CN101058818A - Hydantoinase gene, coded amino acid and application thereof - Google Patents

Abstract

The invention provides a hydantoin enzyme gene, an amino acid of the gene code, a recombinant plasmid with the gene and the gene application. The code amino acid sequence of hydantoin enzyme gene is different from the present known source gene, the expression product of the hydantoin enzyme gene with hydroxybenzene hydantoin as the substrate shows high enzymatic activity, the specific activity is higher than the hydantoin enzyme from Agrobacterium sp. KNK712 in the present industrial production.

Description

A kind of Hydantoinase gene and amino acids coding thereof and application

Technical field

The invention belongs to the genetically engineered field, specifically, relate to a kind of Hydantoinase gene and amino acids coding thereof and application.

Background technology

D-amino acid and derivative thereof are the important raw and processed materials of medicine and field of food, are widely used in the intermediate of semisynthetic antibiotics, polypeptide hormone, pyrethroid, sterilant and sweeting agent etc.Wherein, D-D-pHPG (D-pHPG) is the important source material that there is SKF-60771 etc. synthetic semi-synthetic Broad spectrum antibiotics amoxycilline Trihydrate bp, S 578, cefoperazone, cephalo Luo Qi.Shortcomings such as the traditional processing technology of D-D-pHPG need be by chemosynthesis and fractionation (building-up process of D-D-pHPG as shown in Figure 1), and it is low that this technology exists yield, and environmental pollution is serious.

Since the last century the eighties, enzyme process prepares the research of D-D-pHPG and uses developed, its process is as follows: at first utilize chemical means to synthesize D, L-para hydroxybenzene glycolylurea (D, L-HPH), utilize biological enzyme to be translated into the D-D-pHPG then as the substrate of biological enzyme effect.Generally speaking, earlier obtain D by oxoethanoic acid, phenol, urea condensation, acyl benzene glycolylurea (pHPH) in the L-para hydroxybenzene second, pHPH can be by D-glycolylurea enzyme (D-hydantoinase, E.C.3.5.2.2) the stereospecificity hydrolysis generates D-N-carboxamide D-pHPG (D-CpHPG), at alkaline condition or exist under the condition of racemase, further transformed thereby do not turned to the D-type by the L-type enantiomorph racemization of open loop.By this enzymatic asymmetric hydrolysis reaction, can make the pHPH that mixes racemization be converted into D-HPG fully.Now, the report that carries out this reaction at industrial existing employing immobilized D-glycolylurea enzyme or immobilized cell.

Production by Enzymes D-D-pHPG has important use to be worth industrial, therefore it is produced the research of used relevant enzyme and transforms significant.And but D-glycolylurea enzyme catalysis 5 ' list substitutes the open loop of glycolylurea or dihydrouracil, form the intermediate of carbamyl class, this intermediate product can be degraded to the sweet amino acid of D-para hydroxybenzene by carbamyl hydrolysis enzyme, becomes the important intermediate of semi-synthetic β-Nei Xiananleikangshengsu.Thereby, the research of D-glycolylurea enzyme is become one of hot research problem of relevant industries gradually.

In recent years, people are from Pseudomonas fluorescens DSM84, Bacillus stearothermophilisNS1122A, Pseudomonas putida CCRC12857, Agrobacterium sp.KNK712, Agrobacteriumradiobacter NRRL B11291 clones the gene of coding glycolylurea enzyme, and it has been carried out the molecular biology transformation, to improve involved enzyme character.Yet, do not see the report that has the clone to derive from the glycolylurea enzyme of Jannaschia sp.CCS1 up to now as yet.

Summary of the invention

The objective of the invention is to, the Hydantoinase gene of a kind of Jannaschia of deriving from sp.CCS1 is provided.

Another object of the present invention is, a kind of described Hydantoinase gene amino acids coding is provided.

Another object of the present invention is, a kind of the have Hydantoinase gene of the aminoacid sequence shown in the coding SEQ ID NO:2 and a kind of recombinant plasmid of carrier DNA sequence are provided.

Last purpose of the present invention is, a kind of application of described Hydantoinase gene is provided.

For achieving the above object, the present invention obtains Hydantoinase gene by PCR method from Jannaschia sp.CCS1 genome amplification.

The dna fragmentation of the glycolylurea enzyme that will obtain after will increasing again is connected with carrier pET28a, thereby obtains the recombinant plasmid pETJ2 of glycolylurea enzyme.

The recombinant plasmid of described glycolylurea enzyme is transferred among the E.coli BL21 (DE3), and with described engineering bacteria BL21 (DE3)/pETJ2 fermentation culture, thereby described Hydantoinase gene amino acids coding obtained.

With the expression product of the Hydantoinase gene that obtained, with the para hydroxybenzene glycolylurea as substrate, preparation D-D-pHPG, and compare with the glycolylurea enzyme that derives from Agrobacterium sp.KNK712.

Hydantoinase gene amino acid sequence coded provided by the invention is different from the glycolylurea enzyme in present known source, and it is lived showing higher enzyme during as substrate with the para hydroxybenzene glycolylurea, apparently higher than the glycolylurea enzyme of Agrobacterium sp.KNK712.

Though, in the present invention, during construction of expression vector, that use is pET28a, still for the staff of this area, uses other carrier to make up corresponding expression vector, as the pET system, the pSU system, pTrc system, pMW system, the pKK system, the expression vector that RSF1010 etc. are commonly used also is conspicuous, so also should belong to scope of the present invention.Same, staff for this area, the corresponding expression vectors that builds is transformed into other microorganism, in Rhodopseudomonas (Pseudomonas), Flavobacterium (Flavobacterium), bacillus (Bacillus), Serratia (Serratia), Agrobacterium (Agrobacterium), corynebacterium (Corynebacterium) or brevibacterium sp (Brevibacterium) etc., obtain corresponding transformant microorganism, also be conspicuous, so also should belong to scope of the present invention.

Description of drawings

Fig. 1 is the synthetic synoptic diagram of D-D-pHPG.

Fig. 2 is the structure synoptic diagram of new glycolylurea expression of enzymes carrier pETJ2.

Fig. 3 crosses the electrophorogram of expressing the glycolylurea enzyme in intestinal bacteria.

Wherein each swimming lane is respectively: 1 is BL21 (the DE3)/pETJ2 before inducing; 2 are BL21 (the DE3)/pETJ2 after inducing; 3 is albumen Marker.

Fig. 4 utilizes HPLC to detect the graphic representation that the glycolylurea enzyme reaction generates product.

Embodiment

Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples only are used to the present invention is described but not are used to limit scope of the present invention.

The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, the condition described in " molecular cloning experiment guide (third edition) " (work such as J. Sa nurse Brooker, 2003) is carried out.

Embodiment 1, contain the expression vector of goal gene and the structure of engineering bacteria

1.1, design of primers

The genome sequence of the Jannaschia sp.CCS1 that announces according to Genebank, design primer A and B, its sequence is as follows:

Primer A:5 '-AGGGGGATCCATGAGCAAGGTGATCAAGGG-3 ';

Primer B:5 '-CTAGAAGCTTTCAAACCCCCGCCGGAATG-3 '.

1.2, pcr amplification

With A and B is primer, is template with the genomic dna that derives from Jannaschia sp.CCS1, the pcr amplification goal gene.

Reaction system: 1 * PCR Buffer for KOD plus, 2mM MgSO ₄, each 0.4 μ M of primer A and primer B, dJannaschia sp.CCS1 genomic dna 100ng, 0.2mM dNTP, 1U KOD plus/50 μ l.

Reaction conditions: 94 ℃ of 5min, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1.5min, 30 circulations; 72 ℃ of 10min.

Reaction is pressed operational manual after finishing, and the PCR product is reclaimed the product of the about 1.5kb of test kit recovery purifying with the glue of Hua Shun company.

Check order to reclaiming product, sequencing result shows that the gene that amplification obtains has the dna sequence dna shown in the SEQ ID NO:3, and its corresponding amino acid sequence is shown in SEQ ID NO:2.

As Query, in GenBank, carry out homology search with the aminoacid sequence (SEQ ID NO:1) of the D-glycolylurea enzyme that derives from Agrobacterium sp.KNK712.The result shows, this aminoacid sequence (SEQ ID NO:2) is 40% with the homology of the aminoacid sequence (Q409E0) with SEQ ID NO:1 record, according to the BLAST interpretation of result of NCBI, find the conserved domain that there is the glycolylurea enzyme in it, infer that it may have the function of glycolylurea enzyme.

1.3, carrier construction and engineering bacteria

Building process as shown in Figure 2, concrete operations are as follows:

With reference to operational manual, respectively pcr amplification product and pET28a carrier (available from Novagen company) are carried out double digestion with BamHI (available from MBI company) and HindIII (available from MBI company), adopt the damping fluid of the double digestion damping fluid R of MBI company as endonuclease reaction.

After enzyme is cut, product detects with 1% agarose gel electrophoresis, and the purpose fragment is reclaimed in rubber tapping, and (PCR product enzyme is cut the back clip size and is about 1.5kb, the pET28a enzyme is cut the back size and is about 5.4kb), then with reference to operational manual, cut the back fragment with the enzyme that the glue of Hua Shun company reclaims that test kit reclaims respectively and the enzyme of purified pcr product is cut back fragment and carrier pET28a.

Get PCR product enzyme and cut back fragment and each 4 μ l of pET28a carrier segments, 1 μ l T4 ligase enzyme damping fluid and 1 μ l T4 ligase enzyme, in 16 ℃, connection is spent the night, and results connect product, are expression vector pETJ2.

Get 5 μ l and connect product transformed into escherichia coli DH5 α competent cell, 37 ℃ of overnight incubation.

Picking list bacterium colony inserts in the liquid nutrient medium, and in 37 ℃, 250rpm cultivated after 6-8 hour, extracting plasmid, and checking clone.Choose enzyme and cut the correct cloning and sequencing of checking, whether suddenly change with the checking sequence.

Sequencing result shows that the sequence of goal gene is not undergone mutation.

The expression vector pETJ2 that builds is transformed host bacterium E.coli BL21 (DE3) (available from Novagen company), engineering bacteria called after BL21 (the DE3)/pETJ2 that obtains.

Though, in the present embodiment, during construction of expression vector, that use is pET28a, still for the staff of this area, uses other carrier to make up corresponding expression vector, as the pET system, the pSU system, pTrc system, pMW system, the pKK system, the expression vector that RSF1010 etc. are commonly used also is conspicuous, so also should belong to scope of the present invention.Same, staff for this area, the corresponding expression vectors that builds is transformed into other microorganism, in Rhodopseudomonas (Pseudomonas), Flavobacterium (Flavobacterium), bacillus (Bacillus), Serratia (Serratia), Agrobacterium (Agrobacterium), corynebacterium (Corynebacterium) or brevibacterium sp (Brevibacterium) etc., obtain corresponding transformant microorganism, also be conspicuous, so also should belong to scope of the present invention.

The expression of embodiment 2, goal gene

Single bacterium colony of picking E.coli BL21 (DE3)/pETJ2 inserts and contains in the LB liquid nutrient medium of 50 μ g/ml kantlex, and 37 ℃, 200rpm, overnight incubation.

BL21 (the DE3)/pETJ2 of incubated overnight inserted by 1% inoculum size contain in the LB liquid nutrient medium of 50 μ g/ml kantlex, 37 ℃, 200rpm, when being cultured to OD600 ≈ 0.6, adding final concentration is the IPTG of 0.25mM, in 20 ℃, inducing culture 5 hours.

Bacterium liquid after 12000rpm is centrifugal 2 minutes, is abandoned supernatant.

To precipitate with the Tris-HCl damping fluid of 50mM pH8.0 resuspended, ultrasonication.With the cell after the fragmentation, by the proteic expression amount of polyacrylamide gel electrophoresis analysis purposes, the result as shown in Figure 3.

According to the result of Fig. 3 as seen, the expression amount of target protein accounts for 30% of total protein, can be beneficial to the immobilization of follow-up zymoprotein and the suitability for industrialized production that transforms continuously by at intestinal bacteria reorganization overexpression J2.

The mensuration that embodiment 3, target protein enzyme are lived

BL21 (the DE3)/pETJ2 of incubated overnight among the embodiment 2 inserted by 1% inoculum size contain in the TB liquid nutrient medium of 50 μ g/ml kantlex, 37 ℃, 200rpm, when being cultured to OD600 ≈ 0.6, adding final concentration is the IPTG of 0.25mM, in 20 ℃, and inducing culture 5 hours.

Collect bacterium liquid, centrifugal, supernatant discarded, and in-20 ℃ of freeze thawing once, use the Tris-HCl damping fluid of 50mM pH8.0 that thalline is resuspended then.

Thalline after resuspended is joined in the Tris-HCl damping fluid that contains 1% para hydroxybenzene glycolylurea, pH 8.0, and 40 ℃ were reacted 10 minutes, added 3.7% HCl termination enzyme reaction and mixing.

With above-mentioned reaction solution centrifugal 5 minutes in 12000rpm, get the amount (result is as shown in Figure 4) of utilizing HPLC analytical reaction product to generate after 4 times of the supernatant dilutions, calculate enzyme and live, the result is as shown in table 1.

Enzyme work calculation method:

Table 1, target protein enzyme activity determination result

	O.D.600	μmol/min/ml/OD
			BL21/pETJ2	2.2±0.1	0.016±0.002

Embodiment 4, target protein are than the mensuration of living

BL21 (the DE3)/pETJ2 of incubated overnight among the embodiment 2 inserted by 1% inoculum size contain in the TB liquid nutrient medium of 50 μ g/ml kantlex, 37 ℃, 200rpm, when being cultured to OD600 ≈ 0.6, adding final concentration is the IPTG of 0.25mM, in 20 ℃, and inducing culture 5 hours.

Collect bacterium liquid, centrifugal, supernatant discarded uses the Tris-HCl damping fluid of 50mM pH8.0 that thalline is resuspended then.Resuspended bacterium hydraulic pressure is pressed broken wall, adopt the GE Ni Sepharose High Performance of company purifying target protein, and measure target protein concentration with the Bradford method.

Is final concentration 0.06mg/mL with target protein with the Tris-HCl of 50mM pH8.0 dilution, joins in the Tris-HCl damping fluid that contains 1% para hydroxybenzene glycolylurea pH 8.0, and 40 ℃ of reactions 30 minutes add 3.7% HCl and end enzyme reaction and mixing.

With above-mentioned reaction solution centrifugal 5 minutes in 12000rpm, get the amount (result is as shown in Figure 4) of utilizing HPLC analytical reaction product to generate after 10 times of the supernatant dilutions, calculate enzyme than living, the result is as shown in table 2.

Use the same method can from contain the engineering bacteria (CGMCCNo.0520.2) that derives from Agrobacterium sp.KNK712 glycolylurea enzyme, express, glycolylurea enzyme that purifying obtains deriving from Agrobacterium sp.KNK712, and under above-mentioned same condition, measure its activity and ratio and live.

Table 2, target protein are than measurement result alive

	Than μ mol/ alive (min.mg albumen)
		Agrobacterium sp.KNK712	2.0±0.02
J2	9.1±0.4

According to experimental result in the foregoing description, can infer the albumen of aminoacid sequence with SEQ ID NO:2, have the function of glycolylurea enzyme, be a kind of glycolylurea enzyme therefore; And glycolylurea enzyme of the present invention is different from the glycolylurea enzyme in present known source, it is with the para hydroxybenzene glycolylurea during as substrate, showing higher ratio lives, it is about 4 times of the glycolylurea enzyme that derives from Agrobacterium sp.KNK712 than living, therefore, in the industrial production of utilization double-enzyme method production D-pHPG, more wide utilization prospect can be arranged.

Sequence table

＜110〉Shanghai Inst. of Life Science, CAS

＜120〉a kind of glycolylurea enzyme

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Claims (24)

1, a kind of Hydantoinase gene is characterized in that, described Hydantoinase gene derives from Jannaschia sp.CCS1.

2, Hydantoinase gene as claimed in claim 1 is characterized in that, the amino acid of described genes encoding has the aminoacid sequence shown in the SEQ IDNO:2.

3, Hydantoinase gene as claimed in claim 1 or 2 is characterized in that, described gene has the dna sequence dna shown in the SEQ ID NO:3.

4, as each described Hydantoinase gene amino acids coding of claim 1-3.

5, amino acid as claimed in claim 4 is characterized in that, described amino acid has the aminoacid sequence shown in the SEQ ID NO:2.

6, amino acid as claimed in claim 5 is characterized in that, described amino acid is coded by the Hydantoinase gene with the dna sequence dna shown in the SEQ ID NO:3.

7, a kind of recombinant plasmid is characterized in that, described recombinant plasmid has Hydantoinase gene and a kind of carrier DNA sequence of the aminoacid sequence shown in the coding SEQ ID NO:2.

8, recombinant plasmid as claimed in claim 7 is characterized in that, described Hydantoinase gene has the dna sequence dna shown in the SEQ ID NO:3.

9, recombinant plasmid as claimed in claim 7 is characterized in that, described carrier comprises: pET system, pSU system, pTrc system, pMW system, pKK system, RSF1010.

10, recombinant plasmid as claimed in claim 9 is characterized in that, carrier is pET28a.

11, as the application of each described Hydantoinase gene of claim 1-3, it is characterized in that, be used to prepare the glycolylurea enzyme.

12, application as claimed in claim 11 is characterized in that, may further comprise the steps:

A, construction recombination plasmid;

B, conversion host bacterium;

C, fermentation culture obtain the glycolylurea enzyme.

13, application as claimed in claim 12 is characterized in that, described recombinant plasmid has Hydantoinase gene and a kind of carrier DNA sequence of the aminoacid sequence shown in the coding SEQ ID NO:2.

14, recombinant plasmid as claimed in claim 13 is characterized in that, described Hydantoinase gene has the dna sequence dna shown in the SEQ ID NO:3.

15, recombinant plasmid as claimed in claim 14 is characterized in that, described carrier comprises: pET system, pSU system, pTrc system, pMW system, pKK system, RSF1010.

16, recombinant plasmid as claimed in claim 15 is characterized in that, carrier is pET28a.

As claim 11 or 12 described application, it is characterized in that 17, described glycolylurea enzyme has the aminoacid sequence shown in the SEQ ID NO:2.

18, the application that is used to prepare the D-D-pHPG as each described Hydantoinase gene of claim 1-3.

19, application as claimed in claim 18 is characterized in that, may further comprise the steps:

A, construction recombination plasmid;

B, conversion host bacterium;

C, fermentation culture, preparation glycolylurea enzymic fermentation liquid;

D, utilize fermented liquid with D, L-para hydroxybenzene glycolylurea substrate conversion is the D-D-pHPG.

20, application as claimed in claim 19 is characterized in that, described recombinant plasmid has Hydantoinase gene and a kind of carrier DNA sequence of the aminoacid sequence shown in the coding SEQ ID NO:2.

21, recombinant plasmid as claimed in claim 20 is characterized in that, described Hydantoinase gene has the dna sequence dna shown in the SEQ ID NO:3.

22, recombinant plasmid as claimed in claim 21 is characterized in that, described carrier comprises: pET system, pSU system, pTrc system, pMW system, pKK system, RSF1010.

23, recombinant plasmid as claimed in claim 22 is characterized in that, carrier is pET28a.

As claim 18 or 19 described application, it is characterized in that 24, described glycolylurea enzyme has the aminoacid sequence shown in the SEQ ID NO:2.

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