CN102808007A - Method for preparing S-phenyl-L-cysteine by enzymatic conversion method - Google Patents
Method for preparing S-phenyl-L-cysteine by enzymatic conversion method Download PDFInfo
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- CN102808007A CN102808007A CN2012102516241A CN201210251624A CN102808007A CN 102808007 A CN102808007 A CN 102808007A CN 2012102516241 A CN2012102516241 A CN 2012102516241A CN 201210251624 A CN201210251624 A CN 201210251624A CN 102808007 A CN102808007 A CN 102808007A
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- phenyl
- halfcystine
- serine
- cysteine
- feed liquid
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- 238000006243 chemical reaction Methods 0.000 title claims abstract description 88
- 238000000034 method Methods 0.000 title claims abstract description 28
- 230000002255 enzymatic effect Effects 0.000 title claims abstract description 8
- XYUBQWNJDIAEES-QMMMGPOBSA-N (2r)-2-amino-3-phenylsulfanylpropanoic acid Chemical compound OC(=O)[C@@H](N)CSC1=CC=CC=C1 XYUBQWNJDIAEES-QMMMGPOBSA-N 0.000 title abstract description 5
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims abstract description 70
- 239000007788 liquid Substances 0.000 claims abstract description 37
- 229960001153 serine Drugs 0.000 claims abstract description 35
- 102000004190 Enzymes Human genes 0.000 claims abstract description 25
- 108090000790 Enzymes Proteins 0.000 claims abstract description 25
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims abstract description 18
- 229940024606 amino acid Drugs 0.000 claims abstract description 18
- 150000001413 amino acids Chemical class 0.000 claims abstract description 18
- 229960004799 tryptophan Drugs 0.000 claims abstract description 17
- 238000006911 enzymatic reaction Methods 0.000 claims abstract description 15
- 238000000926 separation method Methods 0.000 claims abstract description 5
- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 claims description 36
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 27
- 235000001014 amino acid Nutrition 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 14
- 239000006035 Tryptophane Substances 0.000 claims description 14
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 6
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- 241000589614 Pseudomonas stutzeri Species 0.000 claims description 6
- 230000000968 intestinal effect Effects 0.000 claims description 6
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 5
- 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 claims description 5
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 5
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- 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 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 2
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- 239000006071 cream Substances 0.000 claims description 2
- 239000008103 glucose Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 2
- 229920002114 octoxynol-9 Polymers 0.000 claims description 2
- 235000019319 peptone Nutrition 0.000 claims description 2
- -1 polyoxyethylene octyl phenyl ether Polymers 0.000 claims description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 2
- 229920000053 polysorbate 80 Polymers 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 239000012138 yeast extract Substances 0.000 claims description 2
- 239000004201 L-cysteine Substances 0.000 claims 4
- 238000002425 crystallisation Methods 0.000 abstract description 13
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- RZWQDAUIUBVCDD-UHFFFAOYSA-M sodium;benzenethiolate Chemical compound [Na+].[S-]C1=CC=CC=C1 RZWQDAUIUBVCDD-UHFFFAOYSA-M 0.000 abstract 1
- 239000012530 fluid Substances 0.000 description 49
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 36
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 26
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 24
- 238000003828 vacuum filtration Methods 0.000 description 24
- 238000005406 washing Methods 0.000 description 24
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 14
- 238000000967 suction filtration Methods 0.000 description 14
- 238000001816 cooling Methods 0.000 description 13
- 150000008550 L-serines Chemical class 0.000 description 12
- 230000008021 deposition Effects 0.000 description 12
- 238000010438 heat treatment Methods 0.000 description 12
- 210000001082 somatic cell Anatomy 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 239000012954 diazonium Substances 0.000 description 6
- 150000001989 diazonium salts Chemical class 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 238000005903 acid hydrolysis reaction Methods 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 238000006555 catalytic reaction Methods 0.000 description 4
- ZPIRTVJRHUMMOI-UHFFFAOYSA-N octoxybenzene Chemical compound CCCCCCCCOC1=CC=CC=C1 ZPIRTVJRHUMMOI-UHFFFAOYSA-N 0.000 description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 4
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 4
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 229910052802 copper Inorganic materials 0.000 description 3
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- 238000009776 industrial production Methods 0.000 description 3
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- VZXPDPZARILFQX-BYPYZUCNSA-N O-acetyl-L-serine Chemical compound CC(=O)OC[C@H]([NH3+])C([O-])=O VZXPDPZARILFQX-BYPYZUCNSA-N 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 2
- 229940045803 cuprous chloride Drugs 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
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- 235000010265 sodium sulphite Nutrition 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- QAGYKUNXZHXKMR-UHFFFAOYSA-N CPD000469186 Natural products CC1=C(O)C=CC=C1C(=O)NC(C(O)CN1C(CC2CCCCC2C1)C(=O)NC(C)(C)C)CSC1=CC=CC=C1 QAGYKUNXZHXKMR-UHFFFAOYSA-N 0.000 description 1
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- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 description 1
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- FQXBMKZVLSACGS-UHFFFAOYSA-N n,n-dimethylmethanamine;hexadecane;hydrobromide Chemical compound Br.CN(C)C.CCCCCCCCCCCCCCCC FQXBMKZVLSACGS-UHFFFAOYSA-N 0.000 description 1
- QAGYKUNXZHXKMR-HKWSIXNMSA-N nelfinavir Chemical compound CC1=C(O)C=CC=C1C(=O)N[C@H]([C@H](O)CN1[C@@H](C[C@@H]2CCCC[C@@H]2C1)C(=O)NC(C)(C)C)CSC1=CC=CC=C1 QAGYKUNXZHXKMR-HKWSIXNMSA-N 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biology, and in particular relates to a method for preparing S-phenyl-L-cysteine by an enzymatic conversion method. The method comprises the following steps of: mixing wet thalli or crude enzyme liquid with L-tryptophan synthase activity and L-serine-containing mixed amino acid feed liquid serving as a raw material, adding proper amount of phenthiol or sodium thiophenolate, performing enzymatic reaction at the temperature of between 25 and 55 DEG C under the condition that the pH value is 6 to 11, and separating by an isoelectric point crystallization process to obtain the S-phenyl-L-cysteine serving as a conversion product. By the method, the problems of the separation and comprehensive utilization of L-serine in the mixed amino acid feed liquid are solved, and the high-added value S-phenyl-L-cysteine is obtained, so the method has the advantages of wide source of the raw material, low cost, simplicity of operation, short enzymatic conversion time, low production cost and the like.
Description
One, technical field
The invention belongs to biological technical field, be specifically related to the enzymatic conversion preparation method of S-phenyl-L-halfcystine.
Two, background technology
S-phenyl-L-halfcystine is the amino acid that a kind of nonprotein is formed; Its with sulfydryl have the important physical function, S-phenyl-L-halfcystine be the treatment AIDS-treating medicine naphthalene Fei Nawei (anti-hiv protease suppressor factor Nelfinavir) synthesis material (US5484926; EP0889036).S-phenyl-L-halfcystine can be used for cosmetics additive, field of medicaments, field of food and feed additive field etc., has very widely to use.
Preparing method according to present reported in literature S-phenyl-L-halfcystine mainly contains chemical synthesis and enzyme transforming process.
1, chemical synthesis
The chemical synthesis of S-phenyl-L-halfcystine is mainly utilized the reaction of copper catalysis L-halfcystine and diazonium salt of aniline; Obtain S-phenyl-L-halfcystine at last; The reactions step of S-phenyl-L-halfcystine chemosynthesis is many, and operation easier is big, is not suitable for large-scale industrial production.
Wang Jianwu etc. (Chinese pharmaceutical chemistry magazine, 2000, (10) 4:283-284) adopt copper catalysis L-halfcystine and the synthetic S-phenyl of diazonium salt of aniline reaction-L-halfcystine, and aniline is dissolved in the concentrated hydrochloric acid; Drip Sodium Nitrite and obtain diazonium salt, add cysteine hydrochloride and cuprous chloride, the reaction back adds the sodium sulphite suction filtration in stink cupboard; Transfer pH6 with ammoniacal liquor, white crystal is separated out, and filters; Oven dry, productive rate is 60%, purity is 95%.
(CN 200410089193.9,2005) such as high beautiful hats become diazonium salt of aniline with Sodium Nitrite with aniline reaction, diazonium salt of aniline is reacted with L-halfcystine solution again; Add cuprous chloride; Carry out the decopper(ing) reaction with sodium sulphite then, suction filtration is transferred pH6; Filtering drying obtains bullion S-phenyl-L-halfcystine, productive rate 50%.
(CN 03128884.7 for Zhang Guanyong etc.; 2003) with the complex compound of L-cysteine hydrochloride and solubility cuprous salt reaction generation copper, generate S-phenyl-L-halfcystine with the diazonium salt of aniline reaction then, add dissolvable sulfide solution and carry out the decopper(ing) reaction; Cooling; Filter, add ammoniacal liquor in the filtrating and neutralize, adopt ethanol and water mixed liquid bullion S-phenyl-L-halfcystine recrystallization.
2, enzyme transforming process
Specificity is strong, reaction conditions is gentle, advantages of environment protection comes into one's own day by day because of having for enzymatic conversion method; The Enzymatic transformation method has advantages such as the end product accumulation volume is high, reaction time is short, the separation purification is easy; It is the cheap S-of production phenyl-L-halfcystine effective means; The investigator utilizes the tryptophane synth(et)ase enzyme process to prepare S-phenyl-L-halfcystine both at home and abroad, and Production by Enzymes S-phenyl-L-halfcystine has become the direction of people's concern in recent years and research.
Thomas HP Maier (Nature Biotechnology, 21 (4): 422-427,2003) is a substrate with thiophenol and O-acetyl-L-Serine, and pH transfers to 6.7, and CysM enzyme enzyme process prepares S-phenyl-L-halfcystine, and productive rate is 72%.
European patent (EP1247869-A, 2002) has reported that employing contains O-acetyl-L-Serine, thiophenol and product CysM bacterial strain DH5 α/pFL145 cell suspension and prepares S-phenyl-L-halfcystine, and pH transfers to 6.7, and yield is 65%.
It is substrate with L-Serine and the indoles that kilnitamin contains that burnt celebrating just waits (Bioresource Technology, 102:3554-3557,2011), has synthesized the L-tryptophane with the tryptophane synth(et)ase enzyme process.But not only can catalysis L-Serine and the synthetic L-tryptophane of indoles according to the bibliographical information tryptophane synth(et)ase, can also react generation S-phenyl-L-halfcystine with thiophenol by catalysis pure article L-Serine.
Japanese Patent (JP10001468A, 1996; JP9313195-A, 1997; JP9084592-A; 1997) and USP (US5756319-A; 1998) reported that with DL-serine or L-Serine be substrate; With the thiophenol is the sulfydryl donor, and the tryptophane synth(et)ase enzyme process synthesizes S-phenyl-L-halfcystine, and the report that above tryptophane synth(et)ase enzyme process synthesizes S-phenyl-L-halfcystine is a substrate with pure article of DL-serine or the pure article of L-Serine all.
At present, China S-phenyl-L-halfcystine production is mainly derived from chemical synthesis, this method technical maturity, but the chemical synthesis reactions step is many, and operation easier is big, and production cost is high, is not suitable for large-scale industrial production.And China can produce the kilnitamin feed liquid that is rich in the L-Serine that can not directly be used for the food and medicine aspect in a large number in the amino acid industrial production on the other hand, and this kilnitamin feed liquid direct separation prepares L-Serine cost height, efficient is low.
Three, summary of the invention
The problem that the present invention need solve provides a kind of method for preparing S-phenyl-L-halfcystine efficiently, cheaply.The present invention is a substrate with the kilnitamin feed liquid that is rich in the L-Serine in the amino acid industry, and the tryptophane synth(et)ase enzyme process synthesizes S-phenyl-L-halfcystine.
The present invention can realize through following technical scheme:
The enzymatic conversion preparation method of S-phenyl-L-halfcystine the steps include:
(1) has active intestinal bacteria ATCC15489 of tryptophane synth(et)ase or Pseudomonas aeruginosa CGMCC NO:1.1129 or Pseudomonas stutzeri CGMCC NO:1.202 or subtilis CGMCC NO:1.1628; In substratum, cultivate, produce the wet thallus that contains tryptophane synth(et)ase.
The wet thallus or the crude enzyme liquid that (2) will contain tryptophane synth(et)ase mix with the kilnitamin feed liquid that contains the L-Serine, add Vitazechs and tensio-active agent and thiophenol or thiophenol sodium again, at 25～55 ℃, carry out enzymatic reaction under pH 6～11 conditions; Reaction generates S-phenyl-L-halfcystine.
Culture medium carbon source in the above-mentioned steps (1) adopts glucose and SANMALT-S and sucrose and/or fructose, and total carbon source quality concentration is 5～50 g/L in the substratum; Nitrogenous source adopts Carnis Bovis seu Bubali cream and yeast extract paste and steeping water and peptone and/or soya-bean cake hydrolyzed solution, and total nitrogen source quality concentration is 1～30 g/L in the substratum.
The kilnitamin feed liquid that contains the L-Serine in the above-mentioned steps (2) is the reaction solution of Keratin sulfate hydrolyzed solution or silk hydrolyzed solution or the synthetic L-Serine of enzyme process; Total aminoacid content is 1%～50% weight percent in the kilnitamin feed liquid, and wherein L-Serine content in total aminoacid is 5%～95% weight percent.
Add an amount of tensio-active agent tween-80 or hexadecane trimethyl ammonium bromide (CTAB) or polyoxyethylene octyl phenyl ether (OP) in the conversion fluid of above-mentioned steps (2), its concentration is 0.005 g/L～5.0 g/L.
The present invention compared with prior art has following advantage:
(1) the present invention adopts intestinal bacteria ATCC15489 or Pseudomonas aeruginosa CGMCC NO:1.1129 or Pseudomonas stutzeri CGMCC NO:1.202 or the subtilis CGMCC NO:1.1628 that contains tryptophane synth(et)ase; In preferred substratum, cultivate and to efficiently express tryptophane synth(et)ase; Making enzyme process synthesize S-phenyl-L-halfcystine has higher catalytic rate and transformation efficiency, and wherein L-Serine molar yield reaches more than 84%.
(2) to adopt the cheap kilnitamin feed liquid that contains the L-Serine be reaction raw materials in the present invention; Made full use of industrial by-products; Solved the difficult problem of L-Serine high efficiency separation in the kilnitamin feed liquid; Also producing S-phenyl-L-halfcystine for large-scale low-cost simultaneously provides the more raw material of horn of plenty, has had good economic benefit and social benefit.
Advantages such as (3) the synthetic S-phenyl of enzyme process-L-halfcystine has the reaction conditions gentleness, and the enzyme stereoselectivity is strong, and catalytic efficiency (is high, and cost is low, and technical process is simple are fit to suitability for industrialized production.
Four, embodiment
Following examples only are used for the present invention is specified, but protection scope of the present invention is not limited in following examples.
Embodiment one
1. with the centrifugal 20 g wet thallus that obtain of 1000 mL intestinal bacteria (ATCC15489) fermented liquids; Join in the 500 mL conversion fluids; The hair acid hydrolysis liquid (amino acid total content 15%), 19 g thiophenols, 0.2 g/L Vitazechs and the 0.005 g/L OP that contain 17.5 g L-Serines in the conversion fluid, 8.5,37 ℃ of enzymatic reaction 15 h of pH; S-phenyl-L-halfcystine is 29.2 g in the conversion fluid of reaction end back, is 89% to L-Serine molar yield.
2. with centrifugal 15 min of conversion fluid 4000 r/min, remove somatic cells, supernatant is transferred pH 0.5 with 6 mol/L hydrochloric acid, heating, decolorizing with activated carbon; Suction filtration, filtrating is transferred pH 3.0 with 5 mol/L NaOH, and cooling is separated out deposition, vacuum filtration; Pure water washing, dry 27.7 g S-phenyl-L-halfcystine bullion, through sour solution-off look, in and crystallization; Vacuum filtration, the washing, dry 26.4 g S-phenyl-L-halfcystine elaboration, specific rotation=+ 74
o(
c=1,1.5 mol/L sulfuric acid).
Embodiment two
1. with the centrifugal 20 g wet thallus that obtain of 1000 mL Pseudomonas aeruginosas (CGMCC NO:1.1129) fermented liquid; Join in the 500 mL conversion fluids; The silk hydrolyzed solution (amino acid total content 30%), 42 g thiophenols, 0.2 g/L Vitazechs and the 5 g/L tween-80s that contain 36 g L-Serines in the conversion fluid, 9.0,25 ℃ of enzymatic reaction 20 h of pH; S-phenyl-L-halfcystine is 58.2 g in the conversion fluid of reaction end back, is 86 % to L-Serine molar yield.
2. with centrifugal 15 min of conversion fluid 4000 r/min, remove somatic cells, supernatant is transferred pH 0.5 with 6 mol/L hydrochloric acid, heating, decolorizing with activated carbon; Suction filtration, filtrating is transferred pH 3.0 with 5 mol/L NaOH, and cooling is separated out deposition, vacuum filtration; Pure water washing, dry 55.3g S-phenyl-L-halfcystine bullion, through sour solution-off look, in and crystallization; Vacuum filtration, the washing, dry 51.9 g S-phenyl-L-halfcystine elaboration, specific rotation=+ 74
o(
c=1,1.5 mol/L sulfuric acid).
Embodiment three
1. with centrifugal wet thallus 18 g that obtain of 1000 mL Pseudomonas stutzeris (CGMCC NO:1.202) fermented liquid; Join in the 500 mL conversion fluids; The enzyme process that contains 12 g L-Serines in the conversion fluid prepares L-Serine feed liquid, 14 g thiophenols, 0.2 g/L Vitazechs and 0.005 g/L tween-80,6.0,55 ℃ of enzymatic reaction 15 h of pH; S-phenyl-L-halfcystine is 19.4 g in the conversion fluid of reaction end back, is 86 % to L-Serine molar yield.
2. with centrifugal 15 min of conversion fluid 4000 r/min, remove somatic cells, supernatant is transferred pH 0.5 with 6 mol/L hydrochloric acid, heating, decolorizing with activated carbon; Suction filtration, filtrating is transferred pH 3.0 with 5 mol/L NaOH, and cooling is separated out deposition, vacuum filtration; Pure water washing, dry 17.7 g S-phenyl-L-halfcystine bullion, through sour solution-off look, in and crystallization; Vacuum filtration, the washing, dry 16.1 g S-phenyl-L-halfcystine elaboration, specific rotation=+ 73
o(
c=1,1.5 mol/L sulfuric acid).
Embodiment four
1. with the centrifugal 20 g wet thallus that obtain of 1000 mL subtilises (CGMCC NO:1.1628) fermented liquid; Join in the 500 mL conversion fluids; The enzyme process that contains 48 g L-Serines in the conversion fluid prepares L-Serine feed liquid, 56 g thiophenols, 0.2 g/L Vitazechs and 0.5 g/L tween-80,9.0,35 ℃ of enzymatic reaction 20 h of pH; S-phenyl-L-halfcystine is 79.4 g in the conversion fluid of reaction end back, is 88 % to L-Serine molar yield.
2. with centrifugal 15 min of conversion fluid 4000 r/min, remove somatic cells, supernatant is transferred pH 0.5 with 6 mol/L hydrochloric acid, heating, decolorizing with activated carbon; Suction filtration, filtrating is transferred pH 3.0 with 5 mol/L NaOH, and cooling is separated out deposition, vacuum filtration; Pure water washing, dry 75.4 g S-phenyl-L-halfcystine bullion, through sour solution-off look, in and crystallization; Vacuum filtration, the washing, dry 71.6 g S-phenyl-L-halfcystine elaboration, specific rotation=+ 73
o(
c=1,1.5 mol/L sulfuric acid).
Embodiment five
1. with the centrifugal 20 g wet thallus that obtain of 1000 mL Pseudomonas stutzeris (CGMCC NO:1.202) fermented liquid; Join in the 500 mL conversion fluids; The hair acid hydrolysis liquid (amino acid total content 50%), 70 g thiophenols, 0.2 g/L Vitazechs and the 5 g/L CTAB that contain 60 g L-Serines in the conversion fluid, 9,35 ℃ of enzymatic reaction 20 h of pH; S-phenyl-L-halfcystine is 94.6 g in the conversion fluid of reaction end back, is 84 % to L-Serine molar yield.
2. with centrifugal 15 min of conversion fluid 4000 r/min, remove somatic cells, supernatant is transferred pH 0.5 with 6 mol/L hydrochloric acid, heating, decolorizing with activated carbon; Suction filtration, filtrating is transferred pH 3.0 with 5 mol/L NaOH, and cooling is separated out deposition, vacuum filtration; Pure water washing, dry 89.8 g S-phenyl-L-halfcystine bullion, through sour solution-off look, in and crystallization; Vacuum filtration, the washing, dry 85.4 g S-phenyl-L-halfcystine elaboration, specific rotation=+ 73
o(
c=1,1.5 mol/L sulfuric acid).
Embodiment six
1. with the centrifugal 18 g wet thallus that obtain of 1000 mL Pseudomonas stutzeris (CGMCC NO:1.202) fermented liquid; Join in the 500 mL conversion fluids; The hair acid hydrolysis liquid (amino acid total content 20%), 28 g thiophenols, 0.2 g/L Vitazechs and the 5 g/L OP that contain 24 g L-Serines in the conversion fluid, 11,35 ℃ of enzymatic reaction 16 h of pH; S-phenyl-L-halfcystine is 40.2 g in the conversion fluid of reaction end back, is 89 % to L-Serine molar yield.
2. with centrifugal 15 min of conversion fluid 4000 r/min, remove somatic cells, supernatant is transferred pH 0.5 with 6 mol/L hydrochloric acid, heating, decolorizing with activated carbon; Suction filtration, filtrating is transferred pH 3.0 with 5 mol/L NaOH, and cooling is separated out deposition, vacuum filtration; Pure water washing, dry 38.2 g S-phenyl-L-halfcystine bullion, through sour solution-off look, in and crystallization; Vacuum filtration, the washing, dry 36.3 g S-phenyl-L-halfcystine elaboration, specific rotation=+ 74
o(
c=1,1.5 mol/L sulfuric acid).
Embodiment seven
1. with the centrifugal 18 g wet thallus that obtain of 1000 mL intestinal bacteria (ATCC15489) fermented liquids; Join in the 500 mL conversion fluids; The silk hydrolyzed solution (amino acid total content 15%), 21 g thiophenols, 0.2 g/L Vitazechs and the 0.5 g/L OP that contain 18 g L-Serines in the conversion fluid, 9.0,35 ℃ of enzymatic reaction 15 h of pH; S-phenyl-L-halfcystine is 29 g in the conversion fluid of reaction end back, is 86 % to L-Serine molar yield.
2. with centrifugal 15 min of conversion fluid 4000 r/min, remove somatic cells, supernatant is transferred pH 0.5 with 6 mol/L hydrochloric acid, heating, decolorizing with activated carbon; Suction filtration, filtrating is transferred pH 3.0 with 5 mol/L NaOH, and cooling is separated out deposition, vacuum filtration; Pure water washing, dry 27.5 g S-phenyl-L-halfcystine bullion, through sour solution-off look, in and crystallization; Vacuum filtration, the washing, dry 26.4 g S-phenyl-L-halfcystine elaboration, specific rotation=+ 73
o(
c=1,1.5 mol/L sulfuric acid).
Embodiment eight
1. with the centrifugal 18 g wet thallus that obtain of 1000 mL Pseudomonas aeruginosas (CGMCC NO:1.1129) fermented liquid; Join in the 500 mL conversion fluids; The hair acid hydrolysis liquid (amino acid total content 10%), 16.8 g thiophenol sodium, 0.2 g/L Vitazechs and the 0.5 g/L CTAB that contain 12 g L-Serines in the conversion fluid, 9.0,35 ℃ of enzymatic reaction 15 h of pH; S-phenyl-L-halfcystine is 19.3 g in the conversion fluid of reaction end back, is 86 % to L-Serine molar yield.
2. with centrifugal 15 min of conversion fluid 4000 r/min, remove somatic cells, supernatant is transferred pH 0.5 with 6 mol/L hydrochloric acid, heating, decolorizing with activated carbon; Suction filtration, filtrating is transferred pH 3.0 with 5 mol/L NaOH, and cooling is separated out deposition, vacuum filtration; Pure water washing, dry 17.3 g S-phenyl-L-halfcystine bullion, through sour solution-off look, in and crystallization; Vacuum filtration, the washing, dry 16.5 g S-phenyl-L-halfcystine elaboration, specific rotation=+ 74
o(
c=1,1.5 mol/L sulfuric acid).
Embodiment nine
1. with the centrifugal 20 g wet thallus that obtain of 1000 mL subtilises (CGMCC NO:1.1628) fermented liquid; Join in the 500 mL conversion fluids; The silk hydrolyzed solution (amino acid total content 20 %), 33.6 g thiophenol sodium, 0.2 g/L Vitazechs and the 5 g/L tween-80s that contain 24 g L-Serines in the conversion fluid, 9.0,35 ℃ of enzymatic reaction 18 h of pH; S-phenyl-L-halfcystine is 38.2 g in the conversion fluid of reaction end back, is 85 % to L-Serine molar yield.
2. with centrifugal 15 min of conversion fluid 4000 r/min, remove somatic cells, supernatant is transferred pH 0.5 with 6 mol/L hydrochloric acid, heating, decolorizing with activated carbon; Suction filtration, filtrating is transferred pH 3.0 with 5 mol/L NaOH, and cooling is separated out deposition, vacuum filtration; Pure water washing, dry 36.2 g S-phenyl-L-halfcystine bullion, through sour solution-off look, in and crystallization; Vacuum filtration, the washing, dry 34.4 g S-phenyl-L-halfcystine elaboration, specific rotation=+ 73
o(
c=1,1.5 mol/L sulfuric acid).
Embodiment ten
1. with the centrifugal 18 g wet thallus that obtain of 1000 mL intestinal bacteria (ATCC15489) fermented liquids; Join in the 500 mL conversion fluids; The hair acid hydrolysis liquid (amino acid total content 30%), 50.4 g thiophenol sodium, 0.2 g/L Vitazechs and the 5 g/L tween-80s that contain 36 g L-Serines in the conversion fluid, 9.0,35 ℃ of enzymatic reaction 20 h of pH; S-phenyl-L-halfcystine is 57.1 g in the conversion fluid of reaction end back, is 85 % to L-Serine molar yield.
2. with centrifugal 15 min of conversion fluid 4000 r/min, remove somatic cells, supernatant is transferred pH 0.5 with 6 mol/L hydrochloric acid, heating, decolorizing with activated carbon; Suction filtration, filtrating is transferred pH 3.0 with 5 mol/L NaOH, and cooling is separated out deposition, vacuum filtration; Pure water washing, dry 54.2 g S-phenyl-L-halfcystine bullion, through sour solution-off look, in and crystallization; Vacuum filtration, the washing, dry 51.5 g S-phenyl-L-halfcystine elaboration, specific rotation=+ 73
o(
c=1,1.5 mol/L sulfuric acid).
Embodiment 11
1. with the centrifugal 2 g wet thallus that obtain of 100 mL subtilises (CGMCC NO:1.1628) fermented liquid; Join in the 50 mL conversion fluids; The silk hydrolyzed solution (amino acid total content 1%), 1.4g thiophenol, 0.2 g/L Vitazechs and the 0.005 g/L CTAB that contain 1.2 g L-Serines in the conversion fluid, 9.0,35 ℃ of enzymatic reaction 5 h of pH; S-phenyl-L-halfcystine is 1.94 g in the conversion fluid of reaction end back, is 86 % to L-Serine molar yield.
2. with centrifugal 15 min of conversion fluid 4000 r/min, remove somatic cells, supernatant is transferred pH 0.5 with 6 mol/L hydrochloric acid, heating, decolorizing with activated carbon; Suction filtration, filtrating is transferred pH 3.0 with 5 mol/L NaOH, and cooling is separated out deposition, vacuum filtration; Pure water washing, dry 1.82 g S-phenyl-L-halfcystine bullion, through sour solution-off look, in and crystallization; Vacuum filtration, the washing, dry 1.63 g S-phenyl-L-halfcystine elaboration, specific rotation=+ 73
o(
c=1,1.5 mol/L sulfuric acid).
Embodiment 12
1. with the centrifugal 20 g wet thallus that obtain of 1000 mL Pseudomonas aeruginosas (CGMCC NO:1.1129) fermented liquid; Join in the 500 mL conversion fluids; The enzyme process that contains 24 g L-Serines in the conversion fluid prepares L-Serine feed liquid, 28 g thiophenols, 0.2 g/L Vitazechs and 5 g/L tween-80s, 9.0,35 ℃ of enzymatic reaction 18 h of pH; S-phenyl-L-halfcystine is 38.7 g in the conversion fluid of reaction end back, is 86 % to L-Serine molar yield.
2. with centrifugal 15 min of conversion fluid 4000 r/min, remove somatic cells, supernatant is transferred pH 0.5 with 6 mol/L hydrochloric acid, heating, decolorizing with activated carbon; Suction filtration, filtrating is transferred pH 3.0 with 5 mol/L NaOH, and cooling is separated out deposition, vacuum filtration; Pure water washing, dry 36.7 g S-phenyl-L-halfcystine bullion, through sour solution-off look, in and crystallization; Vacuum filtration, the washing, dry 34.9 g S-phenyl-L-halfcystine elaboration, specific rotation=+ 73
o(
c=1,1.5 mol/L sulfuric acid).
Claims (5)
1. the enzymatic conversion preparation method of S-phenyl-L-halfcystine is characterized in that being made up of following steps:
(1) has the active bacterial strain of tryptophane synth(et)ase and in substratum, cultivate, produce the wet thallus that contains tryptophane synth(et)ase;
The wet thallus or the crude enzyme liquid that (2) will contain tryptophane synth(et)ase mix with the kilnitamin feed liquid that contains the L-Serine; Add Vitazechs and tensio-active agent and thiophenol or thiophenol sodium again; At 25～55 ℃; Carry out enzymatic reaction under pH 6～11 conditions, the separation of isoelectric point crystallizing method obtains S-phenyl-L-halfcystine.
2. S-phenyl according to claim 1-L-cysteine by using enzyme conversion preparation method is characterized in that the described bacterial strain of step (1) is intestinal bacteria ATCC15489 or Pseudomonas aeruginosa CGMCC NO:1.1129 or Pseudomonas stutzeri CGMCC NO:1.202 or subtilis CGMCC NO:1.1628.
3. S-phenyl according to claim 1-L-cysteine by using enzyme conversion preparation method is characterized in that the described substratum of step (1) is glucose and SANMALT-S and sucrose and/or fructose, and total carbon source quality concentration is 5～50 g/L in the substratum; Nitrogenous source adopts Carnis Bovis seu Bubali cream and yeast extract paste and steeping water and peptone and/or soya-bean cake hydrolyzed solution, and total nitrogen source quality concentration is 1～30 g/L in the substratum.
4. S-phenyl according to claim 1-L-cysteine by using enzyme conversion preparation method; It is characterized in that the kilnitamin feed liquid of the described L-of the containing Serine of step (2) derives from the reaction solution of Keratin sulfate hydrolyzed solution or silk hydrolyzed solution or the synthetic L-Serine of enzyme process; Total aminoacid content is 1%～50% weight percent in the kilnitamin feed liquid, and wherein L-Serine content in total aminoacid is 5%～95% weight percent.
5. S-phenyl according to claim 1-L-cysteine by using enzyme conversion preparation method; It is characterized in that the described tensio-active agent of step (2) is tween-80 or cetyl trimethylammonium bromide or polyoxyethylene octyl phenyl ether, its concentration is 0.005 g/L～5.0 g/L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103194501A (en) * | 2013-03-29 | 2013-07-10 | 凯莱英医药集团（天津）股份有限公司 | Method for synthetizing chiral cyclic alkyl amino acid by amino transferase |
| CN104017838A (en) * | 2014-06-18 | 2014-09-03 | 宿州学院 | Enzymatic conversion preparation method of 2-methyl-L-tryptophan |
| CN108913727A (en) * | 2018-06-20 | 2018-11-30 | 安徽瑞然生物药肥科技有限公司 | A kind of culture medium |
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| US5756319A (en) * | 1995-07-18 | 1998-05-26 | Mitsui Toatsu Chemicals, Inc. | Production process of S-phenyl-L-cysteine |
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| US5756319A (en) * | 1995-07-18 | 1998-05-26 | Mitsui Toatsu Chemicals, Inc. | Production process of S-phenyl-L-cysteine |
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| GENHAI ZHAO等: "Enzymatic synthesis of L-tryptophan fromhair acid hydrolysis industries wastewater with tryptophan synthase", 《BIORESOURCE TECHNOLOGY》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103194501A (en) * | 2013-03-29 | 2013-07-10 | 凯莱英医药集团（天津）股份有限公司 | Method for synthetizing chiral cyclic alkyl amino acid by amino transferase |
| CN103194501B (en) * | 2013-03-29 | 2015-05-27 | 凯莱英医药集团（天津）股份有限公司 | Method for synthetizing chiral cyclic alkyl amino acid by amino transferase |
| CN104017838A (en) * | 2014-06-18 | 2014-09-03 | 宿州学院 | Enzymatic conversion preparation method of 2-methyl-L-tryptophan |
| CN104017838B (en) * | 2014-06-18 | 2016-08-31 | 宿州学院 | A kind of enzymatic conversion preparation method of 2-methyl-L-tryptophan |
| CN108913727A (en) * | 2018-06-20 | 2018-11-30 | 安徽瑞然生物药肥科技有限公司 | A kind of culture medium |
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