CN114276947A - Method for preparing L-cysteine by enzymatic conversion and application - Google Patents
Method for preparing L-cysteine by enzymatic conversion and application Download PDFInfo
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- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 title claims abstract description 138
- 239000004201 L-cysteine Substances 0.000 title claims abstract description 69
- 235000013878 L-cysteine Nutrition 0.000 title claims abstract description 69
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- 238000006243 chemical reaction Methods 0.000 title description 63
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a method for preparing L-cysteine by bioconverting DL-ATC by using Pseudomonas putida YD 1205. The invention provides a novel Pseudomonas putida YD1205(Pseudomonas putida YD1205), a method for generating L-cysteine by biotransformation of DL-ATC (DL-2-amino-delta 2-thiazoline-4-carboxylic acid) by using wet thalli obtained by culturing the strain can biotransform DL-ATC with the concentration of 30 g/L-70 g/L, thereby greatly improving the production efficiency of the L-cysteine and enabling the product concentration to reach 40 g/L.
Description
Technical Field
The invention relates to the technical field of amino acid preparation by a biological enzyme method, in particular to a method for preparing L-cysteine by biotransformation of DL-ATC by using pseudomonas putida YD1205 and application thereof.
Background
L-cysteine is only one common amino acid with a side chain containing sulfhydryl group, is an important food and drug raw material, and is widely applied to the fields of medicine manufacture, health food production, food preservation, food addition, feed addition, pet food, essence and spice, cosmetic production and the like.
Traditionally, the production of L-cysteine mainly depends on a hair hydrolysis method, and although the process is relatively simple, the yield is low, the energy consumption is high, a large amount of irritant gas is generated in the hydrolysis process, the waste liquid is difficult to treat, and the environmental pollution is serious. In recent years, the enzyme conversion method has been increasingly emphasized due to the advantages of good stereoselectivity, capability of obtaining a single configuration product, high product purity, mild reaction conditions, environmental friendliness and the like. The production of L-cysteine by an enzyme conversion method can be realized by adopting different substrates, wherein the research on microbial conversion by taking DL-2-amino-delta 2-thiazoline-4-carboxylic acid (DL-ATC) as a substrate is most popular.
In 2008, plum blossom and the like synthesize L-cysteine by converting DL-ATC with a Pseudomonas TS1138 strain by an enzyme method, and the optimal DL-ATC concentration for converting L-cysteine is obtained by experiments and is 9g/L (plum blossom, Huang Lei, Huai, and the like, the influence of DL-ATC on the L-cysteine produced by the TS1138 strain by the enzyme method is reported by Tianjin scientific and technical university, 2008,023(002): 27-29.).
2.047g/L L-cysteine (CN 100467587C) was obtained by bioconversion using 1% DL-ATC as a substrate using wet cells or crude enzyme solution of Pseudomonas zjwp-14 such as Wangpen in 2009. Chenning et al used another Pseudomonas putida TS-1138 as enzyme source, and maintained DL-ATC at 0.6% concentration as substrate by feeding stream, and synthesized 5.70g/l L-cysteine (CN 101348809A) by biotransformation.
Although the production capacity of the L-cysteine is increased to a certain extent through optimization of a biological process and genetic operation, the production capacity is limited by low strain conversion rate and low enzyme activity of the strain, the L-cysteine produced by catalysis with the DL-ATC as a substrate through an enzyme method is still a low-concentration reaction, and the substrate concentration is usually 10 g/L-15 g/L, so that the production efficiency of the L-cysteine is low, the production cost is high, the amount of generated wastewater is large, and the application of the L-cysteine in industrial production is greatly limited.
Therefore, the establishment of a high-performance strain and a method for maximizing the initial DL-ATC substrate concentration of the enzymatic reaction of the strain so as to improve the production efficiency of L-cysteine is a problem to be solved urgently in the industrial application of the field.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, one purpose of the invention is to find a novel Pseudomonas putida YD1205(Pseudomonas putida YD1205), and a method for generating L-cysteine by biotransformation of DL-ATC (DL-2-amino-delta 2-thiazoline-4-carboxylic acid) by using wet thalli obtained by culturing the strain realizes great improvement of the production efficiency of the L-cysteine, and the product concentration can reach more than 40 g/L.
To this end, the invention provides in a first aspect a Pseudomonas putida YD1205(Pseudomonas putida YD 1205). According to an embodiment of the invention, the Pseudomonas putida YD1205 has a preservation number of CCTCC NO: M2015080.
Pseudomonas putida YD1205(Pseudomonas putida YD1205) has been deposited in China center for type culture Collection (CCTCC, address No. 299 of one of the eight branches of the Wuchang district, Wuhan city, Hubei) 3/2015, and the number of the deposit is registered in the collection center as CCTCC NO: M2015080. The strain is obtained by the inventor through ultraviolet irradiation, microwave mutagenesis, gamma ray irradiation mutagenesis treatment and DL-ATC substrate resistance screening of alternative strains.
The inventor obtains a pseudomonas putida YD1205 strain through artificial mutagenesis and screening of the strain, the strain has high enzyme production efficiency, and the pseudomonas putida YD1205 strain is applied to a reaction for producing L-cysteine by using DL-ATC as a substrate and performing enzyme conversion reaction by using wet thalli obtained by culturing the pseudomonas putida YD1205 as an enzyme source and using DL-2-amino-delta 2-thiazoline-4-carboxylic acid (DL-ATC) as a substrate to obtain L-cysteine or further prepare the L-cystine through oxidation. The enzyme produced by the pseudomonas putida YD1205 improves the amount of DL-ATC converted in unit time, can catalyze and convert DL-ATC with the concentration of 30-70g/L, and improves the production efficiency of L-cysteine or L-cystine by 2-3 times.
The second aspect of the invention provides the usage of the pseudomonas putida YD1205 strain or the bacterial suspension or the culture solution thereof in the preparation of L-cysteine or L-cystine.
In a third aspect, the invention provides a method for preparing L-cysteine. According to an embodiment of the invention, the method comprises:
(1) carrying out fermentation culture on the pseudomonas putida in the first aspect so as to obtain a culture solution or thallus;
(2) and mixing the culture solution or the thallus with a DL-ATC disperse system, and carrying out enzyme conversion reaction to obtain a product L-cysteine.
The production of L-cysteine by microbial enzyme method conversion with DL-ATC as reaction substrate is mainly completed by three enzymes, namely ATC racemase, L-ATC hydrolase and S-carbamoyl-L-cysteine amide hydrolase or N-carbamoyl-L-cysteine amide hydrolase. The production of L-cysteine by enzymatic conversion presents two difficulties: firstly, the substrate concentration is lower; secondly, the L-ATC hydrolase is unstable. On one hand, because the enzyme participating in the way of producing L-cysteine by enzyme conversion belongs to an induced enzyme, the concentration of a substrate is limited to be low, and the enzyme cannot be induced to produce more enzyme, on the other hand, the existing pseudomonas enzyme-producing strain has low enzyme activity and low production efficiency of L-cysteine.
The inventor obtains Pseudomonas putida YD1205(Pseudomonas putida YD1205) from alternative strains through ultraviolet irradiation, microwave mutagenesis and gamma ray irradiation mutagenesis treatment and screening, and the strain is applied to the production of L-cysteine, so that the catalytic efficiency of enzymatic conversion can be greatly improved, and the yield of the L-cysteine is improved.
Specifically, the invention provides a novel preparation method for converting DL-ATC into L-cysteine by enzyme. The method specifically comprises the following steps: taking wet thalli obtained by culturing pseudomonas putida YD1205 as an enzyme source, carrying out enzyme conversion reaction on an enzymatic culture medium taking DL-2-amino-delta 2-thiazoline-4-carboxylic acid (DL-ATC) as a substrate under a certain condition to obtain a reaction liquid containing L-cysteine, further filtering or centrifuging, separating and recovering the wet thalli, extracting to prepare the L-cysteine, or further oxidizing to prepare the L-cystine. The method has the following effects: (1) the enzyme produced by the pseudomonas putida YD1205 improves the amount of converted DL-ATC in unit time, the efficiency of catalyzing DL-ATC substrate to produce L-cysteine is high, and the production efficiency of L-cysteine or L-cystine is improved by 2-3 times; (2) the enzymatic culture medium has simple components, and the use cost of materials is reduced; (3) the amount of wastewater generated by the enzymatic reaction is reduced, and the difficulty and cost of the post-treatment are reduced.
According to an embodiment of the invention, the concentration of DL-ATC is from 30g/L to 70g/L, preferably from 50g/L to 70 g/L.
According to an embodiment of the invention, the DL-ATC dispersion further comprises auxiliary materials.
According to an embodiment of the invention, the auxiliary material is selected from Mn metal salts.
According to an embodiment of the invention, the Mn of the Mn metal salt is Mn2+The concentration of (A) is 0.3-0.5 g/L.
According to the embodiment of the invention, the mass ratio of the thalli to DL-ATC is (4-12): (30-70).
According to a preferred embodiment of the present invention, the mass ratio of the bacterial cells to DL-ATC is 1 (6-8). According to an embodiment of the invention, the DL-ATC dispersion is prepared by:
and (3) adding DL-ATC into water at the temperature of 60-80 ℃ for dissolving, and then cooling to the temperature of the enzyme conversion reaction so as to obtain a DL-ATC disperse system.
In the pathway of synthesizing L-cysteine by enzymatically converting DL-ATC, the hydrolase used is an inducible enzyme, which is induced by DL-ATC, and on the one hand, higher concentration of DL-ATC induces the Pseudomonas putida of the present invention to produce more enzyme. The high-efficiency strain can efficiently catalyze DL-ATC in a saturated state, and simultaneously the DL-ATC can induce the strain to produce enzyme. On the other hand, the enzyme produced by the pseudomonas putida YD1205 provided by the invention can catalyze more DL-ATC in unit time, thereby improving the yield of L-cysteine.
According to the embodiment of the invention, the temperature of the enzyme conversion reaction is 25-40 ℃, and preferably 30-40 ℃.
According to an embodiment of the invention, the rotation speed of the enzyme conversion reaction is 150-250 rpm, preferably 200-250 rpm.
According to the embodiment of the invention, the enzymatic conversion reaction time is 11-13 h.
The enzyme conversion reaction speed is 150-250 rpm, the generation of the L-cysteine can be further promoted, and if the speed is further increased, the L-cysteine is oxidized into the L-cystine.
In a fourth aspect, the invention provides a process for the preparation of L-cystine. According to an embodiment of the invention, the method comprises:
oxidizing the L-cysteine produced by the production method described in the second aspect to obtain L-cystine.
The fifth aspect of the invention provides the use of the pseudomonas putida YD1205 strain of the first aspect or a bacterial suspension or culture solution thereof in the preparation of a biological agent for preparing L-cysteine or L-cystine.
In a sixth aspect, the invention provides a biological agent. According to an embodiment of the present invention, the biological agent comprises pseudomonas putida YD1205 strain of the first aspect or a bacterial suspension thereof or a culture solution thereof.
The implementation of the method of the invention has the following effects:
(1) the enzyme produced by the pseudomonas putida YD1205 improves the amount of converted DL-ATC in unit time, the efficiency of catalyzing DL-ATC substrate to produce L-cysteine is high, and the production efficiency of L-cysteine or L-cystine is improved by 2-3 times;
(2) the enzymatic culture medium has simple components, and the use cost of materials is reduced;
(3) the amount of wastewater generated by the enzymatic reaction is reduced, and the difficulty and cost of the post-treatment are reduced.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Preservation information:
the strain name: pseudomonas putida YD1205
Pseudomonas putide YD1205
The preservation date is as follows: 3 months and 3 days in 2015
The preservation unit: china center for type culture Collection
The preservation number is: CCTCC NO: M2015080
Address: university of Wuhan, China
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows HPLC profiles of DL-ATC, L-cysteine, L-cystine in the standard;
FIG. 2 shows HPLC profiles of DL-ATC, L-cysteine, L-cystine in samples obtained by preparation of example 3;
FIG. 3 shows the changes of DL-ATC, L-cysteine, L-cystine in samples of different reaction durations;
FIG. 4 shows the variation of the length of the enzymatic conversion of L-cysteine in the strains before and after mutagenesis.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
In one embodiment of the present invention, the present invention provides a method for preparing L-cysteine or L-cystine by enzymatically converting DL-ATC, comprising the steps of:
(1) dissolving 30-70g of DL-ATC in warm water at 60-80 ℃, continuously stirring, cooling to below 35 ℃, adding auxiliary materials, diluting to 1L with water, adjusting the pH to 8.0, and using the solution as an enzymatic culture medium (providing carbon and nitrogen sources for survival of bacteria) and the DL-ATC as a reaction substrate;
(2) blowing nitrogen into the enzymatic culture medium for more than 30min to fully replace the air in the system;
(3) adding wet thalli obtained by culturing pseudomonas putida YD1205 as an enzyme source into an enzymatic culture medium, wherein the added mass is 4-12 g;
(4) keeping a certain enzymatic reaction condition, continuously introducing nitrogen into the reaction system, and carrying out an enzymatic conversion reaction to obtain a reaction solution containing L-cysteine;
(5) monitoring the concentration of related products by HPLC, filtering or centrifuging after the reaction is finished, separating and recovering wet thalli, and extracting to prepare L-cysteine or further oxidizing to prepare L-cystine.
In one embodiment of the invention, the enzymatic medium DL-ATC is initially present at a concentration of 30 to 70 g/L. The enzymatic media also contains adjuvants including, but not limited to, MnSO4、MnCl2Or Mn2+One or a combination of a plurality of metal salts, and the concentration is 0.3-0.5 g/L.
In one embodiment of the present invention, the enzyme-derived wet cells can be prepared as follows:
seed culture: pseudomonas putida YD1205 strain stored in a low-temperature refrigerator at-80 ℃ with glycerol was inoculated on LB slant medium and cultured overnight at 30 ℃.
② shake flask culture: a ring of pseudomonas putida YD1205 is picked by an inoculating ring and transferred to a shake flask culture medium, the temperature is 30 ℃, the rotating speed of a shaking table is 120rpm, and the culture is carried out for 12 to 18 hours. The shake flask culture medium comprises 0.5% of yeast powder, 0.5% of peptone, 1% of sodium chloride and pH7.0.
③ fermentation culture: inoculating the shake flask culture solution with the inoculation amount of 0.5-10%, and performing multistage amplification culture in a fermentation culture medium, wherein each stage of culture is performed for 5-9 hours at the rotation speed of 120-180 rpm and at the temperature of 25-37 ℃ to obtain the fermentation enzyme solution. The fermentation medium comprises 4% of corn steep liquor, 0.05% of dipotassium phosphate, 0.75% of monosodium glutamate, 0.05% of magnesium sulfate and pH 7.5-8.0.
Fourthly, the fermentation enzyme liquid is centrifuged or filtered, and the wet thalli are collected and frozen for preservation.
In one embodiment of the invention, the mass ratio of the enzyme-derived wet bacteria to DL-ATC in the enzymatic culture medium is 1 (6-8).
In one embodiment of the present invention, the enzymatic reaction is carried out at a temperature of 30 to 40 ℃ and a rotation speed of 200 to 250 rpm. The enzymatic reaction lasts for 11-13 h.
In one embodiment of the invention, the course of the enzymatic reaction is monitored by HPLC. The chromatographic conditions are as follows: a Dionex Ultimate 3000 high performance liquid chromatograph, a normal phase silica gel bonded glycol chromatographic column, a mobile phase of acetonitrile-0.01M ammonium dihydrogen phosphate solution (pH 3.0) (80:20), a detection wavelength of 200nm, a flow rate of 1.5ml/min, a column temperature of 35 ℃ and a sample introduction amount of 20 μ L.
In one embodiment of the invention, the reaction solution is centrifuged or filtered to obtain an L-cysteine solution and enzyme source wet bacteria, wherein the L-cysteine solution is further extracted and refined, and the enzyme source wet bacteria are recycled for the next time, wherein the recycling times are 1-2 times.
In one embodiment of the present invention, the present invention provides a method for preparing L-cysteine or L-cystine by enzymatically converting DL-ATC, comprising the steps of:
(1) dissolving 50-70 g of DL-ATC in warm water at 60-80 ℃, continuously stirring, cooling to below 35 ℃, adding auxiliary materials, diluting to 1L with water, and adjusting the pH to 8.0 to serve as an enzymatic culture medium; (2) blowing nitrogen into the enzymatic culture medium for more than 30min to fully replace the air in the system;
(3) adding wet thalli obtained by culturing pseudomonas putida YD1205 as an enzyme source into an enzymatic culture medium, wherein the added mass is 6-12 g; (4) keeping the rotation speed of 200-250 rpm and the temperature of 30-40 ℃, continuously introducing nitrogen into the reaction system, and performing enzyme conversion reaction to obtain a reaction solution containing L-cysteine;
(5) monitoring the concentration of the relevant product by HPLC; and after the reaction is carried out for 11-13 hours, filtering or centrifuging, separating and recovering wet thalli, extracting and preparing L-cysteine, or further oxidizing to prepare L-cystine.
"Dispersion" means a system in which a substance or substances are highly dispersed in another substance (referred to as a dispersion medium), and "DL-ATC dispersion" as used herein includes DL-ATC solutions and DL-ATC suspensions.
In the present invention, the "concentration of DL-ATC" refers to the ratio of the weight of DL-ATC in the dispersion to the total volume of the suspension when the suspension is changed from a DL-ATC solution to a suspension due to a change in temperature.
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: acquisition of Pseudomonas putida YD1205
1. Mutagenesis and selection of strains
The inventor selects and separates the strain from soil near an industrial garden for producing DL-ATC in a pond town of yellow stone city in Hubei province, and sequentially carries out ultraviolet irradiation, microwave mutagenesis and gamma ray irradiation composite mutagenesis treatment, wherein the ultraviolet irradiation power is 18W, the irradiation distance is 35cm, and the irradiation time is 180 s; inducing low fire grade by microwave, and carrying out ice bath treatment for 150 s; co60The gamma irradiation dose was about 1.5 Kgy. And (3) carrying out substrate resistance screening on the treated strains by using a culture medium with DL-ATC as a unique nitrogen source. The screening medium used contained: glycerol 205mM, DL-ATC 0.5% -3% (w/v), KH2PO4 44mM、Na2HPO4 106mM、MgSO4 2mM、CoCl2-6H2O 0.63μM、MnSO4 0.4μM、CuCl2 0.3μM、H3BO3 0.8μM、Na2MoO4 0.22μM、ZnSO4 0.69μM、FeSO4 5μM、CaCl2 0.68μM、Na2SeO3 0.41μM、NiCl2 0.2μM、 Kao&Michayluk vitamin solution 1% (v/v, Sigma), agar 2% (w/v).Preparing a flat plate by using the culture medium, culturing for 2 days at 30 ℃, and screening strains according to the growth conditions of bacterial colonies with different concentrations of DL-ATC to obtain a strain YD 1205.
2. Acquisition and identification of Pseudomonas putida YD1205 Strain
The 16S rRNA sequence of the strain obtained by screening was shown in SEQ ID NO. 1, and it was found to have 98.9% homology with some bacteria in the genus Pseudomonas, such as Pseudomonas monteilii, by aligning with the 16S rRNA sequence in the gene bank. Based on the above results, the selected strain was identified as belonging to Pseudomonas putida (Pseudomonas putida) YD 1205.
The nucleotide sequence of 16S rRNA is as follows:
GAGCGTGCGCAGCTACACATGCAAGTCGAGCGGATGACGGGAGCTTGCTCCCTG ATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAAC GTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTC GGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAATGGCT CACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGA GACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAA AGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTA AGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCGACAGAATA AGCACCGGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAA TCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAATCC CCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGTAGGGCAGAGGG TGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGC GAAGGCGACCACCTGGGCTCATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAA CAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATC CTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCG CAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGT TTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCA GAGATGGATTGGTGCCTTCGGGAACTCTGACACAGGTGCTGCATGGCTGTCGTCAGCT CGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTAC CAGCACGTAATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGT GGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATG GTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCTCACAAAACCGATCGT AGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCG AATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCA TGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGAGGACGGTACCATCAT GATCATGGCC。
3. preservation of Pseudomonas putida YD1205
The strain YD1205, belonging to Pseudomonas putida (Pseudomonas putide), has been deposited in China center for type culture Collection (CCTCC, with the address of 299 eighth Lot in Wuchang district, Wuhan City, Hubei) 3.3.3.3.2015, and has been registered with the number of CCTCC NO: M2015080 by the preservation center.
Pseudomonas putida YD1205 also has the following colonial characteristics: the bacillus pumilus is a non-zymocyte, is a bacillus pumilus individual, has the diameter of about 0.5-1.0um and the length of about 2um, and can grow rapidly, and a punctate, round, moist and white colony can grow after being subjected to planar culture at 30 ℃ for 24 hours.
Example 2: culture of Pseudomonas putida YD1205 and obtaining of thallus
Pseudomonas putida YD1205 strain stored in a low-temperature refrigerator at-80 ℃ with glycerol was inoculated on LB slant medium and cultured overnight at 29 ℃. A ring of pseudomonas putida YD1205 was picked with an inoculating loop and transferred to a shake flask culture medium, and cultured at 29 ℃ and 120rpm of a shaking table for 15 hours. The components of the culture medium are 0.5 percent of yeast powder, 0.5 percent of peptone, 1 percent of sodium chloride and pH7.0. Taking shake flask culture solution strain to perform 2-stage fermentation tank amplification culture, wherein the inoculum size is 5%, and the culture time is 37 ℃, and each stage culture time is 5-9 hours, so as to obtain fermentation enzyme solution. The fermentation medium comprises corn steep liquor 4%, dipotassium hydrogen phosphate 0.05%, monosodium glutamate 0.75%, magnesium sulfate 0.05%, and pH7.5-8.0. And centrifuging or filtering the fermentation enzyme liquid, collecting wet thalli, and freezing and preserving.
Example 3: preparation of L-cysteine
Adding 60g DL-ATC into 70 deg.C water, stirring to dissolve completely, cooling to below 35 deg.C, adding 0.4g Mn SO4The volume is determined to be 1L, and the pH value is adjusted to 8.0 by NaOH solution. After nitrogen gas was blown into the above medium for 30min to displace the air inside the system, 9g of wet cells obtained by culturing Pseudomonas putida YD1205 in example 2 were added, and nitrogen gas was continuously blown into the reaction system to conduct an enzymatic reaction while maintaining the reaction temperature at 35 ℃ and the stirring speed at 200 rpm. Sampling after reacting for 8h and detecting by using a high performance liquid chromatographThe product concentration was compared to a standard control and calculated by appearance. The DL-ATC, L-cysteine and L-cystine profiles of the standard control and the sample are shown in FIG. 1 and FIG. 2. Wherein, the peak No. 1 is the peak of L-cysteine, the peak No. 2 is the peak of DL-ATC, and the peak No. 3 is the peak of L-cystine. After that, the product concentration was measured by sampling every 1h, and the reaction results are shown in FIG. 3. Indicating that the product concentration did not change substantially after 13 h.
Example 4: investigation of substrate concentration
Adding 30, 40, 50, 60, 70, 80, 90g DL-ATC into 70 deg.C water, stirring to dissolve completely, cooling to below 35 deg.C, adding 0.4g MnSO4The volume is determined to be 1L, and the pH value is adjusted to 8.0 by NaOH solution. After the medium was purged with nitrogen for 30min to replace the air in the system, 4.5, 6, 7, 9, 10, 11 and 13g of wet cells obtained by the culture of Pseudomonas putida YD1205 in example 2 were added, and the reaction system was continuously purged with nitrogen while maintaining the reaction temperature at 35 ℃ and stirring at 200rpm to carry out the enzymatic reaction. After the reaction is completed, sampling and detecting the product concentration, wherein the reaction results are as follows: TABLE 1
| Substrate DL-ATC concentration (g/L) | L-cysteine (g/L) | Conversion rate |
| 30 | 22.92 | 92.26％ |
| 40 | 30.20 | 91.18％ |
| 50 | 38.03 | 91.87％ |
| 60 | 45.33 | 91.24％ |
| 70 | 47.67 | 82.24％ |
| 80 | 39.98 | 60.35％ |
| 90 | 33.95 | 45.56％ |
The conversion in table 1 was calculated in the following manner: actual measured mass of L-cysteine ÷ mass of theoretically produced L-cysteine X100% calculated on the total amount of substrate added
The results in Table 1 show that higher product conversions can be achieved at substrate concentrations of 30-70 g/L.
Example 5: study on the amount of pseudomonas putida YD1205
Adding 60g DL-ATC into 70 deg.C water, stirring to dissolve completely, cooling to below 35 deg.C, adding 0.4g MnSO4The volume is determined to be 1L, and the pH value is adjusted to 8.0 by NaOH solution. After nitrogen gas was blown into the above medium for 30min to displace the air inside the system, 6 to 15g of wet cells obtained by the culture of Pseudomonas putida YD1205 in example 2 were added, and nitrogen gas was continuously blown into the reaction system while maintaining the reaction temperature at 35 ℃ and the stirring speed at 200rpm to carry out the enzymatic reaction. After 12h of reaction, respectively sampling and detecting the product concentration and the reaction result conditionThe following were used:
TABLE 2
| Pseudomonas putida YD1205 dosage (g) | L-cysteine (g/L) |
| 6 | 36.25 |
| 6.8 | 38.45 |
| 7.5 | 42.58 |
| 8.6 | 44.76 |
| 10 | 44.97 |
| 12 | 44.83 |
| 15 | 45.19 |
The results in Table 2 show that the yield of L-cysteine can be higher by adding 7.5-10 g of Pseudomonas putida YD1205 wet bacteria into the reaction system, the product concentration of L-cysteine can reach more than 40g/L, the reaction can be completed, and when the bacteria amount is continuously increased, the substrate reaction is basically saturated, and the product concentration is not obviously improved.
Example 6: investigation of transformation temperature
Adding 60g DL-ATC into 70 deg.C water, stirring to dissolve completely, cooling to below 35 deg.C, adding 0.4g MnSO4The volume is determined to be 1L, and the pH value is adjusted to 8.0 by NaOH solution. After nitrogen gas was blown into the above medium for 30min to displace the air inside the system, 9g of wet cells obtained by the culture of Pseudomonas putida YD1205 in example 2 were added, and nitrogen gas was continuously blown into the reaction system to conduct the enzymatic reaction while maintaining the reaction temperature at 20 to 45 ℃ and the stirring speed at 200 rpm. After reacting for 12h, respectively sampling and detecting the product concentration, wherein the reaction results are as follows:
TABLE 3
The results in Table 3 show that the conversion temperature of the enzymatic conversion reaction is preferably 25-40 ℃, which is favorable for the production of L-cysteine.
Example 7: exploration of conversion speed
Adding 60g DL-ATC into 70 deg.C water, stirring to dissolve completely, cooling to below 35 deg.C, adding 0.4g MnSO4The volume is determined to be 1L, and the pH value is adjusted to 8.0 by NaOH solution. Nitrogen gas was blown into the above medium for 30min to displace the air inside the system, 9g of wet cells obtained by the culture of Pseudomonas putida YD1205 in example 2 were added, and nitrogen gas was continuously blown into the reaction system to conduct the enzymatic reaction while maintaining the reaction temperature at 35 ℃ and the stirring speed at 100-350 rpm. After reacting for 12h, respectively sampling and detecting the product concentration, wherein the reaction results are as follows:
TABLE 4
The results in Table 4 show that the conversion speed is in the range of 150-250 rpm, the generation of L-cysteine can be promoted, and if the conversion speed is further increased, the L-cysteine is oxidized into L-cystine.
Example 8 Recycling of cells
Adding 60g DL-ATC into 70 deg.C water, stirring to dissolve completely, cooling to below 35 deg.C, adding 0.4g MnSO4The volume is determined to be 1L, and the pH value is adjusted to 8.0 by NaOH solution. And blowing nitrogen into the culture medium for 30min, replacing the air in the system, adding 9g of wet thalli centrifugally recovered from the reaction liquid, continuously introducing nitrogen into the reaction system, respectively maintaining the reaction temperature at 35 ℃, and stirring at the rotating speed of 200rpm, and carrying out enzymatic reaction. After 12h of reaction, a sample was taken to determine the product concentration. After completion of the first enzymatic conversion reaction, the cells were recovered and subjected to the same reaction conditions as described above to evaluate the conversion rate of wet cells for recycling. The reaction results were as follows:
TABLE 5
| Recovery of wet cells | L-cysteine (g/L) |
| 1 | 37.46 |
| 2 | 35.29 |
| 3 | 14.14 |
The results in Table 5 show that the enzyme conversion reaction using the wet cells of the present invention can be recycled once, and the conversion rate of the product can be maintained high even when the enzyme conversion reaction is used for the second time, but the conversion rate is affected when the enzyme conversion reaction is used for the third time.
Example 9
The reaction solution obtained in any of examples 3 to 9 was centrifuged, oxygen was introduced into the supernatant obtained by the centrifugation for 30 minutes, the pH was adjusted to about 5.0, and absolute alcohol was added thereto to a final concentration of 30%, followed by standing for 5 hours. The reaction mixture was filtered and the solids collected. And (3) washing the solid with hot water, drying, and carrying out optical rotation detection on the sample to obtain the specific rotation degree of-215 which is similar to the L-cystine standard substance of-220.
Example 10
Wet cells of the original wild type strain (WT) were prepared according to the same method as that for the culture of Pseudomonas putida in example 2, and L-cysteine was prepared by the enzymatic conversion reaction using the wet cells, and the procedure and conditions of the enzymatic conversion reaction were exactly the same as those in example 3. Respectively counting the product concentration in the process of carrying out enzyme conversion reaction by using the pseudomonas putida YD1205 and the wild type strain, sampling every 1h after 8h of reaction to detect the product concentration, wherein the reaction result is as shown in figure 4, and the comparison result of the WT strain and the mutagenized pseudomonas putida YD1205 shows that after 8h of enzymatic reaction, the yield of L-cysteine generated by converting DL-ATC by the pseudomonas putida YD1205 reaches 40g/L and is improved by 2 times compared with the original strain 13 g/L.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Hubei Daihe Biotechnology Ltd
<120> method for preparing L-cysteine by enzymatic conversion and use thereof
<130> BI3211714
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1440
<212> DNA
<213> Pseudomonas sp YD-1205
<400> 1
gagcgtgcgc agctacacat gcaagtcgag cggatgacgg gagcttgctc cctgattcag 60
cggcggacgg gtgagtaatg cctaggaatc tgcctggtag tgggggacaa cgtttcgaaa 120
ggaacgctaa taccgcatac gtcctacggg agaaagcagg ggaccttcgg gccttgcgct 180
atcagatgag cctaggtcgg attagctagt tggtggggta atggctcacc aaggcgacga 240
tccgtaactg gtctgagagg atgatcagtc acactggaac tgagacacgg tccagactcc 300
tacgggaggc agcagtgggg aatattggac aatgggcgaa agcctgatcc agccatgccg 360
cgtgtgtgaa gaaggtcttc ggattgtaaa gcactttaag ttgggaggaa gggcagtaag 420
ttaatacctt gctgttttga cgttaccgac agaataagca ccgggctaac tctgtgccag 480
cagccgcggt aatacagagg gtgcaagcgt taatcggaat tactgggcgt aaagcgcgcg 540
taggtggttc gttaagttgg atgtgaaatc cccgggctca acctgggaac tgcatccaaa 600
actggcgagc tagagtaggg cagagggtgg tggaatttcc tgtgtagcgg tgaaatgcgt 660
agatatagga aggaacacca gtggcgaagg cgaccacctg ggctcatact gacactgagg 720
tgcgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc gtaaacgatg 780
tcaactagcc gttggaatcc ttgagatttt agtggcgcag ctaacgcatt aagttgaccg 840
cctggggagt acggccgcaa ggttaaaact caaatgaatt gacgggggcc cgcacaagcg 900
gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggcct tgacatgcag 960
agaactttcc agagatggat tggtgccttc gggaactctg acacaggtgc tgcatggctg 1020
tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgta acgagcgcaa cccttgtcct 1080
tagttaccag cacgtaatgg tgggcactct aaggagactg ccggtgacaa accggaggaa 1140
ggtggggatg acgtcaagtc atcatggccc ttacggcctg ggctacacac gtgctacaat 1200
ggtcggtaca gagggttgcc aagccgcgag gtggagctaa tctcacaaaa ccgatcgtag 1260
tccggatcgc agtctgcaac tcgactgcgt gaagtcggaa tcgctagtaa tcgcgaatca 1320
gaatgtcgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt 1380
gggttgcacc agaagtagct agtctaacct tcggaggacg gtaccatcat gatcatggcc 1440
Claims (10)
1. Pseudomonas putida YD1205(Pseudomonas putida YD1205) is characterized in that the preservation number is CCTCC NO: M2015080.
2. The use of Pseudomonas putida YD1205 strain or its bacterial suspension or its culture solution of claim 1 in the preparation of L-cysteine or L-cystine.
3. A method for preparing L-cysteine, which comprises the following steps:
(1) fermenting and culturing the pseudomonas putida of claim 1 to obtain a culture solution or bacteria;
(2) and mixing the culture solution or the thallus with a DL-ATC disperse system, and carrying out enzyme conversion reaction to obtain a product L-cysteine.
4. The method of claim 3, wherein the concentration of DL-ATC is 30 to 70g/L, preferably 50 to 70 g/L.
5. The method of claim 3, wherein the DL-ATC dispersion further comprises excipients;
optionally, the adjuvant is selected from Mn metal salts;
optionally, Mn in the Mn metal salt2+The concentration of (b) is 0.3-0.5 g/L.
6. The method according to claim 3, wherein the mass ratio of the cells to DL-ATC is (4-12): (30-70), preferably 1 (6-8).
7. The method of claim 6, wherein the DL-ATC dispersion is prepared by:
adding DL-ATC into water at the temperature of 60-80 ℃ for dissolving, and then cooling to the temperature of enzyme conversion reaction so as to obtain a DL-ATC disperse system;
optionally, the temperature of the enzyme conversion reaction is 25-40 ℃, and preferably 30-40 ℃;
optionally, the rotation speed of the enzyme conversion reaction is 150-250 rpm, preferably 200-250 rpm;
optionally, the enzyme conversion reaction time is 11-13 h.
8. A method for preparing L-cystine, characterized in that it comprises:
oxidizing L-cysteine produced by the production method according to any one of claims 3 to 7 to obtain L-cystine.
9. Use of pseudomonas putida YD1205 strain or a bacterial suspension thereof or a culture solution thereof according to claim 1, for preparing a biological agent, wherein the biological agent is used for preparing L-cysteine or L-cystine.
10. A biological agent comprising the Pseudomonas putida YD1205 strain of claim 1 or a bacterial suspension thereof or a culture solution thereof.
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