JPH0346115B2 - - Google Patents
Info
- Publication number
- JPH0346115B2 JPH0346115B2 JP9947782A JP9947782A JPH0346115B2 JP H0346115 B2 JPH0346115 B2 JP H0346115B2 JP 9947782 A JP9947782 A JP 9947782A JP 9947782 A JP9947782 A JP 9947782A JP H0346115 B2 JPH0346115 B2 JP H0346115B2
- Authority
- JP
- Japan
- Prior art keywords
- cystine
- cysteine
- culture solution
- atc
- culture
- 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.)
- Expired
Links
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 claims description 37
- 229960003067 cystine Drugs 0.000 claims description 37
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 35
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 27
- 235000018417 cysteine Nutrition 0.000 claims description 27
- 244000005700 microbiome Species 0.000 claims description 14
- 239000004158 L-cystine Substances 0.000 claims description 9
- 235000019393 L-cystine Nutrition 0.000 claims description 9
- 230000003698 anagen phase Effects 0.000 claims description 7
- 235000001014 amino acid Nutrition 0.000 claims description 6
- 150000001413 amino acids Chemical class 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 230000032683 aging Effects 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 239000004201 L-cysteine Substances 0.000 claims description 4
- 235000013878 L-cysteine Nutrition 0.000 claims description 4
- 239000011593 sulfur Substances 0.000 claims description 4
- 229910052717 sulfur Inorganic materials 0.000 claims description 4
- VHPXSBIFWDAFMB-UHFFFAOYSA-N 2-amino-Delta(2)-thiazoline-4-carboxylic acid Chemical compound NC1=[NH+]C(C([O-])=O)CS1 VHPXSBIFWDAFMB-UHFFFAOYSA-N 0.000 claims description 3
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 2
- CDMKLKAZVMTVHX-UHFFFAOYSA-N 4,5-dihydro-1,3-thiazol-3-ium-4-carboxylate Chemical compound OC(=O)C1CSC=N1 CDMKLKAZVMTVHX-UHFFFAOYSA-N 0.000 claims 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 claims 1
- 239000000243 solution Substances 0.000 description 35
- 238000006243 chemical reaction Methods 0.000 description 15
- 108090000790 Enzymes Proteins 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 10
- 230000000694 effects Effects 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- 239000002609 medium Substances 0.000 description 6
- 230000005070 ripening Effects 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241001600125 Delftia acidovorans Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241000589565 Flavobacterium Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000192041 Micrococcus Species 0.000 description 3
- 241000589516 Pseudomonas Species 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000012264 purified product Substances 0.000 description 3
- 241000590020 Achromobacter Species 0.000 description 2
- 241001453369 Achromobacter denitrificans Species 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 241000193764 Brevibacillus brevis Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 241000588914 Enterobacter Species 0.000 description 2
- 241000588698 Erwinia Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 241000191938 Micrococcus luteus Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 241000607715 Serratia marcescens Species 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 239000012736 aqueous medium Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 229910001410 inorganic ion Inorganic materials 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000009423 ventilation Methods 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
- 241000588986 Alcaligenes Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000186146 Brevibacterium Species 0.000 description 1
- 241000125240 Dimorpha Species 0.000 description 1
- 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 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-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
- 241000588915 Klebsiella aerogenes Species 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 241000721603 Mycoplana Species 0.000 description 1
- 241001135976 Mycoplana dimorpha Species 0.000 description 1
- 241000588701 Pectobacterium carotovorum Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000192023 Sarcina Species 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 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 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 241000319304 [Brevibacterium] flavum Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000003679 aging effect Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 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
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940092559 enterobacter aerogenes Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000002816 microbial assay Methods 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
- 239000011707 mineral Substances 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- DUIOPKIIICUYRZ-UHFFFAOYSA-N semicarbazide Chemical compound NNC(N)=O DUIOPKIIICUYRZ-UHFFFAOYSA-N 0.000 description 1
- UQDJGEHQDNVPGU-UHFFFAOYSA-N serine phosphoethanolamine Chemical compound [NH3+]CCOP([O-])(=O)OCC([NH3+])C([O-])=O UQDJGEHQDNVPGU-UHFFFAOYSA-N 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
本発明はＬ−含硫アミノ酸、即ちＬ−システイ
ンおよびＬ−シスチンの製造法に関する。
従来、Ｌ−システイン（以下、システインと略
す）およびＬ−シスチン（以下、シスチンと略
す）は毛髪等のシステインやシスチンの含量の高
い天然物を鉱酸で加水分解し、生じたアミノ酸混
液よりシスチンとして単離取得し、システインは
このようにして得られたＬ−シスチンを還元して
製造されていた。
これに対し、本出願人においては既に、２−ア
ミノ−チアゾリン−４−カルボン酸（以下、
ATCと記す。）を原料とし、微生物の作用を利用
してシステイン又はシスチンを製造する方法（特
開昭51−136619号公報参照）を開発した。
この方法に於ては、ATCを含硫アミノ酸に変
換する能力を有する微生物が酵素源として利用さ
れ、この酵素は誘導酵素であるため、酵素誘導剤
として原料のATCが必要とされている。しかし
ながらATCは高価であり、原料の１部を酵素誘
導剤として使用することは経済的でなく、この方
法を工業的に実施し、含硫アミノ酸を安価に製造
するためには、酵素誘導剤として使用するATC
の量を削減し、かつ菌体の酵素活性を高くするこ
とが必要とされていた。そこで本発明者等は培養
液の酸素活性を高める方法を開発することを目的
として種々研究を重ねた結果、ATCの培地への
添加時期を微生物の対数増殖期、特に対数増殖期
の後半にすることによつてATCが大巾に削減で
きること、更に、培養して得られた培養液を30〜
45℃に少くとも１時間以上保持して熟成すること
によりATC水解活性が20〜40％増大することを
発見した。本発明はこの発見に基づいて完成され
たものである。
本発明は次の(A)、(B)、(C)及び(D)の各工程を含有
してなる含硫アミノ酸の製造法に関する。
(A) ２−アミノ−チアゾリン−４−カルボン酸を
水解してシステイン又はシスチンを生成する能
力を有する微生物を栄養培地で培養し、対数増
殖期にATCを添加して培養する工程
(B) 培養液を30〜45℃に少なくとも１時間以上保
持して熟成する工程
(C) 熟成した培養液に含まれる微生物をATCに
作用せしめてシステイン又はシスチンを生成せ
しめる工程
(D) 生成したシステイン又はシスチンを採取する
工程
上記(A)工程に於て使用する微生物は、アクロモ
バクター、アルカリゲネス、バチルス、ブレビバ
クテリウム、エンテロバクター、エルビニア、エ
ツシエリヒア、フラボバクテリウム、ミクロコツ
カス、ミコプラナ、シユードモナス、サルシナあ
るいはセラチアの各属に属し、ATCを水解して
システイン又はシスチンを生成する能力を有する
微生物であり、その他自然界に存在する野生株、
公的な微生物保存機関に保存されている菌株或い
はそれらより人工的に変異誘導した微生物群よ
り、ATCからのシスチン又は（及び）システイ
ン生産能の有無を調べることによつて分離採取さ
れるものも使用できる。
より具体的に例示するならば次の如き菌株があ
げられる：サルチナ・ルテアAJ−1217（Sarcina
lutea）ATCC272、アクロモバクター・デルマル
バアAJ−1983（Achremebaeter delmarvae）
FERM−P3483、アルカリゲネス・デニトリフイ
カンスAJ−2553（Alcaligenes denitrificans）
ATCC15173、バチルス・ブレビスAJ−1982
（Bacillus brevis）ATCC8185、ブレビバクテリ
ウム・フラバムAJ−1516（Brevibaeterium
flavum）ATCC13826、エンテロバクター・アエ
ロゲネスAJ−2643（Enterobacter
aerogenesIAM1019）FERM−P2764FERM BP
−321、エルビニア・カロトボラAJ−2753
（Erwinia carotovoraCCM873）FERM−P2766、
シユードモナス・チアゾリノフイラムAJ−3854
（Pseudomonas thiazolinophilum）FERM−
P2810FERM BP−322、シユードモナス・デス
モリチカAJ2368（P.demolytica）FERM−
P2816FERM BP−323、エツシエリヒア・コリ
カAJ−2592（Escherichiacoli IAM1132）FERM
−P2763、ミクロコツカス・ソドネンシスAJ−
1753（Micrococcus sodonensis）ATCC11880、
ミコプラナ・ジモルフアAJ−2809（Mycoplana
dimorpha）ATCC4249、セラチア・マルセツセ
ンスAJ−2698（Serratia marcescens IAM1206）
FERM−P2765、フラボバクテリウム・アシドフ
イクムAJ−2494（Flavobacterium acidoficum）
ATCC8366、プソドモナス・オバリスAJ−2236、
（Pseudomenas ovalis IAM1454）FERM−
P2762FERM BP−1328等が挙げられる。
微生物を培養するための培地は炭素源、窒素
源、無機イオン、更に要すれば有機微量栄養素を
適宜含有する培地であり、例えば、炭素源として
グルコース、シユクロース、キシロース、糖蜜等
の糖類、酢酸等の有機酸、エタノール、グリセロ
ール、メタノール等のアルコール類など、窒素源
として硫酸アンモニウム、塩化アンモニウムな
ど、有機栄養源として、酵母エキス、ペプトン、
肉エキス、コーン・ステイープリカーなど、無機
イオンとして、マグネシウム、鉄、マンガン、カ
リウム、ナトリウム、リン酸などのイオンが適宜
用いられる。
微生物の培養法は常法に従つて行えば良く、上
記培地のPHを６〜９とし、20〜40℃で好気的に培
養し、対数増殖期、特に対数増殖期の後半、更に
具体的には後期第１表に示す組成の培地を用いて
30℃で４〜８時間培養した時点、菌体濃度で示せ
ば培養液を1/26に希釈した液の562nｍに於る吸
光度が0.4〜0.7に達した時点で、培養液にATCを
0.05〜0.6％、望ましくは0.05〜0.1％添加し、更
に培養して酵素活性の高い培養液を得る。このよ
うに対数増殖期を選択することによつて酵素誘導
体のATCの量を1/5〜1/10に削減することができ
る。
(B)工程に於ては、上記培養液を30〜45℃の温度
に少くとも1.0時間以上、望ましくは1.0〜3.0時間
保持して培養液の熟成を行う。この熟成期間中は
培養液をゆるやかに撹拌することが望ましく、こ
の熟成工程を導入することにより培養液の酸素活
性は20〜40％増大し、かつ、安定した酵素活性が
得られる。
(C)工程は上記熟成した培養液に含まれる微生物
をATCに作用せしめてシステインはシスチンを
生成せしめる工程であり、この工程に於ては、熟
成した培養液に含まれる微生物を酵素源として使
用する。即ち、熟成した培養液、培養液から分離
した分離菌体、洗滌生菌体、凍結乾燥体、アセト
ン乾燥菌体、物理的、化学的もしくは生化学的に
破壊された菌体、抽出液、粗精製物、精製物、精
製蛋白標晶、または菌体もしくは精製処理物の固
定化物などを酵素源として使用する。
原料であるATCは合成法にて供給されるもの
で、Ｄ−体、Ｌ−体、ラセミ体のいずれもが使用
される。
基質濃度は、バツチ式、連続式によつて異なる
が、バツチ式では一般に水性媒質中0.1〜30％、
好ましくは0.5〜10％程度で連続式では、これよ
りやや濃度を低下させた方が好ましい。
反応は、普通、水性媒質中で15〜60℃、好まし
くは30℃〜50℃附近で、PH＝６〜10、好ましくは
7.0〜9.5附近で行なわれる。反応時間は、静置、
撹拌、流下等の手段あるいは酵素標品の形態、力
価によつて異なつてくるので一様ではないが、バ
ツチ法では通常10分〜72時間程度である。
反応が進行すると、反応液中にシステインとシ
スチンが共存するのであるが、通常システインは
空気中の酸素により酸化されてシステンに変化し
易く、時間がたつにつれてシスチンの量が増大す
る。しかしながら、反応条件等の変更によつてシ
ステインとシスチンの濃度比を変えることも可能
である。なお、反応液にヒドロキシルアミンやセ
ミカルバジドを添加することによつてシステイン
及び／またはシスチンの収率を向上することがで
きる。
(D)工法に於るシステイン及びシスチンの採取法
は常法に従つて行えば良く、例えば、通気酸化に
より大部分をシスチンとし、その難溶性を利用し
て晶析する方法によりＬ−シスチンを得ることが
できる。また、システインにする場合は電気還元
による通常の方法で処理して、システインとして
採取することができる。
シスチンとシステインの定量は、液体クロマト
グラフイーと微生物定量法によつて行われる。後
者は乳酸菌ロイコノストツク・チトロボラム
ATCC8081を用いる方法であるが、この菌はシス
チンとシステインの双方によつて何等の生育を与
えられるので両アミノ酸の和を定量することにな
り、通常シスチンとして表示される。なお、この
菌はＤ体のシスチン、システインでは生育できな
い。
以下、実施例にて説明する。
実施例 １
第１表に示す組成の培地1.0を2.0容のジヤ
ーフアーメンターに張り込み、120℃で15分間加
熱・滅菌した。
The present invention relates to a method for producing L-sulfur-containing amino acids, namely L-cysteine and L-cystine. Conventionally, L-cysteine (hereinafter abbreviated as cysteine) and L-cystine (hereinafter abbreviated as cystine) have been produced by hydrolyzing cysteine such as hair or natural products with a high cystine content with mineral acids, and using the resulting amino acid mixture to obtain cystine. Cysteine was isolated and obtained as L-cystine, and cysteine was produced by reducing L-cystine thus obtained. In contrast, the present applicant has already developed 2-amino-thiazoline-4-carboxylic acid (hereinafter referred to as
It is written as ATC. ) as a raw material and developed a method for producing cysteine or cystine by utilizing the action of microorganisms (see Japanese Patent Application Laid-Open No. 136619/1983). In this method, a microorganism having the ability to convert ATC into a sulfur-containing amino acid is used as an enzyme source, and since this enzyme is an inducible enzyme, the raw material ATC is required as an enzyme inducer. However, ATC is expensive, and it is not economical to use part of the raw material as an enzyme inducer. ATC to use
There was a need to reduce the amount of bacteria and increase the enzyme activity of bacterial cells. Therefore, the present inventors have conducted various studies with the aim of developing a method to increase the oxygen activity of the culture solution, and as a result, the timing of adding ATC to the culture medium is set to be the time of adding ATC to the culture medium during the logarithmic growth phase of the microorganism, especially in the latter half of the logarithmic growth phase. In particular, ATC can be significantly reduced, and the culture solution obtained by culturing can be
It has been discovered that ATC hydrolysis activity increases by 20-40% by aging at 45°C for at least 1 hour. The present invention was completed based on this discovery. The present invention relates to a method for producing a sulfur-containing amino acid, which comprises the following steps (A), (B), (C), and (D). (A) A step of culturing a microorganism that has the ability to hydrolyze 2-amino-thiazoline-4-carboxylic acid to produce cysteine or cystine in a nutrient medium, and adding ATC during the logarithmic growth phase. (B) Cultivation A process of aging the liquid by maintaining it at 30 to 45°C for at least 1 hour (C) A process of allowing microorganisms contained in the aged culture liquid to act on ATC to produce cysteine or cystine (D) A process of producing cysteine or cystine Collection process The microorganisms used in the above step (A) include Achromobacter, Alcaligenes, Bacillus, Brevibacterium, Enterobacter, Erwinia, Ethusierichia, Flavobacterium, Micrococcus, Mycoplana, Pseudomonas, Sarcina, and Serratia. A microorganism that belongs to the genus ATC and has the ability to hydrolyze ATC to produce cysteine or cystine, and other wild strains that exist in nature.
There are also those that are isolated and collected from bacterial strains stored in public microbial repositories or microorganisms artificially mutated from them by examining the presence or absence of cystine from ATC or (and) cysteine production ability. Can be used. More specific examples include the following strains: Sarcina lutea AJ-1217 (Sarcina lutea AJ-1217).
lutea) ATCC272, Achromobacter delmarvae AJ-1983 (Achremebaeter delmarvae)
FERM−P3483, Alcaligenes denitrificans AJ−2553 (Alcaligenes denitrificans)
ATCC15173, Bacillus brevis AJ-1982
(Bacillus brevis) ATCC8185, Brevibacterium flavum AJ-1516 (Brevibaeterium
flavum) ATCC13826, Enterobacter aerogenes AJ-2643 (Enterobacter
aerogenesIAM1019) FERM−P2764FERM BP
−321, Erwinia carotovora AJ−2753
(Erwinia carotovoraCCM873) FERM−P2766,
Pseudomonas thiazolinophyllum AJ-3854
(Pseudomonas thiazolinophilum) FERM−
P2810FERM BP−322, Pseudomonas desmolytica AJ2368 (P.demolytica) FERM−
P2816FERM BP−323, Escherichia coli AJ−2592 (Escherichia coli IAM1132) FERM
−P2763, Micrococcus sodonensis AJ−
1753 (Micrococcus sodonensis) ATCC11880,
Mycoplana dimorpha AJ-2809
dimorpha) ATCC4249, Serratia marcescens AJ-2698 (Serratia marcescens IAM1206)
FERM-P2765, Flavobacterium acidoficum AJ-2494 (Flavobacterium acidoficum)
ATCC8366, Psodomonas obalis AJ-2236,
(Pseudomenas ovalis IAM1454) FERM−
Examples include P2762FERM BP-1328. The medium for culturing microorganisms is a medium containing a carbon source, a nitrogen source, inorganic ions, and, if necessary, organic micronutrients as appropriate.For example, as a carbon source, sugars such as glucose, sucrose, xylose, and molasses, acetic acid, etc. organic acids, alcohols such as ethanol, glycerol, and methanol; nitrogen sources such as ammonium sulfate and ammonium chloride; organic nutritional sources such as yeast extract, peptone,
Ions such as magnesium, iron, manganese, potassium, sodium, and phosphoric acid are appropriately used as inorganic ions in meat extracts, corn staple liquor, and the like. The culture of microorganisms can be carried out according to conventional methods. For the latter stage, use a medium with the composition shown in Table 1.
After culturing at 30°C for 4 to 8 hours, when the absorbance at 562 nm of a 1/26 dilution of the culture solution reached 0.4 to 0.7, ATC was added to the culture solution.
Add 0.05 to 0.6%, preferably 0.05 to 0.1%, and further culture to obtain a culture solution with high enzyme activity. By selecting the logarithmic growth phase in this manner, the amount of ATC in the enzyme derivative can be reduced to 1/5 to 1/10. In step (B), the culture solution is maintained at a temperature of 30 to 45° C. for at least 1.0 hours or more, preferably 1.0 to 3.0 hours to ripen the culture solution. During this ripening period, it is desirable to gently stir the culture solution, and by introducing this ripening step, the oxygen activity of the culture solution increases by 20 to 40%, and stable enzyme activity can be obtained. Step (C) is a step in which the microorganisms contained in the aged culture solution are made to act on ATC to produce cystine, and in this step, the microorganisms contained in the aged culture solution are used as an enzyme source. do. That is, aged culture solution, isolated bacterial cells separated from the culture solution, washed viable bacterial cells, freeze-dried cells, acetone-dried bacterial cells, physically, chemically or biochemically destroyed bacterial cells, extracts, crude Purified products, purified products, purified protein standards, or immobilized products of bacterial cells or purified products are used as enzyme sources. ATC, which is a raw material, is supplied by a synthetic method, and all of the D-form, L-form, and racemic form are used. The substrate concentration differs depending on the batch type or continuous type, but in the batch type it is generally 0.1 to 30% in the aqueous medium.
Preferably, it is about 0.5 to 10%, and in a continuous system, it is preferable to lower the concentration slightly. The reaction is usually carried out in an aqueous medium at 15-60°C, preferably around 30-50°C, and at a pH of 6-10, preferably
It is held around 7.0-9.5. Reaction time: standing still;
The batch method usually takes about 10 minutes to 72 hours, although it is not uniform because it depends on the means of stirring, flowing down, etc., and the form and potency of the enzyme preparation. As the reaction progresses, cysteine and cystine coexist in the reaction solution, but normally cysteine is easily oxidized by oxygen in the air and converted to cysteine, and the amount of cystine increases over time. However, it is also possible to change the concentration ratio of cysteine and cystine by changing reaction conditions and the like. Note that the yield of cysteine and/or cystine can be improved by adding hydroxylamine or semicarbazide to the reaction solution. (D) Collection of cysteine and cystine in the construction method can be carried out according to conventional methods. For example, most of the cysteine is converted to cystine by aerated oxidation, and L-cystine is obtained by crystallizing by taking advantage of its poor solubility. Obtainable. In addition, when producing cysteine, it can be collected as cysteine by processing it by the usual method of electroreduction. Quantification of cystine and cysteine is performed by liquid chromatography and microbial assay. The latter is the lactic acid bacterium Leuconostocci titroborum.
This method uses ATCC8081, but since this bacterium depends on both cystine and cysteine for growth, the sum of both amino acids is quantified, and is usually expressed as cystine. Note that this bacterium cannot grow on D-form cystine or cysteine. Examples will be described below. Example 1 A 2.0-volume jar fermentor was filled with 1.0 of a medium having the composition shown in Table 1, and sterilized by heating at 120° C. for 15 minutes.
【表】
これにシユードモナス・デスモリチカ
FERM−P2816FERM BP−323の種培養液50ml
を接種し、30℃にて通気撹拌培養（1/2VVM、
600rpm）を開始した。培養期間中培養液のPHは
6.5にコントロールし、培養開始時、及び一定時
間培養後、培養液にDL−ATCを0.1％添加して
培養を続け、培養開始後12時間で培養を終了し
た。得られた培養液にDL−ATC5.0％、KH2PO4
0.1％、FeSO4・7H2O 0.03％を添加し、PHを8.0
に調節後、30℃に24時間保持して反応を行つた。
反応終了後各反応液を通気下で激しく撹拌しシス
テインを酸化させてシスチンに変換せしめた。
この反応液に6N−NaOH溶を加えてシスチン
を溶解し、CPK−08（強酸性カチオン交換樹脂）
カラムを用いる高速液体クロマトグラフイーにて
シスチンを定量した。その結果を第２表に示す。[Table] This is Pseudomonas desmolytica
FERM−P2816FERM BP−323 seed culture solution 50ml
was inoculated and cultured with aeration at 30℃ (1/2VVM,
600rpm). During the culture period, the pH of the culture solution is
6.5, 0.1% DL-ATC was added to the culture solution at the start of culture and after culture for a certain period of time, and culture was continued, and the culture was terminated 12 hours after the start of culture. DL-ATC5.0%, KH 2 PO 4 was added to the obtained culture solution.
Add 0.1%, FeSO4.7H2O 0.03%, pH to 8.0
After adjusting the temperature to 30°C, the reaction was carried out by holding at 30°C for 24 hours.
After the reaction was completed, each reaction solution was vigorously stirred under ventilation to oxidize cysteine and convert it to cystine. Add 6N-NaOH solution to this reaction solution to dissolve cystine, and use CPK-08 (strongly acidic cation exchange resin).
Cystine was quantified by high performance liquid chromatography using a column. The results are shown in Table 2.
【表】
第２表に示すように、菌体濃度が0.35〜0.60の
時期、即ち、対数増殖期間中に添加する場合に最
も活性が高いことが確認された。同様の方法で、
培養６時間後にATCを添加し再に４時間通気撹
拌して1.0の培養液を得た。この培養液を５ml
宛に分けて、夫々10℃から60℃の温度に１時間保
持して熟成を行つた。この熟成培養液に下記第３
表に示す基質溶液5.0mlを加えて混合し、30℃に
３時間保持して酸素反応を行つた。[Table] As shown in Table 2, it was confirmed that the activity was highest when added during the period when the bacterial cell concentration was 0.35 to 0.60, that is, during the logarithmic growth period. In a similar way,
After 6 hours of culturing, ATC was added and stirred again for 4 hours to obtain a 1.0 culture solution. 5ml of this culture solution
The mixture was divided into portions and kept at a temperature of 10°C to 60°C for 1 hour for ripening. Add the following 3rd layer to this aging culture solution.
5.0 ml of the substrate solution shown in the table was added and mixed, and the mixture was kept at 30°C for 3 hours to perform an oxygen reaction.
【表】
反応終了後、6N−NaOH溶液を5.0ml添加して
良く撹拌し、高速液体クロマトグラフイーにてシ
ステイン及びシスチンの生成量を定量した。その
結果を第４表に示す。[Table] After the reaction was completed, 5.0 ml of 6N-NaOH solution was added and stirred well, and the amount of cysteine and cystine produced was determined by high performance liquid chromatography. The results are shown in Table 4.
【表】
第４表に示すように、熟成温度は30〜45℃の間
が良いことが確認された。
同様にして上記培養液を40℃に一定時間保持
し、保持時間と熟成効果との関係を調べた。その
結果を第５表に示す[Table] As shown in Table 4, it was confirmed that the ripening temperature is preferably between 30 and 45°C. Similarly, the above culture solution was held at 40°C for a certain period of time, and the relationship between the holding time and the ripening effect was investigated. The results are shown in Table 5.
【表】【table】
【表】
第５表に示す結果から保持時間は１〜４時間で
充分の熟成効果が得られることがわかる。
実施例 ２
実施例１と同様の方法でシユードモナス・デス
モリチカFERM−P2816FERM BP−323を培養
し、培養６時間培養後、DL−ATCを0.1％添加
して、再に４時間通気撹拌培養を行つた。得られ
た培養液を40℃に３時間保持して熟成を行い、次
いで遠心分離して菌体を集めた。菌体を0.1Mリ
ン酸緩衝液で洗浄しこれを第３表に示す基質溶液
1.0に懸濁液を36℃に24時間保持して反応を行
つた。反応液を通気下で激しく撹拌してシステイ
ンを酸化してシスチン変換せしめ、結晶として析
出せしめた。析出した結晶を濾別し、1N−HC1
に溶解し、6N−NaOHを加えて中和し、シスチ
ンを再結せしめ、8.5ｇのシスチンの結晶を採取
した。この結晶はNMRスペクトル、Ｘ線回析
像、比旋光度のデータからＬ−シスチンと同定さ
れた。尚、旋光度純度は94.5％であつた。
実施例 ３
第１表に示す組成の培地を調製し、500ml容振
盪フラスコに50ml宛分注し、120℃にて10分間加
熱した。この培地に下記第６表に示す試験菌株を
１白金耳接種し30℃で24時間振盪培養を行つた。
培養開始時及び対数増殖期（培養開始後8.0時間）
にDL−ATCを0.1％添加し更に6.0時間培養を行
つた。対数増殖期にDL−ATCを添加して得られ
た培養液を２分し、一方については40℃に３時間
保持して熟成を行つた。夫々の培養液5.0mlに第
３表の基質溶液5.0mlを混合し、30℃に4.0時間保
持して反応を行つた。各反応液中にシステイン及
びシスチンを高速液体クロマトグラフイーで定量
した。その結果を第６表に示す。[Table] From the results shown in Table 5, it can be seen that a sufficient aging effect can be obtained with a holding time of 1 to 4 hours. Example 2 Pseudomonas desmolytica FERM-P2816FERM BP-323 was cultured in the same manner as in Example 1, and after 6 hours of incubation, 0.1% DL-ATC was added and cultured again with aeration for 4 hours. . The resulting culture solution was maintained at 40° C. for 3 hours for ripening, and then centrifuged to collect the bacterial cells. Wash the bacterial cells with 0.1M phosphate buffer and add the substrate solution shown in Table 3.
The reaction was carried out by maintaining the suspension at 36° C. for 24 hours. The reaction solution was vigorously stirred under ventilation to oxidize and convert cysteine to precipitate crystals. The precipitated crystals were filtered and 1N-HC1
6N-NaOH was added to neutralize the cystine, and cystine was re-consolidated, and 8.5 g of cystine crystals were collected. This crystal was identified as L-cystine based on the NMR spectrum, X-ray diffraction image, and specific rotation data. The optical rotation purity was 94.5%. Example 3 A culture medium having the composition shown in Table 1 was prepared, dispensed into 500 ml shaking flasks to 50 ml, and heated at 120° C. for 10 minutes. One platinum loopful of the test strains shown in Table 6 below was inoculated into this medium and cultured with shaking at 30°C for 24 hours.
At the start of culture and logarithmic growth phase (8.0 hours after start of culture)
0.1% DL-ATC was added to the cells and cultured for an additional 6.0 hours. The culture solution obtained by adding DL-ATC during the logarithmic growth phase was divided into two parts, and one part was kept at 40°C for 3 hours to ripen. 5.0 ml of the substrate solution shown in Table 3 was mixed with 5.0 ml of each culture solution, and the mixture was kept at 30° C. for 4.0 hours to carry out the reaction. Cysteine and cystine in each reaction solution were quantified by high performance liquid chromatography. The results are shown in Table 6.
Claims (1)
る含硫アミノ酸の製造方法。 (A) ２−アミノ−チアゾリン−４−カルボン酸を
水解してＬ−システイン又はＬ−シスチンを生
成する能力を有する微生物を栄養培地で培養
し、対数増殖期に２−アミノ−チアゾリン−４
−カルボン酸を添加して培養する工程。 (B) (A)工程で得られた培養液を30〜45℃に少なく
とも1.0時間以上保持して熟成する工程。 (C) 熟成培養液に含まれる微生物を２−アミノ−
チアゾリン−４−カルボン酸に作用させてＬ−
システイン又はＬ−シスチンを生成せしめる工
程。 (D) Ｌ−システイン又はＬ−シスチンを採取する
工程。[Scope of Claims] 1. A method for producing a sulfur-containing amino acid, comprising the following steps (A), (B), (C), and (D). (A) A microorganism that has the ability to hydrolyze 2-amino-thiazoline-4-carboxylic acid to produce L-cysteine or L-cystine is cultured in a nutrient medium, and during the logarithmic growth phase, 2-amino-thiazoline-4
- A step of adding carboxylic acid and culturing. (B) A step of aging the culture solution obtained in step (A) by maintaining it at 30 to 45°C for at least 1.0 hour. (C) Microorganisms contained in the aging culture solution are treated with 2-amino-
L- by acting on thiazoline-4-carboxylic acid
A step of producing cysteine or L-cystine. (D) Step of collecting L-cysteine or L-cystine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9947782A JPS58216692A (en) | 1982-06-10 | 1982-06-10 | Preparation of amino acid containing sulfur |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9947782A JPS58216692A (en) | 1982-06-10 | 1982-06-10 | Preparation of amino acid containing sulfur |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS58216692A JPS58216692A (en) | 1983-12-16 |
| JPH0346115B2 true JPH0346115B2 (en) | 1991-07-15 |
Family
ID=14248386
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9947782A Granted JPS58216692A (en) | 1982-06-10 | 1982-06-10 | Preparation of amino acid containing sulfur |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58216692A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106676157A (en) * | 2017-02-20 | 2017-05-17 | 南华大学 | Application of legionella thalli in preparing amino acid profile transformation agent |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102007007333A1 (en) | 2007-02-14 | 2008-08-21 | Wacker Chemie Ag | Process for the purification of L-cysteine |
-
1982
- 1982-06-10 JP JP9947782A patent/JPS58216692A/en active Granted
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106676157A (en) * | 2017-02-20 | 2017-05-17 | 南华大学 | Application of legionella thalli in preparing amino acid profile transformation agent |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS58216692A (en) | 1983-12-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4197754B2 (en) | Method for producing lactic acid or succinic acid | |
| EP0046284B1 (en) | Method for preparing 2,5-diketo-d-gluconic acid and microorganisms for carrying out the method | |
| JPH0342074B2 (en) | ||
| US5916782A (en) | Process for producing aspartase and process for producing L-aspartic acid | |
| JPH0346115B2 (en) | ||
| JP4198147B2 (en) | Method for producing organic acid | |
| US5288620A (en) | Process for the production of optically active 2-hydroxy-4-phenyl-3-butenoic acid | |
| JP3709007B2 (en) | Process for producing fumaric acid | |
| JPS6036446A (en) | Preparation of l-alpha-amino acid | |
| JP2518218B2 (en) | Method for producing microbial cell | |
| US3138540A (en) | Manufacture of glutamic acid by fermentation | |
| EP0295622B1 (en) | Process for producing l-tryptophan | |
| JP4269416B2 (en) | Process for producing α-halo-α, β-saturated carbonyl compound | |
| JPS58116690A (en) | Preparation of d-beta-hydroxyamino acid | |
| JPH0349555B2 (en) | ||
| JPS608119B2 (en) | Method for producing L-sulfur-containing amino acids | |
| JPS63219389A (en) | Production of di-d-fructosylfuranose 1,2':2,3' dianhydride | |
| JPS6117475B2 (en) | ||
| JPH0525476B2 (en) | ||
| JPS6128398A (en) | Preparation of l-valine | |
| JPH02257874A (en) | Bacterial strain of genus rhodococcus and production of 2-hydroxybutyric acid using the same | |
| JPH08154692A (en) | Production of d-2-aminobutyric acid | |
| JPH0353916B2 (en) | ||
| JPH0329395B2 (en) | ||
| JPS61257196A (en) | Racemization of 2-oxo-oxazolidine-4-carboxylic acid |