CN108949844A - A kind of enzymatic conversion preparation method of S- methyl-Lcysteine - Google Patents
A kind of enzymatic conversion preparation method of S- methyl-Lcysteine Download PDFInfo
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- CN108949844A CN108949844A CN201810634882.5A CN201810634882A CN108949844A CN 108949844 A CN108949844 A CN 108949844A CN 201810634882 A CN201810634882 A CN 201810634882A CN 108949844 A CN108949844 A CN 108949844A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
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- C12P13/12—Methionine; Cysteine; Cystine
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Abstract
The invention discloses a kind of enzymatic conversion preparation methods of S- methyl-Lcysteine, will have the active bacterial strain of tryptophan synthetase cultivation and fermentation in the medium, and obtain the wet thallus containing tryptophan synthetase after fermentation liquid centrifugation；Conversion fluid will be added in above-mentioned wet thallus, include: one of the feed liquid containing Serine, phosphopyridoxal pyridoxal phosphate and methyl mercaptan, methanol, ethyl alcohol, ether, acetone in the conversion liquid；Enzymatic reaction, the isolated S- methyl-Lcysteine of isoelectric point crystallizing method are carried out under the conditions of 25~55 DEG C, pH 6~11.The present invention uses the specific bacterial strain containing tryptophan synthetase, culture can be with high efficient expression tryptophan synthetase in preferred culture medium, enzymatic clarification S- methyl-Lcysteine is set to have higher catalytic rate and conversion ratio, Serine molar yield reaches 95% or more, enzymatic clarification reaction condition is mild, enzyme stereoselectivity is strong, high catalytic efficiency.
Description
Technical field
The present invention relates to a kind of method for transformation of S- methyl-Lcysteine more particularly to a kind of S- methyl-
The enzymatic conversion preparation method of L-cysteine.
Background technique
S- methyl-Lcysteine is a kind of amino acid of nonprotein composition, and S- methyl-Lcysteine is a kind of medicine
Object intermediate can be used as the oxidation that antioxidant inhibits protein and peptide, also have a certain effect in biological components analysis.
" chemistry world " is disclosed using L-cysteine as raw material in 2003,7:370-372, and trimethyl phosphate is methyl
Change the method that reagent has synthesized S- methyl-Lcysteine.One kind is disclosed in United States Patent (USP) US6147257,2000. with diformazan
The method that base carbonic ester makees methylating reagent prepares S- methyl-Lcysteine complex steps, and the reaction time is long.
Currently, the production of S- methyl-Lcysteine is mainly derived from chemical synthesis, reactions steps of this method is more, operation
Difficulty is big, high production cost, is not suitable for large-scale industrial production.And another aspect China amino acids industry production in, meeting
Generate the hair acid hydrolysis liquid rich in Serine in terms of largely cannot be directly used to food and medicine, the kilnitamin material
Liquid, which is directly separated, prepares that Serine is at high cost, low efficiency.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of enzyme process of S- methyl-Lcysteine
Conversion preparation method improves the catalytic rate and conversion ratio of reaction.
The present invention is achieved by the following technical solutions, the present invention the following steps are included:
(1) there will be the active bacterial strain of tryptophan synthetase cultivation and fermentation in the medium, be contained after fermentation liquid centrifugation
The wet thallus of tryptophan synthetase；
(2) conversion fluid will be added in above-mentioned wet thallus, include: the feed liquid containing Serine, phosphoric acid pyrrole in the conversion liquid
It trembles one of aldehyde and methyl mercaptan, ether, methanol, ethyl alcohol, acetone；It is anti-that enzymatic is carried out under the conditions of 25~55 DEG C, pH 6~11
It answers, the isolated S- methyl-Lcysteine of isoelectric point crystallizing method.
In the step (1), there is the active bacterial strain of tryptophan synthetase to be selected from Escherichia coli ATCC15489, verdigris false
Monad CGMCC NO:1.1129, Pseudomonas stutzeri CGMCC NO:1.202, bacillus subtilis CGMCC NO:1.1628
One of.
In the step (1), the carbon source in culture medium uses one of glucose, maltose, sucrose, fructose or more
Kind, total carbon source mass concentration is 10~40g/L in culture medium；Nitrogen source in culture medium using beef extract, yeast extract, corn pulp,
One of peptone, soya-bean cake hydrolyzate are a variety of, and total nitrogen source mass concentration is 5~35g/L in culture medium.
In the step (2), the concentration of Serine is 0.5~40.5g/L in the feed liquid containing Serine.
The feedstream origins containing Serine are in hair acid hydrolysis liquid or pure Serine liquid；It is total in hair acid hydrolysis liquid
The weight percent of amino acid be 5%~45%, wherein weight percent of the Serine in total amino acid be 10%~
90%.
In the step (2), the ether, methanol, ethyl alcohol or acetone, concentration are 0.001g/L~4.0g/L.
In the step (2), the concentration of phosphopyridoxal pyridoxal phosphate is 0.2~0.8g/L, the concentration of methyl mercaptan is 0.22~
18.5g/L。
In the step (2), the specific method is as follows for isoelectric point crystallizing method:
3000~5000r/min of conversion fluid after enzymatic reaction is centrifuged 10~20min, removes somatic cells；Heating turns
Change liquid, using decolorizing with activated carbon, filter, filtrate is adjusted to pH 6~8, stands and precipitating is precipitated, and vacuum filtration dries to obtain S- methyl-
L-cysteine crude product, using ethanol washing, vacuum filtration dries to obtain S- methyl-Lcysteine fine work.
The present invention has the advantage that the present invention uses the specific bacterial strain containing tryptophan synthetase compared with prior art,
Culture can be such that enzymatic clarification S- methyl-Lcysteine has higher in preferred culture medium with high efficient expression tryptophan synthetase
Catalytic rate and conversion ratio, wherein Serine molar yield reaches 95% or more；
The present invention uses the hair acid hydrolysis liquid containing Serine for reaction raw materials, cheap, and takes full advantage of work
Industry by-product solves the problems, such as that Serine efficiently separates in kilnitamin feed liquid, while also raw for large-scale low-cost
It produces S- methyl-Lcysteine and provides more abundant raw material, with good economic efficiency and social benefit；
Enzymatic clarification S- methyl-Lcysteine has reaction condition mild, and enzyme stereoselectivity is strong, high catalytic efficiency,
At low cost, the advantages that process flow is simple, is suitble to industrialized production.
Specific embodiment
It elaborates below to the embodiment of the present invention, the present embodiment carries out under the premise of the technical scheme of the present invention
Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation
Example.
Embodiment 1
The present embodiment the following steps are included:
(1) by 1000mL bacillus subtilis CGMCC NO:1.1628, cultivation and fermentation, fermentation liquid are centrifuged in the medium
It to 20g wet thallus, is added in 500mL conversion fluid, methyl mercaptan, the 0.2g/L of Serine, 4.81g in conversion fluid containing 11g
Phosphopyridoxal pyridoxal phosphate and 0.5g/L ethyl alcohol, pH8.0,37 DEG C of enzymatic reaction 20h, after reaction half Guang of S- methyl-L- in conversion fluid
Propylhomoserin is 13.3g, is 99% to Serine molar yield；
(2) conversion fluid 4000r/min is centrifuged 15min, removes somatic cells, heating, decolorizing with activated carbon filters, filtrate
Adjust pH 7 or so, stand be precipitated precipitating, vacuum filtration, dry 11.5g S- methyl-Lcysteine crude product, mass concentration are
95% ethanol washing, vacuum filtration, dries to obtain 10.3g S- methyl-Lcysteine fine work, purity 99.9%.
Embodiment 2
The present embodiment the following steps are included:
(1) by 1000mL Pseudomonas stutzeri CGMCC NO:1.202, cultivation and fermentation, fermentation liquid are centrifuged in the medium
It to wet thallus 20g, is added in 500mL conversion fluid, the hair acid hydrolysis liquid of the Serine in conversion fluid containing 21g, 9.62g
Methyl mercaptan, 0.2g/L phosphopyridoxal pyridoxal phosphate and 0.01g/L acetone, pH8.0,37 DEG C of enzymatic reaction 15h, after reaction in conversion fluid
S- methyl-Lcysteine is 26.1g, is 97% to Serine molar yield；
(2) conversion fluid 5000r/min is centrifuged 10min, removes somatic cells, heating, decolorizing with activated carbon filters, filtrate
Adjust pH 7 or so, stand be precipitated precipitating, vacuum filtration, dry 25.8g S- methyl-Lcysteine crude product, mass concentration are
95% ethanol washing, vacuum filtration, dries to obtain 22.3g S- methyl-Lcysteine fine work, purity 99.9%.
Embodiment 3
The present embodiment the following steps are included:
(1) by 1000mL pseudomonas aeruginosa CGMCC NO:1.1129, cultivation and fermentation, fermentation liquid are centrifuged in the medium
It to 15g wet thallus, is added in 500mL conversion fluid, Serine, 4.81g methyl mercaptan, 0.2g/L phosphorus in conversion fluid containing 11g
Sour pyridoxal and 0.5g/L ethyl alcohol, 8.0,37 DEG C of enzymatic reaction 16h of pH, after reaction half Guang ammonia of S- methyl-L- in conversion fluid
Acid is 12.7g, is 95% to Serine molar yield；
(2) conversion fluid 3000r/min is centrifuged 20min, removes somatic cells, heating, decolorizing with activated carbon filters, filtrate
Adjust pH 7 or so, stand be precipitated precipitating, vacuum filtration, dry 10.7g S- methyl-Lcysteine crude product, mass concentration are
95% ethanol washing, vacuum filtration, dries to obtain 9.9g S- methyl-Lcysteine fine work, purity 99.9%；
Embodiment 4
The present embodiment the following steps are included:
(1) by 1000mL Escherichia coli ATCC15489, cultivation and fermentation, fermentation liquid are centrifuged to obtain the wet bacterium of 12g in the medium
Body is added in 500mL conversion fluid, the hair acid hydrolysis liquid of the Serine in conversion fluid containing 21g, 9.62g methyl mercaptan,
The phosphopyridoxal pyridoxal phosphate of 0.2g/L and the methanol of 0.005g/L, 8.0,35 DEG C of enzymatic reaction 16h of pH, after reaction in conversion fluid
S- methyl-Lcysteine is 25.9g, is 96% to Serine molar yield；
(2) conversion fluid 4000r/min is centrifuged 10min, removes somatic cells, heating, decolorizing with activated carbon filters, filtrate
Adjust pH 7 or so, stand be precipitated precipitating, vacuum filtration, dry 23.1g S- methyl-Lcysteine crude product, mass concentration are
95% ethanol washing, vacuum filtration, dries to obtain 21.1g S- methyl-Lcysteine fine work, purity 99.9%.
Claims (8)
1. a kind of enzymatic conversion preparation method of S- methyl-Lcysteine, which comprises the following steps:
(1) there will be the active bacterial strain of tryptophan synthetase cultivation and fermentation in the medium, and obtain ammonia containing color after fermentation liquid centrifugation
The wet thallus of acid enzyme；
(2) conversion fluid will be added in above-mentioned wet thallus, include: the feed liquid containing Serine, phosphopyridoxal pyridoxal phosphate in the conversion liquid
And methyl mercaptan, one of ether, methanol, ethyl alcohol, acetone；At 25~55 DEG C, enzymatic reaction is carried out under the conditions of pH6~11, etc.
The electricity point isolated S- methyl-Lcysteine of crystallisation.
2. a kind of enzymatic conversion preparation method of S- methyl-Lcysteine according to claim 1, which is characterized in that
In the step (1), there is the active bacterial strain of tryptophan synthetase to be selected from Escherichia coli ATCC15489, pseudomonas aeruginosa
CGMCC NO:1.1129, Pseudomonas stutzeri CGMCC NO:1.202, one in bacillus subtilis CGMCC NO:1.1628
Kind.
3. a kind of enzymatic conversion preparation method of S- methyl-Lcysteine according to claim 1, which is characterized in that
In the step (1), medium component includes carbon source material, nitrogen source, 2.2g/L citric acid, 2.8g/L ammonium sulfate, 5.0g/
L K2HPO4、2.5g/L MgSO4、0.04g/L CaCl2、0.001g/L CoCl2、0.0002g/L MnC4H6O4·4H2O.Culture
Carbon source in base uses one of glucose, maltose, sucrose, fructose or a variety of, and total carbon source mass concentration is in culture medium
10~40g/L；Nitrogen source in culture medium using one of beef extract, yeast extract, corn pulp, peptone, soya-bean cake hydrolyzate or
A variety of, total nitrogen source mass concentration is 5~35g/L in culture medium.
4. a kind of enzymatic conversion preparation method of S- methyl-Lcysteine according to claim 1, which is characterized in that
In the step (2), the concentration of Serine is 0.5~40.5g/L in the feed liquid containing Serine.
5. a kind of enzymatic conversion preparation method of S- methyl-Lcysteine according to claim 4, which is characterized in that
The feedstream origins containing Serine are in hair acid hydrolysis liquid or pure Serine liquid；Total amino acid in hair acid hydrolysis liquid
Weight percent is 5%~45%, and wherein weight percent of the Serine in total amino acid is 10%~90%.
6. a kind of enzymatic conversion preparation method of S- methyl-Lcysteine according to claim 1, which is characterized in that
In the step (2), the ether, methanol, ethyl alcohol or acetone, concentration are 0.001g/L~4.0g/L.
7. a kind of enzymatic conversion preparation method of S- methyl-Lcysteine according to claim 1, which is characterized in that
In the step (2), the concentration of phosphopyridoxal pyridoxal phosphate is 0.2~0.8g/L, and the concentration of methyl mercaptan is 0.22~18.5g/L.
8. a kind of enzymatic conversion preparation method of S- methyl-Lcysteine according to claim 1, which is characterized in that
In the step (2), the specific method is as follows for isoelectric point crystallizing method:
3000~5000r/min of conversion fluid after enzymatic reaction is centrifuged 10~20min, removes somatic cells；Thermal conversion liquid,
It using decolorizing with activated carbon, filters, filtrate is adjusted to pH 6~8, stands and precipitating is precipitated, and vacuum filtration dries to obtain half Guang of S- methyl-L-
Propylhomoserin crude product, using ethanol washing, vacuum filtration dries to obtain half Guang ammonia fine work of S- methyl-L-.
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| CN110205346A (en) * | 2019-07-02 | 2019-09-06 | 武汉轻工大学 | A kind of preparation method of Serine |
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| CN110205346A (en) * | 2019-07-02 | 2019-09-06 | 武汉轻工大学 | A kind of preparation method of Serine |
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Application publication date: 20181207 |