CN105950595B - (-)-gamma-lactam enzyme, gene, mutant, carrier and its preparation and application - Google Patents
(-)-gamma-lactam enzyme, gene, mutant, carrier and its preparation and application Download PDFInfo
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Abstract
The present invention relates to a kind of (-)-gamma-lactam enzyme, its gene and mutant, recombinant expression plasmid and recombinant expression transformants containing the gene and mutant, the preparation of the recombination (-)-gamma-lactam enzyme, and should application of (-)-gamma-lactam enzyme in preparation (+)-gamma-lactam.Compared with prior art, (-)-gamma-lactam enzyme of the invention has the characteristics that enzymatic activity is high, concentration of substrate tolerance is good, preparing (+)-gamma-lactam using the enzymatic has the advantages such as reaction condition is mild, concentration of substrate is high, catalyst amount is few, therefore has a good application prospect in homocyclic nucleus glycoside pharmaceutical intermediate (+)-gamma-lactam industrial production.
Description
Technical field
The invention belongs to technical field of bioengineering, more particularly, to a kind of (-)-gamma-lactam enzyme, its gene and mutation
Body, recombinant expression plasmid and recombinant expression transformants containing the gene and mutant, the recombination (-)-gamma-lactam enzyme system
It is standby, and should application of (-)-gamma-lactam enzyme in preparation (+)-gamma-lactam.
Background technique
Gamma-lactam is that the abbreviation of compound 2- azabicyclic [2.2.1] hept- 5- alkene -3- ketone (is also commonly called as Wen Sinei
Ester), molecular formula C6H7NO.Two enantiomters of gamma-lactam are all useful medicine intermediates, wherein (-)-γ-
Lactams can be used for synthesizing antiretroviral drugs Abacavir and anti-influenza virus medicament Peramivir；And (+)-γ-is interior
Amide can be used for synthetic chemokines receptor CCR2 antagonist MK-0812 and depeptidyl peptidase inhibitors Metro Li Ting.
(+)-gamma-lactam or (-)-gamma-lactam are prepared using biological catalysis, are generally adopted by hydrolase, mesh
It is preceding that (+)-gamma-lactam enzyme is studied than wide, many preparations for having been used for (-)-gamma-lactam.Such as: source
It can be effectively catalyzed in (+)-gamma-lactam enzyme of Comamonas acidovorans and split 4.6M (500g L-1) raceme
Gamma-lactam, remaining (-)-gamma-lactam ee value > 98% (Bioorg.Med.Chem., 1999,7,2163-2168).
200g L-1Cellulose fixed (+)-gamma-lactam enzyme from Thermophilic Bacteria Sulfolobus solfataricus can have
The catalysis of effect ground splits 0.6M (65g L-1) racemization gamma-lactam, the conversion ratio of racemization gamma-lactam reaches 49.5% when 9h,
The ee value of remaining (-)-gamma-lactam can reach 99.5%.By (+)-gamma-lactam of Sulfolobus solfataricus
After enzyme is fixed on E. coli cell surface by surface of E. coli display technique, 40g L-1Recombinant cell can be catalyzed
0.92M(100g L-1) racemization gamma-lactam (Appl.Microbiol.Biotechnol., 2014,98,6991-7001) water
Solution is split, and conversion ratio reaches 49.5% when 4h, and the ee value of (-)-gamma-lactam can reach 99.5%.250g L-1(+)-γ-interior
Amide immobilised enzymes RutB can be catalyzed 2.0M (218g L-1) racemization gamma-lactam Hydrolysis Resolution, 4h conversion ratio can reach
50%, the ee value of (-)-gamma-lactam can reach 99.5% (Appl.Microbiol.Biotechnol., 2015,99,4691-
4700).From Nocardia farcinica amidase NfpolyA on pure enzyme carrying capacity be 0.25g L-1When, it can be catalyzed
Hydrolyze 0.46M (50g L-1) substrate (ChemCatChem, 2014,6,2517-2521).10g L-1Bacillius
Subtilis168/pMA5-delm lyophilized cells can hydrolyze 0.92M (100g L-1) racemization gamma-lactam, 22.5h conversion ratio
Can reach 55.2%, the ee value of (-)-gamma-lactam reach 99% (Appl.Biochem.Biotechnol., 2015,176,
1687–1699).Compared with (+)-gamma-lactam enzyme, the research of (-)-gamma-lactam enzyme is less, and (-)-reported at present
The research emphasis of gamma-lactam enzyme focuses primarily upon the characterization of zymologic property, the less preparation applied to (+)-gamma-lactam.
It splits and disappears in 0.5mL reaction system from (-)-gamma-lactam enzyme of Bradyrhizobium japonicum USDA 6
Gamma-lactam is revolved, after 37 DEG C of reactions for 24 hours, ee value > 99% (Chinese invention patent, publication number CN of (+)-gamma-lactam
103966192A)。30g L-1Commercialized Lipolase (CAL-B) can split 0.2M in isopropyl ether-water two-phase system
(22.2g L-1) racemization gamma-lactam (Eur.J.Org.Chem., 2008,5263-5268；Patent of invention WO 2009/
007759A1).(-)-gamma-lactam enzyme from Aureobacterium sp. through glutaraldehyde cross-linking, it is anti-in serialization
0.46M (50g L can be split in answering-1) racemization gamma-lactam, 96h conversion ratio can reach 50%, the ee of (+)-gamma-lactam
It is worth > 99% (Tetrahedron-Asymmetry, 1993,4,1117-1128).These enzymes stereoselectivity all with higher,
When reaction conversion ratio reaches 50%, ee value > 99% of remaining (+)-gamma-lactam.Nevertheless, due to these enzymatic activitys
Low, concentration of substrate poor resistance, therefore, it is difficult to large-scale applications.
Compared with conventional chemical synthesis, biocatalysis racemization gamma-lactam Hydrolysis Resolution prepares (+)-gamma-lactam
Method mild, environmental-friendly, simple operation and other advantages with reaction condition, but the biocatalysis of (+)-gamma-lactam at present
Synthetic method is only limitted to laboratory scale, haves the defects that enzymatic activity is low, catalyst usage amount is big, production concentration is not high, uncomfortable
Close industrialized production.Active high, stability is good and is resistant to high concentration product/product enzyme therefore, it is necessary to screening, to meet work
The demand of industry metaplasia production (+)-gamma-lactam.
Summary of the invention
The present invention prepares the deficiency on (+)-gamma-lactam for enzyme process in the prior art, provides a kind of catalytic activity
(-)-gamma-lactam enzyme SvGL and its mutant high, thermal stability is good, enantioselectivity is high and substrate tolerance is high, contains
There are the recombinant expression plasmid and recombinant expression transformants of the enzyme and mutant gene, recombinates the system of SvGL and its mutant enzyme powder
It is standby, and the application in preparation (+)-gamma-lactam.
The purpose of the present invention can be achieved through the following technical solutions:
One of technical solution:
A kind of (-)-gamma-lactam enzyme is following (a) or protein (b):
Protein (a): the protein that the amino acid sequence as shown in SEQ ID No.2 in sequence table is constituted；
Protein (b): pass through one or several amino acid substitutions and (-)-gamma-lactam in the amino acid sequence of (a)
The derived protein that enzymatic activity improves.
The preparation of the protein (a) can be from streptomyces viridochromogenes Streptomyces viridochromogenes
It separates and obtains in CGMCC 4.692, or separate and obtain from the transformant that recombinant expression is somebody's turn to do (-)-gamma-lactam enzyme, it can also
To be artificial synthesized acquisition.
The streptomyces viridochromogenes Streptomyces viridochromogenes CGMCC 4.692 is this laboratory
It is bought from China General Microbiological culture presevation administrative center, and voluntarily preservation.In the present invention, inventor is to laboratory preservation
More than 100 strain bacteriums are cultivated, and medium centrifugal takes appropriate resting cell, are suspended in the phosphorus containing 10mM racemization gamma-lactam
In sour potassium buffer (100mM, pH 7.0), shaking reaction detects the conversion ratio of hydrolysis substrate, to these bacterial strain catalytic waters
The activity of solution reaction is evaluated, and wherein Streptomyces viridochromogenes CGMCC 4.692 is shown best
Reaction effect.And then using shotgun to (-)-of Streptomyces viridochromogenes CGMCC 4.692
Gamma-lactam enzyme is cloned, and is obtained recombination (-)-gamma-lactam enzyme with high activity, is named as SvGL, ammonia
Base acid sequence is as shown in SEQ ID No.2 in sequence table.
The protein (b) is on the basis of protein (a) by obtained from one or several amino acid substitutions
The derived protein that (-)-gamma-lactam enzymatic activity significantly improves.High activity and highly-solid selectively (-)-γ-are obtained in screening
On the basis of lactamase SvGL, inventor carries out protein transformation to wild type SvGL, to further increase the activity of the enzyme.
Wild type SvGL is subjected to Homology search in Genebank database, the sequence of acquisition is subjected to homologous comparison,
Fixed point saturation mutation carried out to conserved positions, to the 188th on the basis of finding the amino acid sequence shown in SEQ ID No.2
After phenylalanine carries out single-point replacement, (-)-gamma-lactam enzymatic activity with higher.Furthermore pass through the active pocket to SvGL
Half design and rational and single-point saturation mutation are carried out with the amino acid residue near substrate channels, and discovery is shown in the SEQ ID No.2
188th phenylalanine of amino acid sequence carries out carrying out single-point to the 130th leucine on the basis of single-point replacement replacing
After changing, equally there is higher (-)-gamma-lactam enzymatic activity.Wherein, by of amino acid sequence shown in SEQ ID No.2
188 phenylalanine residues replace with the mutant protein SvGL of trp residue acquisitionF188WIt is catalyzed (-)-gamma-lactam water
The activity of solution improves 1.3 times.The 188th phenylalanine residue of amino acid sequence shown in SEQ ID No.2 is replaced with into color
Histidine residue, the 130th leucine residue replace with the mutant protein SvGL of isoleucine residues acquisitionL130I/F188WCatalysis
The activity of (-)-gamma-lactam hydrolysis improves 2.2 times, by the 188th phenylpropyl alcohol ammonia of amino acid sequence shown in SEQ ID No.2
Sour residue replaces with trp residue, the 130th leucine residue replaces with tyrosine residue, the mutant protein of acquisition
SvGLL130Y/F188WThe activity of catalysis (-)-gamma-lactam hydrolysis improves 3.2 times.
Wherein the determination of activity of (-)-gamma-lactam enzyme carries out at 30 DEG C, reaction system: 10mM kaliumphosphate buffer
The racemization gamma-lactam of (pH 7.0), 10mM and suitable (-)-gamma-lactam enzyme pass through liquid chromatogram after reacting 20min
(HPLC) reduction amount of gamma-lactam is measured so that it is determined that initial reaction rate.The definition of one enzyme-activity unit (U) is above-mentioned
Under reaction condition, enzyme amount required for the gamma-lactam of 1.0 μm of ol of catalyzing hydrolysis per minute.
The two of technical solution:
A kind of isolated nucleic acid, the nucleic acid are the nucleic acid of coding (-)-gamma-lactam enzyme as described in technical solution one.Tool
Body, be the nucleic acid of following (1) or (2):
(1) nucleic acid of the composition of the nucleotide sequence as shown in SEQ ID No.1 in sequence table；
(2) nucleic acid of following protein (a) or protein (b) is encoded:
Protein (a): by the protein that amino acid sequence shown in SEQ ID No.2 forms in sequence table；
Protein (b): pass through one or several amino acid substitutions and (-)-gamma-lactam in the amino acid sequence of (a)
The derived protein that enzymatic activity improves.
The preparation method of nucleic acid of the present invention is this field customary preparation methods, and the preparation method is preferably: from
The nucleic acid molecules of coding SvGL are extracted in Streptomyces viridochromogenes CGMCC 4.692, or pass through gene
Clone technology obtains the genomic nucleic acid molecule of coding SvGL and its mutant, or is compiled by artificial complete sequence synthetic method
The nucleic acid molecules of code SvGL and its mutant.
The method of the genomic nucleic acid molecule of the present invention that coding SvGL and its mutant are obtained by gene clone technology
Are as follows: with forward primer (Primer F) 5 '-CGGAATTCATGCCGTACATCACCGTG-3 ' (EcoR I), reverse primer
(Primer R)5’-CCCAAGCTTTCACTTCTCCAGGAAGGC-3 ' (Hind III), using round pcr to technical solution one
The SvGL of middle acquisition and its gene DNA sequence of mutant are expanded.
PCR system (50 μ L): 0.25 5 μ L, dNTP Mix of μ L, 10 × Buffer of rTaq, 4 μ L, template plasmid is about
2 μ L, Primer R of 100ng, Primer F 2 μ L, ddH2O complements to 50 μ L.
PCR response procedures: (1) 98 DEG C of denaturation 3min；(2) 98 DEG C of denaturation 30s；(3) 55 DEG C of annealing 30s；(4) 72 DEG C of extensions
1min；Step (2)~(4) carry out 30 circulations, last 72 DEG C of extensions 10min, 4 DEG C of preservation products altogether.
The three of technical solution:
The recombinant expression plasmid of nucleic acid sequence of the one kind comprising (-)-gamma-lactam enzyme of the invention.
The nucleic acid sequence of (-)-gamma-lactam enzyme gene of the invention can be connected to respectively by it by conventional method in that art
It is built-up on kind conventional plasmid carrier.The preferred pET series plasmids of plasmid, more preferable plasmid pET28a.(-)-γ-
Lactamase gene can be operatively connected to be suitble to the downstream of the regulating and controlling sequence of expression, with (-)-γ-interior acyl described in realization
The inducible expression of amine enzyme.
The four of technical solution:
Recombinant expression transformants of the one kind comprising (-)-gamma-lactam enzyme gene of the invention or its recombinant expression plasmid.
The recombination table can be made by converting recombinant expression plasmid of the invention into suitable host cell in it
Up to transformant.The host cell can be the various conventional host cells of this field, on condition that the recombinant expression can be made to carry
Body steadily voluntarily replicates, and (-)-gamma-lactam enzyme gene entrained by it can be by effective expression.The preferred large intestine bar of the present invention
Bacterium, more preferable e. coli bl21 (DE3) or bacillus coli DH 5 alpha.
The five of technical solution:
A kind of preparation method recombinating (-)-gamma-lactam enzyme comprising following steps: cultivate recombinant expression of the invention
Transformant is recombinated (-)-gamma-lactam enzyme.Wherein, cultivating culture medium used in the recombinant expression transformants can be selected from
The conventional medium of this field, on condition that recombinant expression transformants can be made to grow and generate (-)-γ of the present invention-interior acyl
Amine enzyme.The concrete operations of other culture transformant can be carried out by this field routine operation.Preferably, by above-mentioned technical proposal structure
The recombination bacillus coli containing (-)-gamma-lactam enzyme gene built, is seeded to containing 50 μ g mL-1The LB of kanamycin sulfate is trained
Support base (peptone 10g L-1, yeast extract 5g L-1, NaCl g L-1, pH 7.0) in, it is equipped with by the inoculum concentration access of 1% (v/v)
In the 500mL triangular flask of 100mL LB culture medium, it is placed in 37 DEG C, 180rpm shaking table shaken cultivation, as the OD of culture solution600Reach
When 0.6, the isopropyl-β-D-thiogalactoside (IPTG) of final concentration of 0.2mM is added as inducer, 16 DEG C of inductions are for 24 hours
Afterwards, by medium centrifugal, cell is collected, and twice with brine, obtains resting cell.Resulting resting cell is hanged
Float in the buffer (pH 8.0) of 10mL, the ultrasonication in ice bath, supernatant is collected by centrifugation to get recombination (-)-γ-is arrived
The crude enzyme liquid of lactamase.
(-)-gamma-lactam enzyme that the present invention obtains contains histidine tag in N-terminal, therefore it is pure to use nickel column to carry out albumen
Change.The following are buffer formulations: A liquid: Tris-HCl buffer (20mM, pH 8.0), containing 0.5M NaCl, 20mM imidazoles and
The glycerol of 10% (w/v)；B liquid: Tris-HCl buffer (20mM, pH 8.0) contains 0.5M NaCl, 500mM imidazoles and 10%
(w/v) glycerol.The crude enzyme liquid of (-)-gamma-lactam enzyme of expression is loaded in nickel column, elutes foreign protein with A liquid first,
Target protein then is eluted with B liquid, according to the protein of the collection purifying of SDS-PAGE detection, is added final concentration of 20%
(w/v) glycerol, saves backup in -70 DEG C.
The six of technical solution:
(-)-gamma-lactam enzyme of the invention is applied to the hydrolysis of catalytic racemization gamma-lactam to prepare optics
Active (+)-gamma-lactam, (+)-gamma-lactam, which can be used as, prepares the use of homocyclic nucleus glycoside pharmaceutical intermediate.
The substrate of hydrolysis is racemization gamma-lactam.Reaction condition for example reaction temperature, concentration of substrate, buffer composition,
PH, enzyme dosage etc. can be optimized by the normal condition of the such reaction in this field, be selected.
Such as reaction temperature be 30 DEG C when, racemization gamma-lactam concentration of substrate be 10mM, investigated SvGLL130Y/F188W(refer to
Be that the 188th phenylalanine of the amino acid sequence as shown in SEQ ID No.2 in sequence table replaces with tryptophan, while
130 leucines replace with the protein that gained amino acid sequence is constituted after tyrosine) activity in different pH buffers.Institute
Buffer solution system are as follows: sodium citrate buffer solution (pH 5.0-6.0)；Sodium phosphate buffer (pH 6.0-8.5)；Tris-HCl
Buffer (pH 8.0-9.0) and Gly-NaOH buffer (9.0-12.0).The results are shown in Table 1, SvGLL130Y/F188WIt is most suitable
PH is 8.0 or so.
Table 1 (-)-gamma-lactam enzyme SvGLL130Y/F188WActivity in different pH buffers
The condition of typical enzymatic racemization gamma-lactam hydrolysis is as follows: 0.2~1mg being recombinated SvGL or its mutant is molten
Solution is in 10ml buffer, the preferred kaliumphosphate buffer of pH 7.0, and the concentration that substrate racemic gamma-lactam is added is 1~
4M, reaction solution are sufficiently mixed reaction.Reaction carries out at 20 DEG C~70 DEG C, and preferably 30 DEG C.Extracted after reaction with methylene chloride
It takes 4 times, combining extraction liquid adds anhydrous sodium sulfate to be dried overnight.Rotary evaporation, which removes solvent, can be obtained (+)-gamma-lactam.
Compared with prior art, the positive effect of the present invention is that: use (-)-gamma-lactam enzyme of the invention
SvGL and its mutant catalysis (-)-gamma-lactam hydrolysis, preparation optical activity (+)-gamma-lactam have catalyst activity
Height, the few significant advantage of dosage.For substrate gamma-lactam at concentrations up to 4M, catalyst usage amount is 0.2g L-1When, reaction 11h turns
For rate close to 50%, the separation yield of product is higher than 47.5%.Relative to other preparation methods, institute is prepared using the method for the present invention
(-)-gamma-lactam enzyme have catalyst amount is few, concentration of substrate is high, reaction condition is mild, optical purity of products is good, work
Skill is environmental-friendly, easy to operate, is easy to the advantages such as industry amplification, therefore have good prospects for commercial application.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.
Embodiment 1 (-)-gamma-lactam enzyme SvGL gene clone
Bacterial strain Streptomyces viridochromogenes CGMCC 4.692 is cultivated in LB culture medium,
Using high salt method high-purity, the genome DNA of large fragment, it is dissolved in TE buffer (pH8.0), is placed in -20 DEG C of preservations,
" fine works molecular biology experiment guide " of the specific method with reference to volumes such as F Ao Sibai.
Partially digested using total DNA progress of the enzyme Sau3AI in limiting to extraction, the DNA fragmentation after digestion, which passes through electrophoresis, to carry out
Purifying, the segment of about 4~6kb is recycled using glue recovery purifying kit, and the DNA of recycling is dissolved in TE buffer (10mM, pH
8.0) in, -20 DEG C of preservations are placed in.
It is attached by following reaction system and carrier pUC118:
16 DEG C incubate 6 hours, take 10 μ L enzyme-linked products convert 200 μ L bacillus coli DH 5 alpha competent cells (TaKaRa,
Code:D9057), picking monoclonal contains 100 μ g mL to added with 300 μ L-1The deep-well plates of the LB culture medium of ampicillin, 37
DEG C shaken cultivation is stayed overnight, and 50 μ L of inoculation are to containing 100 μ g mL added with 600 μ L-1The second level deep hole of the LB culture medium of ampicillin
The IPTG of final concentration of 0.2mM is added after 37 DEG C of shaken cultivation 3h for plate, and 16 DEG C of inductions are for 24 hours.50 μ L bacterium solutions are taken to be added to 300 μ L
In reaction mixture containing final concentration of 10mM racemization gamma-lactam, 37 DEG C of oscillating reactions 1h are added isometric methanol and terminate
Reaction, thin plate chromatography detect the generation of product, and the definition for having obvious product to generate is positive clone molecule.Positive clone molecule is entrusted
Shanghai Sani Bioisystech Co., Ltd carries out sequencing, analyzes the DNA sequence dna of acquisition, to open reading therein
Frame coded sequence is expressed, reacts and compare, final to obtain the nucleotide sequence as shown in SEQ ID NO.1, according to the nucleosides
(-)-gamma-lactam enzyme that the sequence is expressed is named as by the amino acid sequence that acid sequence is speculated as shown in SEQ ID NO.2
SvGL。
With forward primer (Primer F) 5 '-CGGAATTCATGCCGTACATCACCGTG-3 ', reverse primer (Primer
R)5’-CCCAAGCTTTCACTTCTCCAGGAAGGC-3 ', using polymerase chain reaction (PCR) technology in positive clone molecule
SvGL nucleotide sequence expanded, the DNA fragmentation containing SvGL gene order of acquisition is used into EcoR I and Hind respectively
III double digestion is then attached with the plasmid pET28a for also passing through EcoR I and Hind III double digestion, obtains plasmid
pET28a-SvGL.The plasmid pET28a-SvGL of acquisition is transformed into E. coli BL21, and is spread evenly across and contains
There are 50 μ g mL-1The LB agar plate of kanamycins.After 37 DEG C are incubated overnight, selecting monoclonal and serving extra large Sani's biotechnology has
Limit company is sequenced, as a result correctly.
The fixed point saturation mutation of 2 188 phenylalanines of embodiment
UsingII Site-Directed Mutagenesis Kit(Stratagene,Catalog#
200522) scheme is operated.Design the primer containing catastrophe point: forward primer (Primer F) 5'-GCCGTCCGCA
ACAGCNNKAACGTCGCCGCGGGC-3' and reverse primer (Primer R) 5'-GCCCGCGGCGACGTTMNNGCTGTTGCG
GACGGC-3'.PCR reaction system (50 μ L): template 0.5~20ng, 5 μ L 10 × KOD plus buffer, 5 μ L dNTP are (each
2.0mM), 2 μ L MgSO4(25mM), a pair of of each 1 μ L of mutant primer (20 μM), 1 unit KOD enzyme (TOYOBO CO.,
LTD., Osaka, Japan), add sterile purified water to 50 μ L.Wherein the template is the plasmid pET28a- that embodiment 1 obtains
SvGL.PCR response procedures: (1) 94 DEG C of denaturation 5min；(2) 94 DEG C of denaturation 30sec, (3) 55 DEG C of annealing 1min, (4) 68 DEG C extend
7min, step (2)~(4) carry out 30 circulations, last 68 DEG C of extensions 10min, 4 DEG C of preservation products altogether.Obtained PCR is expanded to produce
Object is spread evenly across in 37 DEG C of Transformed E .coli BL21 competent cells after restriction endonuclease Dpn I digests 2h containing 50 μ g
mL-1The LB agar plate of kanamycins.After 37 DEG C are incubated overnight, monoclonal is selected, carries out Activity determination, wherein active soprano
It serves extra large Sani Biotechnology Co., Ltd to be sequenced, the results showed that the 188th phenylalanine replaces with tryptophan, is ordered
Entitled SvGLF188W。
The fixed point saturation mutation of 3 130 leucines of embodiment
UsingII Site-Directed Mutagenesis Kit(Stratagene,Catalog#
200522) scheme is operated.Design the primer containing catastrophe point: forward primer (Primer F) 5'-TCGCTGGAGC
CCTGCNNKCTCAAGTCCGACGAC-3' and reverse primer (Primer R) 5'-GTCGTCGGACTTGAGMNNGCAGGGCTC
CAGCGA-3'.PCR reaction system (50 μ L): template 0.5~20ng, 5 μ L 10 × KOD plus buffer, 5 μ L dNTP are (each
2.0mM), 2 μ L MgSO4(25mM), a pair of of each 1 μ L of mutant primer (20 μM), 1 unit KOD enzyme (TOYOBO CO.,
LTD., Osaka, Japan), add sterile purified water to 50 μ L.Wherein the template is the plasmid pET28a- that embodiment 2 obtains
SvGLF188W.PCR response procedures: (1) 94 DEG C of denaturation 5min；(2) 94 DEG C of denaturation 30sec, (3) 55 DEG C of annealing 1min, (4) 68 DEG C
Extend 7min, step (2)~(4) carry out 30 circulations, last 68 DEG C of extensions 10min, 4 DEG C of preservation products altogether.What amplification obtained
PCR product 37 DEG C through restriction endonuclease Dpn I digest 2h after Transformed E .coli BL21 competent cells, and be spread evenly across containing
50μg mL-1The LB agar plate of kanamycins.It after 37 DEG C are incubated overnight, select monoclonal and carries out Activity determination, wherein activity is most
High person serves extra large Sani Biotechnology Co., Ltd and is sequenced, the results showed that leucine point in highest two mutant of activity
Tyrosine and isoleucine are not sported, is respectively designated as SvGLL130Y/F188WAnd SvGLL130I/F188W。
The preparation and determination of activity of 4 recombinase of embodiment
The recombination bacillus coli inoculation of expression wild type and mutant SvGL that such as embodiment 1-3 the method is obtained
To containing 50 μ g mL-1In the LB culture medium of kanamycin sulfate, 37 DEG C of shaken cultivations are stayed overnight, and are accessed by the inoculum concentration of 1% (v/v)
In 2L triangular flask equipped with 600mL LB culture medium, 37 DEG C, 180rpm shaking table shaken cultivation are set, as the OD of culture solution600Reach
When 1.5, the IPTG of final concentration of 0.2mM is added as inducer, after 16 DEG C of inductions for 24 hours, by medium centrifugal, collects cell,
And twice with brine, resting cell is obtained.Resulting resting cell is suspended in kaliumphosphate buffer (20mM, pH
7.0) in, high-pressure homogenization crusher machine is freeze-dried up to corresponding recombination enzyme powder.
Protein purification is carried out using nickel column.The following are buffer formulations: A liquid: Tris-HCl buffer (20mM, pH
8.0), the glycerol containing 0.5M NaCl, 20mM imidazoles and 10% (w/v)；B liquid: Tris-HCl buffer (20mM, pH 8.0),
Glycerol containing 0.5M NaCl, 500mM imidazoles and 10% (w/v).By the crude enzyme liquid loading of (-)-gamma-lactam enzyme of expression
Onto nickel column, foreign protein is eluted with A liquid first, then elutes target protein with B liquid, the collection detected according to SDS-PAGE
The protein of purifying is added the glycerol of final concentration of 20% (w/v), saves backup in -70 DEG C.
With kaliumphosphate buffer (100mM, pH 7.0) 0.1mg mL will be diluted to by the pure enzyme of ni-sepharose purification-1, draw
10 μ L enzyme solutions are added in the kaliumphosphate buffer (100mM, pH7.0) that 1mL contains 10mM racemization gamma-lactam, 30 DEG C,
1000rpm shaking reaction 20min, is added the extraction of 600 μ L ethyl acetate, adds anhydrous sodium sulfate to be dried overnight, HPLC
The reduction amount of (Chiralpark AS-H) analysis measurement gamma-lactam is so that it is determined that initial reaction rate.One enzyme-activity unit
(U) definition is under the above conditions, to hydrolyze enzyme amount required for the gamma-lactam of 1.0 μm of ol per minute.Respectively recombinate pure enzyme
Rate activity is listed in Table 2 below.
The pure activity ratio of 2 wild type (-) of table-gamma-lactam enzyme and its mutant compared with
5 temperature of embodiment is to recombination (-)-gamma-lactam enzyme SvGLL130Y/F188WActive influence
With kaliumphosphate buffer (100mM, pH 7.0) 0.5mg mL will be diluted to by the pure enzyme of ni-sepharose purification-1, draw
100 μ L enzyme solutions are added in the kaliumphosphate buffer (100mM, pH 7.0) that 0.5mL contains 100mM racemization gamma-lactam, not equality of temperature
It spends under (20~70 DEG C), 1000rpm shaking reaction 20min is added the extraction of 600 μ L ethyl acetate, adds anhydrous sodium sulfate dried
At night, the reduction amount of HPLC (Chiralpark AS-H) analysis measurement gamma-lactam is so that it is determined that initial reaction rate.As a result such as
Shown in table 3, SvGLL130Y/F188WThe catalytic activity highest at 40 DEG C.
Table 3 (-)-gamma-lactam enzyme SvGLL130Y/F188WActivity at different temperatures
Embodiment 6pH is to recombination (-)-gamma-lactam enzyme SvGLL130Y/F188WActive influence
0.2mg is recombinated into SvGLL130Y/F188WEnzyme is added in 10mL kaliumphosphate buffer (100mM, pH 7.0), is added
(+)-gamma-lactam mixes reaction to final concentration of 3.4M at 40 DEG C.500 μ L are sampled after reaction 8h, 600 μ L acetic acid are added
Ethyl ester extraction, adds anhydrous sodium sulfate to be dried overnight, chiral HPLC (Chiralpark AS-H) analysis measurement conversion ratio and three-dimensional choosing
Selecting property.Concrete analysis condition are as follows: Detection wavelength 230nm, 30 DEG C of column temperature, mobile phase is acetonitrile: isopropanol (9:1, v/v), flow velocity
For 0.8mL min-1.The results are shown in Table 4, and under 5.0-7.0 reaction condition of pH, the pH of buffer does not have the conversion ratio of reaction
Apparent to influence, as pH > 7, reaction rate starts sharply to decline.
4 difference pH of table is to recombinase SvGLL130Y/F188WIt is catalyzed the influence of (+)-gamma-lactam hydrolysis
The hydrolysis of 7 recombinase SvGL catalytic racemization body gamma-lactam of embodiment
Enzyme powder, 1M is lyophilized in recombination E.coli BL21 (DE3) pET28a-SvGL that 0.1mg such as embodiment 4 is obtained
(1.1g) raceme gamma-lactam is added in 10mL kaliumphosphate buffer (100mM, pH 7.0), and reaction is mixed at 40 DEG C.
5 μ L of timing sampling is added the extraction of 600 μ L ethyl acetate, adds anhydrous sodium sulfate to be dried overnight, chiral HPLC (Chiralpark
AS-H) analysis measurement conversion ratio and stereoselectivity.Concrete analysis condition are as follows: Detection wavelength 230nm, 30 DEG C of column temperature, mobile phase
For acetonitrile: isopropanol (9:1, v/v), flow velocity are 0.8mL min-1.8h is converted, reaction conversion ratio is close to 50%, residue (+)-γ-
The optical purity of lactams is higher than 99%.
8 recombinase SvGL of embodimentL130Y/F188WThe hydrolysis of catalytic racemization body gamma-lactam
Recombination E.coli BL21 (DE3) pET28a-SvGL that 0.2mg such as embodiment 4 is obtainedL130Y/F188WLyophozyme
Powder, 4M (4.4g) racemization gamma-lactam are added in 10mL kaliumphosphate buffer (100mM, pH 7.0), are mixed at 40 DEG C anti-
It answers.5 μ L of timing sampling is added the extraction of 600 μ L ethyl acetate, adds anhydrous sodium sulfate to be dried overnight, chiral HPLC (Chiralpark
AS-H) analysis measurement conversion ratio and stereoselectivity.Concrete analysis condition are as follows: Detection wavelength 230nm, 30 DEG C of column temperature, mobile phase
For acetonitrile: isopropanol (9:1, v/v), flow velocity are 0.8mL min-1.11h is converted, reaction conversion ratio terminates reaction close to 50%,
Reaction solution is extracted 4 times with 10mL methylene chloride, separates organic phase, and anhydrous sodium sulfate is added and is dried overnight, rotary evaporation removes molten
Agent, recrystallization obtain 2.1g (+)-gamma-lactam, separate yield 47.7%, optical purity 99%.
9 recombinase SvGL of embodimentL130Y/F188WThe hydrolysis of catalytic racemization body gamma-lactam
Recombination E.coli BL21 (DE3) pET28a-SvGL that 2mg such as embodiment 5 is obtainedL130Y/F188WEnzyme powder, 2M is lyophilized
(22g) raceme gamma-lactam is added in 100mL kaliumphosphate buffer (100mM, pH 7.0), and reaction is mixed at 40 DEG C.
5 μ L of timing sampling is added the extraction of 600 μ L ethyl acetate, adds anhydrous sodium sulfate to be dried overnight, chiral HPLC (Chiralpark
AS-H) analysis measurement conversion ratio and stereoselectivity.Concrete analysis condition are as follows: Detection wavelength 230nm, 30 DEG C of column temperature, mobile phase
For acetonitrile: isopropanol (9:1, v/v), flow velocity are 0.8mL min-1.6h is converted, reaction conversion ratio terminates reaction, instead close to 50%
It answers liquid 100mL methylene chloride to extract 4 times, separates organic phase, anhydrous sodium sulfate is added and is dried overnight, rotary evaporation removes molten
Agent, recrystallization obtain 10.5g (+)-gamma-lactam, separate yield 47.7%, optical purity 99%.
10 recombinase SvGL of embodimentL130Y/F188WThe hydrolysis of catalytic racemization body gamma-lactam
Recombination E.coli BL21 (DE3) pET28a-SvGL that 20mg such as embodiment 5 is obtainedL130Y/F188WFreeze-drying enzyme powder,
3.4M (370g) raceme gamma-lactam is added in 1L kaliumphosphate buffer (100mM, pH 7.0), is mixed at 40 DEG C anti-
It answers.5 μ L of timing sampling is added the extraction of 600 μ L ethyl acetate, adds anhydrous sodium sulfate to be dried overnight, chiral HPLC (Chiralpark
AS-H) analysis measurement conversion ratio and stereoselectivity.Concrete analysis condition are as follows: Detection wavelength 230nm, 30 DEG C of column temperature, mobile phase
For acetonitrile: isopropanol (9:1, v/v), flow velocity are 0.8mL min-1.8h is converted, reaction conversion ratio terminates reaction, instead close to 50%
It answers liquid 1L methylene chloride to extract, separates organic phase, anhydrous sodium sulfate is added and is dried overnight, rotary evaporation removes solvent, ties again
Crystalline substance obtains 180g (+)-gamma-lactam, separates yield 48.6%, optical purity 99%.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Claims (8)
1. a kind of (-)-gamma-lactam enzyme, which is characterized in that it is following (a) or protein (b):
(a): the 188th phenylalanine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into tryptophan, while the 130th
Position leucine replaces with the protein of amino acid sequence composition after isoleucine；
(b): the 188th phenylalanine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into tryptophan, while the 130th
Position leucine replaces with the protein of amino acid sequence composition after tyrosine.
2. a kind of isolated nucleic acid, which is characterized in that the nucleic acid is to encode (-)-gamma-lactam as described in claim 1
The nucleic acid molecules of enzyme.
3. a kind of recombinant expression plasmid comprising nucleic acid as claimed in claim 2.
4. a kind of recombinant expression transformants comprising recombinant expression plasmid as claimed in claim 3.
5. a kind of preparation method of (-)-gamma-lactam enzyme as described in claim 1, which is characterized in that comprise the following steps: training
Recombinant expression transformants as claimed in claim 4 are supported, (-)-gamma-lactam enzyme of recombinant expression is obtained.
6. a kind of (-)-gamma-lactam enzymatic racemization gamma-lactam enantioselective hydrolysis, preparation (+)-gamma-lactam
Using, which is characterized in that (-)-gamma-lactam enzyme is following protein:
(1) protein that the amino acid sequence as shown in SEQ ID No.2 is constituted；
(2) the 188th phenylalanine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into amino acid sequence after tryptophan
The protein of composition；
(3) the 188th phenylalanine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into tryptophan, while the 130th
Leucine replaces with the protein of amino acid sequence composition after isoleucine；
(4) the 188th phenylalanine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into tryptophan, while the 130th
Leucine replaces with the protein of amino acid sequence composition after tyrosine.
7. application according to claim 6, which comprises the following steps: use described (-)-gamma-lactam
Enzyme is as catalyst, catalytic racemization gamma-lactam enantioselective hydrolysis, then extracted from reaction solution, purify it is unreacted
(+)-gamma-lactam of high-optical-purity.
8. application according to claim 7, which is characterized in that the enzymatic racemization gamma-lactam mapping of (-)-gamma-lactam
The condition of selective hydrolysis are as follows: the concentration of (-)-gamma-lactam enzyme is 1~100mg/L, the concentration of racemization gamma-lactam
For 1~4M, reaction temperature is 20~80 DEG C, and reaction solution pH is 5~9.
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