF THE TECHNIQUE

p-galactosidase (EC 3.2.1.23), also called lactase, has as its primary function the hydrolysis of lactose into D-galactose and D-glucose. Therefore, the fundamental application of p-galactosidases is limited to the field of dairy products and byproducts. Furthermore, this enzyme has transgalactosylase activity and is used for the synthesis of galactosyl-oligosaccharides, compounds with growing interest in the agri-food and pharmaceutical areas.

As a non-enzymatic alternative to hydrolysis with p-galactosidases, chemical hydrolysis can be used, but it produces undesirable effects (e.g.: loss of nutrients, organoleptic defects, etc.)

P-galactosidases are used in the agri-food industry to reduce the lactose content of dairy products and derivatives, which has a wide variety of interest.

Industrially, two types of p-galactosidases are of increasing importance in processes: thermostable and cold-resistant. The use of thermostable enzymes at high temperatures is related to the decrease in the viscosity of the substrate and the reduction of the possibility of microbial contamination. Cold-active enzymes make it possible to apply them in the treatment of milk and dairy products under conditions that preserve the flavor and nutritional characteristics of these foods.

The production of dairy products with reduced levels of lactose is attributed to three main advantages: (a) obtaining products for population groups with lactose intolerance; (b) formation of galatooligosaccharides (GOS), which promote the growth of intestinal microflora; and (c) improvement in the technological characteristics of dairy products.

The hydrolysis of lactose with p-galactosidase has its greatest focus on the development of milk and dairy derivatives that are free or have reduced content of this sugar, enabling their consumption by lactose intolerant individuals.

On the other hand, the high concentration of lactose in non-fermented dairy products, such as ice cream and condensed milk, can lead to excessive crystallization of lactose, resulting in products with a sandy or rough texture. In this way, hydrolysis improves the absorption capacity and creaminess, and these products become more digestible.

Another perspective of the use of this enzyme is the production of galactooligosaccharides, carbohydrates considered prebiotics and that have recognized technological and nutritional properties. Among the advantages associated with the synthesis of these carbohydrates from lactose is the reduction in the caloric content of the food, the reduction of lactose intolerance and three other important effects coming from the synergy between GOS and probiotics, namely That is, in the modulation of the immune system, intestinal transit and stimulation of nutrient assimilation.

Despite the efforts made to date, there is still a need for thermostable p-galactosidase enzymes with improved activity.

EXPLANATION OF THE INVENTION

The present inventors have found a new enzyme beta-galactosidase (BWβg1) that shows to be stable and efficient in the production of GOS at elevated temperatures, compatible with sterilization treatments of dairy products, significantly reducing the risk of contamination by adventitious microbial growth.

The data provided below also show that the enzyme of the invention has a high affinity for its natural substrate, lactose. And, on the other hand, the enzyme of the invention is highly efficient in the production of GOS, producing significant quantities of galactooligosaccharides in a short period of time. In addition, it is versatile in terms of the nature of the substrate, since high yields are achieved starting from lactose or milk.

Therefore, the enzyme of the invention plays a double role: it hydrolyzes lactose and, at the same time, synthesizes GOS. Both activities, also, at low and high temperatures.

The good catalytic capacity of the beta-galactosidase of the invention, therefore, has a surprising impact on the final properties of the product since the enzyme is capable of degrading lactose, which can have a detrimental effect, and incorporating GOS, with a beneficial effect, to the product. Therefore, the enzyme of the invention makes it possible to obtain products with beneficial health properties.

In relation to the above, it is notable that this double function is exhibited by the enzyme under the same conditions of temperature and pH, that is, in a single incubation step of the enzyme with the substrate, the reduction in the content of lactose and GOS production.

Surprisingly, the optimal temperature and pH conditions make the enzyme of the invention usable in a pasteurization process. As shown below, the researchers carried out the pasteurization of a dairy product in the presence of the enzyme of the invention and two commercial enzymes: only in the case of the enzyme of the invention, a remarkable production was achieved simultaneously of GOS and a significant reduction in lactose.

The above represents a substantial improvement in the agri-food sector, particularly dairy products, since the enzyme of the invention allows the hydrolysis of lactose, the production of GOS to be carried out in a single step under conditions of temperature, time and pH. and the destruction of microorganisms.

Surprisingly, the inventors have also found that this enzyme is highly active at low temperatures, between 3 and 10°C. Advantageously, working at these temperatures prevents the growth of microorganisms.

It is the first time that a beta-galactosidase enzyme with such an activity profile under such different temperature conditions has been reported, making this enzyme a highly versatile tool.

The versatility, as well as the high efficiency in GOS synthesis and lactose hydrolysis, make the enzyme of the invention a very attractive tool in the agri-food sector, particularly dairy products.

Thus, in a first aspect the present invention provides an enzyme having p-galactosidase activity, which is selected from (a) an amino acid sequence consisting of the sequence SEQ ID NO: 1; and (b) an amino acid sequence encoded by the nucleotide sequence SEQ ID NO: 2, wherein the enzyme is capable of hydrolyzing lactose and producing galactooligosaccharides simultaneously at a temperature between 3 and 10 ° C or between 55 and 89 ° C .

In one embodiment, the enzyme has an amino acid sequence identity of at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or is 100% with the sequence SEQ ID NO: 1. In one embodiment, the enzyme has 100% sequence identity with the sequence SEQ ID NO: 1.

In one embodiment, the enzyme has a nucleotide sequence identity of at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or is 100% with the sequence SEQ ID NO: 2. In one embodiment, the enzyme has 100% sequence identity with the sequence SEQ ID NO: 2.

In the present invention, the term "identity" refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. If, in the optimal alignment, a position in a first sequence is occupied by the same residue as the corresponding position in the second sequence, the sequences exhibit identity with respect to that position. The level of identity between two sequences (or "percent sequence identity") is measured as a ratio of the number of identical positions shared by the sequences with regarding size of the sequences (i.e., percentage sequence identity = (number of identical positions / total number of positions) x 100).

Various mathematical algorithms are known to rapidly obtain optimal alignment and calculate identity between two or more sequences and are incorporated into several available software programs. Examples of such programs include MATCH-BOX, MULTAIN, GCG, FASTA, and ROBUST, among others, for sequence analysis. Preferred software analysis programs include the ALIGN, CLUSTAL W and BLAST programs (e.g., BLAST 2.1, BL2SEQ and later versions thereof).

For amino acid sequence analysis, weight matrices are used, such as BLOSUM matrices (e.g., BLOSUM45, BLOSUM50, BLOSUM62, and BLOSUM80 matrices), Gonnet matrices, or PAM matrices (e.g., PAM30, PAM70, PAM120 matrices). , PAM160, PAM250 and PAM350), in order to determine identity.

BLAST programs provide analysis of at least two amino acid sequences, either by aligning a selected sequence against multiple sequences in a database (e.g., GenSeq), or, with BL2SEQ, between two selected sequences. BLAST programs are preferably modified with low-complexity filtering programs such as DUST or SEG programs, which are preferably integrated into the operations of the BLAST program. If gap costs (or gap scores) are used, the gap cost is preferably set between approximately -5 and -15. Similar per-gap parameters can be used with other programs as appropriate. BLAST programs and the principles underlying them are further described in, for example, Altschul et al., "Basic local alignment search tool", 1990, J. Mol. Biol, v. 215, pages 403-410.

For analysis of multiple sequences, the CLUSTAL W program can be used. The CLUSTAL W program is desirably run using “dynamic” (vs. “fast”) parameters. Amino acid sequences are evaluated using a variable set of BLOSUM matrices depending on the level of identity between the sequences. The CLUSTAL W program and the underlying operating principles are further described in, for example, Higgins et al., "CLUSTAL V: improved software for multiple sequence alignment", 1992, CABIOS, 8(2), pages 189-191.

In an embodiment of the first aspect of the invention, the enzyme has activity in a pH range between 6 and 9, and in a temperature range between 3 and 10°C and/or between 60 and 89°C. In another embodiment of the first aspect of the invention, the enzyme has activity at a pH between 6.5 and 8 and a temperature range between 4 and 9°C and/or between 75 and 85°C.

For the purposes of the present invention, any given range includes the values of the lower and upper ends of the range.

In a second aspect, the present invention provides a recombinant nucleotide sequence comprising a nucleotide sequence encoding the enzyme of the invention, operably linked to an expression promoter.

In one embodiment, the recombinant nucleotide sequence comprises a nucleotide sequence that encodes a sequence that has at least 85% identity with the sequence SEQ ID NO: 2. In another embodiment of the second aspect of the invention the nucleotide sequence encodes a sequence that has an identity of at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or is 100% with the sequence SEQ ID NO: 2 In another embodiment of the second aspect of the invention, the nucleotide sequence that encodes the enzyme corresponds to the sequence SEQ ID NO: 2.

Illustrative and non-limiting examples of expression promoters are the promoters of: LTR, SV40, lac or trp of E. coli, PL of phage lambda and others known to control the expression of genes in eukaryotic, prokaryotic cells or viruses.

In a third aspect the present invention provides an expression construct comprising the recombinant nucleotide sequence of the second aspect of the invention.

The constructs comprise a vector, such as a plasmid, into which the enzyme sequence is inserted in a forward or reverse orientation. In an embodiment of the third aspect, the gene construct is an expression vector.

Illustrative and non-limiting examples of commercial vectors are: pQE70, pQE60, pQE-9 (Qiagen), pD10, psiX174, pBluescript II KS; pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK ₂ 23-3, pKK ₂ 33-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, μMSG, pSVL (Pharmacia).

Vectors include chromosomal, non-chromosomal and synthetic DNA sequences, for example, derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowlpox virus and pseudorabies. However, any other vector can be used as long as it is replicable and viable in the host.

The expression vector also provides a ribosome binding site for translation initiation and a transcription terminator. The vector may also include sequences appropriate to amplify expression.

Additionally, the expression vectors may contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. .coli.

Insertion of the gene of the present invention into a vector, insertion of the selection marker gene (if necessary) and insertion of a promoter (if necessary), and the like, can be carried out by a recombination DNA technology. standard.

In a fourth aspect the present invention provides a recombinant host cell transformed with the recombinant nucleic acid of the second aspect of the invention or the construct of the third aspect of the invention.

Illustrative and non-limiting examples of suitable host cells are: bacterial cells, such as Escherichia coli, Streptomyces, Bacillus subtilis; fungi such as yeast; insect cells such as Dmsophila S2 and Spodoptera Sf9; animal such as CHO, COS or Bowes melanoma; adenovirus; plant cells, etc. The selection of a suitable host is part of the knowledge of the person skilled in the art.

The transformed host cell can be obtained by transfection or transformation, using the aforementioned vector of the present invention. Transfection and transformation can be carried out, for example, by a calcium phosphate coprecipitation method, electroporation, lipofection, microinjection, a Hanahan method, a lithium acetate method, protoplast-polyethylene glycol method, and the like.

Another aspect of the invention is to provide a method for producing a pgalactosidase. In one embodiment of the production method of the invention, pgalactosidase is produced using the transformant mentioned above. In the production method of this embodiment, the transformant is grown under conditions such that the encoded enzyme is produced.

Culture methods and culture conditions are not particularly limited as long as the desired p-galactosidase protein can be produced. That is, the methods and culture conditions suitable for cultivating the microorganisms to be used can be appropriately adjusted to the conditions under which p-galactosidase is produced. Liquid culture or solid culture can be used as a culture method, in particular liquid culture.

As a medium, any medium in which the transformants to be used can grow can be used. For example, a medium supplemented with a carbon source such as glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses and organic acid; and further, a nitrogen source such as ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate or peptone, yeast extract, corn steep liquor, casein hydrolyzate, bran and meat extract; and further, an inorganic salt such as potassium salt, magnesium salt, sodium salt, phosphate salt, manganese salt, iron salt and zinc salt, and the like can be used. In order to promote the growth of the transformants to be used, vitamins, amino acids and the like can be added to the medium. The medium is grown under aerobic conditions such that the pH of the medium is adjusted, for example, to about 3 to 10 (preferably about 7 to 8), and the culture temperature is generally about 10 ° C to 50 ° C (preferably approximately 20°C to 37°C) for 1 to 7 days (preferably 3 to 4 days). An example of the culture method may include a shaking culture method and a submerged aerobic culture method. Once the cells are transformed with the gene construction, these are cultured until reaching a desired density and activation of the expression promoter is induced, through routine methods.

The transformed cells can be collected by centrifugation, disrupted by chemical or physical means, and the resulting crude oil subjected to purification.

The enzyme can be recovered and purified from recombinant cell culture media by methods including ammonium sulfate or ethanol precipitation, chromatographic separation, and ultrafiltration. Folding steps can also be incorporated to complete the configuration of the mature protein. Finally, purification can be carried out by high performance liquid chromatography (HPLC).

The p-galactosidase of the present invention is provided in the form of, for example, an enzyme preparation. The enzyme preparation may contain, in addition to an active ingredient (p-galactosidase of the present invention), excipient, buffering agents, suspending agents, stabilizer, preservatives, antiseptics, physiological saline solution and the like. Examples of the excipient may include lactose, sorbitol, D-mannitol, sucrose and the like. Examples of the buffering agent may include phosphate, citrate, acetate and the like. Examples of the stabilizer may include propylene glycol and ascorbic acid and the like. Examples of the preservative may include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methyl paraben and the like.

Alternatively, the enzyme may be immobilized on a suitable solid phase material such as an ion exchange material, such as DEAE-cellulose or similar material, to provide immobilized enzyme composition products.

Alternatively, the enzyme, optionally in combination with excipient, buffering agents, suspending agents, stabilizer, preservatives, antiseptics, physiological saline and the like, can be frozen or lyophilized. Advantageously, thanks to the thermostability profile shown by the enzyme of the invention, the resulting material can be stored or transported to the laboratory or environment in which the reaction will subsequently be carried out. Freeze-dried material provides a more convenient way to store and transport as no refrigeration is required.

In a fifth aspect, the present invention provides a dairy product comprising the enzyme as defined in the first aspect, the recombinant nucleotide sequence according to the second aspect, the expression construct according to the third aspect or the recombinant host cell according to the fourth aspect. aspect. In one embodiment, the dairy product comprises the inactivated enzyme. This embodiment will correspond to the final product, once the lactose hydrolysis/GOS production process has been carried out and the inactivation of the enzyme has been carried out using any of the routine protocols.

In the context of the present invention, the term "inactivated enzyme" means that the enzyme has been denatured, modified or degraded and has lost its betagalactosidase activity.

From the characterization incorporated below, the GOS produced by the enzyme of the invention, of 3 and 4 units, have been reported in the state of the art to play a beneficial role in health.

Therefore, the enzyme, recombinant sequence, expression construct or host cell described above can be used to produce a mixture of galactooligosaccharides that can form part of a product to improve intestinal health. Said product can be selected from the group consisting of dairy products (for example liquid milk, dry milk powder such as whole milk powder, skimmed milk powder, fat-enriched skimmed milk powder, whey powder, infant formulas , formula, ice cream, yogurt, cheese, fermented milk products), beverages such as fruit juice, baby food, cereals, bread, biscuits, sweets, cakes, food supplements, dietary supplements, animal food, food for poultry or of course any other food or drink. The presence of galacto-oligosaccharides in such products has the advantage of increasing the growth of health-promoting Bifidobacterium in the product or in the intestinal flora of the consumer after ingestion of the product or both.

In a sixth aspect, the invention provides the use of the enzyme, the recombinant nucleotide sequence, the expression construct or the recombinant host cell of the invention, for the production of galactooligosaccharides from lactose.

In another aspect, the invention provides the use of the enzyme, the recombinant nucleotide sequence, the expression construct or the recombinant host cell of the invention, to increase the amount of GOS in a lactose-based product.

In a seventh aspect, the invention provides a process for producing galactooligosaccharides comprising the step of incubating the enzyme or transformed host cell as defined above with a lactose-based product at a pH between 6.5 and 8.0. In one embodiment, this incubation step is carried out at a temperature between 55 and 89°C or between 65 and 85°C or between 70 and 75°C, and at a pH between 6.5 and 8. The time of the step The incubation period will be necessary to achieve the desired amount of GOS and will depend, among other factors, on the nature of the substrate. The person skilled in the art can stop the incubation at any time by inactivating the enzyme, for example by raising the temperature above 90°C, for example between 90-100°C. In another embodiment, before and/or after the step of High temperature incubation, the method comprises cooling the reaction medium (which includes the lactose-based product and the enzyme) to a temperature between 3 and 10°C, in particular between 5 and 9°C, in particular 8°C. C for a period of time between 10 and 48 hours, particularly between 15 and 30 hours, at a pH between 6.5 and 8. Alternatively, in another embodiment, the incubation step is carried out at a temperature between 3 and 10 °C, between 5 and 9°C, or at 8°C.

In another aspect, the invention provides a process for increasing the content of galactooligosaccharides in a dairy product, comprising the step of incubating the enzyme or transformed host cell as defined above with a lactose-based product at a pH between 6.5 and 8.0. In one embodiment, incubation is carried out at a temperature between 55 and 89°C or between 65 and 85°C or between 70 and 75°C, at a pH between 6.5 and 8. The time of the incubation step It will be necessary to achieve the desired amount of GOS and will depend, among other factors, on the nature of the substrate. The person skilled in the art can stop the incubation at any time by inactivating the enzyme, for example by raising the temperature above 90°C, for example between 90-100°C. In another embodiment, before and/or after the high temperature incubation step, the procedure comprises cooling the reaction medium (which includes the lactose-based product and the enzyme) to a temperature between 3 and 10°C, in particular between 5 and 9°C, in particular 8°C, at a pH between 6.5 and 8, for a period of time between 10 and 48 hours, particularly between 15 and 30 hours. Alternatively, in another embodiment, the incubation step is carried out at a temperature between 3 and 10°C, between 5 and 9°C, or at 8°C.

In the present invention, "lactose-based product" has the same meaning as "lactose-containing substrate" and "dairy product" and refers to any product, natural or synthetic, that incorporates lactose in its composition or, alternatively, consists of lactose. in lactose. The lactose-based product can be selected from commercial lactose, whole milk, semi-skimmed milk, skimmed milk, whey, fat-enriched skimmed milk and filtered whey. These dairy products can be obtained from cows, buffaloes , sheep, camels, llamas or goats.Fortified skimmed milk is defined as whole milk that has been skimmed to remove the milk fat, which is later replaced by adding oil or vegetable fat.

In an eighth aspect, the present invention provides the use of the enzyme, recombinant nucleotide sequence, expression construct or recombinant host cell of the invention, as defined above, in the hydrolysis of lactose.

In another aspect, the present invention provides the use of the enzyme, the recombinant nucleotide sequence, the expression construct or the recombinant host cell of the invention, as defined above, to reduce the lactose content in a lactose-based product.

In a ninth aspect, the present invention provides a lactose hydrolysis process comprising the step of incubating the enzyme or the recombinant host cell as defined above, with a lactose-based product, at a pH between 6.5 and 8.0. In one embodiment, the incubation is carried out at a temperature between 55 and 89 ° C or between 65 and 85 or between 70 and 75 ° C, at a pH between 6.5 and 8. The time of the incubation step will be the necessary to reach the desired lactose level and will depend, among other factors, on the nature of the substrate. The person skilled in the art can stop the incubation at any time by inactivating the enzyme, for example by raising the temperature above 90°C, for example between 90-100°C. Alternatively, in another embodiment, the incubation step is carried out at a temperature between 3 and 10°C, between 5 and 9°C, or at 8°C, at pH between 6.5 and 8. In another embodiment, previously and /or after the incubation stage, the The method comprises cooling the reaction medium (which includes the lactose-based product and the enzyme) to a temperature between 3 and 10°C, in particular between 5 and 9°C, in particular 8°C, at a pH between 6.5 and 8, for a period of time between 10 and 48 hours, particularly between 15 and 30 hours.

In another aspect, the present invention provides a method of reducing the lactose content in a dairy product, comprising the step of incubating the enzyme or the recombinant host cell as defined above, with a lactose-based product. , at a pH between 6.5 and 8.0. In one embodiment, the incubation is carried out at a temperature between 55 and 89 ° C or between 65 and 85 or between 70 and 75 ° C, at a pH between 6.5 and 8. The time of the incubation step will be the necessary to achieve the desired amount of GOS and will depend, among other factors, on the nature of the substrate. The person skilled in the art can stop the incubation at any time by inactivating the enzyme, for example by raising the temperature above 90°C, for example between 90-100°C. Alternatively, in another embodiment, the incubation step is carried out carried out at a temperature between 3 and 10°C, between 5 and 9°C, or at 8°C, at a pH between 6.5 and 8. In another embodiment, before and/or after the incubation stage, the procedure comprises cooling the reaction medium (including the lactose-based product and the enzyme) to a temperature between 3 and 10°C, in particular between 5 and 9°C, in particular 8°C, for a period of time between 10 and 48 hours, particularly between 15 and 30 hours, at a pH between 6.5 and 8.

Generally the procedures and apparatus employed for lactose hydrolysis are similar to those commonly used in equivalent enzymatic hydrolysis processes and a wide range of possible systems and procedures will be apparent to those skilled in the art. Therefore, in one embodiment, in a continuous incubation mode, the lactose-containing substrate in solution form is passed to a reactor containing the enzyme or enzymatically active whole cell preparation, preferably in immobilized form. The immobilized enzyme composition or immobilized whole cell preparation, and the glucose and galactose products are recovered downstream of the reactor.

In one embodiment of the methods provided by the present invention, the lactose-based substrate used in the incubation step is liquid.

In another embodiment of the methods provided by the present invention, the lactose-based substrate used in the incubation step is solid. In this embodiment, the enzyme can be incorporated in the form of a liquid enzyme preparation or, alternatively, added in solid form, to subsequently incorporate an aqueous medium at a pH between 6.5 and 8.0.

Furthermore, in view of the high thermal stability the enzyme can be incorporated directly with UHT dairy products before heat treatment and can be advantageously sterilized together with milk during heat treatment.

Thus, in a tenth aspect the present invention provides a process for preparing a sterilized, in particular pasteurized, dairy product, comprising pasteurizing the dairy product, such as milk, in the presence of the enzyme or the recombinant host cell as defined further. above.

In an embodiment of the tenth aspect, the pasteurization step is carried out at a temperature between 55 and 90°C, for a period of time between 5 and 30 seconds.

In another embodiment of the tenth aspect, prior to the pasteurization step, the dairy product, in the presence of the enzyme, is cooled to a temperature between 3 and 10°C, particularly between 4 and 9°C, particularly between 6 and 8.5°C. c.

In another embodiment of the tenth aspect, subsequent to the pasteurization step, the resulting dairy product is cooled to a temperature between 3 and 10°C, particularly between 3.5 and 5°C, particularly at 4°C.

In another embodiment of the tenth aspect, a cooling step is carried out before (between 3-10°C) and after pasteurization (between 4-9°C).

In another embodiment of the tenth aspect, the cooling step(s) is carried out for a period of time between 10 and 48 hours, particularly between 15 and 30 hours.

In one embodiment of the methods of the invention, these optionally comprise one or more of the steps of inactivation and/or elimination of the enzyme, conversion of the resulting product to powder and packaging in liquid or powder form.

As previously explained and demonstrated below, the product resulting from the enzymatic action of the beta-galactosidase of the invention has a low lactose content and is enriched in GOS.

Therefore, in the eleventh aspect, the present invention provides a dairy product obtainable by any of the methods provided by the present invention.

Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. Furthermore, the word “comprises” includes the case “consists of.” For those skilled in the art, other objects, advantages and features of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. Numerical signs relating to the drawings and placed in parentheses in a claim are intended solely to enhance the understanding of the claim, and should not be construed as limiting the scope of protection of the claim. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments indicated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the effect of temperature (T) expressed in degrees Celsius on the relative activity (A) of the enzyme, expressed in % (relative activity: % activity with respect to the maximum activity recorded in the assay). The Y axis represents activity, and the X axis represents temperature.

FIG. 2 represents the relative enzymatic activity (A), expressed in %, as a function of temperature: at 75 °C (a), 65 °C (b) and 55 (°C) and at different times (t) expressed in hours. The “Y” axis represents the relative enzymatic activity and the “Y” axis represents time.

FIG. 3 represents the effect of pH on the relative activity (A) of the enzyme, expressed in %. The Y axis represents the relative activity, and the X axis represents the pH.

FIG. 4 represents the production of GOS by the enzyme of the invention (black bar) and the commercial preparations Biocon (gray bar) and Maxilact (white bar) at 16 and 24 hours of incubation at 8°C and after pasteurization at time zero (HTST 0 h) and 24 hours later at 4°C (HTST 24 h). The stars indicate if there are significant differences applying a t-student of the result obtained with the BWβg1 enzyme with respect to the p-galactosidase from Biocon (gray star) or with the p-galactosidase from Maxilact (white star).

EXAMPLES

1. Cloning, expression and purification of 3-galactosidase

A sequence clone SEQ ID NO: 2, selected by functional search in a plasmid metalibrary constructed from the thermal waters of As Burgas (Ourense), was found to be capable of hydrolyzing X-Gal (5-bromo-4- chloro-indolyl-p-D-galactopyranoside) as it gave rise to a bluish coloration on solid plates of Luria Bertani (LB) medium supplemented with 100 ^g/ml ampicillin and 0.04% X-Gal in N,N-dimethylformamide. The deduced amino acid sequence for said enzyme corresponds to the sequence SEQ ID NO: 1.

The p-galactosidase ORF ( BWpgl) was amplified directly from the positive clone with primers MCA141 (5'CAATTCCCCTCTAGAATGACCCTGCGTGAGTTC3' (SEQ ID NO: 3)) and MCA140 (5'AGTGGTGGTGGTGGTGGTGCACCCTCATACATAGCCTCAC3' (SEQ ID NO: 4)). . For ligation into the pET21a vector, the NEBuilder HiFi DNA assembly kit (New England Biolabs, “NEB”) was used.

The construct was used to transform into E. coli T7 Express (C2566) (NEB) cells by heat shock, following the manufacturer's instructions, and express the protein, for which it was induced with 0.4 mM IPTG for 2 hours at 37°C. .

The cells were then centrifuged (5000 rpm 15 min 4°C) and resuspended in 20 mM sodium phosphate buffer pH 7.2 with 500 mM NaCl and a Complete Mini EDTA-Free Protease Inhibitor Cocktail (Roche). Cell lysis was carried out by sonication on ice with a Vibra Cell sonicator (100W, 5min 2”ON/8”OFF), (Sonics & Materials). The resulting crude extract was heated at 70°C for 10 minutes to denature the E. coli proteins.

Subsequently, the supernatant obtained after centrifugation (14,000 rpm 20 min) was passed through a histidine column HisTrapTM HP column (GEHealthcare). following the manufacturer's instructions and using an ÁKTA chromatography system (GEHealthcare). Briefly, the column was equilibrated with 20mM sodium phosphate buffer pH 7.2, 500mM NaCl and 20mM imidazole and protein elution was carried out with 20mM sodium phosphate buffer pH 7.2, 500mM NaCl and 500mM imidazole.

Those fractions corresponding to the elution peak of the chromatography were selected, subsequently carrying out a p-galactosidase activity test on each of them to check the presence of the enzyme. Selected fractions were concentrated and dialyzed using an Amicon Ultra-15 30,000 MWCO column (Millipore). The concentration of purified protein was quantified following the protocol of the Bio-Rad Protein Assay kit (Bio-Rad), using bovine serum albumin as a standard.

Protein samples at different stages of purification were loaded on a 10% acrylamide SDS-PAGE gel for determination of their molecular weight. The NZYcolour Protein Marker II (Nzytech) was used as a molecular weight marker and protein visualization was carried out by Coomasie blue staining. In this way, it was concluded that the enzyme with betagalactosidase activity had a molecular weight of approximately 75 kDa.

Analysis of the amino acid sequence of the protein

The amino acid sequence deduced from the nucleotide sequence obtained after sequencing the positive clone was compared with other known sequences using a protein-protein local alignment search tool (BLAST) (Altschul et al., "Basic local alignment search tool ", 1990, J. Mol. Biol, v. 215, pages 403-410). The conserved motifs in the sequences were searched using the ScanProsite tool (de Castro et al., 2006, "ScanProsite: Detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins". Nucleic Acids Res., v. 34). Additionally, a multiple sequence alignment was carried out with Clustal Omega (Sievers et al., 2011, Fast, scalable generation of high- quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol., v. 7) and the same program was used to build the phylogenetic trees with other pgalactosidases from known families of Glycosyl Hydrolases (GHs).

With the previous tests, it was concluded that the amino acid sequence of BWbg1 consisted of 664 amino acid residues and that the enzyme under study belongs to the GH-35 family.

Determination of enzymatic activity

The quantification of the enzymatic activity was carried out using the substrate ortonitrophenyl-pD-galactopyranoside (ONPG). Purified protein preparations were diluted in 150 μL of buffer Z (100 mM Na ² HPO ⁴ , 40 mM NaH ² PO ⁴ , 10 mM KCl, 1.6 mM MgSO ⁴ pH 7). After incubating for 5 minutes at 80°C, the reaction was started with the addition of 150 μL of ONPG at a concentration of 4 mg mL-1. To stop the reaction, 100 μL of 1 M Na ² CO ³ was added to 100 μL of the reaction mixture. The o-nitrophenol produced in the reaction was quantified by UV absorbance at 420 nm.

In the pH, optimal temperature and thermostability tests, the enzymatic activity was expressed as a percentage of relative activity, that is, percentage of activity with respect to the maximum activity recorded in the test.

In the ONPG kinetics and lactose hydrolysis assays, p-galactosidase activity was expressed in enzymatic units (U), defined as the amount of enzyme capable of releasing one ^mol of product (o-nitrophenol) per minute (^molmin- 1) in different experimental conditions of temperature and pH. In all cases the measurements were carried out in triplicate.

Kinetics of ONPG and lactose hydrolysis. Determination of affinity for lactose

The kinetic characterization of the enzyme was based on the determination of the pgalactosidase activity of the purified protein at different concentrations of ONPG (0-10 mM) following the protocol described in the previous section. Measurements were carried out in triplicate with 0.3 ^g mL-1 of enzyme. To estimate the values of the kinetic constants, a nonlinear regression fit by least squares was carried out from the Michaelis Menten graphs, with the Prism 6.00 software for Windows (GraphPad Software Inc.).

The kinetics of lactose hydrolysis were carried out by determining the glucose released by the enzyme at different lactose concentrations. To do this, the purified protein was diluted in buffer Z to a concentration of 104.5 ^g/mL. The initial rate was determined in triplicate using 104.5 ^g mL-1 of enzyme and a lactose concentration of 0, 10, 20 and 80 mM. Reaction times were 6–20 min at 80 °C. Finally, the samples were heated at 96 °C for 5 min in order to stop the reaction.

p-galactosidase activity is expressed in enzymatic units (U), defined as the amount of enzyme capable of releasing 1 ^mol of product (D-glucose) per minute under experimental conditions (^mol min -1). Glucose concentration was quantified using the commercial D-Glucose GOD-POD kit (Nzytech). A nonlinear least squares regression fit was used to infer kinetic parameters from the generated Michaelis-Menten plots (via Prism 6).

Table 2. Kinetic values of K ^m and V ^max for the hydrolysis of lactose and ONPG.

Effect of temperature on enzyme stability and activity

The effect of temperature was determined by quantifying the relative activity (percentage of activity with respect to the maximum activity recorded) of the enzyme at 55-90°C with ONPG (4mg mL-1) in buffer Z using the procedure described in section “ Determination of enzymatic activity”.

In a first test, the temperature that provides the optimal activity of the enzyme was determined. Thus, the enzyme was pre-incubated at a final concentration of 113 ^g mL-1 in buffer Z at each of the test temperatures and the activity was measured based on the o-nitrophenol released (as detailed in previous sections). The results were expressed as a percentage of relative activity (percentage of enzymatic activity with respect to the maximum recorded). From the data provided in FIG. 1, it was concluded that the optimal temperature was around 80°C.

Next, a thermostability study of the enzyme was carried out in a temperature range of 55-75°C for 1-2 hours. The results are shown in FIG. 2. From these data it is concluded that the enzyme is extremely active for long periods of time under high temperature conditions, between 55 and 75°C. This is indicative of the high stability of the enzyme of the invention.

Effect of pH on enzymatic activity

To estimate the effect of pH on the enzymatic activity, 113 ^g mL-1 of the enzyme were preincubated at the optimal temperature (80°C) in 20 mM Britton-Robinson buffer (Britton and Robinson, 1931, CXCVIII.— Universal buffer solutions and the dissociation constant of veronal. J. Chem. Soc., v. 0, pages 1456-1462) with different pH values, within the range between 5.0-8.5 adjusted with a 1M NaOH solution using a pH meter. Next, the relative activity against ONPG (4mg mL-1) was quantified. The results are expressed as a percentage of relative activity (percentage of activity with respect to the maximum activity recorded) and are shown in FIG. 3.

As can be seen, the optimum pH for the enzyme of the invention was found between 6.5-7.5.

Production and characterization study at elevated temperatures (70°C)

GOS, glucose, galactose, and lactose concentrations were determined by HPLC (Waters Breeze I HPLC), using a Waters Sugar-Pak column eluted at 90 °C with 0.1 M disodium EDTA in Milli-Q water at a flow rate of 0.5 mL min -1, and a refractive index detector: 2414 (Waters).

To carry out the reactions, 0.034 mg of pure protein was mixed in 0.1 M phosphate buffer (pH 6.8), supplemented with 40% lactose. Samples (500 μL) were incubated at 70°C and 650 rpm. At 0, 0.5, 1, 2, 4, 6 and 24 h, the reaction was stopped by heating to 99 °C to inactivate the enzyme. All samples were stored at −20°C for further analysis.

Carbohydrates were quantified by external calibration using standard solutions of galactose, glucose, lactose, raffinose and stachyose. Total GOS production was estimated by expressing the final concentration as a percentage of the initial lactose concentration (in % w/v).

A maximum GOS production level of 48% was detected using 0.034 mg, with an initial lactose concentration of 400 mg/mL. and after four hours of reaction. The chromatographic profiles obtained by HPLC indicate that the predominant GOS produced by BWμg1 are also trisaccharides followed by tetrasaccharides, which show the best effects.

Production and characterization study at low temperatures (8°C)

The inventors carried out a comparative study with commercial enzymes to verify the difference in efficiency in obtaining a product rich in GOS.

In these experiments, the hydrolysis and GOS production capacity of the p-galactosidase BWβg1 of the invention was compared with the commercial p-galactosidases Biolactase NL Super (Biocon) and Maxilact LGi 5000 (DSM). In order to compare, the same enzymatic units (EU) were used in the three enzymes (34.32 micromoles/min in 50 microliters of double-distilled water), so the commercial preparations had to be diluted one hundred times.

For the experiment, 50 microliters of each of the diluted enzymes (all with the same total EU, as previously indicated) were placed in the presence of 450 microliters of fresh semi-skimmed milk from Milbona (from LIDL). They were incubated at 8°C for 24 hours and a sample was taken after 16 hours and 24 hours. After 24 hours, the pasteurization step was carried out, which consisted of subjecting the sample to 79°C for 17 seconds. A sample was taken immediately after pasteurization (HTST 0 h) and after allowing a period of 24 hours to pass in a refrigerator at 4°C (HTST 24 h). Subsequently, the samples were kept frozen until they were passed through 10K cut filters (to eliminate the enzyme, as well as the proteins and fat from the milk) by centrifuging at 13,000 for 10 minutes. The eluate was diluted tenfold and passed through 0.22 micron filters through a syringe. 15 microliters were loaded into the HPLC to determine the GOS concentration, following the protocol described in the previous section. All samples were performed in triplicate. As a control, a triplicate of fresh semi-skimmed milk with lactose (450 microliters plus 50 microliters of water) and fresh semi-skimmed milk without lactose (450 microliters plus 50 microliters of water), both from the Milbona brand from LIDL, was made.

The beta-galactosidase enzyme of the invention was found to be capable of producing significant and superior amounts of GOS regardless of temperature, whether 79°C (pasteurization temperature) or 8°C (storage temperature), as as shown in Fig. 4.

On the other hand, the inventors found that the enzyme, once the pasteurization process was completed and after a rest period of 24 hours, continued to produce significantly GOS.

Finally, it was found that the BWβg1 enzyme of the invention, even though it is a thermostable enzyme, has activity at the low test temperature, 8°C, and under the experimental conditions tested, it presents a lactose reduction of 63% after 24 hours of incubation at 8°C and subsequent pasteurization, compared to 52.6% of the commercial enzyme of the Biocon brand and 38.5% of the commercial enzyme Maxilact, which represents a significant difference in lactose hydrolysis of the BWβg1 enzyme with regarding the two commercial preparations.

Thus, it was found that the enzyme of the invention was capable of producing practically the same amount of GOS by incubating the substrate with the enzyme in the cold as during the pasteurization process. This is significant for the dual behavior and the extraordinary stability and versatility in such a temperature range.

APPOINTMENT LIST

Altschul, SFet al., "Basic local alignment search tool", J. Mol. Biol., 1990, v. 215, pages 403—410.

Britton, HTS, et al.,"CXCVIII.— Universal buffer solutions and the dissociation constant of veronal", J. Chem. Soc., 1931, v. 0, 1456-1462.

de Castro, E., et al., "ScanProsite: Detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins", Nucleic Acids Res., 2006, v. 34, pages W362-W365.

Sievers, F. et al. "Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega", Mol. Syst. Biol., v. 7, 539.

Claims (17)

1. Enzyme having beta-galactosidase activity, which is selected from (a) an amino acid sequence consisting of the sequence SEQ ID NO: 1; and (b) an amino acid sequence encoded by the nucleotide sequence SEQ ID NO: 2, wherein the enzyme is capable of hydrolyzing lactose and producing galactooligosaccharides simultaneously at a temperature between 3 and 10 ° C or between 55 and 89 ° C . 2. Enzyme according to any of claim 1, consisting of the sequence SEQ ID NO: 1. 3. Enzyme according to any of claims 1-2, which is immobilized on a support, in particular on a solid phase material. 4. Recombinant nucleotide sequence comprising a nucleotide sequence encoding the enzyme as defined in any of claims 1-2, operably linked to an expression promoter. 5. Expression construct comprising the nucleotide sequence of claim 4. 6. Recombinant host cell transformed with the nucleic acid of claim 4 or the expression construct of claim 5. 7. Process for producing galactooligosaccharides, the process comprising the step of incubating the enzyme as defined in any of claims 1-3 or the recombinant host cell of claim 6, with a lactose-based product, at a pH between 6.5 and 8, and at a temperature between 3 and 10°C, or between 55-89°C, where the lactose-based product, or dairy product, is selected from commercial lactose, whole milk, semi-skimmed milk , skimmed milk, whey, fat-enriched skimmed milk and filtered whey. 8. Procedure for increasing the content of galactooligosaccharides in a lactose-based product, the procedure comprising the incubation step of the enzyme is defined in any of claims 1-3 or of the host cell recombinant of claim 6, with the lactose-based product, at a pH between 6.5 and 8, and at a temperature between 3 and 10°C, or between 55-89°C, wherein the lactose-based product, or dairy product, is selected from commercial lactose, whole milk, semi-skimmed milk, skimmed milk, whey, fat-enriched skimmed milk and filtered whey. 9. Lactose hydrolysis procedure, the procedure comprising the step of incubating, in a liquid medium, the enzyme as defined in any of claims 1-3 or the recombinant host cell of claim 6, with a product a lactose base, at a pH between 6.5 and 8 and at a temperature between 3 and 10°C, or between 55-89°C, where the lactose-based product, or dairy product, is selected from commercial lactose, whole milk, semi-skimmed milk, skimmed milk, whey, fat-enriched skimmed milk and filtered whey. 10. Procedure for reducing the lactose content in a dairy product, the procedure comprising the step of incubating the enzyme as defined in any of claims 1-3 or the recombinant host cell of claim 6, with the product a lactose base, at a pH between 6.5 and 8 and at a temperature between 3 and 10°C, or between 55-89°C, where the lactose-based product, or dairy product, is selected from commercial lactose, whole milk, semi-skimmed milk, skimmed milk, whey, fat-enriched skimmed milk and filtered whey. 11. Procedure according to any of claims 7-10, wherein the incubation step is carried out at a temperature between 55 and 89°C and the procedure optionally comprises a cooling step before and/or after the step. incubation, where the temperature in the cooling stage(s) is between 3 and 10°C. 12. Procedure for preparing a pasteurized dairy product comprising pasteurizing the dairy product in the presence of an enzyme as defined in any of claims 1-3 or the recombinant host cell according to claim 6, at a pH between 6.5 and 8, wherein the lactose-based product, or dairy product, is selected from commercial lactose, whole milk, milk semi-skimmed, skimmed milk, whey, fat-enriched skimmed milk and filtered whey. 13. Procedure of claim 12, wherein the pasteurization step is carried out at a temperature between 55 and 89°C, for a period of time between 5 and 30 seconds. 14. Procedure according to any of claims 12-13, wherein prior to the pasteurization step, the dairy product and the enzyme are cooled to a temperature between 3 and 10°C, particularly between 4 and 9°C, particularly between 6 and 8°C. 15. Procedure according to any of claims 12-14, wherein subsequent to the pasteurization step, the resulting dairy product is cooled to a temperature between 3 and 10°C, particularly between 3.5 and 5°C. 16. Method according to any of claims 12-15, wherein the cooling step is carried out for a period of time between 10 and 48 hours, particularly between 15 and 30 hours. 17. Dairy product obtainable by the procedure defined in any of claims 7-16.