JPH0575385B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention provides a method for producing lipases using molds belonging to filamentous fungi, specifically, a method for producing lipases by culturing molds belonging to filamentous fungi in a liquid medium, and producing a large amount of lipases. This relates to a method for stably producing it. [Prior art] Lipase has been produced from animal resources, mainly pancreas, and plant resources, such as castor, for a long time.
40-4422, 42-8912, 56-42266
Publication No. Agricultural and Biological Chemistry
Biological Chemistry) Volume 38, No. 6, 1249 (1974),
Vol. 39, No. 5, p. 1063 (1975), Vol. 33, No. 3, 299
(1969), etc., the genus Aspergillus, the genus Geotricum, and the genus Penicillium.
There are known methods of manufacturing by culturing filamentous fungi such as Rhizopus, Rhizopus, and Mucor. Lipase has been used as a medicine (digestive agent) since ancient times, and is also used to hydrolyze milk fat to produce flavor. In addition, lipoprotein lipase was known as an enzyme in the body of animals, but it is known that similar microbial lipoprotein lipase is produced by Rhizopus and Mucor genus, and it is known that lipoprotein lipase is produced by the genus Rhizopus and Mucor. It is used for measurement. For the production method of microbial lipoprotein lipase, see Japanese Patent Publication No. 41-7836 and No. 58-37834.
Publication No. Agricultural and Biological Chemistry
Biological Chemistry) Vol. 44, No. 4, p. 799 (1980
The method described in 2010) is known. Regarding lysophospholipase, please refer to the Biochemical Journal (Biochemical Journal).
Journal) Vol. 70, p. 559 (1958)
A process using Penicillium notatum is known and can be used to produce glycerophosphocholine from lecithin. [Problems to be Solved by the Invention] With the conventional methods described above, it is difficult to stably produce lipases in large quantities, and therefore lipases are relatively expensive. Therefore, for example, when lipase is used for the above-mentioned purposes such as pharmaceuticals, it is cost-effective even though lipase is relatively expensive, but recently, methods of using lipase for decomposition of fats and oils and transesterification of fats and oils have been introduced. The developed lipases, especially those derived from fungi, have positional specificity to the α-position of triglycerides and are said to be useful for transesterification of oils and fats. Therefore, there is a need for a method for producing lipase that is inexpensive in terms of cost. Therefore, an object of the present invention is to provide a method for producing lipases in large quantities at low cost using simple means. [Means for Solving the Problems] The present invention aims to achieve the above object by culturing a mold that belongs to filamentous fungi and producing lipases in a liquid medium, and obtaining lipases from the culture by using a substrate in the medium. This was achieved by increasing the concentration of lipases in the culture by feeding substrates that can be used by the filamentous fungi to such an extent that the bacterial cell concentration does not increase from the point at which the dissolved oxygen concentration begins to increase as the dissolved oxygen concentration begins to increase. It is something. The method for producing lipases of the present invention will be described in detail below. Lipases produced by the method for producing lipases of the present invention include lipase (enzyme number
3.1.1.3), microbial lipoprotein lipase (enzyme number 3.1.1.34), or lysophosphoripase (enzyme number 3.1.1.5). The filamentous fungi used in the present invention include Aspergillus flavus.
flavus) ATCC 11492, Aspergillus oryzae ATCC 14605, Aspergillus parajiiteicus
parasiticus) ATCC 26850, etc., Beauveria bassiana
Beauveria (Beauveria genus), Geotricum kyandeidam (bassiana) ATCC 26037, 26851, etc.
cand idum) ATCC 34614, Geotricum genus such as Geotricum klebahnii ATCC 2001, Metarhizium genus such as Metarhizium anispliae ATCC 26852, Mucor flavus IAM
6143, Mucor miehei
IAM 9741, Mucor
Javanicus) IAM 6108, 6089, etc., Mucor genus, Paecilomyces
genus Paecilomyces such as (Farinosus) ATCC 26853, Penicillium
caseicolum) ATCC 24936, Penicillium cyclopium (Penicillium cyclopium) ATCC
Penicillium genus such as 34613, Rhizopus delemer
IFO 4697, 4726, 4754, 4771, 4773, ATTC
34612, Rhizopus chinensis
chinensis) FERM 2767, Rhizopus niveus IFO 4759, Rhizopus japonicus IFO 5318,
Rhizopus genus such as 4758, Verticillium lecanii
Verticillium genus, such as ATCC 26854 (Lecanii), etc. In addition, IFO is a strain preserved by the Fermentation Research Institute,
ATCC is a strain preserved in the American Type Culture Collection, FERM is a strain preserved in the Institute of Microbiology, Agency of Industrial Science and Technology, and IAM is a strain preserved in the Institute of Applied Microbiology, the University of Tokyo, all of which are publicly available. The substrates fed in the present invention include glucose,
Water-soluble carbohydrates such as sucrose, maltose, fructose, glycerin, dextrin, and soluble starch; oil-in-water emulsified fats and oils of animal and vegetable oils stably emulsified with surfactants, phospholipids, proteins, etc., and natural oils such as soy milk and milk. emulsified oils and fats; protein decomposition products such as polypeptone, tryptone, yeast extract, fish or meat extract; cornstarch liquor, defatted soybean flour or its extract, casein soda, other water-soluble proteins such as albumin; Mixtures of proteins can be used alone or in appropriate combinations. It is known to culture microorganisms by feeding a substrate, but in the conventional fed-batch culture method, for example, in the production of ban yeast, a sugar solution is continuously fed to cultivate microorganisms while avoiding alcohol fermentation. To propagate
-49792, the purpose of which is to automatically feed sugar to bacteria intermittently to grow the bacterial cells, and the present invention does not The feature is that a much smaller amount of fed-batch is performed than in conventional methods. Therefore, when carrying out the method for producing lipase of the present invention, first, a mold that belongs to the above-mentioned filamentous fungi and produces lipases is cultured on a potato sucrose agar medium or the like, and the bacterial cells are multiplied. Next, inoculate the spores if they form spores, or the bacterial cells if they do not form spores, into a liquid medium in a suitable incubator,
Cultivate. The amount of inoculation is not particularly limited, but in the case of spores it is approximately 10 ⁴ to 10 ⁸ particles/100 ml, and in the case of bacterial cells it is approximately 1 loopful/100 ml. The liquid medium used in the present invention usually includes:
0.01-0.2% by weight of inorganic salts such as KH ₂ PO ₄ and MgSO ₄
In addition, sugars such as glucose, sucrose, and soluble starch may be added in an amount of about 0 to 4% by weight as required, and corn stable liquor, soy milk, soy flour, yeast extract, polypeptone, etc. may be added. Culture at an appropriate temperature, for example 20°C, at a pH of about 5.0 to 6.5.
It may be carried out by shaking culture, aeration stirring culture, etc. at ~33°C. Then, feeding of the substrate is started from the time when the dissolved oxygen concentration starts to rise, and the feeding amount of the substrate is set so that the dissolved oxygen concentration, pH, and amount of bacterial cells remain approximately constant. Generally speaking, when the amount of substrates required is based on saccharides, the amount of protein is a little more, and the amount of emulsified oils and fats is a little less. However, if the addition amount is too small, no effect will be seen, so it may be determined as appropriate depending on the combination of each bacterial strain and substrate, but in practice it is preferably 1 to 6% by weight per hour based on the dry weight of the bacterial cells in the culture system. is preferably about 1.5 to 4.5% by weight per hour. The fed-batch method includes a method of feeding intermittently at intervals of a certain period of time, and a method of almost continuous feeding under computer control using a glucose sensor, a dissolved oxygen concentration sensor, etc. To obtain lipases from the culture, measure the lipase activity at any time during the culture, remove the bacterial cells by a conventional method such as filtration when the lipase activity reaches almost the maximum, and further ultrafiltrate or remove the filtrate. Alternatively, lipases can be recovered by treatment with ammonium sulfate precipitation, organic solvent precipitation with acetone, alcohol, etc., adsorption with an ion exchange resin, or the like. In addition, when the culture time is long or the culture volume is large, the culture solution may be extracted while adding the substrate to perform bacterial cell isolation and subsequent processing. Lipases obtained by the method of the present invention can be used for various purposes in the same manner as lipases obtained by conventional methods. [Example] Examples of the present invention and comparative examples are shown below,
The present invention is not limited to these. In Examples and Comparative Examples, lipase activity and lipoprotein lipase activity were measured as follows. [Measurement of lipase activity] Lipase activity was measured as follows based on the method of Yamada et al., described in Nouka-shi Vol. 36, No. 10, p. 860 (1962). Method: In a 100 ml Erlenmeyer flask, add 4 ml of 0.1M phosphate buffer with pH 6.0 and 5 ml of olive oil emulsion (mixed emulsion of 2% polyvinyl alcohol aqueous solution dissolved at below 80°C and JP olive oil at a ratio of 3:1). In addition,
Preheat at 37±0.2°C for 5 minutes. Add 1 ml of the sample containing lipase to this and react at 37±0.2°C for exactly 30 minutes. Immediately add acetone/ethanol (1:
1) After adding 10ml of liquid to stop the reaction, 0.05N
10ml of caustic soda solution and acetone/ethanol (1:
1) Add 10ml of the solution and back titrate with 0.05N hydrochloric acid solution using phenolphthalene as an indicator. (After adding acetone/ethanol solution to 0.1M phosphate buffer and olive oil emulsion as a blank, 1 ml of sample
Use the one with the added. Calculation formula for lipase activity Lipase activity (U/ml) = Tb - Ts / 0.6 x 10 x f x n Note] Tb...Blank titration value; Ts...Sample titration value; f...0.05N hydrochloric acid titer; n ...Sample dilution factor (For this lipase activity, the amount of enzyme that releases 0.1 μg/min of fatty acids is 1 unit (U).) [Measurement of lipoprotein lipase activity] 2% polyvinyl alcohol solution as the substrate
Add 2.0g of olive oil to 22.5ml and emulsify at 10℃ or below, add 1ml and 50ml of adult bovine serum, and boil at 37℃ for 30 minutes.
Create a sample by incubating for minutes. Add 2 ml of 0.2M phosphate buffer (PH2.0) to 2 ml of this substrate solution and preheat at 37°C for 5 minutes. Add 1 ml of the enzyme solution appropriately diluted with the above buffer and allow to react at 37°C for 10 minutes, and determine the decrease in turbidity by measuring the absorbance at 660 nm. It is generally known that there is a proportional relationship between the amount of turbidity reduction and the amount of fatty acid production, so Dancombe's method [Journal of Biochemistry] was used in advance.
Biochemistry) 88, 8 (1963)], the amount of enzyme can be determined from the results of investigating the relationship between the amount of fatty acids produced and the decrease in turbidity. That is, 1 unit (U) of the enzyme amount shown here is the enzyme amount that releases μeg/min of fatty acid. Example 1 and Comparative Example 1 Glucose 2%, Corn Stap Liquor 10
%, KH ₂ PO ₄ 0.1%, and MgSO ₄ 7H ₂ O 0.05%, pH 5.8 medium 60 was inoculated into 400 ml of the same medium at a rate of 10 ⁶ spores/100 ml, and 28 ℃
Rhizopus delemer IFO cultured for 2 days in
4754 bacteria were inoculated and cultured with aeration at an aeration rate of 1VVM, a temperature of 28°C, and a rotation speed of 250 rpm. The progress of the culture is shown in FIGS. 1 and 2. Figures 1 and 2
In the figure, 1 is a graph showing the relationship between fermentation time and dissolved oxygen concentration (DO, with the saturation value being 100) in the culture solution, 2 is a graph showing the relationship between fermentation time and PH of the culture solution, and 3 is a graph showing the relationship between fermentation time and culture solution PH. Graph showing the relationship between time and bacterial cell concentration (dry weight %) of the culture solution, 4 is a graph showing the relationship between fermentation time and glucose concentration of the culture solution, and 5 is fermentation time and lipase activity (U/ml) It is a graph showing the relationship between Figure 1 shows the case where 32g of a 30W/V% glucose solution was added every hour (12 times, i.e. 11 hours) as the glucose portion immediately after the dissolved oxygen concentration started to rise (Example 1). The culture progress is shown. Moreover, FIG. 2 shows the progress of culture when feeding was not performed (Comparative Example 1). As is clear from FIGS. 1 and 2, Example 1
In both cases of Comparative Example 1, glucose was consumed 20 hours after the start of fermentation, and then assimilation of proteins etc. started, and the amount of bacterial cells continued to increase, but the substrate was consumed 31 hours after the start of fermentation. The dissolved oxygen concentration and pH begin to rise rapidly. However, after the start of feeding the substrate in Example 1, the bacterial cell concentration hardly increases in any case, but in the case of Comparative Example 1, in which the substrate was not fed, the concentration increased 34 hours after the start of fermentation. Maximum lipase activity (261U/
ml) and subsequently decreased due to decomposition, whereas in the case of Example 1 in which the substrate was fed, the dissolved oxygen concentration remained at almost the same level, and after the start of fermentation
The lipase activity reached its maximum (510 U/ml) in 45 hours, and the lipase activity almost doubled by feeding the substrate. Examples 2 to 3 and Comparative Example 2 Rhizopus delemer ATCC 34612 bacteria were mixed with 4% polypeptone, 2% glucose, 0.1% KH ₂ PO ₄ ,
and 400 _ml of medium containing _{MgSO4.7H2O0.05} %
¹⁰ as the number of spores in 2 Erlenmeyer flasks containing 6
cells/100ml, and rotatably cultured at 28℃ for 2 days.
It was used as a seed fungus. Additionally, 100 fermenters each containing 60 of the various media shown in Table 1 below were sterilized at 121°C for 20 minutes. Next, each of these mediums was inoculated with the above-mentioned inoculum and stirred at 250 rpm at 28°C, with an initial aeration rate of 0.5 VVM and a dissolved oxygen concentration (hereinafter referred to as DO) of 1/5. Thereafter, it was cultured as 1VVM. In Comparative Example 2, fed-batch was not carried out, and in Examples 2 and 3, fed-batch was started immediately after the substrate in the medium was consumed by the bacterial cells and DO started to rise, and the feeding was carried out for 9 hours and 30 minutes, respectively. A fed-batch process was conducted for 7 hours. Example 2 uses only glucose (feeding amount
30 g/hour), Example 3 was glucose (fed dose 20
g/hour) and polypeptone (feed amount: 15 g/hour) were each fed as an aqueous solution. The maximum lipase activity in Comparative Example 2 was 204 U/ml, whereas in Example 2,
A maximum lipase activity of 508 U/ml and 427 U/ml in Example 3 was obtained (see Table 1 below). Examples 4 to 7 and Comparative Example 3 Rhizopus delemer IFO 4697 as a bacterial strain
The experiments were carried out in the same manner as in Example 2, except that the bacteria and the various media shown in Table 1 below were used. However, Comparative Example 3 did not use fed-batch, Example 4 used seuucrose (feeding amount 28 g/hour, feeding time 5 hours), and Example 5 used seuucrose (feeding amount 26 g/hour).
Example 6 is emulsified oil (feeding amount 20 g/hour, feeding time 7 hours), Example 7 is polypeptone (feeding amount 45 g/hour, feeding time 7 hours)
were fed together. In the examples, lipase activity was obtained that was 1.7 times as much as that of Comparative Example 3 when the feeding time was short, and about 2 times as much when the feeding time was long (see Table 1 below).

[Table] Examples 8 to 10 and Comparative Examples 4 to 6 Except for using the bacteria shown in the bacterial species column of Table 2 below,
Examples 8 to 10 are the culture conditions of Example 2 and Comparative Example 4.
-6 were carried out under the same culture conditions as Comparative Example 2. Although the feeding start time differed depending on the bacterial species, the feeding time was all 7 hours. The results are shown in the table below.
Shown in 2. No matter which bacteria were used, it was observed that the lipase activity of Examples was improved by about twice as much as that of Comparative Examples by feeding.

[Table] In all Examples, the lipase accumulated in the culture system was removed by removing the bacterial cells by filtration or centrifugation, and the culture filtrate was subjected to ammonium sulfate precipitation with or without ultrafiltration concentration. It can be used after rough purification or purification by conventional means such as organic solvent precipitation method using acetone or alcohol, adsorption method using ion exchange resin, etc. Examples 11-12 and Comparative Examples 7-8 Example 11 and Comparative Example 7 used Geotrichum candidum ATCC 34614, and Example 12 and Comparative Example 8 used Aspergillus oryzae ATCC.
14605 bacterium was used. Medium: 4% rice bran, 5% cornstarch liquor, 1% glucose, KH ₂
One containing 0.1% PO ₄ , 0.05% MgSO ₄ .7H ₂ O, and 0.05% antifoaming agent Adekanol SX 294 (manufactured by Asahi Denka Kogyo Co., Ltd.) (PH 5.8) was used. Each of the above bacteria was grown for 28 hours in a 500 ml Erlenmeyer flask containing 100 ml of the above medium.
The cells were cultured at ℃ for 2 days and used as inoculum. Additionally, 10 jar fermenters each containing the above medium 6 were sterilized at 121°C for 30 minutes. Next, the above-mentioned inoculum was added to each of these sterilized media in terms of the number of spores.
10 ⁶ pieces/100ml were inoculated, and in the example, the substrate was consumed.
Immediately after the DO started to rise, 3 g of glucose was fed every hour for 6 hours, and the culture was terminated 1 hour after the feeding. In the comparative example, feeding was not performed, and the culture was terminated one hour after the DO started to rise. After the culture was completed, the bacterial cells were centrifuged, and the lipase was recovered by a conventional method. Lipase activity 1 hour after DO rise is shown in the table below.
3, and regardless of which bacteria were used, the lipase activity of Examples was found to be about twice as high as that of Comparative Examples by feeding.

〔Effect of the invention〕

According to the method for producing lipases of the present invention, lipases can be produced in large quantities at low cost using simple means.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the progress of culture in Example 1, and FIG. 2 is a graph showing the progress of culture in Comparative Example 1. 1... Graph showing the relationship between fermentation time and dissolved oxygen concentration (DO, saturation value is 100) of the culture solution, 2... Graph showing the relationship between fermentation time and PH of the culture solution, 3... Fermentation Graph showing the relationship between time and bacterial cell concentration (dry weight %) of the culture solution, 4...Graph showing the relationship between fermentation time and glucose concentration of the culture solution, 5...Fermentation time and lipase activity (U/ml ) is a graph showing the relationship between

Claims (1)

[Scope of Claims] 1. When a mold that belongs to filamentous fungi and produces lipases is cultured in a liquid medium and lipases are obtained from the culture, the substrate in the medium is almost consumed and the dissolved oxygen concentration increases. From the beginning, a substrate that can be used by filamentous fungi is added to the extent that the fungal cell concentration does not increase.
A method for producing lipases, which comprises increasing the concentration of lipases in a culture. 2. The substrate according to claim 1, wherein the substrate to be fed is one or a mixture of two or more selected from water-soluble carbohydrates, emulsified oils and fats, protein decomposition products, or proteins. Method for producing lipases. 3. The method for producing lipases according to claim 1, wherein the amount of the substrate fed is 1 to 6% by weight per hour based on the dry weight of the bacterial cells in the culture system. 4 Lipases include lipase (enzyme number 3.1.1.3),
Microbial lipoprotein lipase (enzyme number
3.1.1.34) or lysophosphoripase (enzyme number 3.1.1.5). 5 The filamentous fungus is Aspergillus.
Genus, Beauveria, Geotricum, Metarhizium, Mucor, Paecilomyces, Penicillium, Rhizopus,
2. The method for producing lipases according to claim 1, wherein the lipase is a bacterium belonging to the genus Verticillium. 6. The method for producing lipases according to claim 5, wherein the filamentous fungus is a fungus belonging to either the genus Mucor or the genus Rhizopus. 7. The method for producing lipases according to claim 5, wherein the filamentous fungus is a fungus belonging to the genus Penicillium.