CN110452891B - Penicillium expansum cis-epoxy succinate hydrolase gene and application thereof - Google Patents

Abstract

A penicillium expansum cis-epoxy succinate hydrolase gene and application thereof, belonging to the technical field of biological engineering. The invention provides a penicillium expansum cis-epoxy succinate hydrolase polypeptide and a coding gene thereof on one hand, and provides application of the coding gene in producing L (+) -tartaric acid or salts thereof on the other hand.

Description

Penicillium expansum cis-epoxy succinate hydrolase gene and application thereof

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to a penicillium expansum cis-form epoxy succinate hydrolase gene and application thereof.

Background

Tartaric acid, an alpha-carboxylic acid, also known as 2, 3-dihydroxysuccinic acid or 2, 3-dihydroxysuccinic acid. In 1769, the Swedish chemist Carl Wilhelm Scheele originally found the presence of L (+) -tartaric acid in the leftover crude tartaric acid of wine, hence the name "tartaric acid". L (+) -tartaric acid is widely existed in nature, so it is also called "natural tartaric acid", especially the content of tamarind fruit and grape is high, it is important food emulsifier, beverage acid agent, medicine resolving agent, gypsum retarder, dyeing and printing resist agent, photographic developer and metal polishing agent, and can be extensively used in food industry, medicine chemical industry and building industry. The microbial conversion method is the mainstream method for industrially producing the L (+) -tartaric acid at present, namely, maleic anhydride is taken as a raw material, the cis-epoxy succinic acid or the salt thereof is obtained through hydrolysis and epoxidation, and then the cis-epoxy succinic acid or the salt thereof is biologically catalyzed by a microorganism containing cis-epoxy succinic acid hydrolase to generate the L (+) -tartaric acid or the salt thereof.

Among the microorganisms that have been reported to be useful for producing L (+) -tartaric acid or a salt thereof are: nocardia (Nocardia), Corynebacterium (Corynebacterium), Rhodococcus (Rhodococcus), Rhizobium (Rhizobium), Pseudomonas (Pseudomonas), Achromobacter (Achromobacter), Acetobacter (Acetobacter), Agrobacterium (Agrobacterium), Alcaligenes (Alcaligenes), Acinetobacter (Acinetobacter), Klebsiella (Klebsiella), and Bacillus bifidus (Labrys). Wherein, the gene sequence of cis-epoxy succinic acid hydrolase derived from Nocardia, Rhodococcus and Klebsiella and the coded amino acid sequence thereof are reported, and a genetic engineering bacterium is constructed for producing L (+) -tartaric acid or salts thereof. The reported strains for producing the L (+) -tartaric acid or the salt thereof are all bacteria, and the L (+) -tartaric acid or the salt thereof is not reported to be produced by using fungi.

Generally, the method for producing the L (+) -tartaric acid by the microbial transformation method has the characteristics of good stereospecificity of enzyme, quick enzymatic reaction, high optical purity and yield of products, simple and easy separation and purification of the products and the like. However, since the enzyme system inherent in the microorganism is very complex, the expression level of cis-epoxysuccinate hydrolase is not high, and the activity of the cis-epoxysuccinate hydrolase is also restricted by various factors inside and outside the cell, the production efficiency of the L (+) -tartaric acid is low. The gene engineering technology is utilized to construct the gene engineering bacteria with the cis-form epoxy succinic acid hydrolase gene, so that the high-efficiency expression of the cis-form epoxy succinic acid hydrolase can be realized, and the method becomes the key for industrial production of the L (+) -tartaric acid.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to design and provide an Aspergillus niger cis-epoxy succinate hydrolase gene and a technical scheme for application thereof.

The invention is realized by the following technical scheme:

the penicillium expansum cis-epoxy succinate hydrolase polypeptide is characterized in that the amino acid sequence of the polypeptide is as follows:

1) an amino acid sequence shown as SEQ ID No. 2; or

2) The amino acid sequence shown in SEQ ID No.2 is formed by replacing, deleting and/or adding one or more amino acid residues to form an amino acid sequence with the same function.

The application of the penicillium expansum cis-epoxy succinate hydrolase polypeptide in the production of L (+) -tartaric acid or salts thereof.

The gene for coding the penicillium expansum cis-epoxy succinate hydrolase polypeptide is characterized in that the nucleotide sequence is as follows:

1) the nucleotide shown as SEQ ID No. 1; or

2) The nucleotide sequence shown in SEQ ID No.1 is substituted by one or more nucleotides to obtain a gene sequence for coding the penicillium expansum cis-epoxy succinate hydrolase polypeptide.

The coding gene is applied to the regulation and control of the application of the microorganism in the production of L (+) -tartaric acid or salts thereof.

The recombinant expression vector containing the gene for coding the penicillium expansum cis-epoxy succinate hydrolase polypeptide.

The recombinant microorganism strain for expressing the penicillium expansum cis-epoxy succinate hydrolase polypeptide.

The method for producing the L (+) -tartaric acid or the salt thereof utilizes the penicillium expansum cis-epoxy succinate hydrolase polypeptide to react with the cis-epoxy succinate or the salt thereof.

The method for producing the L (+) -tartaric acid or the salt thereof utilizes the recombinant microorganism strain to react with the cis-epoxy succinic acid or the salt thereof.

The method for producing the L (+) -tartaric acid or the salt thereof is characterized by comprising the following steps:

1) culturing the recombinant microorganism strain in a suitable medium;

2) immobilizing cells, cell extracts or cell-free extracts of the recombinant microorganism strains obtained in the step 1) or cis-epoxysuccinate hydrolase purified by the recombinant microorganism strains on a solid support or embedded in a fixed carrier;

3) reacting the recombinant microorganism strain obtained in step 1) or the preparation obtained in step 2) with cis-epoxysuccinic acid or a salt thereof under conditions suitable for hydrolysis to produce L (+) -tartaric acid or a salt thereof; and

4) optionally recycling the immobilized or embedded enzymes and/or cells.

The invention provides a penicillium expansum cis-epoxy succinate hydrolase polypeptide and a coding gene thereof on one hand, and provides application of the coding gene in producing L (+) -tartaric acid or salts thereof on the other hand.

Drawings

FIG. 1 shows the result of SDS-polyacrylamide gel electrophoresis of the recombinant engineering bacteria of example 4 of the present invention after IPTG induction.

Detailed Description

To further illustrate the technical means and effects of the present invention for achieving the predetermined objects, the following detailed description will be given to the genes encoding cis-epoxysuccinate hydrolase, the polypeptides encoded thereby and the related applications thereof, the specific embodiments, structures, characteristics and effects thereof according to the present invention, in conjunction with the preferred embodiments. The overall idea of the invention comprises the following points: separating and purifying cis-epoxy succinate hydrolase from a penicillium expansum fermentation broth, sequencing the N-terminal and the C-terminal of the cis-epoxy succinate hydrolase, designing degenerate primers according to the amino acid sequences of the N-terminal and the C-terminal, cloning a cis-epoxy succinate hydrolase gene from the penicillium expansum, constructing a prokaryotic expression vector, transforming escherichia coli, culturing the escherichia coli transformed with the cis-epoxy succinate hydrolase gene, and verifying the function of the cis-epoxy succinate hydrolase gene. The foregoing is a single embodiment, and further details are provided in the following description with reference to the figures.

Example 1: separation and purification of penicillium expansum cis-epoxy succinate hydrolase

The invention adopts the strain penicillium expansum (Penicillium expansum) WH-3, the strain is preserved in China general microbiological culture Collection center (CGMCC), the preservation registration number is CGMCC No.16798, and the strain is named as penicillium expansum (CGMCC)Penicillium expansum) WH-3, storage day: 03 h 12.2018, address of depository: beijing, Chaoyang, quarter Xilu No.1 Hospital (zip code: 100101).

Taking Penicillium expansum (Penicillium expansum) WH-3 is preserved in a PDA slant culture medium, and the preparation method of the slant culture medium comprises the following steps: weighing 200g of peeled fresh potatoes, cutting the potatoes into small pieces, putting the small pieces into a pot, adding 1000ml of water, heating to boil, and keeping for 30 min. Then the solution is filtered on a measuring cup by using double-layer gauze while the solution is hot, and the filtrate is left. Adding 20g of glucose and 15-20 g of agar, supplementing the filtrate to 1000ml, and autoclaving at 121 ℃ for 20 min. After the sterilization is finished, pouring the sterilized eggplant-shaped bottle on an ultra-clean bench sterilized by ultraviolet rays, sealing the eggplant-shaped bottle after the slant culture medium in the eggplant-shaped bottle is cooled and solidified, and standing the eggplant-shaped bottle indoors for one night. If no bacteria grow on the slant culture medium, placing the slant culture medium in a refrigerator at 4 ℃ for later use.

1: separation and purification of penicillium expansum cis-epoxy succinate hydrolase

(1) 5mL of spore suspension is added to a slant culture medium full of Penicillium expansum WH-3 dense spores, the spores are scraped by an inoculating shovel to be fully washed, and the mycelia are removed by filtering through sterile three-layer mirror wiping paper. The spore suspension formulation was 0.1% Tween80 and 0.9% NaCl.

(2) Diluting the filtered spore liquid, counting with a blood counting chamber according to 1X10⁶Spores were inoculated at a concentration of one/mL into a 250mL Erlenmeyer flask containing 50mL of seed mediumAnd performing shake culture at 25 ℃ and 150rpm for 24-36 h to obtain the penicillium expansum strain seed solution. The seed culture medium is a self-made PDB culture medium and the preparation method comprises the following steps: weighing 200g of peeled fresh potatoes, cutting the potatoes into small pieces, putting the small pieces into a pot, adding 1000ml of water, heating to boil, and keeping for 30 min. Then the solution is filtered on a measuring cup by using double-layer gauze while the solution is hot, and the filtrate is left. 10g of glucose was added and the filtrate was made up to 1000ml and autoclaved at 121 ℃ for 20 min.

(3) Inoculating the seed solution of the penicillium expansum 20mL into a conical flask 1000mL containing 200mL of enzyme-producing culture medium, and carrying out shake culture at 25 ℃ and 150rpm for 3 days to obtain corresponding penicillium expansum cells and fermentation liquor. The enzyme production culture medium is a yeast extract sucrose (YSM) culture medium, and the preparation method comprises the following steps: weighing 40g of yeast extract and 160g of sucrose, adding distilled water to reach the constant volume of 1000ml, and carrying out autoclaving at 121 ℃ for 20 min.

(4) Filtering to remove thallus to obtain fermentation liquid.

(5) Adding ammonium sulfate into the fermentation liquor to 35% saturation, centrifuging at 10000rpm for 20min, and collecting supernatant 1.

(6) The supernatant 1 was added with ammonium sulfate to 75% saturation, centrifuged at 10000rpm for 20nin, and the precipitate was collected and dissolved in a pre-cooled 0.1mol/L potassium phosphate buffer.

(7) Dialyzing at 4 ℃ in 0.1mol/L potassium phosphate buffer solution for 48h, and replacing the dialyzate for 3-4 times.

(8) The enzyme solution after dialysis is passed through DEAE-Sepharose column, and the effluent liquid from column chromatography has enzyme activity.

(9) Performing ultrafiltration concentration on the effluent of the column to obtain concentrated solution 1, subjecting the concentrated solution 1 to Pheny-Sepharose column, eluting with 0.1mol/L potassium phosphate buffer solution (pH8.0), collecting the effluent with enzyme activity, and performing ultrafiltration concentration to obtain concentrated solution 2.

(10) Passing the concentrated solution 2 through MonoQ HR5/5 column, eluting with 0.1mol/L potassium phosphate buffer solution (pH8.0), collecting eluate with enzyme activity, and ultrafiltering and concentrating to obtain concentrated solution 3.

(11) The concentrated solution 3 was subjected to SDS-polyacrylamide gel electrophoresis and stained with Coomassie Brilliant blue R250, and the result showed that the purified cis-epoxysuccinate hydrolase had a single band and a molecular weight of about 30kDa in the SDS-polyacrylamide gel electrophoresis gel.

2: determination of penicillium expansum cis-epoxysuccinate hydrolase activity

Taking 10 mL of fermentation liquor, adding 1mL of 1mol/L cis-epoxy sodium succinate (pH8.0) solution, reacting for 1h at 37 ℃, and determining the content of tartaric acid in the reaction liquor.

The detection method of the tartaric acid content in the reaction solution comprises the following steps: 2.5ml of 1% ammonium metavanadate solution is placed in a 25ml volumetric flask, a proper amount of the reaction solution is added, 1ml of 1mol/L sulfuric acid is added, distilled water is used for fixing the volume to 25ml, a part of the mixture is uniformly mixed, a spectrophotometer is used for measuring the light absorption value at 480nm, and the content of tartaric acid in the reaction solution is calculated according to the established L (+) -tartaric acid standard curve.

The preparation method of the L (+) -tartaric acid standard curve comprises the following steps: 0.25g L-sodium tartrate solution was dissolved in 25ml of distilled water to prepare a 10 mg/ml L (+) -sodium tartrate solution. 2.5ml of 1% ammonium metavanadate solution was added to 11 25ml volumetric flasks, and then 0ml, 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml, 0.6ml, 0.7ml, 0.8ml, 0.9ml and 1.0ml of the above-mentioned L (+) -sodium tartrate solution were sequentially added to the 11 volumetric flasks, 1ml of 1mol/L sulfuric acid was added thereto, and a volume was determined to 25ml with distilled water, and after mixing, a portion of the mixture was measured for absorbance at 480nm using a spectrophotometer, and an L (+) -tartaric acid calibration curve was prepared.

The enzyme activity unit is defined as: under the above reaction conditions, 1mL of the fermentation broth was used for 1h, and the amount of enzyme required to produce 1. mu. mol of tartaric acid was determined.

Example 2: sequencing the N-and C-termini of the Penicillium expansum cis-epoxysuccinate hydrolase

The N-terminal amino acid sequence and C-terminal amino acid sequence of the penicillium expansum cis-epoxysuccinate hydrolase are detected by the usual method as follows: the cis-epoxysuccinate hydrolase obtained after the separation and purification is subjected to SDS-polyacrylamide gel electrophoresis, and then transferred to a PVDF membrane by Western hybridization. The membrane was stained with Coomassie brilliant blue staining solution, and the band corresponding to cis-epoxysuccinate hydrolase was cleaved and recovered, and then 10 amino acid sequences of the N-terminus and C-terminus were determined by a protein sequencer, respectively. The sequencing result showed that the N-terminal 10 amino acid sequence of the cis-epoxysuccinate hydrolase was MSRDEPPSML (SEQ ID NO:3) and the C-terminal 10 amino acid sequence was ARYFGIESEL (SEQ ID NO: 4).

Example 3: design of degenerate primer and cloning of Penicillium expansum WH-3 cis-epoxy succinate hydrolase gene

Two degenerate primers were designed based on the N-terminal sequence (SEQ ID NO:3), C-terminal sequence (SEQ ID NO:4) and stop codon sequence (TAA/TGA/TAG) not coding for amino acids of the above-mentioned Penicillium expansum cis-epoxysuccinate hydrolase as follows:

primer 1: 5 '-ATGWSSNXGGAYGARCCNCC-3';

primer 2: 5 '-YYANAYYTCNYSYTCNATNCC-3';

wherein R is A/G, Y is C/T, W is A/T, S is G/C, X is A/C, and N is A/G/C/T.

The cloning of the cis-epoxysuccinate hydrolase gene with degenerate primers was performed as follows:

(1) the spore solution was inoculated into a self-made PDB medium, cultured with shaking at 25 ℃ and 150rpm for 48 hours, and the Penicillium expansum pellet was collected, and the genome of Penicillium expansum WH-3 was extracted according to the method described in Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech).

(2) A PCR reaction was carried out using the above genome as a template and primers 1 and 2 as primers. The PCR reaction system is 2 muL of the template obtained in the step 1, 1 muL of Taq DNA polymerase (5 mu m/muL), 5 muL of 10 xPCR buffer, 1 muL of primer 1(10 mumol/muL), 1 muL of primer 2(10 mumol/muL), 1 muL of dNTP (100 mmol/muL), and H₂O39 muL and the total volume is 50 muL.

(3) The 50. mu.L PCR reaction system was subjected to the following PCR reaction procedure:

preheating at 94 deg.C for 5 min; then, the temperature is 94 ℃ for 50s, 46 ℃ for 30s and 72 ℃ for 1min for 30 cycles; finally, extension is carried out for 10min at 72 ℃.

(4) The PCR product was subjected to agarose gel (1%) electrophoresis. Electrophoresis results show that a clear band exists between 750bp and 1000 bp. The band was recovered and ligated with pUC_mT vector ligation, transformation into E.coli DH 5. alpha. and colonies were picked for sequencing validation. Sequencing knotAs shown in SEQ ID NO 1, the length of the nucleic acid is 801 nucleotides, wherein ATG is an initiation codon and TGA is a termination codon. The nucleotide sequence shown in SEQ ID NO:1 was translated into an amino acid sequence using DNAStar software to obtain the amino acid sequence shown in SEQ ID NO:2 (i.e., the amino acid sequence of cis-epoxysuccinate hydrolase of the present invention). Comparing the coding gene and amino acid sequence of the cis-epoxy succinate hydrolase with the reported cis-epoxy succinate hydrolase sequence respectively, finding that the similarity between the coding gene and the amino acid sequence of the cis-epoxy succinate hydrolase does not exceed 50 percent, thereby indicating that the coding gene of the cis-epoxy succinate hydrolase obtained by the invention is a novel gene.

Example 4: construction of penicillium expansum cis-epoxysuccinate hydrolase gene prokaryotic expression vector and expression of vector in escherichia coli

Designing a forward primer 3 containing a recognition site of a restriction enzyme Nco I and a reverse primer 4 containing a recognition site of a restriction enzyme BamH I according to a sequence shown in SEQ ID NO.1, wherein the sequences of the primer 3 and the primer 4 are respectively as follows:

primer 3:5' -CATGCCATGGATATGTCACGGGATGAACCCC;

primer 4:5' -CGGGATCCTCACAATTCACTTTCTATTCCAAAAT.

And (3) carrying out PCR reaction by taking the genome of the penicillium expansum WH-3 as a template and taking the primer 3 and the primer 4 as primers. The PCR reaction system is 2 muL of the template obtained in the step 1 of the embodiment 3, 1 muL of Taq DNA polymerase (5 mu m/muL), 5 muL of 10 XPCR buffer, 1 muL of primer 3(10 mumol/muL), 1 muL of primer 4(10 mumol/muL), 1 muL of dNTP (100 mmol/muL), and H₂O39 muL and the total volume is 50 muL.

The 50. mu. LPCR reaction system described above was subjected to the following PCR reaction procedure:

preheating at 94 deg.C for 5 min; then, the temperature is 94 ℃ for 50s, 50 ℃ for 30s, and 72 ℃ for 1min for 30 cycles; finally, extension is carried out for 10min at 72 ℃.

Recovering the PCR product and mixing with pUC_mT vector ligation, transformation into E.coli DH 5. alpha. and colonies were picked for sequencing validation. The sequencing result is compared with the coding gene of cis-epoxysuccinate hydrolase (as shown in SEQ ID NO: 1)Shown), the results show: the coding gene of cis-epoxy succinate hydrolase containing recognition sites of restriction enzymes Nco I and BamH I is successfully inserted into pUC_m-a T vector. Then, pUC containing cis-epoxy succinate hydrolase gene was treated with restriction enzymes Nco I and BamH I, respectively_mDouble digestion of the-T vector and the pET-15b expression vector with T₄The DNA ligase enzyme links the enzyme-cut cis-epoxy succinate hydrolase gene with the enzyme-cut pET-15b vector, the ligation product is transformed into escherichia coli BL21(DE3), and colonies are picked for sequencing. And comparing the sequencing result with the cis-epoxy succinate hydrolase gene, and completely matching the sequences, thereby successfully constructing the pET-15b recombinant vector containing the cis-epoxy succinate hydrolase gene and the genetic engineering bacteria containing the pET-15b recombinant vector.

As shown in FIG. 1, after IPTG induction and SDS-polyacrylamide gel electrophoresis, the genetically engineered strain showed a significant band at about 30kDa (lane 1) showing a protein size corresponding to that calculated from the amino acid sequence of cis-epoxysuccinate hydrolase; coli BL21(DE3) containing only pET-15b vector without any foreign nucleotide insert was used as a control and no corresponding protein band (lane 2), indicating that the recombinant protein encoded by cis-epoxysuccinate hydrolase gene can be expressed efficiently after IPTG induction.

Example 5: the genetic engineering bacteria of the invention are utilized to prepare L (+) tartaric acid

The genetically engineered bacterium described in example 4 was further transferred to 1L LB medium after 12 hours of shaking culture at 37 ℃ in LB medium, and after about 2 hours of shaking culture at 37 ℃ and 200rpm, 0.1M IPTG was added and further shaking culture at 37 ℃ and 200rpm was continued for 8 hours. The LB culture medium has the formula: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0. Then adding 10g cis-disodium epoxy succinate into the culture solution, continuing to oscillate at 37 ℃ and 200rpm for 12h, and adding excessive CaCl₂In the aqueous solution, a precipitate was formed. Filtering the precipitate, washing the precipitate with water to obtain 14.0g calcium tartrate, and performing acidolysis with sulfuric acid and anion and cation exchange column on the calcium tartrateRefining, concentrating, crystallizing, separating and drying to obtain 6.5g of solid product. Detected by a Nicolet-Nexus670 Fourier transform infrared spectrometer, detected by a hydrogen spectrum and a carbon spectrum of a Bruker Avance DMX500 nuclear magnetic resonance instrument for nuclear magnetic resonance and Bruker Esquire 3000^plusDetecting by a mass spectrometer, and determining that the solid product is tartaric acid. The specific optical rotation of the solid product is alpha detected by a WZZ-2B polarimeter]

= +12.1 °, demonstrating that the solid product is dextro-tartaric acid, i.e., L (+) -tartaric acid, and has a purity of 99.9%.

The cis-epoxysuccinic acid disodium in the embodiment may be replaced by cis-epoxysuccinic acid or salts thereof with various cations, including but not limited to ammonium ion, potassium ion, magnesium ion, calcium ion, and the like.

Example 6: the method for preparing L (+) -tartaric acid by embedding the genetically engineered bacteria in the invention with kappa-carrageenan

The culture solution of example 5 was centrifuged and the genetically engineered bacterial cells were collected, 10g of the genetically engineered bacterial cells were added to 100mL of 40g/L kappa-carrageenan solution, mixed well at 42 ℃ and cooled at 4 ℃, after solidification, soaked in 0.3M potassium chloride solution at 4 ℃ for 10h, the gel was cut into small pieces and stored at 4 ℃. 30g of the immobilized cells were put into 30mL of 1.0M cis-disodium epoxysuccinate solution, reacted at 37 ℃ for 24 hours, and then the immobilized cells were recovered by filtration. Adding CaCl2 water solution into the filtrate, stirring, filtering, washing the precipitate, adding sulfuric acid to hydrolyze the precipitate, refining with anion and cation exchange column, concentrating, crystallizing, and oven drying to obtain L (+) -tartaric acid 3.8 g.

The recovered immobilized cells were put into 30ml of 1.0M cis-disodium epoxysuccinate solution again, reacted at 37 ℃ for 24 hours, and then the immobilized cells were recovered by filtration. Adding CaCl into the filtrate₂And (3) fully stirring the aqueous solution, filtering, washing the precipitate, adding sulfuric acid to hydrolyze the precipitate, refining by an anion-cation exchange column, concentrating, crystallizing and drying to obtain 3.9g of L (+) -tartaric acid.

The immobilized cells can be used repeatedly.

The cis-epoxysuccinic acid disodium in the embodiment may be replaced by cis-epoxysuccinic acid or salts thereof with various cations, including but not limited to ammonium ion, potassium ion, magnesium ion, calcium ion, and the like.

While the invention has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Zhejiang science and technology institute

<120> penicillium expansum cis-epoxy succinate hydrolase gene and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 801

<212> DNA

<213> Penicillium expansum (Penicillium expansum)

<400> 1

atgtcacggg atgaaccccc ttcaatgcta tttttcgatg tcttggggac aattgtggaa 60

tggagatatt gcatcgcgaa cgaactgaat gccgccgcac aaaaggttct acaagaccaa 120

gctcgatctc cgagtcccga tatgcgagca cgtgtctccg atatgtccgc ttcgagctgg 180

caggagatct ccgaggaatg gcatcggtca tatatgaatt ttggagacaa ttacgataca 240

tctaagccct ttatgtcagt agatgagtac aaccgaattt cgctggaaga tattctcacc 300

aaatggcttc tccgtgattt atttaacgag gacgatctca aacatttgac gctcgcttgg 360

catcgacttg actcataccc tgacagtgcg ccgggtcttt cgttgctgaa tactaaattt 420

tcgacgtcta cattatccaa tgggaatgtt aaacttctcg aagacctaca aggacacaat 480

tctttgcctt tcacgcacat taccagtgca gagcacttca gggcttacaa accctcacca 540

gaggtctatg atggtgctgc tcgcagattc ggtttcaaga attcgcaatg ttgtctcgtc 600

gcagcccatc ttgaggactt gcaagctgct aagaaatgcg ggtttcaaac aatctatctc 660

gagagagagt tggaagaagc ttgggatagt agagacgttg cacgagctag agaagaaggt 720

gttgtcgatg tttgggttgg agttggaggc tcgggtctga ttgaagttgc tcgatatttt 780

ggaatagaaa gtgaattgtg a 801

<210> 2

<211> 266

<212> PRT

<213> Penicillium expansum (Penicillium expansum)

<400> 2

Met Ser Arg Asp Glu Pro Pro Ser Met Leu Phe Phe Asp Val Leu Gly

1 5 10 15

Thr Ile Val Glu Trp Arg Tyr Cys Ile Ala Asn Glu Leu Asn Ala Ala

20 25 30

Ala Gln Lys Val Leu Gln Asp Gln Ala Arg Ser Pro Ser Pro Asp Met

35 40 45

Arg Ala Arg Val Ser Asp Met Ser Ala Ser Ser Trp Gln Glu Ile Ser

50 55 60

Glu Glu Trp His Arg Ser Tyr Met Asn Phe Gly Asp Asn Tyr Asp Thr

65 70 75 80

Ser Lys Pro Phe Met Ser Val Asp Glu Tyr Asn Arg Ile Ser Leu Glu

85 90 95

Asp Ile Leu Thr Lys Trp Leu Leu Arg Asp Leu Phe Asn Glu Asp Asp

100 105 110

Leu Lys His Leu Thr Leu Ala Trp His Arg Leu Asp Ser Tyr Pro Asp

115 120 125

Ser Ala Pro Gly Leu Ser Leu Leu Asn Thr Lys Phe Ser Thr Ser Thr

130 135 140

Leu Ser Asn Gly Asn Val Lys Leu Leu Glu Asp Leu Gln Gly His Asn

145 150 155 160

Ser Leu Pro Phe Thr His Ile Thr Ser Ala Glu His Phe Arg Ala Tyr

165 170 175

Lys Pro Ser Pro Glu Val Tyr Asp Gly Ala Ala Arg Arg Phe Gly Phe

180 185 190

Lys Asn Ser Gln Cys Cys Leu Val Ala Ala His Leu Glu Asp Leu Gln

195 200 205

Ala Ala Lys Lys Cys Gly Phe Gln Thr Ile Tyr Leu Glu Arg Glu Leu

210 215 220

Glu Glu Ala Trp Asp Ser Arg Asp Val Ala Arg Ala Arg Glu Glu Gly

225 230 235 240

Val Val Asp Val Trp Val Gly Val Gly Gly Ser Gly Leu Ile Glu Val

245 250 255

Ala Arg Tyr Phe Gly Ile Glu Ser Glu Leu

260 265

<210> 3

<211> 10

<212> PRT

<213> Penicillium expansum (Penicillium expansum)

<400> 3

Met Ser Arg Asp Glu Pro Pro Ser Met Leu

1 5 10

<210> 4

<211> 10

<212> PRT

<213> Penicillium expansum (Penicillium expansum)

<400> 4

Ala Arg Tyr Phe Gly Ile Glu Ser Glu Leu

1 5 10

Claims (9)

1. A penicillium expansum cis-epoxysuccinate hydrolase polypeptide, wherein the amino acid sequence of the polypeptide is: the amino acid sequence shown in SEQ ID No. 2.

2. The application of the penicillium expansum cis-epoxy succinate hydrolase polypeptide in the production of L (+) -tartaric acid or salts thereof, wherein the amino acid sequence of the polypeptide is as follows: the amino acid sequence shown in SEQ ID No. 2.

3. A gene encoding a penicillium expansum cis-epoxysuccinate hydrolase polypeptide according to claim 1, characterized in that its nucleotide sequence is: the nucleotide shown as SEQ ID No. 1.

4. An application of a coding gene in regulating and controlling microorganism to produce L (+) -tartaric acid or salt thereof, wherein the nucleotide sequence of the coding gene is as follows: the nucleotide shown as SEQ ID No. 1.

5. A recombinant expression vector comprising the coding gene of claim 3.

6. A recombinant microorganism strain expressing the penicillium expansum cis-epoxysuccinate hydrolase polypeptide according to claim 1.

7. A process for producing L (+) -tartaric acid or a salt thereof, comprising reacting the penicillium expansum cis-epoxysuccinate hydrolase polypeptide of claim 1 with cis-epoxysuccinate or a salt thereof.

8. A method for producing L (+) -tartaric acid or a salt thereof, characterized by reacting the recombinant microorganism strain of claim 6 with cis-epoxysuccinic acid or a salt thereof.

9. The method for producing L (+) -tartaric acid or a salt thereof according to claim 8, comprising:

1) culturing the recombinant microorganism strain in a suitable medium;

2) immobilizing cells, cell extracts or cell-free extracts of the recombinant microorganism strains obtained in the step 1) or cis-epoxysuccinate hydrolase purified by the recombinant microorganism strains on a solid support or embedded in a fixed carrier;

3) reacting the recombinant microorganism strain obtained in step 1) or the preparation obtained in step 2) with cis-epoxysuccinic acid or a salt thereof under conditions suitable for hydrolysis to produce L (+) -tartaric acid or a salt thereof; and

4) optionally recycling the immobilized or embedded enzymes and/or cells.

Images