CN112813012A - Genetically engineered bacterium, preparation method thereof and application thereof in cysteine production - Google Patents
Genetically engineered bacterium, preparation method thereof and application thereof in cysteine production Download PDFInfo
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- CN112813012A CN112813012A CN202011500021.1A CN202011500021A CN112813012A CN 112813012 A CN112813012 A CN 112813012A CN 202011500021 A CN202011500021 A CN 202011500021A CN 112813012 A CN112813012 A CN 112813012A
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- gene
- cysteine
- genetically engineered
- serine
- trpba
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Abstract
The invention discloses a gene engineering bacterium and an application thereof in cysteine production, wherein the gene engineering bacterium comprises a host cell and a target gene, and the target gene is inserted into an episomal plasmid; or inserted into both the plasmid and the genome; the target gene inserted into the plasmid is mainly composed of a trpBA gene and a trpB gene which are connected in order; the target gene inserted into the genome is the trpBA gene. The trpBA gene and the trpB gene are inserted into a host cell to obtain the trpBA gene and trpB gene over-expressed gene engineering bacteria, and the engineering bacteria can express alpha subunit and beta subunit in a specific molar ratio, so that the reaction for synthesizing L-cysteine by an enzymatic method has higher catalytic rate and conversion rate, wherein the enzymatic conversion time is shortened to 2-8h, and the molar conversion rate of L-serine reaches more than 95%.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a genetic engineering bacterium and application thereof in cysteine production.
Background
L-cysteine is a naturally occurring amino acid and one of non-essential amino acids of the human body, and a sulfhydryl group carried by the L-cysteine has an important physiological function. The L-cysteine and the derivatives thereof can be used in the fields of medicine, cosmetics, food, feed additives and the like, and have very wide application.
According to the current literature reports, the preparation method of L-cysteine mainly comprises an extraction method, a chemical synthesis method and an enzyme conversion method. Among them, the preparation of L-cysteine by enzymatic conversion is becoming the mainstream technology.
The reaction equation of L-cysteine prepared by the enzymatic conversion method is as follows:
the enzyme most studied in the enzymatic conversion method is tryptophan synthase. Tryptophan synthase is a heterogeneous tetramer having an α β α subunit structure, wherein the α, β subunits are encoded by trpA and trpB genes, respectively. The tryptophan synthetase not only can catalyze L-serine and indole to synthesize L-tryptophan, but also can catalyze the reaction of the L-serine and sodium hydrosulfide to generate L-cysteine.
Ken-ichi Ishiwata (Journal of Fermentation and Bioengineering,67(3): 169-172,1989) uses L-serine and sodium hydrosulfide as substrates, and tryptophan synthase synthesizes L-cysteine by an enzymatic method, wherein the L-cysteine in a reaction solution reaches 114g/L under the optimal conditions, and the conversion rate is 47% of that of L-tryptophan synthesized by the enzymatic method by using L-serine and indole as substrates. Japanese patent (JP 62215396-A, 1987; JP 63007790-A,1988) reports a process for enzymatically synthesizing L-cysteine by using L-serine as a substrate and hydrogen sulfide or hydrosulfide or sulfide as a mercapto donor. Japanese patent (JP 62019098-A,1987) discloses the preparation of D-serine by converting L-serine to L-cysteine using a tryptophan synthase using DL-serine and a sulfide as substrates and isolating the same.
The patent with application publication number CN102517352B mixes wet thallus or crude enzyme liquid with L-tryptophan synthetase activity with mixed amino acid feed liquid containing L-serine, adds a proper amount of hydrosulfide or sulfide, carries out enzymatic reaction under the conditions of 25-55 ℃ and pH 6-11, leads L-cysteine generated by the reaction to be oxidized by air or dropwise added with hydrogen peroxide, separates by an isoelectric point crystallization method to obtain L-cystine, and prepares the L-cysteine by electrolytic reduction. Wherein the molar conversion rate of the L-serine reaches more than 80 percent.
At present, in the process for synthesizing L-cysteine by using a tryptophan synthase enzyme method, the enzyme conversion time and the yield of L-cysteine still have room for further improvement.
Disclosure of Invention
The invention provides a gene engineering bacterium and an application thereof in cysteine production, wherein the gene engineering bacterium can control the subunit molar ratio of tryptophan synthetase by regulating and controlling the expression levels of exogenously introduced trpBA genes and trpB genes, thereby shortening the enzyme conversion time and improving the yield of cysteine.
The specific technical scheme is as follows:
the invention provides a genetically engineered bacterium, wherein a target gene is inserted into an episomal plasmid; alternatively, the gene of interest is inserted into both the episomal plasmid and the genome of the host cell;
wherein the target gene inserted into the episomal plasmid is mainly composed of a trpBA gene and a trpB gene which are linked in this order; the target gene inserted into the genome is the trpBA gene;
the nucleotide sequence of the trpBA gene is shown in SEQ ID NO.1, and the nucleotide sequence of the trpB gene is shown in SEQ ID NO. 2.
The gene engineering bacteria of the present invention enhance the expression of the trpBA gene and the trpB gene as compared with unmodified bacteria by increasing the copy number of the trpBA gene and the trpB gene or modifying the expression control sequences of the trpBA gene and the trpB gene.
Further, the host cell is Escherichia coli. Preferably, the host cell is e.coli BL21(DE 3).
Further, the molar ratio of alpha subunit and beta subunit of the tryptophan synthetase expressed by the genetic engineering bacteria is 1: 1.25 to 1.5. It has been unexpectedly found that the molar ratio of alpha subunit to beta subunit of tryptophan synthase can be changed by increasing the copy number of the trpBA gene and the trpB gene or modifying the expression control sequences of the trpBA gene and the trpB gene when the molar ratio of alpha subunit to beta subunit of tryptophan synthase is 1: 1.25-1.5, the enzyme conversion time can be effectively shortened, and the yield of cysteine is improved.
Further, when the target gene is inserted into an episomal plasmid, an SD sequence is also linked between the trpBA gene and the trpB gene; the nucleotide sequence of the SD sequence is shown as SEQ ID NO. 3.
Further, the free plasmid is PET-30 a; the integration site of the target gene inserted into the host cell genome is a Tyrp gene, a pstG gene or an Icd gene.
The invention also provides a gene engineering bacterium, which is named as Escherichia coli (Escherichia coli) Nhuicd A3, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.21194, the preservation date is 11 and 16 days in 2020, and the preservation address is No.3 of West Lu 1 in North Cheng of the rising area in Beijing.
The invention also provides the application of the genetically engineered bacterium in the production of L-cysteine.
The invention also provides a preparation method of the L-cysteine, which comprises the following steps:
(1) culturing the genetically engineered bacteria, inducing enzyme expression, and preparing wet bacteria or crude enzyme solution of the genetically engineered bacteria;
(2) adding L-serine and a sulfur-containing compound into the wet thallus or the crude enzyme solution in the step (1) to carry out enzymatic reaction to obtain L-cysteine;
the molar ratio of alpha subunit and beta subunit of the tryptophan synthetase expressed in the wet thallus or the crude enzyme solution is 1: 1.25 to 1.5;
(3) oxidizing the L-cysteine in the step (2) and separating the oxidized L-cysteine from the reaction liquid to obtain L-cystine; the L-cystine is reduced by electrolysis to obtain the L-cysteine.
Further, the culture medium formula for culturing the genetically engineered bacteria comprises a carbon source and a nitrogen source;
the carbon source is at least one of glucose, maltose, sucrose and fructose; taking the culture medium as a reference, wherein the mass concentration of the total carbon source is 1-20 g/L; the nitrogen source is at least one of beef extract, yeast extract, corn steep liquor, peptone and soybean cake hydrolysate; the mass concentration of the total nitrogen source is 1-30 g/L based on the culture medium.
Further, the source of the L-serine includes, but is not limited to, keratin hydrolysate, silk hydrolysate, reaction liquid for synthesizing the L-serine by an enzyme method, and fermentation liquid for synthesizing the L-serine by a fermentation method.
Further, the sulfur-containing compound is at least one of sodium hydrosulfide, sodium sulfide, ammonium hydrosulfide, ammonium sulfide and hydrogen sulfide; the molar ratio of the sulfur-containing compound to the serine is 0.9-1.2: 1.
preferably, the temperature of the enzymatic reaction is 25-40 ℃, and the pH value of the reaction solution is 7.5-9.
Preferably, the mass ratio of the wet cells or the crude enzyme solution to the L-serine is 0.25-0.5: 1; the molar ratio of the sodium hydrosulfide to the L-serine is 1-1.2: 1.
The invention also provides a more specific process for preparing cysteine by an enzyme method, which takes fermentation liquor and sodium hydrosulfide for synthesizing L-serine by a fermentation method as an example:
filtering the fermentation liquor of serine to obtain a fermentation supernatant, adding wet thalli or crude enzyme liquid, carrying out catalytic reaction with the fermentation liquor (sodium hydrosulfide is required to be added into the system at the temperature of 37 ℃ and the pH value of between 7 and 9), oxidizing the fermentation liquor by air (the pH value is 8.5 to 9 and the temperature is 60 ℃), adjusting the pH value to 11, removing thalli through a ceramic membrane, adding hydrochloric acid to adjust the pH value to about 5, obtaining crude cystine through centrifugal separation, dissolving the crude cystine through hydrochloric acid, further decoloring and sterilizing the solution, adding acid to crystallize the crude cystine to obtain A4 hydrochloride, and entering an electrolysis link. Finally, crystallizing to obtain the L-cysteine monohydrate hydrochloride.
Compared with the prior art, the invention has the following beneficial effects:
(1) the trpBA gene and the trpB gene are inserted into a host cell to obtain the trpBA gene and trpB gene over-expressed gene engineering bacteria, and the engineering bacteria can express alpha subunit and beta subunit in a specific molar ratio, so that the reaction for synthesizing L-cysteine by an enzymatic method has higher catalytic rate and conversion rate, wherein the enzymatic conversion time is shortened to 2-8h, and the molar conversion rate of L-serine reaches more than 95%.
(2) The invention also provides a preparation method of the L-cysteine, which does not need to add pyridoxal phosphate and a surfactant.
(3) The invention improves the utilization efficiency of wet thalli or crude enzyme liquid.
(4) According to the invention, the target gene is integrated into the genome of the host cell, so that the biomass of the host cell is increased by 15-16%.
Drawings
FIG. 1 is a schematic diagram showing the structure of the final expression vector B (PET-30a-TrpBA-SD-TrpB) in example 1.
FIG. 2 is a schematic structural diagram of the Ptarget-Tyrp sgRNA expression vector in example 2.
FIG. 3 is a schematic diagram of the structure of vector A used for cloning a targeting fragment in example 2.
Detailed Description
The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.
The vectors referred to in the following examples are: PET-30a commercial vector, Crispr-Cas9 plasmid system (P-target, P-Cas); the sequences used were the native TrpBA and TrpB genes of E.coli BL-21.
EXAMPLE 1 preparation of genetically engineered bacterium E.coli BL21(DE3)/PET-30a-TrpBA-SD-TrpB in which target Gene is overexpressed in plasmid
1. Construction of an overexpression plasmid
Constructing a strategy: the TrpBA gene (the nucleotide sequence is shown as SEQ ID NO. 1) is inserted into a multi-cloning site of the PET-30a plasmid, and is connected in series with an SD sequence (the nucleotide sequence is shown as SEQ ID NO. 3) from other commercial vectors, and the TrpB gene (the nucleotide sequence is shown as SEQ ID NO. 2) in the natural TrpBA gene is added behind the SD sequence to carry out tandem expression of the TrpBA and the TrpB. The promoter is induced by T7 promoter, and the inducer is IPTG or lactose.
The method comprises the following specific steps:
firstly, a genome of an Escherichia coli K-12 strain (including a cereal bar genome) is extracted by using a genome extraction kit, and a TrpBA whole gene sequence with the length of 2000bp (shown as SEQ ID NO. 1) is cloned in the Escherichia coli K-12 genome. Designing a primer 1(trpB-F-BamH1) and a primer 2(trpA-RSD-EcoR1) according to the nucleotide sequence shown in SEQ ID NO.1, introducing BamHI and EcoRI cutting sites into the primers, carrying out amplification, cutting the primers 2 with an element SD sequence expressed in tandem, using BamHI and EcoRI to cut, and connecting the primers to a PET-30a vector to obtain a vector A.
Primer 1(trpB-F-BamH1) F: CGGGATCCATGACAACATTACTTAACCCCTATTTTGGTGAG, respectively;
primer 2(trpA-RSD-EcoR1) R: CGGAATTCATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTTAACTGCGCGTCG, respectively;
then, the TrpB sequence was cloned with primer 3(trpB-F-EcoR1) and primer 4(trpB-R-Xho1), digested with EcoRI and Xho I, ligated into vector A, resulting in the final vector B, i.e.: PET-30a-TrpBA-SD-TrpB (shown in FIG. 1).
2. Preparation of competent cells
(1) Taking out the preserved BL-21 strain from a refrigerator at the temperature of-80 ℃, dipping the strain liquid by using an inoculating loop, streaking on an LB solid culture medium, and culturing in an incubator at the temperature of 37 ℃ overnight.
(2) The next day, a single colony was picked from the LB solid medium and placed in LB medium, and cultured overnight at 37 ℃ (about 16 h).
(3) Transferring the bacterial liquid into a fresh 50mL LB liquid culture medium according to the ratio of 1: 100-1: 50, carrying out shaking amplification culture at 37 ℃, measuring OD600 every 20-30 min after the culture liquid begins to be turbid, and stopping culture when the OD600 value is 0.3-0.5;
(4) transferring the bacterial liquid into a centrifugal tube precooled on ice, carrying out ice bath for 30min, and centrifuging for 10min at 4000r/min at 4 ℃.
(5) Discard the supernatant and add 1mL of precooled CaCl2The solution gently suspends the cells, and the cells are centrifuged at 4000r/min for 10min at 4 ℃.
(6) Repeating the step (4) once.
(7) The supernatant was discarded and 100. mu.L of precooled CaCl was added2The solution, carefully suspended cells, i.e. made competent cell suspension.
(8) The prepared competent cell suspension can be directly used for transformation experiments, or can be added with equal volume of 20% glycerol (sterilized), mixed uniformly and then subpackaged in a 1.5mL centrifuge tube and stored at-80 ℃.
3. Preparation of genetically engineered bacteria
Storing competent cells of Escherichia coli BL21(DE3) at-80 deg.C in ice bath at 0 deg.C for 10min, and dissolving on ice; sucking about 1 mu L of PET-30a-TrpBA-SD-TrpB vector plasmid after sequencing verification, adding the plasmid into a competent liquid, gently mixing uniformly, placing on ice for 30min, carrying out heat shock on the plasmid for 90s in a water bath at 42 ℃, transferring to an ice bath for 5min, adding 1mL of LB culture medium, allowing the plasmid to recover for 45min in a shaking table at 37 ℃, sucking 50 mu L of the plasmid, plating the plasmid in a solid LB culture medium, carrying out overnight culture (16h) at 37 ℃, and selecting a single clone to culture to finally obtain the genetically engineered bacterium E.coli BL21(DE3)/PET-30 a-TrpBA-SD-TrpB.
Example 2 preparation of genetically engineered bacterium E.coli BL21(DE3)/Cas9 BAB with target Gene integrated into genome
Constructing a strategy: extracting the genome of Escherichia coli K-12 strain by using genome extraction kit, cloning the front and rear homology arms of T primer yrp with primer V and primer VI, and primer VII and VIII, respectively having lengths of 647bp and 456bp, cloning TrpBA whole gene sequence (2000bp) in Escherichia coli K-12 genome with primer I and primer II (primer I contains T7 promoter), performing enzyme digestion with corresponding enzyme, and then connecting into PET-30a vector according to the front homology arm, T7 promoter and TrpBA gene, to obtain vector A' (shown in figure 3), and cloning the targeting fragment with a length of 3100bp by using primer IX and primer X.
The method comprises the following specific steps:
(1) designing primers (a primer I and a primer II), extracting an escherichia coli genome, and cloning a TrpBA gene on the genome by using a primer PCR (polymerase chain reaction) as shown in SEQ ID No. 1;
primer I (trpB-F-EcoRI) F: CCGGAATTCTAATACGACTCACTATAGGATGACAACATTACTTAACCCCTATTTTGGTGAG, respectively;
primer II (trpA-R-HindIII) R: CCCAAGCTTTAACTGCGCGTCGCCGCTTTCATC are provided.
(2) Determining gene integration site Tyrp (or pstG or Icd gene can be selected) (fermentation OD value can be increased after knockout), designing P-target primer gRNA (primer III, primer IV), cloning the gRNA into a vector through circular PCR, transforming into escherichia coli, and extracting plasmid (shown in figure 2);
primer III (Tyrp gRNA) F: GGAGGTGTACCAGCATGTTCGTTTTAGAGCTAGAAATAGCAAG;
primer IV (Tyrp gRNA) R: GAACATGCTGGTACACCTCCACTAGTATTATACCTAGGACTGAGC.
(3) Designing a targeting segment: PCR cloning a front homologous arm (647bp) (a primer V and a primer VI) and a rear homologous arm (456bp) (a primer VII and a primer VIII) of a Tyrp integration site on an escherichia coli genome, connecting the front homologous arm, a T7 promoter and a TrpBA gene and the rear homologous arm into a PET30A plasmid through enzyme digestion connection, cloning a targeting fragment through a primer IX and a primer X, cutting glue and recovering to obtain a high-concentration PCR fragment;
primer V (previous homology arm Tyrp BamHI) F: CGCGGATCCAAGAACTCTCTACCCTCGACACAC, respectively;
primer VI (pro homology arm tyrp EcoRI) R: CCGGAATTCGTGTAGCACATCAACGCCCAAAGC, respectively;
primer VII (rear homology arm Tyrp HindIII) F: CCCAAGCTTAGATACCGGTCTTGGCACGCTG, respectively;
primer VIII (rear homology arm tyrp XhoI) R: CCGCTCGAGAACACCCAGCGTAGCTTACGAAC, respectively;
primer IX (targeting primer F): AAGAACTCTCTACCCTCGACACAC, respectively;
primer X (targeting primer R): AACACCCAGCGTAGCTTACGAAC are provided.
(4) Preparing competence: transforming 1 μ L of P-Cas9 plasmid (temperature sensitive, Kan resistant; Kan working concentration is 50mg/L) into competent Escherichia coli BL-21, coating Kan plate, and culturing at 30 deg.C for 12 h; selecting a single colony, inoculating the single colony in a 5mL LB test tube (containing Kan), and culturing for 12h at 30 ℃; inoculating the seed solution into a 50mL LB liquid shake flask according to the inoculation amount of 1%, culturing at 30 ℃ until OD is 0.1-0.15, and adding L-arabinose (final concentration: 20-30 mM). Then culturing at 30 ℃ until OD is 0.6; the E.coli was then made competent.
The specific method for making escherichia coli competent comprises the following steps: individual clones were picked, inoculated into 5mL of LB medium containing Kan, and cultured overnight at 30 ℃. The cultured strain was inoculated in 50mL of LB medium containing Kan at 1% on the next day, and shake-cultured at 30 ℃ until OD 600. apprxeq.0.1. (about 50 min). Inducer L-arabinose (final concentration: 20-30mM) was added, and the culture was continued until OD 600. apprxeq.0.6. And attention is paid to timely measuring the OD value, so that the OD value is prevented from being too high. (about 1 h) the culture was transferred to a 50mL centrifuge tube (25mL) and cooled in ice bath for 10 min. The competent cells were kept close to 0 ℃ in the subsequent steps. All centrifuge tubes need to be pre-chilled on ice before adding cells. Centrifuge at 4 ℃ 3000g for 10min and discard the supernatant. The liquid is removed as clean as possible, and part of the cells can be lost in the removal process. 15mL of ice-cold calcium chloride solution was added and the cells were resuspended on ice. And (5) uniformly mixing. Centrifuge at 4 ℃ 3000g for 10min and discard the supernatant. 2mL of ice-cold 15% glycerol calcium chloride solution was added, and the cells were resuspended in ice and 200. mu.L of the suspension was dispensed. Storing at-80 deg.C.
(5) Gene integration: pTarget-Tyrp (spectinomycin resistance; working concentration 50mg/L) plasmid (about 30ng, content requirement is not high) and targeting fragment (3100bp) are transferred into the above-mentioned Escherichia coli competent cells together.
(6) Removing the P-target plasmid: taking a positive monoclonal, inoculating the positive monoclonal into a 5mL LB test tube containing Kan liquid, adding IPTG (final concentration: 0.3mM), and culturing at 30 ℃ for 12 h; inoculating the cultured bacterial liquid into a 5mL LB test tube without the antibiotic liquid, and culturing at 37 ℃ for 12-24 h; coating a non-resistant LB plate, selecting single colonies, culturing each single colony on a Kan plate and a spectinomycin plate at the same time for 12h at 37 ℃.
(7) Selecting single clone, carrying out colony PCR verification, selecting the bacterium integrated with TrpBA to prepare competence, transferring the PET30A-TrpBA plasmid into over-expression, and carrying out fermentation verification.
Example 3 preparation of genetically engineered bacterium E.coli BL21(DE3)/PET BAB Cas9 BAB with target genes both overexpressed in plasmid and integrated into genome
The method of example 1 was used to prepare the plasmid PET-30A-TrpBA-SD-TrpB, the method of example 2 was used to prepare the bacterium integrating the TrpBA gene and PET-30A-TrpBA, the bacterium integrating the TrpBA gene and PET-30A-TrpBA was made competent, the plasmid PET-30A-TrpBA-SD-TrpB was transformed into the bacterium, and the co-expression bacterium, e.coli BL21(DE3)/PET BAB 9 BAB, which had both free expression of PET-30A-TrpBA-SD-TrpB and integration of the gene was obtained after culturing.
The inventor has deposited the strain, the name of the strain is Escherichia coli (Escherichia coli) Nhuicd A3, the preservation unit is China general microbiological culture Collection center, the preservation number is CGMCC No.21194, the preservation date is 11-16-11-2020-year, and the preservation address is No.3 of Xilu 1. Beijing, Chaoyang district, Beijing.
Example 4 preparation of Wet cells and crude enzyme solution
(1) E.coli BL21(DE3), the strain E.coli BL21(DE3)/PET-30a-TrpBA-SD-TrpB prepared in example 1, the integrated E.coli BL21(DE3)/Cas9 BAB strain prepared in example 2 and the E.coli BL21(DE3)/PET BAB Cas9 BAB co-expression strain prepared in example 3 are taken, inoculated to a seed culture medium for activation and then fermented and cultured.
(2) Seed culture medium: 10.0g of yeast extract powder, 16.0g of peptone, 5.0g of sodium chloride and 1.0g of glucose, adjusting the pH to 7.4, sterilizing at 121 ℃ for 20 min.
(3) Fermentation medium: 8.0-11 g of glucose, 4.0g of disodium hydrogen phosphate, 2.54g of monopotassium phosphate, 3.0g of sodium chloride, 2.5-4.0 g of ammonium sulfate, 2.1g of citric acid, 5.0g of yeast extract powder, 0.4-0.6 g of magnesium sulfate heptahydrate, 0.30g of ferrous sulfate heptahydrate, 0.5mL of foam killer and 1mL of antibiotic, adding water to a constant volume of 1L, adjusting the pH value to 7.0-7.4, and performing steam sterilization for 15-20 min.
(4) Controlling fermentation culture conditions: controlling the pressure of an air tank at 0.05 +/-0.002 MPa, pH7.0 +/-0.02, initially stirring at a rotating speed of 300rpm and dissolved oxygen of 20%, inoculating the seed solution into a fermentation tank according to an inoculation amount of 8%, initially culturing at a temperature of 37 ℃ until the OD600 value is 40-50, adjusting to 26-32 ℃, simultaneously adding an inducer to continue culturing for 16-18 h, wherein the induction concentration of IPTG is 0.3-1 mM, feeding sugar according to dissolved oxygen feedback, and adding the inducer at a constant speed of 20 g/h. .
(5) Preparation of wet cells: and (4) centrifuging the fermentation liquor obtained in the step (4), wherein the centrifugation conditions are as follows: and (3) the temperature is 3-8 ℃, the rotating speed is 4000-6000 rpm, the time is 40-60 min, and the supernatant is removed to obtain wet thalli which are stored at low temperature for later use. Detection method of biomass: the fermentation liquor of unit volume is centrifuged, and wet thalli obtained by removing supernatant is dried at 65 ℃ to constant weight.
(6) The detection method of enzyme activity comprises the following steps: 8g of pure serine, 0.5g of wet cells, and 10g of 47% sodium hydrosulfide solution were weighed out, and pure water was added to the weighed total amount of 100 g. Stirring and heating to 37 ℃ to start reaction; taking 0.1g of reaction solution after 1 hour, adding 50g of phosphoric acid aqueous solution with the pH value of 2.7 for dilution, filtering a sample by using a 0.22 mu m microporous filter membrane, detecting the content of cysteine by using a high performance liquid chromatography, and calculating the enzyme activity. The enzyme activity was defined as the millimole of cysteine produced per gram of wet cells per hour in U under the above conditions.
(7) Preparation of crude enzyme solution: and (3) washing the wet bacteria collected in the step (5) for 1-3 times by using pure water or PBS (pH 7.4) buffer solution, resuspending, placing the centrifugal tube filled with the bacteria suspension in an ice bath, and performing ultrasonic crushing under the condition that an ultrasonic instrument probe is placed 1cm below the liquid level, the power is 455W, the ultrasonic is carried out for 4s, the interval is 4s, the total time is 30-40 min, and the temperature is 0-4 ℃. The disrupted cell solution was centrifuged at 12000rpm at 4 ℃ for 15min to remove insoluble cell debris, and the supernatant was collected as a crude enzyme solution.
(8) The detection method of the subunit ratio comprises the following steps: diluting the crude enzyme solution to OD (OD) of 3-5, mixing with 5-O-Adingbuffer according to a ratio of 1:4, standing at 100 ℃ for 10min, cooling, centrifuging at 12000rpm, taking 20 mu L of supernatant, running SDS-PAGE protein gel, electrically transferring the protein onto an NC membrane, dyeing with ponceau after membrane transfer is successful, washing with deionized water for 3-4 times until the red color is basically faded, and taking a picture. And finally, importing the picture into Image J software for gray value calculation. Molar ratio-grey value ratio/subunit molecular weight size ratio. The molar ratio of the alpha subunit to the beta subunit was calculated based on the gray scale values and the results are shown in the following table.
Example 5 Effect of molar ratio of alpha subunit to beta subunit on enzyme Activity
Purification process of α and β subunits: pretreatment of the affinity column: the preservation solution containing 1mL of his. bind resin medium was filled into Econo-eolun m Chromatography column, after the resin had completely precipitated, 20% alcohol in the column was first eluted, 10 column volumes (10mL) of deionized water were added to wash off the residual alcohol, and after the deionized water had completely eluted, 10 column volumes of binding buffer were added to equilibrate the column.
(1) Purification of TRPSA: 5mL of the supernatant of example 4(7) was added to the pretreated affinity column and allowed to flow out slowly and naturally; 10mL of binding buffer (50mM NaH) was used first2PO4300mM NaCI, 10mM imidazole, pH8.0) to elute unbound hetero-proteins; a further 10mL of rinsing buffer A (50mM NaH) was used2PO4300mM NaCl, 50mM imidazole, pH8.0) through the column to elute the hetero-protein non-specifically bound to the affinity column; then 5mL of rinsing buffer A and 5mL of rinsing buffer B (50mM NaH) were used2PO4300mM NaCI, 70mM imidazole, pH8.0) to elute the hetero-protein non-specifically bound to the affinity column; then 4mL of rinsing buffer B is used for passing through the column to elute the hybrid protein which is not specifically combined with the affinity column; finally, 5mL of elution buffer (50mM NaH) was used2PO4300mM NaCl, 300mM imidazole, pH8.0) to elute the protein of interest. The collected target protein solution was filled into a dialysis bag and dialyzed against 5mM Tris & HCl buffer solution of pH7.9 to remove salts. The protein concentration after dialysis was determined according to the Bradford method. And (3) ultrafiltration concentration: centrifuging at 4000rpm and 4 deg.C, removing impurities with 10KD filter membrane, and collecting protein under the filter membrane. Finally, concentrate to 1.5mL protein.
(2) Purification of TRPSB: the removal of the contaminating proteins was performed as above, and the desired protein was eluted with 5mL of elution buffer. The collected target protein solution was filled into a dialysis bag and dialyzed against 5mM Tris.HCl buffer solution of pH7.9 to remove salts. The protein concentration after dialysis was determined according to the Bradford method. And (3) ultrafiltration concentration: centrifuging at 4000rpm and 4 deg.C, removing impurities with 30KD filter membrane, and collecting protein under the filter membrane. Finally, concentrate to 1mL protein. (the concentration of the alpha subunit is 46.38ug/mL, and the concentration of the beta subunit is 96.03ug/mL)
0.08g of pure serine, 320ul of beta-subunit enzyme solution and 0.1g of 47% sodium hydrosulfide solution are weighed, alpha subunits with the beta subunit molar numbers of 0.4, 0.6, 1, 1.2 and 1.4 times are respectively added, and then pure water is respectively added to the total weight of 1 g. After reacting for 1h at 37 ℃, 0.1g of reaction solution is taken, 50g of phosphoric acid aqueous solution with pH value of 2.7 is added for dilution, a sample is filtered by a 0.22 mu m microporous filter membrane, and the cysteine content is detected by high performance liquid chromatography.
Molar ratio of alpha subunit to beta subunit | Relative content of cysteine |
1:0.4 | 88% |
1:0.6 | 92% |
1:0.8 | 95% |
1:1 | 100% |
1:1.2 | 120% |
1:1.4 | 116% |
1:1.6 | 112% |
EXAMPLE 5 Synthesis of cysteine catalyzed by genetically engineered bacterium E.coli BL21(DE3)/PET-30a-TrpBA-SD-TrpB
The crude enzyme solution of the genetically engineered bacterium E.coli BL21(DE3)/PET-30a-TrpBA-SD-TrpB prepared in example 4 is taken, the crude enzyme solution is added according to the proportion of 8g L-serine: 3.4g wet thalli, the addition amount of 40% sodium hydrosulfide is 12g, the volume of constant volume is 100mL, the catalytic reaction process is maintained at 37 ℃, the process is required to be stirred, the reaction time is 3 hours, the conversion rate of serine is 99% after the reaction is finished, the yield of cysteine is 90%, then the temperature is increased to 90 ℃, the temperature is kept for 15min, and the wet thalli are inactivated. Aerating and oxidizing to obtain a cystine suspension, adding a sodium hydroxide solution, adjusting the pH to 11, removing bacterial residues through a membrane or centrifugation, adding hydrochloric acid to adjust the pH to about 5, crystallizing to obtain a cystine solid, dissolving the solid with concentrated hydrochloric acid, and electrolyzing to obtain cysteine monohydrate hydrochloride.
Example 6 Synthesis of L-cysteine catalyzed by genetically engineered bacterium E.coli BL21(DE3)/Cas9 BAB
The crude enzyme solution of the genetically engineered bacterium E.coli BL21(DE3)/Cas9 BAB prepared in example 4 is taken, the crude enzyme solution is added according to the proportion of 8g L-serine to 3.4g of wet bacteria, the adding amount of 40% sodium hydrosulfide is 12g, the volume of constant volume is 100mL, the catalytic reaction process is maintained at 37 ℃, the process is stirred, the reaction time is 4.5h, the reaction is finished by taking the serine conversion rate of 99% as an index, the cysteine yield is 85%, then the temperature is raised to 90 ℃, the temperature is kept for 15min, and the wet bacteria are inactivated. Aerating and oxidizing to obtain a cystine suspension, adding a sodium hydroxide solution, adjusting the pH to 11, removing bacterial residues through a membrane or centrifugation, adding hydrochloric acid to adjust the pH to about 5, crystallizing to obtain a cystine solid, dissolving the solid with concentrated hydrochloric acid, and electrolyzing to obtain cysteine monohydrate hydrochloride.
Example 7 genetically engineered bacterium E.coli BL21(DE3)/PET BAB Cas9 BAB catalyzed synthesis of L-cysteine
The crude enzyme solution of the genetically engineered bacterium E.coli BL21(DE3)/PET BAB Cas9 BAB prepared in example 4 is taken, wet bacteria are put in according to the proportion of 8g L-serine: 3.4g, the addition amount of 40% sodium hydrosulfide is 12g, the volume of constant volume is 100mL, the catalytic reaction process is maintained at 37 ℃, the process is stirred, the reaction time is 2h, the conversion rate of serine is 99% after the reaction is finished, the yield of cysteine is 98%, the temperature is raised to 90 ℃, the temperature is kept for 15min, and the wet bacteria are inactivated. Aerating and oxidizing to obtain a cystine suspension, adding a sodium hydroxide solution, adjusting the pH to 11, removing bacterial residues through a membrane or centrifugation, adding hydrochloric acid to adjust the pH to about 5, crystallizing to obtain cystine solid, dissolving the solid with concentrated hydrochloric acid, and electrolyzing to obtain 11.8g of cysteine monohydrate hydrochloride.
Example 8 genetically engineered bacterium E.coli BL21(DE3)/PET BAB Cas9 BAB catalyzed synthesis of cysteine
The crude enzyme solution of the genetically engineered bacterium E.coli BL21(DE3)/PET BAB Cas9 BAB prepared in example 4 is taken, wet bacteria are put in according to the proportion of 8g L-serine: 3.4g, the addition amount of 40% sodium hydrosulfide is 12g, the volume of constant volume is 100mL, the catalytic reaction process is respectively maintained at 20, 25, 30, 37 and 40 ℃, the process needs stirring, the reaction time is 2 hours, the conversion rate of serine after reaction is respectively 88%, 93%, 97%, 99% and 96%, the yield of cysteine reaches 84%, 90%, 93%, 94% and 91%, then the temperature is raised to 90 ℃ and the temperature is kept for 15min, and the wet bacteria are inactivated. Aerating and oxidizing to obtain a cystine suspension, adding a sodium hydroxide solution, adjusting the pH to 11, removing bacterial residues through a membrane or centrifugation, adding hydrochloric acid to adjust the pH to about 5, crystallizing to obtain a cystine solid, dissolving the solid with concentrated hydrochloric acid, and electrolyzing to obtain cysteine monohydrate hydrochloride.
Example 9 Synthesis of cysteine catalyzed by genetically engineered bacterium E.coli BL21(DE3)/PET BAB Cas9 BAB
The crude enzyme solution of the genetically engineered bacterium E.coli BL21(DE3)/PET BAB Cas9 BAB prepared in example 4 is taken, wet bacteria are added according to the proportion of 8g L-serine: 3.4g, the addition amount of 40% sodium hydrosulfide is 12g, the volume of constant volume is 100mL, the catalytic reaction process is maintained at 37 ℃, the reaction is carried out under the conditions of pH7.5, 8, 8.5 and 9 respectively, the process needs to be stirred, the reaction time is 2h, the serine conversion rate of the reaction end is 90%, 95%, 99% and 94%, the cysteine yield reaches 85%, 91%, 95% and 90%, and then the temperature is raised to 90 ℃ and the temperature is kept for 15min, and the wet bacteria are inactivated. Aerating and oxidizing to obtain a cystine suspension, adding a sodium hydroxide solution, adjusting the pH to 11, removing bacterial residues through a membrane or centrifugation, adding hydrochloric acid to adjust the pH to about 5, crystallizing to obtain a cystine solid, dissolving the solid with concentrated hydrochloric acid, and electrolyzing to obtain cysteine monohydrate hydrochloride.
Example 10 catalysis of genetically engineered bacterium E.coli BL21(DE3)/PET BAB Cas9 BAB to synthesize cysteine
Crude enzyme liquid of the genetically engineered bacterium E.coli BL21(DE3)/PET BAB Cas9 BAB prepared in example 4 is taken, wet thalli are put into the enzyme liquid according to the proportion of 8g L-serine to 3.4g, and the weight ratio is calculated according to the following steps: adding sodium hydrosulfide according to the molar ratio of 1:0.9, 1:1, 1:1.1 and 1:1.2, wherein the volume of constant volume is 100mL, the catalytic reaction process is maintained at 37 ℃, the process needs stirring, the reaction time is 2 hours, the serine conversion rate at the end of the reaction is 89%, 93%, 98% and 99%, the cysteine yield reaches 85%, 90%, 96% and 97%, then heating to 90 ℃, preserving the temperature for 15min, and inactivating wet bacteria. Aerating and oxidizing to obtain a cystine suspension, adding a sodium hydroxide solution, adjusting the pH to 11, removing bacterial residues through a membrane or centrifugation, adding hydrochloric acid to adjust the pH to about 5, crystallizing to obtain a cystine solid, dissolving the solid with concentrated hydrochloric acid, and electrolyzing to obtain cysteine monohydrate hydrochloride.
Example 11 Synthesis of cysteine catalyzed by genetically engineered bacterium E.coli BL21(DE3)/PET BAB Cas9 BAB
The crude enzyme solution of the genetically engineered bacterium E.coli BL21(DE3)/PET BAB Cas9 BAB prepared in example 4 is taken, wet bacteria are put in according to the proportion of 8g L-serine: 2.0g, 3.4g and 4.0g, the addition amount of 40% sodium hydrosulfide is 12g, the volume of constant volume is 100mL, the catalytic reaction process is maintained at 37 ℃, the process is stirred, the reaction time is 2 hours, the conversion rates of serine after reaction are respectively 93%, 98% and 99%, the yield of cysteine reaches 87%, 96% and 97%, then the temperature is raised to 90 ℃, the temperature is kept for 15min, and the wet bacteria are inactivated. Aerating and oxidizing to obtain a cystine suspension, adding a sodium hydroxide solution, adjusting the pH to 11, removing bacterial residues through a membrane or centrifugation, adding hydrochloric acid to adjust the pH to about 5, crystallizing to obtain a cystine solid, dissolving the solid with concentrated hydrochloric acid, and electrolyzing to obtain cysteine monohydrate hydrochloride.
Sequence listing
<110> Zhejiang New Hecheng composition Ltd
SHANGYU NHU BIOCHEMICAL INDUSTRY Co.,Ltd.
HEILONGJIANG XINHECHENG BIOTECHNOLOGY Co.,Ltd.
<120> genetically engineered bacterium, preparation method thereof and application thereof in cysteine production
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2000
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgacaacat tacttaaccc ctattttggt gagtttggcg gcatgtacgt gccacaaatc 60
ctgatgcctg ctctgcgcca gctggaagaa gcttttgtca gtgcgcaaaa agatcctgaa 120
tttcaggctc agttcaacga cctgctgaaa aactatgccg ggcgtccaac cgcgctgacc 180
aaatgccaga acattacagc cgggacgaac accacgctgt atctcaagcg tgaagatttg 240
ctgcacggcg gcgcgcataa aactaaccag gtgctggggc aggcgttgct ggcgaagcgg 300
atgggtaaaa ccgaaatcat cgccgaaacc ggtgccggtc agcatggcgt ggcgtcggcc 360
cttgccagcg ccctgctcgg cctgaaatgc cgtatttata tgggtgccaa agacgttgaa 420
cgccagtcgc ctaacgtttt tcgtatgcgc ttaatgggtg cggaagtgat cccggtgcat 480
agcggttccg cgacgctgaa agatgcctgt aacgaggcgc tgcgcgactg gtccggtagt 540
tacgaaaccg cgcactatat gctgggcacc gcagctggcc cgcatcctta tccgaccatt 600
gtgcgtgagt ttcagcggat gattggcgaa gaaaccaaag cgcagattct ggaaagagaa 660
ggtcgcctgc cggatgccgt tatcgcctgt gttggcggcg gttcgaatgc catcggcatg 720
tttgctgatt tcatcaatga aaccaacgtc ggcctgattg gtgtggagcc aggtggtcac 780
ggtatcgaaa ctggcgagca cggcgcaccg ctaaaacatg gtcgcgtggg tatctatttc 840
ggtatgaaag cgccgatgat gcaaaccgaa gacgggcaga ttgaagaatc ttactccatc 900
tccgccggac tggatttccc gtctgtcggc ccacaacacg cgtatcttaa cagcactgga 960
cgcgctgatt acgtgtctat taccgatgat gaagcccttg aagccttcaa aacgctgtgc 1020
ctgcacgaag ggatcatccc ggcgctggaa tcctcccacg ccctggccca tgcgttgaaa 1080
atgatgcgcg aaaacctgga taaagagcag ctactggtgg ttaacctttc cggtcgcggc 1140
gataaagaca tcttcaccgt tcacgatatt ttgaaagcac gaggggaaat ctgatggaac 1200
gctacgaatc tctgtttgcc cagttgaagg agcgcaaaga aggcgcattc gttcctttcg 1260
tcacgctcgg tgatccgggc attgagcagt cattgaaaat tatcgatacg ctaattgaag 1320
ccggtgctga cgcgctggag ttaggtatcc ccttctccga cccactggcg gatggcccga 1380
cgattcaaaa cgccactctg cgcgcctttg cggcaggtgt gactccggca caatgttttg 1440
aaatgctggc actgattcgc cagaaacacc cgaccattcc cattggcctg ttgatgtatg 1500
ccaatctggt gtttaacaaa ggcattgatg agttttatgc ccagtgcgaa aaagtcggcg 1560
tcgattcggt gctggttgcc gatgtgccag ttgaagagtc cgcgcccttc cgccaggccg 1620
cgttgcgtca taatgtcgca cctatcttca tctgcccgcc aaatgccgat gacgacctgc 1680
tgcgccagat agcctcttac ggtcgtggtt acacctattt gctgtcacga gcaggcgtga 1740
ccggcgcaga aaaccgcgcc gcgttacccc tcaatcatct ggttgcgaag ctgaaagagt 1800
acaacgctgc acctccattg cagggatttg gtatttccgc cccggatcag gtaaaagcag 1860
cgattgatgc aggagctgcg ggcgcgattt ctggttcggc cattgttaaa atcatcgagc 1920
aacatattaa tgagccagag aaaatgctgg cggcactgaa agtttttgta caaccgatga 1980
<210> 2
<211> 1194
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgacaacat tacttaaccc ctattttggt gagtttggcg gcatgtacgt gccacaaatc 60
ctgatgcctg ctctgcgcca gctggaagaa gcttttgtca gtgcgcaaaa agatcctgaa 120
tttcaggctc agttcaacga cctgctgaaa aactatgccg ggcgtccaac cgcgctgacc 180
aaatgccaga acattacagc cgggacgaac accacgctgt atctcaagcg tgaagatttg 240
ctgcacggcg gcgcgcataa aactaaccag gtgctggggc aggcgttgct ggcgaagcgg 300
atgggtaaaa ccgaaatcat cgccgaaacc ggtgccggtc agcatggcgt ggcgtcggcc 360
cttgccagcg ccctgctcgg cctgaaatgc cgtatttata tgggtgccaa agacgttgaa 420
cgccagtcgc ctaacgtttt tcgtatgcgc ttaatgggtg cggaagtgat cccggtgcat 480
agcggttccg cgacgctgaa agatgcctgt aacgaggcgc tgcgcgactg gtccggtagt 540
tacgaaaccg cgcactatat gctgggcacc gcagctggcc cgcatcctta tccgaccatt 600
gtgcgtgagt ttcagcggat gattggcgaa gaaaccaaag cgcagattct ggaaagagaa 660
ggtcgcctgc cggatgccgt tatcgcctgt gttggcggcg gttcgaatgc catcggcatg 720
tttgctgatt tcatcaatga aaccaacgtc ggcctgattg gtgtggagcc aggtggtcac 780
ggtatcgaaa ctggcgagca cggcgcaccg ctaaaacatg gtcgcgtggg tatctatttc 840
ggtatgaaag cgccgatgat gcaaaccgaa gacgggcaga ttgaagaatc ttactccatc 900
tccgccggac tggatttccc gtctgtcggc ccacaacacg cgtatcttaa cagcactgga 960
cgcgctgatt acgtgtctat taccgatgat gaagcccttg aagccttcaa aacgctgtgc 1020
ctgcacgaag ggatcatccc ggcgctggaa tcctcccacg ccctggccca tgcgttgaaa 1080
atgatgcgcg aaaacctgga taaagagcag ctactggtgg ttaacctttc cggtcgcggc 1140
gataaagaca tcttcaccgt tcacgatatt ttgaaagcac gaggggaaat ctga 1194
<210> 3
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aataattttg tttaacttta agaaggagat atacat 36
<210> 4
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cgggatccat gacaacatta cttaacccct attttggtga g 41
<210> 5
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cggaattcat gtatatctcc ttcttaaagt taaacaaaat tattttaact gcgcgtcg 58
<210> 6
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ccggaattct aatacgactc actataggat gacaacatta cttaacccct attttggtga 60
g 61
<210> 7
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
cccaagcttt aactgcgcgt cgccgctttc atc 33
<210> 8
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ggaggtgtac cagcatgttc gttttagagc tagaaatagc aag 43
<210> 9
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gaacatgctg gtacacctcc actagtatta tacctaggac tgagc 45
<210> 10
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cgcggatcca agaactctct accctcgaca cac 33
<210> 11
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ccggaattcg tgtagcacat caacgcccaa agc 33
<210> 12
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cccaagctta gataccggtc ttggcacgct g 31
<210> 13
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ccgctcgaga acacccagcg tagcttacga ac 32
<210> 14
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
aagaactctc taccctcgac acac 24
<210> 15
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
aacacccagc gtagcttacg aac 23
Claims (10)
1. A gene engineering bacterium comprises a host cell and a target gene transferred into the host cell, and is characterized in that the target gene is inserted into an episomal plasmid; alternatively, the gene of interest is inserted into both the episomal plasmid and the genome of the host cell;
wherein the target gene inserted into the episomal plasmid is mainly composed of a trpBA gene and a trpB gene which are linked in this order; the target gene inserted into the genome is the trpBA gene;
the nucleotide sequence of the trpBA gene is shown in SEQ ID NO.1, and the nucleotide sequence of the trpB gene is shown in SEQ ID NO. 2.
2. The genetically engineered bacterium of claim 1, wherein the host cell is escherichia coli.
3. The genetically engineered bacterium of claim 1, wherein the molar ratio of the alpha subunit to the beta subunit of the tryptophan synthase expressed by the genetically engineered bacterium is 1: 1.25 to 1.5.
4. The genetically engineered bacterium of claim 1, wherein when the gene of interest is inserted into an episomal plasmid, an SD sequence is further ligated between the trpBA gene and the trpB gene; the nucleotide sequence of the SD sequence is shown as SEQ ID NO. 3.
5. A genetically engineered bacterium is named as Escherichia coli (Escherichia coli) Nhuicd A3, the preservation unit is China general microbiological culture Collection center, the preservation number is CGMCC No.21194, the preservation date is 11-16-month-2020, and the preservation address is No.3 of Xilu 1. Beijing, Chaoyang, and the Yangxi district, Beijing.
6. Use of the genetically engineered bacterium of any one of claims 1 to 5 in the production of L-cysteine.
7. A preparation method of L-cysteine is characterized by comprising the following steps:
(1) culturing the genetically engineered bacteria of any one of claims 1 to 5, inducing enzyme expression, and preparing wet cells or crude enzyme solutions of the genetically engineered bacteria;
(2) adding L-serine and a sulfur-containing compound into the wet thallus or the crude enzyme solution in the step (1) to carry out enzymatic reaction to obtain L-cysteine;
the molar ratio of alpha subunit and beta subunit of the tryptophan synthetase expressed in the wet thallus or the crude enzyme solution is 1: 1.25 to 1.5;
(3) oxidizing the L-cysteine in the step (2) and separating the oxidized L-cysteine from the reaction liquid to obtain L-cystine; the L-cystine is reduced by electrolysis to obtain the L-cysteine.
8. The method of claim 7, wherein the medium for culturing the genetically engineered bacteria comprises a carbon source and a nitrogen source;
the carbon source is at least one of glucose, maltose, sucrose and fructose; taking the culture medium as a reference, wherein the mass concentration of the total carbon source is 1-20 g/L; the nitrogen source is at least one of beef extract, yeast extract, corn steep liquor, peptone and soybean cake hydrolysate; taking the culture medium as a reference, wherein the mass concentration of the total nitrogen source is 1-30 g/L;
the source of the L-serine comprises, but is not limited to, keratin hydrolysate, silk hydrolysate, reaction liquid for synthesizing the L-serine by an enzyme method and fermentation liquid for synthesizing the L-serine by a fermentation method;
the sulfur-containing compound is at least one of sodium hydrosulfide, sodium sulfide, ammonium hydrosulfide, ammonium sulfide and hydrogen sulfide; the molar ratio of the sulfur-containing compound to the serine is 0.9-1.2: 1.
9. the method for producing L-cysteine according to claim 7, wherein the temperature of the enzymatic reaction is 25 ℃ to 40 ℃ and the pH of the reaction solution is 7.5 to 9.
10. The method for producing L-cysteine according to claim 7, wherein the mass ratio of the wet cell or crude enzyme solution to L-serine is 0.25 to 0.5: 1; the molar ratio of the sodium hydrosulfide to the L-serine is 1-1.2: 1.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN117230050A (en) * | 2023-11-10 | 2023-12-15 | 北京量维生物科技研究院有限公司 | Tryptophan synthase and application of mutant thereof in production of cysteine and cystine |
WO2024013258A1 (en) * | 2022-07-15 | 2024-01-18 | Cysbio Aps | Production of cysteine or cystine from serine in fermentation medium |
Citations (1)
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CN102517352A (en) * | 2011-12-28 | 2012-06-27 | 南京大学 | Method for preparing L-cysteine through enzymatic conversion |
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2020
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102517352A (en) * | 2011-12-28 | 2012-06-27 | 南京大学 | Method for preparing L-cysteine through enzymatic conversion |
Non-Patent Citations (1)
Title |
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赵静静: "结核分枝杆菌H27Rv色氨酸合成酶α和β亚基的酶学性质及结构分析", 《复旦大学硕士学位论文》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2024013258A1 (en) * | 2022-07-15 | 2024-01-18 | Cysbio Aps | Production of cysteine or cystine from serine in fermentation medium |
CN117230050A (en) * | 2023-11-10 | 2023-12-15 | 北京量维生物科技研究院有限公司 | Tryptophan synthase and application of mutant thereof in production of cysteine and cystine |
CN117230050B (en) * | 2023-11-10 | 2024-01-26 | 北京量维生物科技研究院有限公司 | Tryptophan synthase and application of mutant thereof in production of cysteine and cystine |
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