JPH044888A - Production of amino acid by fermentation - Google Patents
Production of amino acid by fermentationInfo
- Publication number
- JPH044888A JPH044888A JP10451190A JP10451190A JPH044888A JP H044888 A JPH044888 A JP H044888A JP 10451190 A JP10451190 A JP 10451190A JP 10451190 A JP10451190 A JP 10451190A JP H044888 A JPH044888 A JP H044888A
- Authority
- JP
- Japan
- Prior art keywords
- fermentation
- trehalase
- amino acid
- culture
- 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.)
- Granted
Links
- 150000001413 amino acids Chemical class 0.000 title claims abstract description 19
- 238000004519 manufacturing process Methods 0.000 title claims description 6
- 238000000855 fermentation Methods 0.000 title abstract description 22
- 230000004151 fermentation Effects 0.000 title abstract description 22
- 102100029677 Trehalase Human genes 0.000 claims abstract description 23
- 108010087472 Trehalase Proteins 0.000 claims abstract description 23
- 244000005700 microbiome Species 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims description 11
- 229940024606 amino acid Drugs 0.000 description 15
- 235000001014 amino acid Nutrition 0.000 description 15
- 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 14
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 11
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 11
- 239000008103 glucose Substances 0.000 description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 7
- 238000012258 culturing Methods 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 241000186216 Corynebacterium Species 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 241000186249 Corynebacterium sp. Species 0.000 description 5
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 238000000108 ultra-filtration Methods 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 4
- 239000004472 Lysine Substances 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 235000019698 starch Nutrition 0.000 description 4
- 239000008107 starch Substances 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- 108010073771 Soybean Proteins Proteins 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
- 230000001580 bacterial effect Effects 0.000 description 3
- 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 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 229960002989 glutamic acid Drugs 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 235000013379 molasses Nutrition 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 235000019157 thiamine Nutrition 0.000 description 3
- 239000011721 thiamine Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 241000186226 Corynebacterium glutamicum Species 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-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
- 241001465754 Metazoa Species 0.000 description 2
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 238000005903 acid hydrolysis reaction Methods 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 210000004602 germ cell Anatomy 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 239000006456 gs medium Substances 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 235000019710 soybean protein Nutrition 0.000 description 2
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 2
- 229960003495 thiamine Drugs 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 235000016068 Berberis vulgaris Nutrition 0.000 description 1
- 241000335053 Beta vulgaris Species 0.000 description 1
- 241000186146 Brevibacterium Species 0.000 description 1
- 241000186031 Corynebacteriaceae Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- QLULGSLAHXLKSR-UHFFFAOYSA-N azane;phosphane Chemical compound N.P QLULGSLAHXLKSR-UHFFFAOYSA-N 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- LBVWYGNGGJURHQ-UHFFFAOYSA-N dicarbon Chemical compound [C-]#[C+] LBVWYGNGGJURHQ-UHFFFAOYSA-N 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 229940049906 glutamate Drugs 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229910001410 inorganic ion Inorganic materials 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000013587 production medium Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 229940001941 soy protein Drugs 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 150000003625 trehaloses Chemical class 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
[産業上の利用分野〕
本発明は発酵法によるアミノ酸の製造法に関するもので
ある。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing amino acids by fermentation.
[従来の技術]
グルコース、シュークロース、廃糖蜜等を用いるアミノ
酸発酵においては培養液中にトレノ\ロスも生成される
ことが知られている(Biosci。[Prior Art] It is known that in amino acid fermentation using glucose, sucrose, blackstrap molasses, etc., treno\loss is also produced in the culture solution (Biosci.
Rep、、 vol、5. No、6. pp 509
−515.1985)。しかし、このトレハロースに着
目して何らかの処理、あるいは活用することは知られて
いない。Rep,, vol, 5. No, 6. pp509
-515.1985). However, it is not known that this trehalose can be processed or utilized in any way.
トレハロースを発酵菌に資化させることができれば目的
アミノ酸の発酵収率を高められる可能性がある。If trehalose can be assimilated by fermenting bacteria, it is possible to increase the fermentation yield of the target amino acid.
本発明者らは上記目的を達成するべく鋭意検討の結果、
トレハロースをD−グルコースに分解するトレハラーゼ
に着目するに至った。そして、トレハラーゼを著聞生産
する微生物の取得に成功し、このトレハラーゼをアミノ
酸発酵の培養液に加えることによって培養液中に生成蓄
積されたトレハロースを有効利用して目的アミノ酸の発
酵収率を高めうることを見出して本発明を完成するに至
った。As a result of intensive studies to achieve the above object, the inventors of the present invention found that
We have now focused on trehalase, which decomposes trehalose into D-glucose. Furthermore, we succeeded in obtaining a microorganism that significantly produces trehalase, and by adding this trehalase to the culture solution for amino acid fermentation, we can effectively utilize the trehalose produced and accumulated in the culture solution to increase the fermentation yield of the target amino acid. This discovery led to the completion of the present invention.
すなわち、本発明は、微生物を用いるアミノ酸製造方法
において、該微生物の培養液中にトレハラーゼを存在せ
しめることを特徴とするアミノ酸の製造法に関するもの
である。That is, the present invention relates to a method for producing an amino acid using a microorganism, which is characterized in that trehalase is present in the culture solution of the microorganism.
トレハラーゼ(E、C,3,2,1,28)は2.2’
−1−レバロースを分解してD−グルコースを生成する
酵素であり、昆虫、動物、微生物などにその存在が知ら
れている。トレハラーゼは安価なものが好ましく、その
点で本発明者らが開発したコリネバクテリウム属細菌の
産生ずる1・1ツバラーゼ(特開昭62−275682
’3公報)は特に好ましい。Trehalase (E, C, 3, 2, 1, 28) is 2.2'
It is an enzyme that decomposes -1-levalose to produce D-glucose, and its existence is known in insects, animals, microorganisms, etc. An inexpensive trehalase is preferable, and from this point of view, the present inventors have developed 1.1 tubelase produced by a bacterium of the genus Corynebacterium (Japanese Unexamined Patent Publication No. 62-275682).
'3) is particularly preferred.
コリネバクテリウム属細菌を培養してl・レハラゼ4取
得する方法は公知の方法に従えばよい。A known method may be used to obtain L.rehalase 4 by culturing Corynebacterium bacteria.
すなわち、トレハラーゼ産生能を有するコリネバクテリ
ウム属細菌をグルコース、シュークロース、可溶性デン
プン等炭素源、硫安、塩安、燐安等の無機物あるいは酵
母エキス、肉エキス、大豆粕加水分解物、ペプトン等の
有機物の窒素源、ビタミン等の微量有機栄養物、リン酸
カリ、硫酸マグネシラl、等の無機塩を含む液体培地に
培養する。培養条件もフリ不ハクテリウム属細菌を培養
する通常の条件でよいが、本発明者らが先に開発したコ
リネバクテリウム・エスピーに一512株(FERM
P−8485)の場合には高温菌であるので40〜60
°C程度で培養するのがよい。培養は常法に従って通気
撹拌条件下で行ない、通常はトレハラーゼの蓄積が最高
に達するかあるいは経済的に最も好ましい蓄積量に達し
た時点で培養を終了する。培養終了後は培養液をそのま
ま、あるいは濃縮するだけで酵素源として使用してもよ
く、あるいは菌体分に1、限外濾過、ゲル濾過、イオン
交換クロマトグラフィ、硫安性を法等の公知の酵素精製
法によりさらに精製して使用することもできる。l・レ
バラーゼが菌体内酵素の場合には菌体を用い、あるいは
菌体を破壊して酵素抽出してこれを利用するごとはいう
までもない。本発明の方法で使用されるトレハラーゼは
微生物由来に限定されるものではなく、動植物由来のも
のであってもよい。また、遺伝子工学的手法等で調製さ
れたものであってもよい。That is, Corynebacterium bacteria capable of producing trehalase are mixed with carbon sources such as glucose, sucrose, and soluble starch, inorganic substances such as ammonium sulfate, ammonium chloride, and ammonium phosphorus, or yeast extract, meat extract, soybean meal hydrolyzate, and peptone. The cells are cultured in a liquid medium containing an organic nitrogen source, trace organic nutrients such as vitamins, and inorganic salts such as potassium phosphate and magnesyl sulfate. The culture conditions may be the usual conditions for culturing bacteria of the genus FERM, but the present inventors have previously developed Corynebacterium sp.
In the case of P-8485), it is a thermophilic bacterium, so the
It is best to culture at around °C. Cultivation is carried out under aeration and agitation according to a conventional method, and is usually terminated when the accumulation of trehalase reaches its maximum or the most economically preferable accumulation amount. After culturing, the culture solution can be used as it is, or just concentrated and used as an enzyme source, or it can be used as an enzyme source by using a known enzyme method such as ultrafiltration, gel filtration, ion exchange chromatography, ammonium sulfate chromatography, etc. It can also be used after further purification by a purification method. It goes without saying that if L-levarase is an intracellular enzyme, the bacterial cells are used, or the bacterial cells are destroyed and the enzyme is extracted for use. The trehalase used in the method of the present invention is not limited to those derived from microorganisms, but may be derived from animals or plants. Alternatively, it may be prepared by genetic engineering techniques or the like.
アミノ酸発酵の種類は特に限定されるものではなく、例
としてグルタミン酸発酵、リジン発酵、グルタミン発酵
、スI/オニン発酵、アミノ酸発酵、フェニルアラニン
発酵、バリン発酵、イソロイシン発酵等を例として挙げ
ることができる。発酵菌の種類もトレハ[l−スを生成
するものであれば特に限定されない。コリネバクテリウ
ム属細菌又はブレビバクテリウム属細菌等のいわゆるコ
リネ型細菌、例えばコリネバクテリウム・リリュウム、
コリネハクゾリウム・グルタミン酸、ブレビバクテリウ
ム・ラクトファーメンタム等に対して本発明の方法は特
に有効である。The type of amino acid fermentation is not particularly limited, and examples include glutamic acid fermentation, lysine fermentation, glutamine fermentation, suI/onine fermentation, amino acid fermentation, phenylalanine fermentation, valine fermentation, isoleucine fermentation, etc. The type of fermenting bacteria is not particularly limited as long as it produces trehalose. So-called coryneform bacteria such as bacteria of the genus Corynebacterium or bacteria of the genus Brevibacterium, such as Corynebacterium lilyum,
The method of the present invention is particularly effective against Corynebacterium glutamate, Brevibacterium lactofermentum, and the like.
アミノ酸発酵方法はトレハラーゼを添加するほかは公知
の方法に従って行なえばよく、これらの菌を培養する培
地には炭素源、窒素源、無機イオン及び必要に応じその
他の有機微量栄養素を含有する通常の培地が用いられる
。The amino acid fermentation method may be carried out according to a known method except for adding trehalase, and the culture medium for culturing these bacteria may be an ordinary medium containing a carbon source, a nitrogen source, inorganic ions, and other organic micronutrients as necessary. is used.
炭素源の例としては、グルコース、シュークロース、フ
ラクト−ス、ケーンモラセス、ビートモラセス、澱粉加
水分解物などの炭水化物を挙げることができる。窒素源
としてはアンモニアガス、アンモニア水、アンモニウム
塩、尿素等が好適である。培養は、好気的条件で行ない
、通常、培養のコントロールp)Iを4から9の間に温
度を25°Cから39°Cの間に調節する。しかしなが
ら、培養時間の短縮、雑菌防止等の点で40〜60°C
程度の高温発酵を行なうことは好ましく、その場合には
コリネバクテリウム・エスピーに一512株等の産生ず
る至適温度が40〜60°Cにあるような耐熱性トレハ
ラーゼは特に好ましい。Examples of carbon sources include carbohydrates such as glucose, sucrose, fructose, cane molasses, beet molasses, and starch hydrolysates. Suitable nitrogen sources include ammonia gas, aqueous ammonia, ammonium salts, urea, and the like. The cultivation is carried out under aerobic conditions, with the temperature usually adjusted between 25°C and 39°C during cultivation control p)I between 4 and 9. However, in order to shorten culture time and prevent bacteria, it is necessary to
It is preferable to carry out fermentation at a certain high temperature, and in that case, a thermostable trehalase whose optimum temperature for production is 40 to 60°C, such as Corynebacterium sp. 1512 strain, is particularly preferable.
1−レバラーゼの添加量は各発酵の種類と方法、トレハ
ラーゼの種類等を考慮して設定されるが通常0.5〜1
00ユニット/−程度である。添加時期は培養に先立っ
て予め培地に加えておいてもよく、あるいは培養中に1
回あるいは数回に分けて添加してもよい。トレハラーゼ
は固定化して使用することもできる。この固定化酵素は
アミノ酸発酵菌を固定化して使用する場合にそれに混合
してもよく、発酵槽に投入して使用することもできる。1-The amount of levalase added is determined taking into consideration the type and method of each fermentation, the type of trehalase, etc., but is usually 0.5 to 1.
It is about 00 units/-. It may be added to the medium in advance prior to culturing, or it may be added once during culturing.
It may be added at one time or in several parts. Trehalase can also be used in an immobilized state. This immobilized enzyme may be mixed with an immobilized amino acid-fermenting bacterium when used, or it may be used by adding it to a fermenter.
かくして1ないし7日間も培養すれば培地中に著量のア
ミノ酸が生成される。培養液よりアミノ酸を採取する方
法は通常の方法で行なう。Thus, if the culture is continued for 1 to 7 days, a significant amount of amino acids will be produced in the medium. Amino acids are collected from the culture solution using conventional methods.
[作用]
トレハロースはD−グルコースが1.1 結合した形の
非還元性の二炭糖であり、−船釣にはコリネ型細菌はト
レハロースを資化する力が弱く、この生成はとりもなお
さずグルコース等炭素源のアミノ酸への変換率を低めて
いる。アミノ酸発酵の培養液中に生成するトレハロース
をトレハラーゼで処理することにより、効率的にグルコ
ースに変換し、アミノ酸の収率を高めることが出来る。[Effect] Trehalose is a non-reducing dicarbon sugar in which 1.1 D-glucose is linked. This reduces the conversion rate of carbon sources such as glucose into amino acids. By treating trehalose produced in the culture solution of amino acid fermentation with trehalase, it is possible to efficiently convert it to glucose and increase the yield of amino acids.
実施例1
トレハラーゼはコリネバクテリウl、・エスピーに一5
12株から得られたものを使用した。その調製法は以下
の通りである。Example 1 Trehalase is derived from Corynebacterium sp.
Those obtained from 12 strains were used. Its preparation method is as follows.
まずに−512株(FERM P−8485)を1%可
溶性澱粉、1%グルコース、1%ポリペプトン、0.5
%酵母エキス、0.1%に211P04および0.02
%Mg5O,・71120を含有する液体培地に植菌し
、55°Cにて72時間好気的に培養した。得られた培
養液を11000Orp、0°Cにて10分間遠心分離
して菌体を除き、上澄液を得た。ついで、該上澄液を平
均分子量10000の限外濾過膜を用いて約10倍に濃
縮し、10mM燐酸緩衝液(pl+7)で−夜透析した
。この透析後に生じる沈澱を遠心分離で除き、得られた
上澄液を10mM燐酸緩衝液(pH7)で平衡化したD
EAE−トヨパール650Mカラムに吸着させ、0〜0
.7MのNaC1を含む上記と同様な緩衝液の濃度勾配
法によって酵素を溶出させた。溶出した活性画分を集め
分画分子量10000の限外濾過膜を用いて濃縮し、さ
らに10mM燐酸緩衝液(pH7)で−夜透析した。得
られた活性画分を10mM燐酸緩衝液(pH7)で平衡
化したDEAEトヨパールバック6508カラムに吸着
させO〜0.5M NaC1を含有する上記と同様な緩
衝液の濃度勾配法によって酵素を溶出させた。活性画分
を分画分子量10000の限外濾過膜で濃縮し、あらか
しめ0、IM NaClを含む上記緩衝液で平衡化した
アサヒバツクG5−520 Pカラムを用い、0.IM
NaC1を含む上記緩衝液で溶出した。得られた活性
画分を分画分子量10000の限外濾過膜で400ユニ
ツト/戚になるよう濃縮し、酵素溶液とした。First, -512 strain (FERM P-8485) was mixed with 1% soluble starch, 1% glucose, 1% polypeptone, and 0.5
% yeast extract, 0.1% to 211P04 and 0.02
The cells were inoculated into a liquid medium containing %Mg5O, 71120, and cultured aerobically at 55°C for 72 hours. The resulting culture solution was centrifuged at 11,000 Orp and 0°C for 10 minutes to remove bacterial cells and obtain a supernatant. Then, the supernatant was concentrated about 10 times using an ultrafiltration membrane with an average molecular weight of 10,000, and dialyzed against 10 mM phosphate buffer (pl+7) overnight. The precipitate generated after this dialysis was removed by centrifugation, and the resulting supernatant was equilibrated with 10 mM phosphate buffer (pH 7).
Adsorbed on EAE-Toyopearl 650M column, 0 to 0
.. The enzyme was eluted using a gradient method using the same buffer as above containing 7M NaCl. The eluted active fractions were collected, concentrated using an ultrafiltration membrane with a molecular weight cutoff of 10,000, and further dialyzed overnight against 10 mM phosphate buffer (pH 7). The obtained active fraction was adsorbed onto a DEAE Toyo Pearlvac 6508 column equilibrated with 10 mM phosphate buffer (pH 7), and the enzyme was eluted using a concentration gradient method using the same buffer as above containing 0 to 0.5 M NaCl. I let it happen. The active fraction was concentrated using an ultrafiltration membrane with a molecular weight cutoff of 10,000, and then concentrated using an Asahi Back G5-520 P column equilibrated with the above buffer containing 0.0, IM NaCl. IM
Elution was performed with the above buffer containing NaCl. The obtained active fraction was concentrated using an ultrafiltration membrane with a molecular weight cutoff of 10,000 to a concentration of 400 units/relative to give an enzyme solution.
グルコース5g/面、尿素0.4g/d1、KH2P0
.0.1g/df!、、MgSO4・711200.0
4g/d1、FeSO4・711z01mg/社、Mn
SO4・nHzo 1mg/d1、サイアミン・)IC
120μg/社、ビオチン30μg/dll、大豆蛋白
酸加水分解液90mg#J!(全窒素として)を含む種
母培地をpH7,0に調節し、その50rdを500d
容肩付フラスコに入れて加熱殺菌した。これにコリネバ
クテリウム・グルタミクムAJ 11441(FERM
P−5138)を接種し、31.5°Cに保ちつつ1
5時間振盪培養した。Glucose 5g/side, Urea 0.4g/d1, KH2P0
.. 0.1g/df! ,,MgSO4・711200.0
4g/d1, FeSO4・711z01mg/company, Mn
SO4・nHzo 1mg/d1, thiamine・)IC
120μg/company, biotin 30μg/dll, soybean protein acid hydrolyzate 90mg#J! (as total nitrogen) was adjusted to pH 7.0, and the 50rd was adjusted to 500d.
It was placed in a shoulder flask and sterilized by heating. In addition to this, Corynebacterium glutamicum AJ 11441 (FERM
P-5138) and incubated at 31.5°C.
The culture was incubated with shaking for 5 hours.
一方、シュークロース20g/a(総糖量として)KI
I2PO,0,1g/d1、Mg5040.04g/d
1、FeSO4・711201mg/dl、 MnSO
4・4Hz01■/d1、サイアミン・lIc120μ
g/a、ビオチン30μg/a、界面活性剤0.2g/
み、消泡剤0.005d/d1.大豆蛋白酸加水分解液
90■/面(全窒素として)、pt+ 7.0に調整し
た培地285m!を1!容ファーメンタ−に入れ殺菌し
た。冷却後、前述のトレハラーゼを0又は6.7ユニy
ト/mlとなるように添加した。この場合の1ユニツト
は反応温度55°Cで1分間にトレハロースから1μm
olのグルコースを生成する酵素活性である。On the other hand, sucrose 20g/a (as total sugar content) KI
I2PO, 0.1g/d1, Mg5040.04g/d
1.FeSO4・711201mg/dl, MnSO
4.4Hz01■/d1, Thiamin・LC120μ
g/a, biotin 30μg/a, surfactant 0.2g/a
and antifoaming agent 0.005d/d1. Soybean protein acid hydrolysis solution 90cm/plane (as total nitrogen), 285m of culture medium adjusted to pt+ 7.0! 1! The mixture was placed in a fermenter and sterilized. After cooling, add 0 or 6.7 units of trehalase.
It was added so that the amount was 100 g/ml. In this case, one unit is 1 μm from trehalose per minute at a reaction temperature of 55°C.
This is the enzyme activity that produces ol glucose.
これに上記種母培養液を15d接種した。培養は31.
5°Cでアンモニアガスにてpl+ 7.5に保持しつ
つ、通気撹拌下で糖を加えながら行った。培養48時間
後、それぞれの培養液中のL−グルタミン酸とトレハロ
ース量はそれぞれ第1表に示す通りであった。This was inoculated with the above seed culture solution for 15 days. Culture is 31.
The reaction was carried out at 5°C while maintaining the pl+7.5 with ammonia gas and adding sugar under aeration and stirring. After 48 hours of culture, the amounts of L-glutamic acid and trehalose in each culture solution were as shown in Table 1.
第1表
実施例2
トレハラーゼはコリネバクテリウム・エスピーL512
株(FERM P−8485)を下記のGS培地にて5
5°C・72時間培養し、イオン交換樹脂およびゲル濾
過カラムクロマトグラフィーで精製したものを用いた。Table 1 Example 2 Trehalase is Corynebacterium sp. L512
strain (FERM P-8485) in the following GS medium.
The cells were cultured at 5°C for 72 hours and purified using ion exchange resin and gel filtration column chromatography.
GS培地 1% グルコース 1% 可溶性澱粉 1% ポリペプトン 0.5% 酵母エキス 0.1% KzllPo。GS medium 1% glucose 1% soluble starch 1% polypeptone 0.5% yeast extract 0.1% KzllPo.
0.02% Mg5O,・711□0実施例1の菌株
、培地組成及び培養方法を用い、トレハラーゼ無添加の
発酵終了液1ml1に前記のトト・ハラーゼを最終濃度
O及び6,7ユニツト/雌となるように添加し、31
、5 ”Cで7時間反応した。反応後の培養液中の1.
−グルタミン酸とトレハロース量を第2表に示す。0.02% Mg5O,・711□0 Using the strain, medium composition, and culture method of Example 1, the above-mentioned toto halase was added to 1 ml of the fermentation solution without addition of trehalase at a final concentration of O and 6.7 units/female. Add it so that it becomes 31
, 5"C for 7 hours. After the reaction, 1.
- The amounts of glutamic acid and trehalose are shown in Table 2.
第2表
実施例3
トレハラーゼはコリネバクテリウム・エスピー)[−5
12株(FERM p、−8485)を培養して得られ
た培養液(特開昭62−275682号公報)を無菌濾
過して使用した。Table 2 Example 3 Trehalase is Corynebacterium sp.) [-5
A culture solution obtained by culturing 12 strains (FERM p, -8485) (Japanese Unexamined Patent Publication No. 62-275682) was sterile-filtered and used.
ペプトンIg/d1、酵母エギスIg#f、 NaCl
O,5g/み、グルコース0.5(H/み、寒天2g
7dl、pH7,0のUl 成の固体プレーl−にブ1
ノビバクテリウム・ラフ1−ファーメンタムへJ 34
24(FERM P−1711)を植えつけ、温度31
.5°Cで24時間培養し、種母用菌体を得た。Peptone Ig/d1, Yeast Aegis Ig#f, NaCl
O, 5g/mi, glucose 0.5 (H/mi, agar 2g
7 dl, pH 7.0 solid plate of 1-
Novibacterium rough 1-fermentum J 34
24 (FERM P-1711), temperature 31
.. The cells were cultured at 5°C for 24 hours to obtain seed germ cells.
一方、グルコース10g/di、硫安5.5g/d1、
トeSo、−711z0 1mg/d、Mn5Os ・
4H201mg/di!、1(Il、PO40,1g/
d、MgSO40,1g/み、サイアミン・lIc12
0μg/d1、ビオチン50μg/d1、ニコチン酸ア
ミド0.5mg/Li1、大豆蛋白酸加水分解105■
/ミ(全窒素として)を加えpl+ 8.0に調整した
培地20dを500mp、容肩付フラスコに入れて加熱
殺菌1−だ。これに別殺菌したCaC0:+ Igを加
えて、リジン生産用培地とした。このリジン生産用培地
に種母用菌体1白金芽を植えつけた後、ミリポアフィル
タ−で除菌した前述のトレハラーゼを最終濃度O及び6
ユユツ)/dとなるように培養開始時及び24時間目に
添加し、31.5°Cで48時間振盪培養した。それぞ
れの培養液中の1.−リジン量及びトレハロース量を第
3表に示す。On the other hand, glucose 10g/di, ammonium sulfate 5.5g/d1,
ToeSo, -711z0 1mg/d, Mn5Os ・
4H201mg/di! , 1(Il, PO40, 1g/
d, MgSO40, 1g/mi, thiamine lIc12
0μg/d1, biotin 50μg/d1, nicotinamide 0.5mg/Li1, soy protein acid hydrolysis 105■
20 d of culture medium adjusted to pl+ 8.0 by adding /mi (as total nitrogen) was placed in a 500 mp shoulder flask and heat sterilized 1-. Separately sterilized CaC0:+Ig was added to this to prepare a medium for lysine production. After inoculating 1 platinum bud of seed germ cells into this lysine production medium, the aforementioned trehalase, which had been sterilized with a Millipore filter, was added to a final concentration of O and 6.
It was added at the start of the culture and at 24 hours so that the amount was 20%)/d, and cultured with shaking at 31.5°C for 48 hours. 1 in each culture solution. - The amounts of lysine and trehalose are shown in Table 3.
第3表
〔発明の効果〕
本発明によりアミノ酸の発酵収率を高めることができる
。Table 3 [Effects of the Invention] According to the present invention, the fermentation yield of amino acids can be increased.
Claims (1)
の培養液中にトレハラーゼを存在せしめることを特徴と
するアミノ酸の製造法A method for producing an amino acid using a microorganism, the method comprising making trehalase exist in the culture solution of the microorganism.
Priority Applications (1)
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JP10451190A JP2952604B2 (en) | 1990-04-20 | 1990-04-20 | Production of amino acids by fermentation |
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---|---|---|---|
JP10451190A JP2952604B2 (en) | 1990-04-20 | 1990-04-20 | Production of amino acids by fermentation |
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Publication Number | Publication Date |
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JPH044888A true JPH044888A (en) | 1992-01-09 |
JP2952604B2 JP2952604B2 (en) | 1999-09-27 |
Family
ID=14382521
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0537443A1 (en) | 1991-10-18 | 1993-04-21 | Degussa Aktiengesellschaft | Process for the enhancement of the productivity of bacteria excreting amino acids |
FR2747131A1 (en) * | 1996-04-09 | 1997-10-10 | Orsan | PROCESS FOR THE PRODUCTION OF AMINO ACID BY CORYNEBACTERIUM FERMENTATION EXPRESSING TREHALASE ACTIVITY |
JP2009517010A (en) * | 2005-11-28 | 2009-04-30 | ビーエーエスエフ ソシエタス・ヨーロピア | Fermentative production of organic compounds |
WO2009121058A1 (en) * | 2008-03-28 | 2009-10-01 | Novozymes A/S | Producing fermentation products in the presence of trehalase |
CN107058415A (en) * | 2017-03-01 | 2017-08-18 | 中粮生化能源(龙江)有限公司 | It is a kind of to add the method that trehalase improves glutamic acid fermentation saccharic acid conversion ratio |
US9856498B2 (en) | 2012-03-30 | 2018-01-02 | Novozymes A/S | Processes of producing fermentation products |
US10227613B2 (en) | 2012-03-30 | 2019-03-12 | Novozymes A/S | Processes for producing fermentation products |
-
1990
- 1990-04-20 JP JP10451190A patent/JP2952604B2/en not_active Expired - Lifetime
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0537443A1 (en) | 1991-10-18 | 1993-04-21 | Degussa Aktiengesellschaft | Process for the enhancement of the productivity of bacteria excreting amino acids |
FR2747131A1 (en) * | 1996-04-09 | 1997-10-10 | Orsan | PROCESS FOR THE PRODUCTION OF AMINO ACID BY CORYNEBACTERIUM FERMENTATION EXPRESSING TREHALASE ACTIVITY |
WO1997038111A1 (en) * | 1996-04-09 | 1997-10-16 | Orsan | Method for producing an amino acid by fermenting corynebacteria expressing trehalase activity |
JP2009517010A (en) * | 2005-11-28 | 2009-04-30 | ビーエーエスエフ ソシエタス・ヨーロピア | Fermentative production of organic compounds |
WO2009121058A1 (en) * | 2008-03-28 | 2009-10-01 | Novozymes A/S | Producing fermentation products in the presence of trehalase |
US9856498B2 (en) | 2012-03-30 | 2018-01-02 | Novozymes A/S | Processes of producing fermentation products |
US10227613B2 (en) | 2012-03-30 | 2019-03-12 | Novozymes A/S | Processes for producing fermentation products |
US10364445B2 (en) | 2012-03-30 | 2019-07-30 | Novozymes A/S | Processes of producing fermentation products |
US10526620B2 (en) | 2012-03-30 | 2020-01-07 | Novozymes A/S | Processes for producing fermentation products |
US10954533B2 (en) | 2012-03-30 | 2021-03-23 | Novozymes A/S | Processes of producing fermentation products |
US11987831B2 (en) | 2012-03-30 | 2024-05-21 | Novozymes A/S | Processes for producing a fermentation product |
CN107058415A (en) * | 2017-03-01 | 2017-08-18 | 中粮生化能源(龙江)有限公司 | It is a kind of to add the method that trehalase improves glutamic acid fermentation saccharic acid conversion ratio |
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