JPS62208279A - Production of cellulase - Google Patents
Production of cellulaseInfo
- Publication number
- JPS62208279A JPS62208279A JP4958586A JP4958586A JPS62208279A JP S62208279 A JPS62208279 A JP S62208279A JP 4958586 A JP4958586 A JP 4958586A JP 4958586 A JP4958586 A JP 4958586A JP S62208279 A JPS62208279 A JP S62208279A
- Authority
- JP
- Japan
- Prior art keywords
- cellulase
- lactose
- medium
- culture
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010059892 Cellulase Proteins 0.000 title claims abstract description 23
- 229940106157 cellulase Drugs 0.000 title claims abstract description 23
- 238000004519 manufacturing process Methods 0.000 title claims description 5
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 22
- 230000000694 effects Effects 0.000 claims abstract description 22
- 239000008101 lactose Substances 0.000 claims abstract description 22
- 102000005936 beta-Galactosidase Human genes 0.000 claims abstract description 8
- 108010005774 beta-Galactosidase Proteins 0.000 claims abstract description 8
- 241000223259 Trichoderma Species 0.000 claims abstract description 6
- 244000005700 microbiome Species 0.000 claims abstract description 6
- 238000012258 culturing Methods 0.000 claims description 3
- 241000499912 Trichoderma reesei Species 0.000 abstract description 7
- 239000000126 substance Substances 0.000 abstract description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 4
- 229910052799 carbon Inorganic materials 0.000 abstract description 4
- 230000006698 induction Effects 0.000 abstract description 3
- 239000005862 Whey Substances 0.000 abstract description 2
- 102000007544 Whey Proteins Human genes 0.000 abstract description 2
- 108010046377 Whey Proteins Proteins 0.000 abstract description 2
- 239000001963 growth medium Substances 0.000 abstract description 2
- 239000000706 filtrate Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 19
- 239000002609 medium Substances 0.000 description 14
- 102000004190 Enzymes Human genes 0.000 description 12
- 108090000790 Enzymes Proteins 0.000 description 12
- 229940088598 enzyme Drugs 0.000 description 12
- 238000000034 method Methods 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 8
- 229920001817 Agar Polymers 0.000 description 7
- 239000008272 agar Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 235000013619 trace mineral Nutrition 0.000 description 4
- 239000011573 trace mineral Substances 0.000 description 4
- 108010084185 Cellulases Proteins 0.000 description 3
- 102000005575 Cellulases Human genes 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 108010047754 beta-Glucosidase Proteins 0.000 description 2
- 102000006995 beta-Glucosidase Human genes 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 210000004340 zona pellucida Anatomy 0.000 description 2
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- WDMUXYQIMRDWRC-UHFFFAOYSA-N 2-hydroxy-3,4-dinitrobenzoic acid Chemical compound OC(=O)C1=CC=C([N+]([O-])=O)C([N+]([O-])=O)=C1O WDMUXYQIMRDWRC-UHFFFAOYSA-N 0.000 description 1
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 1
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 1
- 241000609240 Ambelania acida Species 0.000 description 1
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
- 244000307700 Fragaria vesca Species 0.000 description 1
- 235000016623 Fragaria vesca Nutrition 0.000 description 1
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 229910017974 NH40H Inorganic materials 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000272534 Struthio camelus Species 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 239000010905 bagasse Substances 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000011419 induction treatment Methods 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- -1 p-nitrophenyl-β-D-glucopyranosyl Chemical group 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000001965 potato dextrose agar Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明はセルラーゼの製法に関する。本明細誓において
セルラーゼとはエキソ−β−グルカナーゼ（ＢＯ２，２
，１，１９）、エンド−β−グルカナーゼ（ＩシＣ３，
２，１，４）及びβ−グルフシダーゼ（ＢＯ３，２，１
，２１）からなる酵素源の総称であり、セルロースをグ
ルコースまで分解する酵素である。DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing cellulases. In this specification, cellulase refers to exo-β-glucanase (BO2,2
, 1, 19), endo-β-glucanase (IC3,
2,1,4) and β-glufusidase (BO3,2,1
, 21), which is an enzyme that decomposes cellulose into glucose.
従来の技術
従来、ラクトースによるセルラーゼの誘導能が高められ
たトリコデルマ・リーセイ（Ｔｒｉｃｈｏ−ｄｅｒｍａ
ｒｅｅｓ＊ｉ） ＭＣＧ−７７（ＮＲＴＬＬ １１２
３６ ） （以下ＭＣＧ−７７と称す）をラクトース１
〜２チの培地に培養してセルラーゼを得る方法は知られ
ている〔特表昭５５−５００８９０号公報、バイオテク
ノロジー・アンｒ・バイオエンジニアリング（Ｂｉｏｔ
ｅｅｈｎｏｌｏｇｙ ａｎｄ Ｂｌｏ＠ｎｇｉｎ＠ｅｒ
ｉｎｇ） ２６゜３８８７ （１９７９） ）。Conventional technology Trichoderma reesei (Trichoderma reesei), which has an enhanced ability to induce cellulase by lactose,
rees*i) MCG-77 (NRTLL 112
36) (hereinafter referred to as MCG-77) to lactose 1
A method for obtaining cellulase by culturing in a medium of ~2.5 mm is known [Japanese Patent Publication No. 500890/1989, Biotechnology Ann R.
eehnology and Blo@ngin@er
ing) 26°3887 (1979)).
発明が解決しようとする問題点
本発明の目的は、改良された高活性のセルラーゼの製法
を提供することにある。Problems to be Solved by the Invention An object of the present invention is to provide an improved method for producing highly active cellulases.
問題点を解決するための手段
本発明により、トリコデルマ属に属し、ラクトースによ
るセルラーゼの誘導能とβ−ガラクトシダーゼ活性とが
高められた微生物を培地に培養し、培養物中にセルラー
ゼを生成蓄積させ、これを採取する工程からなるセルラ
ーゼの製法が提供される。Means for Solving the Problems According to the present invention, a microorganism belonging to the genus Trichoderma and having an enhanced ability to induce cellulase by lactose and β-galactosidase activity is cultured in a medium, and cellulase is produced and accumulated in the culture, A method for producing cellulase is provided, which comprises a step of collecting cellulase.
トリコデルマ属に属し、ラクトースによるセルラーゼの
誘導能とβ−ガラクトシダーゼ活性とが高められた微生
物であればいずれも本発明の目的に用いられる。Any microorganism belonging to the genus Trichoderma that has an enhanced ability to induce cellulase by lactose and β-galactosidase activity can be used for the purpose of the present invention.
実施例に記載された菌株はトリコデルマ・リーセイ（Ｔ
ｒｉｃｈｏｄａｒｍａ ｒ＠５ｓｅｉ） ＬＷ３７６
（ＰＥ几ＭＢＰ−９８０） （以下、ＬＷ３７６と称す
）で、１９８６年２月１日にブダはスト条約の規定によ
り工業技術院微生物工業技術研究所に寄託されている。The bacterial strain described in the Examples is Trichoderma reesei (T
richormar @5sei) LW376
(PE MBP-980) (hereinafter referred to as LW376) was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on February 1, 1986, pursuant to the provisions of the Strike Treaty.
トリコデルマ・リーセイの菌学的性質は、セカンド・イ
ンターナショナル・マイコロジカル・フンブレス（２ｎ
ｄ Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｍｙａｏｌｏｇｉｃ
ａｌＣｏｎｇｒｅｓｓ） 、米国７ｏリダ州タフ／Ｑ、
１９７７年８月、６１８頁に記載されている。The mycological properties of Trichoderma reesei are published by the Second International Mycological Humbres (2n
d International Myaology
alCongress), U.S. 7o Lida, Tough/Q,
August 1977, page 618.
本発明の方法に用いられる菌株は例えば次の方法で得ら
れる。トリコデルマ属に属しかつセルラーゼ生産能を有
する微生物を紫外線照射やニトロソグアニジンのような
変異誘発剤の使用など公知の変異誘導処理をし、処理ず
みの菌株からラクトースによるセルラーゼ誘導能の高い
菌株を選ぶ。このための実用的な手法としては例えば、
親株としてトリコデルマ・リーセイ１３Ｇ−３−７（以
下、ＰＣ−３−７と称す）を用い、ボテトデ中ストロー
ス寒天斜面培地上で２５℃、７日間培養し、胞子を充分
形成させる。生成した胞子を生理的食塩水に懸濁しく約
１〜３ Ｘ １０７個／ＩＭ）、Ｎ−メチル−Ｎｏ−ニ
トロ−Ｎ−二ト△ングアニジンと反応させる（３００μ
ｌ／Ｒ１，ｐＨ７，０，３０℃、３０〜１２０分）。こ
の胞子懸濁液から遠心分離により胞子を集め、よく洗浄
し、平板あたり１００〜３００胞子／ｄＫなるように希
釈し、第１表に示したラクトースを炭素源とする寒天平
板に塗布し、３０℃で２日間培養し、生育してきたコロ
ニーの上に５０ ｎ１Ｍ酢酸緩衝液ｐＨ５，０を含む０
．５％リン酸膨潤セルロース寒天を加えて、４５℃で１
０〜２４時間床温する。セルラーゼを生成しているコロ
ニーのまわシには透明帯が生成する。このに１明帯を基
憩としてセルラーゼ活性の高まった所望の菌株を選別し
た。下記の第２表に示されるように１親株の透明帯形成
能は微弱である。The bacterial strain used in the method of the present invention can be obtained, for example, by the following method. A microorganism belonging to the genus Trichoderma and capable of producing cellulase is subjected to a known mutation induction treatment such as ultraviolet irradiation or the use of a mutagenic agent such as nitrosoguanidine, and a strain with a high ability to induce cellulase by lactose is selected from the treated strains. For example, practical methods for this purpose include:
Trichoderma reesei 13G-3-7 (hereinafter referred to as PC-3-7) was used as a parent strain and cultured on a Strauss agar slant medium in Botetoide at 25° C. for 7 days to allow sufficient spore formation. The generated spores were suspended in physiological saline (approximately 1 to 3 x 107 spores/IM) and reacted with N-methyl-No-nitro-N-ditho△guanidine (300 μl).
l/R1, pH 7, 0, 30°C, 30-120 minutes). Spores were collected from this spore suspension by centrifugation, washed thoroughly, diluted to 100-300 spores/dK per plate, and applied to an agar plate containing lactose as a carbon source as shown in Table 1. After culturing for 2 days at
．． Add 5% phosphoric acid swollen cellulose agar and incubate at 45℃ for 1 hour.
Bring to bed temperature for 0-24 hours. A zona pellucida is formed around colonies that produce cellulase. Then, a desired strain with increased cellulase activity was selected based on the 1 bright zone. As shown in Table 2 below, the ability of the parent strain 1 to form a zona pellucida is weak.
Ｔ、Ｗ３７６株と親株との寒天培地上での性質の比較を
第２表に示す。Table 2 shows a comparison of the properties on the agar medium between the T and W376 strains and the parent strain.
第 １ 表
寒天培地の組成
ラクトース ５ＩＩ酵母エキス
１ｙ
（ＮＨ４）、５ｏ４２ Ｆ
ＫＨ，ＰＯ４４＃
Ｎｏ・トＩＰＯ・
６ ｐＭｇＳＯ４・７Ｈ２０２０
０勇２
ＦｅＳ０４・７Ｈ，Ｏｌ １′９
ＣｓｉＣＡ！２−２Ｈ，０１ｊｉｐ
ミルトリトン１００（牛丼化学薬品（株）＃） ］
、！寒 天 ２
０ Ｆ＋１．Ｂｏ、
１０μｇＭｎＳＯ，’４Ｈ，Ｏ”
１０ μｙＺｎＳＯ４”７
１１，０ ７０μｙＣｕＳＯ
４’ ５Ｈ，０５０ｐｌ／
（Ｎ）１４）、ＭＯ，Ｏ，、・４）１，０
１０１１ｇ水 ｌ ｌ （Ｐ）（５，５）
第 ２ 表
（注）１）＋：集落を形成する。Table 1 Composition of agar medium Lactose 5II yeast extract
1y (NH4), 5o42 F KH, PO44# No・ToIPO・
6 pMgSO4・7H2020
0Yu2 FeS04・7H, Ol 1'9 CsiCA! 2-2H, 01jip Miltriton 100 (Gyudon Chemical Co., Ltd. #) ]
,! Agar 2
0 F+1. Bo,
10μgMnSO,'4H,O''
10μyZnSO4”7
11,0 70μyCuSO
4' 5H,050pl/ (N)14), MO,O,,・4)1,0
1011g water l l (P) (5,5) Table 2 (Note) 1)+: Forms a village.
２）＋：分生子の着生が良い。2) +: Good conidia settlement.
３）−二認められない。3)-2 Not allowed.
４）＋＋−：肉眼で明瞭に認められる。4) ++-: Clearly recognized with the naked eye.
＋：肉眼でわずかに認められる。+: Slightly visible to the naked eye.
本発明における培地の炭素源としては、ラクトース、そ
の含有物が用いられ、さらにこれらト、例工ば、セルロ
ースパクダー、グルコース、セロビオース、２紙、−紋
紙類、オガクズ、ふす１、もみがら、バガス、大豆粕、
コーヒー粕、澱粉等を組み合わせて用いてもよい。ラク
トース含有物としては、例えばホエーがあげられる。As the carbon source of the culture medium in the present invention, lactose and substances containing it are used, and these materials include, for example, cellulose powder, glucose, cellobiose, paper, paper, sawdust, wheat bran, and rice hulls. , bagasse, soybean meal,
Coffee grounds, starch, etc. may be used in combination. Examples of lactose-containing substances include whey.
培地中のラクトース又はラクトース含有物の濃度はラク
トース換算で３〜８％の範囲である。The concentration of lactose or lactose-containing substances in the medium is in the range of 3 to 8% in terms of lactose.
窒素源としては、硫安、硝安などの無機アンモニウム塩
、尿素、アミノ酸、肉エキス、酵母エキス１．ｔ？ Ｉ
Ｊペプトン、蛋白質分解物等の有機窒素含有物が使用さ
れる。無機塩類としては、ＫＨ，ＰＯ２、Ｍｇ８０４−
７Ｈ，０１Ｃａ（Ｊ、”２Ｈ，０、Ｆ’ｅ（Ｊ、−６Ｈ
，０１ＭｎＣ１１・４ Ｈ！Ｏ１Ｚ！ｌ５０４４Ｈ，Ｏ
等が使用される。必要ならば有機微量栄養物を含有する
培地が使用される。前記の菌株の培養は、液体培養のほ
かに固形培養も可能である。液体培養には通常の通気攪
拌培養装置が用いられ、前記培地を使用して、培養温度
２０〜３３℃、好オしくは、２８〜３０゜Ｃ１培養ｐ）
（４〜６で培養すれば４〜１０日閣でセルラーゼ活性は
ｉ＆高となる。ついで、培養液から遠心分離、ｆ過など
の公知の方法によって菌体を除去して上澄液を得る。こ
の上澄液は、このｉｔ粗酵素液として使用することがで
きる。Nitrogen sources include inorganic ammonium salts such as ammonium sulfate and ammonium nitrate, urea, amino acids, meat extract, and yeast extract. T? I
Organic nitrogen-containing substances such as J-peptone and protein decomposition products are used. Inorganic salts include KH, PO2, Mg804-
7H,01Ca(J,"2H,0,F'e(J,-6H
,01MnC11・4 H! O1Z! l5044H,O
etc. are used. If necessary, a medium containing organic micronutrients is used. The above-mentioned bacterial strains can be cultured not only in liquid culture but also in solid culture. A normal aerated stirring culture device is used for liquid culture, and the culture temperature is 20 to 33°C, preferably 28 to 30°C, using the above medium (C1 culture p).
(If cultured for 4 to 6 days, the cellulase activity will be i&high after 4 to 10 days. Then, the bacterial cells are removed from the culture solution by a known method such as centrifugation or filtration to obtain a supernatant. This supernatant can be used as this it crude enzyme solution.
又、得られた上澄液から凍結乾燥、硫安塩析・有機溶剤
による沈澱法など公知の方法を用いることにより粗酵素
剤を得ることができる。Further, a crude enzyme preparation can be obtained from the obtained supernatant liquid by using known methods such as freeze drying, ammonium sulfate salting out, and precipitation using an organic solvent.
次に＃未活性の測定法について説明する。Next, the method for measuring #unactivated will be explained.
（１） エキソ−β−グルカナーゼ活性アビセル８Ｆ
１５０ Ｍ９を基質とし、これに酵素液１．０１．０
．２ Ｍ 、 ｐＨ５，０の酢酸緩衝液４．０−をそれ
ぞれ加え、４５℃で１時間反応させる。(1) Exo-β-glucanase activity Avicel 8F
150 M9 as a substrate, and enzyme solution 1.01.0
．． A 2 M, pH 5.0 acetate buffer 4.0- was added to each, and the mixture was reacted at 45°C for 1 hour.
１００℃、１０分間加熱して反応を停止させる。３゜５
−ジニトロサリチル酸法により還元糖を比色定量する。The reaction is stopped by heating at 100° C. for 10 minutes. 3゜5
- Colorimetric determination of reducing sugars by the dinitrosalicylic acid method.
１分間Ｋ】μｍｏｌのグルコースを遊離する酵素量を１
酵素量位（Ｕｎｉｔ ）と定義する。1 minute K] The amount of enzyme that releases μmol of glucose is 1
Defined as enzyme quantity level (Unit).
（２） エンド−β−グルカナーゼ活性０．２ Ｍ
ＳｐＨ５，０の酢＠緩′４Ｅ液に溶解した１チ力ルボキ
シメチルセルロースナトリウム塩溶液を用意する。これ
に２１＋量の適描に希釈した酵累溶液を加え、４５℃で
３０分間反応させろ。１００℃、】０分間加熱して反応
を停止させた後、３゜５−ジニトロサリチル酸法により
還元糖を比色定量する。１分間に１μｍｏｌのグルコー
スヲ遊離するｎ嵩量を１酵素量位（Ｕｎｉｔ）と定義す
る。(2) Endo-β-glucanase activity 0.2 M
Prepare a 1-tyl carboxymethyl cellulose sodium salt solution dissolved in vinegar @ mild 4E solution with SpH 5.0. Add 21+ volumes of the appropriately diluted fermentation solution to this and react at 45°C for 30 minutes. After stopping the reaction by heating at 100°C for 0 minutes, reducing sugars are determined colorimetrically by the 3°5-dinitrosalicylic acid method. The amount of n volume that releases 1 μmol of glucose per minute is defined as one enzyme amount (Unit).
（３） β−グルコシダーゼ活性
基質液として０．０５Ｍ、 ｐＨ５，０の酢酸緩衝液に
溶解した２ｍＭｐ−ニトロフェニル−β−Ｄ−グルコピ
ラノシｒを用意する。基質液１．０ｕｌｌＩｃ＃＊液２
０μノを加え、４５℃で１０分間反応させる。(3) Prepare 2mM p-nitrophenyl-β-D-glucopyranosyl dissolved in 0.05M, pH 5.0 acetate buffer as a β-glucosidase activity substrate solution. Substrate solution 1.0ulIc#*solution 2
Add 0μ and react at 45°C for 10 minutes.
２．０ ｍｌのＩ Ｆｌ炭酸ナトリワム溶液を加えて反
応を停止する、１分間に１μｔｏｏｌのｐ−＝）ロフェ
ノールを遊離する＃嵩量を１酵素量位（［Ｊｎ ｉ ｔ
）と定義する。Stop the reaction by adding 2.0 ml of I Fl sodium carbonate solution.
).
（４） β−ガラクトシダーゼ
ラクトピラノシｆを用意する。基質液１．０ｍ１Ｃ酵素
液２０μ！を加え、４５℃で１０分間反応さぜろ。(4) Prepare β-galactosidase lactopyranosi f. Substrate solution 1.0ml 1C enzyme solution 20μ! Add and react at 45°C for 10 minutes.
２．０１ＫｔのＩＭＲ酸ナトリクム溶液を加えて反応を
停止する。１分間に１ ｆｉ ｍａｔの０−ニトロフェ
ノールを遊離する酵素量を１酵素量位（［Ｊｎｉｔ）と
定義する。The reaction is stopped by adding 2.01 Kt IMR sodium acid solution. The amount of enzyme that releases 1 fi mat of 0-nitrophenol per minute is defined as 1 enzyme amount ([Jnit).
トリコデルマ・リーセイの変異株のラクトースからのセ
ルラーゼ誘導度は、ラクトース１％を炭素源とする液体
培養を行ない、と清中のエンｒ−β−グルカナーゼ活性
を測定することによって評価される。すなわち、変異株
をボテトデ争ストローズ寒天培地上で２５℃、７日間培
養する。生放した胞子を第３表に示した培地５０ｔｕヲ
含ム３００１１Ｌｔ容量のエルレンマイヤープラスフに
接種し、２８℃、７日間振盪培養する。培養液を遠心分
離し、上清中のエンド−β−グルカナーゼ活性を求める
。その結果を第４表に示す。The degree of cellulase induction from lactose of a Trichoderma reesei mutant strain is evaluated by performing liquid culture using 1% lactose as a carbon source and measuring the enr-β-glucanase activity in the supernatant. That is, the mutant strain is cultured on a Strawberry agar medium at 25° C. for 7 days. The live spores were inoculated into a 30,011 Lt capacity Erlenmeyer plastic tube containing 50 tu of the medium shown in Table 3, and cultured with shaking at 28° C. for 7 days. The culture solution is centrifuged and the endo-β-glucanase activity in the supernatant is determined. The results are shown in Table 4.
第 ３ 表
ラクトース １０．Ｑ ｐポリイブ
トン １．０ ｐ酵母エキス
０．５１ＫＨ２ＰＯ４２，０＃
（ＮＨ４）２Ｓｏ４１．５ 、ｌｉｔＭｔｒＳＯ４−
７７１２００，３１
Ｃ＆Ｃ１２−２夏’ｚｏ
Ｏ，３１／ツイーン８０〔牛丼化学薬
品（株）製〕１．ｏ 尼微量元素液＊１．０ 紅
酒石酸緩衝液 ５０ｍＭ
水 ＩＡ’（ＰＨ４，０）
＊微量元素液
Ｈ３ＢＯ３６１ｎ９
（ＮＨ４）６Ｍｏ、０２４’４Ｈ２０２６１９ＦｅＣ１
３−６Ｈ２０１００ｌｌ９
ＣｕＳＯ４−５Ｈ２０４０Ｑ
ＭｎＧｌ、 −４Ｈ□０
８ ！Ｒ９ＺｏＳＯ４・７Ｈｚ０
２００ Ｒ９水
１００ ｍ第 ４
表
ＬＷ３７６ ２５ＰＣ，−３−７
１５
ＭＣＧ−７７１１
次に実施例を示す。Table 3 Lactose 10. Q p polybutone 1.0 p yeast extract
0.51KH2PO42,0# (NH4)2So41.5, litMtrSO4-
771200,31 C&C12-2 Summer'zo
O, 31/Tween 80 [manufactured by Gyudon Kagakuyaku Co., Ltd.] 1. o Ni trace element solution *1.0 Benitartrate buffer 50mM water IA' (PH4,0) *Trace element solution H3BO361n9 (NH4)6Mo, 024'4H202619FeC1
3-6H20100ll9 CuSO4-5H2040Q MnGl, -4H□0
8! R9ZoSO4・7Hz0
200 R9 water
100m 4th
Table LW376 25PC, -3-7
15 MCG-7711 Next, examples will be shown.
実施例Ｉ
ＬＷ３７６ をポテトデキストローズ寒天培地とで２
５℃、７日間培養した。生成した胞子を第５表の培地５
０１ｎｌを含む３００ｒｎｌエルレンマイヤーフラスコ
に接種し、２８℃で４日間振盪培養した。Example I LW376 was mixed with potato dextrose agar medium.
The cells were cultured at 5°C for 7 days. The generated spores were transferred to medium 5 in Table 5.
A 300rnl Erlenmeyer flask containing 01nl was inoculated and cultured with shaking at 28°C for 4 days.
ついでこの培養液を同組成の培地５００ｄを含む２１エ
ルレンマイヤーフラスコに接［Ｌ、ｚｓ℃で４日間振盪
培養した。さらに１この培養液を第６表の培地２．５１
を含む５１ジャーファーメンタ−に接種して、２８℃で
４日間培養した（回転数： ４５０ ｒｐｍｓ通気：Ｉ
ＶＶＭ）。ｐＨは３ＮＮＨ４０Ｈを添加することによｆ
ｉ、４．０〜４．５の範囲に調整した。This culture solution was then applied to a 21 Erlenmeyer flask containing 500 ml of a medium of the same composition and cultured with shaking at zs°C for 4 days. Furthermore, add this culture solution to the medium 2.51 in Table 6.
The seeds were inoculated into a 51 jar fermentor containing
VVM). The pH was adjusted by adding 3N NH40H.
i was adjusted to a range of 4.0 to 4.5.
第 ５ 表
アビセル １０．ＯＦポリペプトン
１．０１
酵母工牛ス ｏ、ｓ ｇＫＨ２ＰＯ
４２，ＯＦ
（ＮＨ４）２Ｓｏ、 １．５ Ｎ
ＭｇＳＯ４・７１１２０ ０．３
．９Ｃ＆ＩＣ１２・２［ｆ２０
ｏ−３１ツイーン８０（牛丼化学薬品（株）製） １
．０ 尼微量元素液（第３表と同じ） １．０ ｍ
ｌ水 ＩＪ（ｐＨｓ、ｓ ）
第 ６ 表
ラクトース ４０．ＯＩｉポリイブト
ン １．Ｏｉ酵母工中ス
ｏ、ｓ ｙＫＨ２ＰＯ，□、。 １
（Ｎ）（、）２８０４１．５ Ｎ
Ｍｇ８０４−７Ｈ２００，３，９’
ＣａＣＪ ｚ ・２）ｒｚｏ Ｏ
−３１アデカ、ノールＬＧ−１０９
〔旭電化（株）製） ６．Ｏｍ微量元素液
（第３表と同じ） １．Ｑｌｌを水 ｌ ｉ＜ｐＨ
ｓ、ｓ）
４日目に培養液を遠心分離しと清を得た。その上清中の
エンド−β−グルカナーゼ活性は１１０Ｕ７’ｌｊ、工
中ソーβ−グルカナーゼ活性は１５Ｕ／ｉｕ、β−グル
フシダーゼ活性は２．７０Ｌ／ｄ及びβ−ガラクトシダ
ーゼ活性は３．１０７ｍであった。Table 5 Avicel 10. OF Polypeptone 1.01 Yeast engineered beef o,s gKH2PO
42, OF (NH4)2So, 1.5 N
MgSO4・71120 0.3
．． 9C&IC12・2[f20
o-31 Tween 80 (manufactured by Gyudon Chemical Co., Ltd.) 1
．． 0 Ni trace element liquid (same as Table 3) 1.0 m
l Water IJ (pHs, s) Table 6 Lactose 40. OIi polybutone 1. Oi yeast engineering school
o, syKH2PO,□,. 1 (N) (,)28041.5 N Mg804-7H200,3,9' CaCJ z ・2) rzo O
-31 Adeca, Nord LG-109 [manufactured by Asahi Denka Co., Ltd.] 6. Om trace element liquid (same as Table 3) 1. Qll to water l i<pH
s, s) On the fourth day, the culture solution was centrifuged to obtain a supernatant. The endo-β-glucanase activity in the supernatant was 110 U7'lj, the endo-β-glucanase activity was 15 U/iu, the β-glufusidase activity was 2.70 L/d, and the β-galactosidase activity was 3.107 m. .
ついで該上清１ノを硫安７０チ飽和で塩析後、セファデ
ックスＧ−２５（ファルマシア・ファイン・ケミカルズ
社製）を用いて脱塩し、凍結乾燥しＯＩの粗酵素標品な
得た。この標品のエン？−β−グルカナーゼ活性は８．
ＩＵ／１１６９、エキソ−β−グルカナーゼ活性は０，
８６ Ｕ／１９、β−グルコシダーゼ活性は０．１４Ｕ
／１１１９及びβ−ガラクトシダーゼ活性は０．１７０
／ダであった。The supernatant 1 was then salted out with 70% ammonium sulfate, desalted using Sephadex G-25 (manufactured by Pharmacia Fine Chemicals), and lyophilized to obtain a crude enzyme preparation of OI. This standard en? -β-glucanase activity is 8.
IU/1169, exo-β-glucanase activity is 0,
86 U/19, β-glucosidase activity is 0.14 U
/1119 and β-galactosidase activity is 0.170
/ It was da.
対照としてＭＣＧ−７７及びＰＣ−３−７を用い、上記
と同様に培養した。培養液中の酵素活性を第７表に示す
。MCG-77 and PC-3-7 were used as controls and cultured in the same manner as above. Table 7 shows the enzyme activity in the culture solution.
発明の効果
本発明の方法により、高活性のセルラーゼを得ることが
できる。Effects of the Invention Highly active cellulases can be obtained by the method of the present invention.
Claims (2)
ーゼの誘導能とβ−ガラクトシダーゼ活性とが高められ
た微生物を培地に培養し、培養物中にセルラーゼを生成
蓄積させ、培養物からセルラーゼを採取する工程からな
るセルラーゼの製法。(1) From the step of culturing a microorganism belonging to the genus Trichoderma that has enhanced cellulase inducibility and β-galactosidase activity with lactose in a medium, producing and accumulating cellulase in the culture, and collecting cellulase from the culture. A method for producing cellulase.
％（ラクトース換算）である特許請求の範囲第１項記載
の製法。(2) The amount of lactose or its content in the medium is 3 to 8
% (in terms of lactose).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4958586A JPS62208279A (en) | 1986-03-07 | 1986-03-07 | Production of cellulase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4958586A JPS62208279A (en) | 1986-03-07 | 1986-03-07 | Production of cellulase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS62208279A true JPS62208279A (en) | 1987-09-12 |
Family
ID=12835294
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4958586A Pending JPS62208279A (en) | 1986-03-07 | 1986-03-07 | Production of cellulase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62208279A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19735650B4 (en) * | 1997-08-16 | 2007-06-21 | Biopract Gmbh | A method for increasing the cellulolytic activity of Trichoderma reesei mutants in the bioreactor |
| WO2019185680A1 (en) * | 2018-03-28 | 2019-10-03 | Dsm Ip Assets B.V. | Enzyme composition |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6027384A (en) * | 1983-07-27 | 1985-02-12 | Res Assoc Petroleum Alternat Dev<Rapad> | Production of cellulase |
-
1986
- 1986-03-07 JP JP4958586A patent/JPS62208279A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6027384A (en) * | 1983-07-27 | 1985-02-12 | Res Assoc Petroleum Alternat Dev<Rapad> | Production of cellulase |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19735650B4 (en) * | 1997-08-16 | 2007-06-21 | Biopract Gmbh | A method for increasing the cellulolytic activity of Trichoderma reesei mutants in the bioreactor |
| WO2019185680A1 (en) * | 2018-03-28 | 2019-10-03 | Dsm Ip Assets B.V. | Enzyme composition |
| US11193145B2 (en) | 2018-03-28 | 2021-12-07 | Dsm Ip Assets B.V. | Enzyme composition |
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