JP2019017355A - Enzyme manufacturing method - Google Patents

Abstract

To provide a method of recovering a cultured product from an enzyme producing microorganism with ease and at high yield.SOLUTION: The enzyme manufacturing method involves adding polylysine to a cultured product of an enzyme producing microorganism followed by filtering thereof. Preferably, after adding polylysine, the method involves mixing under conditions that do not deactivate the enzyme and that promotes aggregation so as to facilitate filtering. The enzyme production method involves recovering the filtrate after filtration, treating with conventional methods and refining the enzyme. In this enzyme manufacturing method the enzyme is a protease, the concentration of the added polylysine is 0.0001 to 10 mass%, and the enzyme producing microorganism is of the genus Bacillus or is an enzyme producing bacteria belonging to the genus Bacillus.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing an enzyme from a culture.

  Enzymes are produced in the body of animals and plants in addition to those produced by microorganisms, but their industrial production technology is based on means for culturing microorganisms. For example, the genus Bacillus and its related bacteria are widely used as enzyme-producing microorganisms because they secrete enzymes outside the cells. After culturing these microorganisms, it is necessary to efficiently separate the microorganisms and enzymes in the culture, but these microorganisms may produce polymer components in addition to the enzymes, resulting in the culture solution The increase in viscosity often causes problems in the separation of enzymes and microorganisms. For example, a highly viscous culture solution reduces the sedimentation of microorganisms when centrifugation, which is a common cell separation method, is performed. For this reason, in place of filtration using diatomaceous earth, microfiltration using a hollow fiber has been proposed (Patent Document 1).

Japanese Patent Application Laid-Open No. 6-98765

However, microfiltration using a hollow fiber cannot be used for filtration of a highly viscous culture solution, and is not suitable for mass production of enzymes.
Accordingly, an object of the present invention is to provide a technique for efficiently separating an enzyme from a microorganism culture by a simple operation.

  Therefore, the present inventor examined a means for efficiently separating the enzyme and the culture containing the microorganism without inactivating the enzyme. Surprisingly, if polylysine is added to the culture, the microorganism and the viscosity increasing component are added. And the enzyme coagulates and precipitates, and the enzyme is not inactivated and is present in the supernatant. Thus, the filterability is remarkably improved, and it is found that the enzyme can be separated efficiently only by filtering with a normal filtration means. Was completed.

  Accordingly, the present invention provides the following [1] to [5].

[1] A method for producing an enzyme, comprising adding polylysine to a culture of an enzyme-producing microorganism and then filtering.
[2] The production method according to [1], wherein the filtrate is collected after filtration.
[3] The production method according to [1] or [2], wherein the enzyme is a protease.
[4] The production method according to any one of [1] to [3], wherein the added polylysine concentration is 0.0001 to 10% by mass.
[5] The production method according to any one of [1] to [4], wherein the enzyme-producing microorganism is an enzyme-producing bacterium belonging to the genus Bacillus or related to Bacillus.

  ADVANTAGE OF THE INVENTION According to this invention, an enzyme can be isolate | separated simply and efficiently from the culture of an enzyme-producing microorganisms by a normal filtration means, without inactivating an enzyme.

  The enzyme production method of the present invention is characterized by adding polylysine to a culture of enzyme-producing microorganisms and then filtering.

  The enzyme-producing microorganism is not particularly limited as long as it is a microorganism known to produce an enzyme, and examples thereof include the following microorganisms.

Enzyme name: agarase microorganism name: basidiomycete ( Coliolus ) or bacteria ( Bacillus , Pseudomonas )
Enzyme name: Acylase Microorganism name: Filamentous fungi ( Aspergillus ochraceus , Aspergillus melleus )
Enzyme name: α-Actractor decarboxylase (lyase)
Microorganism name: Bacteria ( Bacillus subtilis , Serratia )
Enzyme name: Aminopeptidase Microorganism name: Bacteria ( Aeromonas caviae , Lactobacillus casei , Lactococcus lactis )
Enzyme name: α- amylase microorganisms name: filamentous fungi (Aspergillus aureus, Aspergillus niger, Aspergillus oryzae), bacteria (Alcaligenes latus, Arthrobacter, Bacillus amyloliquefaciens , Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis, Sulfolobus solfataricus) or actinomycetes (Thermomonospora viridis )

Enzyme name: β-amylase microorganism name: filamentous fungi ( Aspergillus oryzae ), actinomycetes ( Streptomyces ) or bacteria ( Bacillus amyloliquefaciens , Bacillus polymyxa , Bacillus subtilis )
Enzyme name: Alginate lyase Microorganism name: Bacteria ( Alteromonas macleodii , Flavobacterium maltivolum , Pseudomonas , Xanthomonas )
Enzyme name: Anthocyanase Microorganism name: Filamentous fungi ( Aspergillus oryzae , Aspergillus niger , Penicillium decumbens )
Enzyme name: Isoamylase Microorganism name: Bacteria ( Bacillus , Flavobacterium odoratum , Pseudomonas amyloderamosa )
Enzyme name: Isomalt dextranase Microorganism name: Bacteria ( Arthrobacter )

Enzyme name: Inulinase Microorganism name: Filamentous fungi ( Aspergillus aculeatus , Aspergillus niger , Aspergillus phoenicis , Penicillium purpurogenum , Trichoderma )
Enzyme name: Invertase Microorganism name: Filamentous fungi ( Aspergillus aculeatus , Aspergillus awamori , Aspergillus niger ), bacteria ( Arthrobacter , Bacillus ) or yeast ( Kluyveromyces lactis , Saccharomyces cerevisiae )
Enzyme name: Urease Microorganism name: Lactobacillus fermentum or bacteria ( Arthrobacter )
Enzyme name: Exomaltotetrahydrase Microorganism name: Bacteria ( Pseudomonas stutzeri )

Enzyme name: Esterase Microorganism name: Filamentous fungi ( Aspergillus ), bacteria ( Pseudomonas ) or yeast ( Candida , Torulopsis )
Enzyme name: Catalase (oxidase)
Microorganism name: Aspergillus aculeatus , Aspergillus awamori , Aspergillus foetidus , Aspergillus niger , Aspergillus phoenicis , Penicillium amagasakiense, bacteria ( Micrococcus lyzodeikticus ) or yeast ( Saccharomyces )
Enzyme name: α-galactosidase Microorganism name: filamentous fungi ( Aspergillus aculeatus , Aspergillus awamori , Aspergillus niger , Aspergillus phoenicis , Mortierella ) or bacteria ( Bacillus stearothermophilus )
Enzyme name: β-galactosidase (lactase)
Microorganism name: filamentous fungi ( Aspergillus oryzae , Penicillium multicolor , Rhizopus oryzae ), bacteria ( Bacillus circulans , Streptococcus ) or yeast ( Kluyveromyces fragillus , Kluyveromyces lactis , Saccharomyces )

Enzyme name: Carboxypeptidase Microorganism name: Filamentous fungi ( Aspergillus ) or yeast ( Saccharomyces cerevisiae )
Enzyme name: Xylanase Microorganism name: Filamentous fungi ( Aspergillus aculeatus , Aspergillus niger , Trichoderma koningii , Trichoderma longibrachiatum reesei , Trichoderma viride )
Enzyme name: chitinase microorganism name: filamentous fungus ( Trichoderma harzianum , Trichoderma reesei ), actinomycetes ( Amycolatopsis orientalis , Streptomyces ) or bacteria ( Aeromonas )
Enzyme name: Chitosanase Microorganism name: Bacteria ( Aeromonas , Bacillus ) or filamentous fungi ( Aspergillus niger , Trichoderma reesei , Trichoderma viride , Verticillium )

Enzyme name: glucanase microorganisms name: filamentous fungi (Aspergillus aculeatus, Aspergillus niger, Humicola insolens, Rhizopus delemar, Trichoderma harzianum, Trichoderma longibrachiatum, Trichoderma viride), Basidiomycetes (Pycnoporus coccineus), bacteria (Arthrobacter, Bacillus subtilis, Pseudomonas paucimobilis ) or Yeast ( Saccharomyces )
Enzyme name: Glucoamylase Microorganism name: Filamentous fungi ( Acremonium , Aspergillus , Humicola grisea , Rhizopus delemar , Rhizopus niveus ), basidiomycetes ( Corticium rolfsii ), bacteria ( Bacillus , Pseudomonas ) or yeast ( Saccharomyces )

Enzyme name: α-glucosidase Microorganism name: Filamentous fungi ( Absidia , Acremonium , Aspergillus ), bacteria ( Bacillus , Pseudomonas ) or yeast ( Saccharomyces )
Enzyme name: β-glucosidase Microorganism name: filamentous fungus ( Aspergillus aculeatus , Aspergillus niger , Aspergillus pulverulentus , Penicillium decumbens , Trichoderma harzianum , Trichoderma longibrachiatum , Trichoderma reesei ) or bacteria ( Bacillus )
Enzyme name: α-glucosyltransferase Microorganism name: Bacteria ( Agrobacterium radiobacter , Arthrobacter , Bacillus , Erwinia , Pimelobacter , Protaminobacter , Pseudomonas , Serratia , Thermus )
Enzyme name: Glucose isomerase Microorganism name: Aspergillus , Actinoplanes missouriensis , Streptomyces griseofuscus , Streptomyces murinus , Streptomyces phaeochromogenes , Streptomyces rubiginosus ) or bacteria ( Bacillus coagulans )
Enzyme name: Glucose oxidase Microorganism name: Filamentous fungi ( Aspergillus aculeatus , Aspergillus niger , Penicillium )

Enzyme name: Glutaminase Microorganism name: Bacillus subtilis , filamentous fungus ( Aspergillus ) or yeast ( Candida )
Enzyme name: Acid phosphatase Microorganism name: Filamentous fungi ( Aspergillus niger , Aspergillus oryzae )
Enzyme name: cyclodextrin glucanotransferase (transferase)
Microorganism name: Bacteria ( Bacillus , Brevibacterium , Corynebacterium )

Enzyme name: cellulase microorganisms name: filamentous fungi (Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus niger, Humicola insolens, Trichoderma harzianum, Trichoderma insolens, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride), Basidiomycetes (Corticium, Irpex , Pycnoporus coccineus ), Actinomyces , Streptomyces, or bacteria ( Bacillus circulans , Bacillus subtillis )
Enzyme name: Tannase Microorganism name: Aspergillus oryzae
Enzyme name: 5'-deaminase Microorganism name: Filamentous fungi ( Aspergillus melleus , Aspergillus oryzae )

Enzyme name: Dextranase Microorganism name: Filamentous fungus ( Chaetomium erraticum , Chaetomium gracile , Penicillium lilacinum )
Enzyme name: Transglucosidase Microorganism name: Filamentous fungi ( Aspergillus niger , Aspergillus usamii ), bacteria ( Sulfolobus solfataricus )
Enzyme name: Transglutaminase Microorganism name: Streptomyces , Streptoverticillium mobaraense or bacteria ( Bacillus )
Enzyme name: Trehalose phosphorylase Microorganism name: Bacteria ( Plesiomonas )
Enzyme name: Narindinase Microorganism name: Filamentous fungi ( Aspergillus usamii , Penicillium decumbens )

Enzyme name: Peroxidase (peroxidase)
Microorganism name: filamentous fungi ( Alternaria , Aspergillus oryzae , Coprinus cinereus , Oidiodendron ) or bacteria ( Bacillus )
Enzyme name: Phytase (phosphohydrolase)
Microorganism name: Aspergillus niger
Enzyme name: Fructosyltransferase Microorganism name: Filamentous fungi ( Aspergillus , Penicillium roqueforti ) or bacteria ( Arthrobacter , Bacillus )
Enzyme name: Pullulanase Microorganism name: Bacteria ( Bacillus , Klebsiella , Sulfolobus solfataricus )

Enzyme name: protease microorganisms name: filamentous fungi (Aspergillus melleus, Aspergillus niger, Aspergillus oryzae, Aspergillus saitoi, Aspergillus, sojae, Monascus pilosus, Monascus purpureus, Mucor circinelloides, Mucor javanicus, Mucor miehei, Mucor rouxii, Penicillium citrinum, Penicillium duponti, Rhizomucor miehei, Rhizopus chinensis, Rhizopus delemar , Rhizopus niveus, Rhizopus oryzae), Basidiomycetes (Pycnoporus coccineus), actinomycetes (Streptomyces), bacteria (Bacillus amyloliquefaciens, Bacillus coagulans J4, Bacillus lentus, Bacillus licheniformis, Bacillus polymixa, Bacillusstearothermophilus, Bacillus subtilis , Bacillus thermoproteolyticus , Pseudomonas paucimobilis ) or yeast ( Saccharomyces )

Enzyme name: pectinase microorganisms name: filamentous fungi (Aspergillus aculeatus, Aspergillus alliaceus, Aspergillus awamori, Aspergillus japonicus, Aspergillus niger, Aspergillus pulverulentus, Aspergillu s usamii, Rhizopus oryzae, Trichoderma), bacteria (Bacillus subtilis), Basidiomycetes (Corticium) or Yeast ( Trichosporon )
Enzyme name: Hesperidinase Microorganism name: Filamentous fungi ( Aspergillus , Penicillium decumbens )

Enzyme name: Peptidase Microorganism name: Filamentous fungi ( Aspergillus niger , Aspergillus oryzae , Aspergillus sojae , Rhizopus oryzae ) or bacteria ( Bacillus , Lactococcus lactis )
Enzyme name: hemicellulase microorganisms name: Bacillus subtilis (Bacillus subtilis), filamentous fungi (Aspergillus aculeatus, Aspergillus awamori, Aspergillus niger, Aspergillus oryzae, Aspergillus usamii, Humicola insolens, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma viride) or Basidiomycetes Fungus ( Corticium , Pycnoporus coccineus )
Enzyme name: Phosphodiesterase Microorganism name: Aspergillus niger , Penicillium citrinum
Enzyme name: Phospholipase Microorganism name: Filamentous fungi ( Aspergillus oryzae , Aspergillus niger ), basidiomycetes ( Corticium ), actinomycetes ( Actinomadura , Nocardiopsis ) or bacteria ( Bacillus )

Enzyme name: Polyphenol oxidase Microorganism name: Filamentous fungi ( Alternaria , Aspergillus niger , Coriolus ) or basidiomycetes ( Cyathus , Polyporus cinereus , Pycnoporus coccineus , Polyporus versicolor , Trametes )
Enzyme name: Maltose phosphorylase Microorganism name: Bacteria ( Plesiomonas )
Enzyme name: Maltotriohydrolase Microorganism name: Filamentous fungi ( Penicillium ) or bacteria ( Bacillus subtilis , Microbacterium )
Enzyme name: Muramidase Microorganism name: Actinomyces , Streptomyces or bacteria ( Bacillus )

Enzyme name: lipase microorganisms name: filamentous fungi (Aspergillus awamori, Aspergillus niger, Aspergillus oryzae, Aspergillus phoenicis, Aspergillus usamii, Geotrichum candidum, Humicola, Mucor javanicus, Mucor miehei, Penicillium camembertii, Penicillium chrysogenum, Penicillium roquefortii, Rhizomucor miehei, Rhizopus delemar , Rhizopus japonicus , Rhizopus miehei , Rhizopus niveus , Rhizopus oryzae ), actinomycetes ( Streptomyces ), bacteria ( Alcaligenes , Arthrobactor , Chromobacterium viscosum , Pseudomonas , Serratia marcescens ) or yeast ( Candida )
Enzyme name: Lipoxygenase Microorganism name: Filamentous fungus ( Rhizopus )
Enzyme name: rennet microorganisms name: yeast (Kluyveromyces lactis), filamentous fungi (. Mucor miehei, Mucor pusillus LINDT , Mucor spp, Rhizomucor miehei), Basidiomycetes (Irpex lacteus) or bacteria (Bacillus cereus, Crypnohectria parasitica, Escherichia coli K-12 etc.)

  Among these enzymes, proteases are preferred, and examples of protease-producing microorganisms include filamentous fungi, basidiomycetes, actinomycetes, bacteria, and yeasts. Bacteria are more preferred. Here, examples of the Bacillus family include Geobacillus, Paenibacillus, Alicyclobacillus, Brevibacillus, Oceanobacillus and the like.

  The culture of an enzyme-producing microorganism is a culture obtained by culturing an enzyme-producing microorganism under conditions where the enzyme is produced, and is a mixture containing the enzyme and the microorganism. For example, the culture which culture | cultivated the microorganisms which belong to a Bacillus genus or a Bacillus related genus on the conditions on which protease is produced is mentioned. The culture is preferably a liquid culture.

Polylysine is added to the culture. Polylysine is usually used as a pH adjusting agent or bacteriostatic agent, but it is not known at all that it can be used for separation of microorganisms and enzymes.
Examples of polylysine include ε-poly-L-lysine and α-poly-L-lysine, and ε-poly-L-lysine used for food is particularly preferred from the viewpoint of safety.

  The addition amount of polylysine is preferably 0.0001% by mass or more and 10% by mass or less, and more preferably 0.0005% by mass or more and 5% by mass or less as a concentration after addition from the viewpoint of microbial aggregation and enzyme selectivity. Preferably, 0.001 mass% or more and 1 mass% or less are more preferable, and 0.005 mass% or more and 0.5 mass% or less are more preferable.

  By adding polylysine, microorganisms and viscosity-increasing components are aggregated and the filterability is remarkably improved. However, a commonly used filter aid may be used in combination. Examples of the filter aid include diatomaceous earth, pearlite, and cellulose, but diatomaceous earth and pearlite are more preferable. 0.01-100 mass parts is preferable with respect to 100 mass parts of culture, 0.05-50 mass parts is more preferable, and 0.1-20 mass parts is further more preferable. The filter aid may be simply added to the culture, but may be precoated on the filter.

  The culture after the addition of polylysine is preferably stirred (preferably 10 minutes to 3 hours) under conditions that do not deactivate the enzyme (for example, normal temperature (5-35 ° C.)). Moreover, you may centrifuge as needed.

  Then, if the culture after polylysine addition is filtered, a precipitation part and a supernatant part can be isolate | separated easily and an enzyme containing liquid will be obtained as a filtrate. Here, examples of the filter used for filtration include filter paper, nitrocellulose, cellulose acetate, polyamide (nylon), polyester (tetron), polypropylene, polyvinylidene chloride, PVDF, PTFE, PAN, and PES. The filtering means may be a pressure type or a suction type, and an industrial filter press device or the like can be used. The viscosity of the obtained filtrate is significantly reduced.

  The obtained enzyme-containing filtrate is prepared by a conventional method such as ammonium sulfate, precipitation with an organic solvent (acetone, ethanol, etc.), ion exchange chromatography, isoelectric chromatography, gel filtration chromatography, hydrophobic chromatography, adsorption column chromatography. Can be purified by means such as chromatography, affinity chromatography, reverse phase column chromatography and the like.

  Examples Next, the present invention will be described with reference to examples, but the present invention is not limited to these examples. In Examples,% indicates mass% unless otherwise specified.

Example 1
Effect of polylysine treatment on the culture solution of Bacillus-related bacteria
Bacillus subtilis var. Natto (NBRC13169), Bacillus amyloliquefaciens (NBRC15535), Geobacillus thermoglucosidasius (NCIMB1195) using a 24 mm test tube, a medium consisting of 3% glycerin, 2% soybean peptone, and 0.3% yeast extract (pH 7. Pre-cultured in 2) by shaking at 30 ° C. for 24 hours. Next, using a 500 mL Erlenmeyer flask, 1% of the preculture solution was inoculated into a medium (pH 7.2) consisting of 4% glycerin, 1% soybean peptone, and 0.5% yeast extract. Cultured by shaking. The culture solution after the culture was adjusted to around pH 7.5, 25% polylysine (manufactured by JNC) was added to a final concentration of 0.02%, and the mixture was stirred at room temperature for 30 minutes. Thereafter, Radiolite # 100 (manufactured by Showa Kagaku Kogyo Co., Ltd.) was used as a precoat. It was spread on No. 2 filter paper (manufactured by Advantech) and subjected to suction filtration under the condition of 1% radiolite # 100 as a body feed to obtain a filtrate. Table 1 shows the results of the clarity (OD ₆₀₀ ) of each culture and filtrate. Moreover, the protease activity of each culture solution and filtrate was measured. Table 2 shows the protease activity of the filtrate when the protease activity of the culture solution is 100%.

  As shown in Table 1, in any culture solution, the clarification degree could be reduced to 5% or less by adding polylysine. Moreover, as shown in Table 2, in any of the filtrates, the protease could be recovered with a high yield of 90% or more.

Example 2
Bacillus subtilis var. Effect of polylysine treatment concentration in natto culture
Bacillus subtilis var. Natto (NBRC13169) was pre-treated by shaking at 30 ° C. for 24 hours in a medium (pH 7.2) consisting of 3% glycerin, 2% soybean peptone, and 0.3% yeast extract using a 24 mm test tube. Cultured. Next, using a 500 mL Erlenmeyer flask, 1% of the preculture solution was inoculated into a medium (pH 7.2) consisting of 4% glycerin, 1% soybean peptone, and 0.5% yeast extract. Cultured by shaking. The culture solution after the culture was adjusted to around pH 7.5, 25% polylysine (manufactured by JNC) was added to a final concentration of 0.02% to 0.1%, and the mixture was stirred at room temperature for 30 minutes. Thereafter, Radiolite # 100 (manufactured by Showa Kagaku Kogyo Co., Ltd.) was used as a precoat. It was spread on No. 2 filter paper (manufactured by Advantech) and subjected to suction filtration under the condition of 1% radiolite # 100 as a body feed to obtain a filtrate. The protease activity of the culture solution and each filtrate was measured. Table 3 shows the protease activity of the filtrate when the protease activity of the culture solution is taken as 100%.

  As shown in Table 3, in any of the filtrates, protease could be recovered with a high yield of 90% or more.

  The protease activity can be calculated as an enzyme activity per solution based on an enzyme amount of 1.0 PU defined in the following method. Add 1 mL of enzyme dilution to 0.6% aqueous casein solution (pH 7.5, 50 mM Tris-HCl buffer containing 2 mM potassium acetate), react at 30 ° C. for 10 minutes, and then trichloroacetic acid reagent (pH 4.0, 8% acetic anhydride). The reaction is stopped by adding 5 mL of sodium, 1.8% trichloroacetic acid, 1.98% acetic acid), and the mixture is further allowed to stand at 30 ° C. for 30 minutes. After filtration, the absorbance at 275 nm is measured. The amount of enzyme that releases an amino acid corresponding to 1 μg of tyrosine per minute under these conditions is defined as 1 PU (Protease Unit).

Claims (5)

  A method for producing an enzyme, comprising adding polylysine to a culture of an enzyme-producing microorganism and then filtering.   The production method according to claim 1, wherein the filtrate is collected after filtration.   The method according to claim 1 or 2, wherein the enzyme is a protease.   The method according to any one of claims 1 to 3, wherein the added polylysine concentration is 0.0001 to 10% by mass.   The production method according to any one of claims 1 to 4, wherein the enzyme-producing microorganism is an enzyme-producing bacterium belonging to the genus Bacillus or the genus Bacillus.