CN105821014A - Symbiobacterium thermophilum meso-diaminopimelate dehydrogenase mutants - Google Patents

Abstract

The invention discloses five symbiobacterium thermophilum meso-diaminopimelate dehydrogenase mutants after coenzyme specific turning. The source of used template enzyme is symbiobacterium thermophilum NADP(H) dependent meso-diaminopimelate dehydrogenase (StDapdh), after enzyme mutation, the amino acid residue with the homology comparison equivalent to the template enzyme site 35 is replaced by glutamic acid which is R35E, the amino acid residue with the homology comparison equivalent to the female parent enzyme site 35 and site 36 are respectively replaced by glutamic acid and valine which are R35E/R36V, the amino acid residue with the homology comparison equivalent to the female parent enzyme site 35 and site 36 are respectively replaced by aspartic acid and valine which are R35d/R36V, the amino acid residue with the homology comparison equivalent to the female parent enzyme site 35 and site 36 are respectively replaced by aspartic acid and glutamine which are R35d/R36Q, the amino acid residue with the homology comparison equivalent to the StDapdh site 35, site 36 and site 76 are respectively replaced by glutamic acid, valine and valine which are R35E/R36V/Y76V, according to the mutant enzyme, the coenzyme specificity is mutated from NADP(H) to NAD(H).

Description

Thermophilic symbiosis bacillusmeso-diaminopimelate dehydrogenase mutant

Technical field

The invention belongs to protein engineering field, relate to a kind of to biocatalyzermesoThe coenzyme Preference of-diaminopimelate dehydrogenase is transformed, and acquisition makesmesoThe coenzyme Preference of-diaminopimelate dehydrogenase becomes the mutant of NAD (H) from NADP (H).

Background technology

D-aminoacid is a kind of alpha-non-natural amino acid, is also important chiral intermediate, food, cosmetics and pharmaceuticals industry extensively apply (Wang Ying, Li Yunzheng,Liaoning chemical industry 2003,32, 58-60.).Known D-amino acid dehydrogenase mostly be memebrane protein (Tanigawa M., Shinohara T.,et al,Amino Acids 2010,38, 247-255; Xu S. J., Ju J. S., Ma Y. H.,Wei Sheng Wu Xue Bao 2007, 47, 634-638; Jones H., Venables W. A.,Biochimie 1983, 65, 177-183; Jones H., Venables W. A.,FEBS Lett. 1983,151, 189-192; Wild J., Obrepalska B.,Mol. Gen. Gent. 1982,186, 405-410; Wild J., Klopotowski T.,Mol. Gen. Gent. 1981, 181, 373-378; Olsiewski P. J., Kaczorowski G. J., Walsh C.,J. Biol. Chem. 1980, 255, 4487-4494.), it is impossible to it is used for commercial production, only hasmeso-diaminopimelate dehydrogenase (DAPDH) (EC 1.4.1.16) and mutant thereof can be utilized for the amino acid whose biosynthesis of D-(Misono H., Soda K.,Journal Biol. Chem. 1980,255, 10599-10605; Vedha-Peters K., Gunawardana M., Rozzell J. D., Novick S. J.,J. Am.n Chem. Soc. 2006,128, 10923-10929; Akita H., Doi K., Kawarabayasi Y., Ohshima T.,Biotechnol . Lett. 2012,34, 1693-1699; Akita H., Suzuki H., Doi K., Ohshima T.,Appl. Microbiol. Biotech. 2013, 98, 1135-1143.).In terms of coenzyme Preference, it is known that DAPDH enzyme and the coenzyme Preference of mutant be NADP (H), there is no the research report of DAPDH family enzyme cofactor Preference transformation the most yet.Compare and coenzyme NAD P (H), coenzyme NAD (H) has more preferable stability, less expensive price and wider array of coenzyme round-robin method, NAD (H) dependent form amino acid dehydrogenase can use hydrogenlyase (FDH) and formic acid ammonia as coenzyme circulating system, and NADP (H) dependent form amino acid dehydrogenase mainly uses glucose dehydrogenase (GDH) and glucose as coenzyme circulating system.Amino acid dehydrogenase needs alkaline pH to carry out catalytic reaction, GDH circulating system use glucose as substrate, the gluconic acid that circulation generates can reduce the pH value of system, the process of impact reaction, and be difficult to remove, also needing additionally to add the ammonium salt needed for reaction, these added ingredient will increase follow-up separation process difficulty；FDH circulating system can use formic acid ammonia as cosubstrate, and wherein ammonia directly participates in synthesis, and formic acid is oxidized to the CO of volatile removing₂, reactant liquor does not has additional impurities, the isolated and purified process of subsequent products will be greatly simplified, during using amino acid dehydrogenase synthesizing amino acid, use NAD (H) to make coenzyme and FDH does blood circulation and will more have application advantage.Therefore, it is thus achieved that the D-amino acid dehydrogenase of NAD (H) dependent form is very important.

We are from thermophilic symbiosis bacillusSymbiobacterium thermophilumIn obtain can reduction amination synthesis D-alanine amino acid dehydrogenase StDapdh(Gao X., Chen X., Liu W., Feng J., Wu Q., Hua L., Zhu D.,Appl. Environ. Microbiol. 2012,78, 8595-8600), this enzyme can be used directly and carry out the amino acid whose synthesis of D-(number of patent application: 201210334554.6), is obtaining X-ray crystal structure (Liu W., Li Z., Huang C. H., Guo R. T., Zhao L., Zhang D., Chen X., Wu Q., Zhu D.,Chembiochem 2014,15, 217-222), expanded by transformation its substrate spectrum to leucine and phenylalanine (Gao X., Huang F., Feng J., Chen X., Zhang H., Wang Z., Wu Q., Zhu D.,Appl. Environ. Microbiol. 2013) and the work such as aminoacid such as Terleu (number of patent application: 201310459718.2) of bigger steric hindrance on the basis of, it is thus achieved that coenzyme Preference is the muton of NAD (H) dependent form, can will increase the feasibility of the actual application of this enzyme further.

Summary of the invention

The present invention is to biocatalyzermesoThe coenzyme Preference of-diaminopimelate dehydrogenase is transformed, and acquisition makesmesoThe coenzyme Preference of-diaminopimelate dehydrogenase becomes the mutant of NAD (H) from NADP (H).

Biocatalyzer used in the present invention, the obtaining step of StDapdh mutant is as follows:

1. With pET32-StDapdh plasmid as template, use Quick Change Mutagenesis Kit mutagenesis kit introduces R35E single-site mutant；

2. With pET32-StDapdh plasmid as template, use Quick Change Mutagenesis Kit mutagenesis kit introduces the sudden change of R35E/R36V two Sites Combination；

3. With pET32-StDapdh plasmid as template, use Quick Change Mutagenesis Kit mutagenesis kit introduces the sudden change of R35D/R36V two Sites Combination；

4. With pET32-StDapdh plasmid as template, use Quick Change Mutagenesis Kit mutagenesis kit introduces the sudden change of R35D/R36Q two Sites Combination；

5. With pET32-StDapdh plasmid as template, use Quick Change Mutagenesis Kit mutagenesis kit introduces the sudden change of R35D/R36Q/Y76V tri-Sites Combination；

6. Constructed engineering bacteria is cultivated, abduction delivering, mutant protein is present in intracellular with soluble form mostly；

7. By Ni-NTA by this mutant protein purification.

Present invention have the advantage that

The inventive method is set out with NADP (H) dependent form amino acid dehydrogenase StDapdh, it is thus achieved that the upset of its coenzyme Preference is the muton enzyme of NAD (H) dependent form.

Accompanying drawing illustrates:

Accompanying drawing 1: corresponding StDapdh muton protein purification SDS-PAGE spectrum.In figure, swimming lane M: protein molecular weight Marker, swimming lane 1 is wild-type enzyme, and swimming lane 2-6 is the muton enzyme of five after purification: R35E, R35E/R36V, R35D/R36V, R35D/R36Q, R35E/R36V/Y76V.

Detailed description of the invention

Further illustrate present invention below by way of specific embodiment, but these embodiments are not construed as limiting the invention.In following Examples, material therefor is unless otherwise indicated, and chemical drugs used, reagent are purchased from Sigma company.DNA and protein Marker is purchased from Fermentas company, and protein purification Ni-NTA filler is purchased from GE.Quick Change Mutagenesis Kit is purchased from Agilent company, and mutant primer requires to be designed according to test kit.Primer synthesis, determined dna sequence are carried out by Jin Weizhi company (Beijing), pET32 plasmid vector, and TOP10, BL21 (DE3) Efficiency Competent is purchased from Novagen.Liquid-phase chromatographic analysis uses Agilent-1200 chromatograph, and chromatographic column is Eclipse XDB-C18 post (4.6 × 150 mm).

Embodiment 1 : gene mutation

UsedStDapdhGene Genbank is AP006840.1, first by this total gene synthesis and be connected on pET32 carrier obtain plasmid: pET32-StDapdh, and in e. coli bl21 (DE3), wild type gene being carried out soluble-expression, the n-end of albumen given expression to is with 6*his tag.The site suddenlyd change as required, with reference to Quick Change Mutagenesis Kit test kit operation instruction, PCR mutant primer used in synthesis table 1, from wild type strain, plasmid DNA is proposed, PCR primer amplification is carried out for template with it, amplification PCR primer out being carried out Dpn1 cutting, carries out nucleic acid recovery and convert TOP10 competence after cutting, picking list bacterium colony sample presentation checks order, for the correct sample that checks order, convert BL21 bacterial strain.

Table 1 : sudden change PCR Primer

WithpET32-StDapdhPlasmid is template, use primer 1 and 2 to introduce R35E single mutation on plasmid, and sudden change afterproduct is converted to escherichia coli TOP10 competence, extract plasmid sequence verification, by checking order, correct Plastid transformation enters in e. coli bl21 (DE3) competence, it is thus achieved that expression strain.Use primer 3 and 4,5 and 6,7 and 8, repeat the above steps respectively, it is thus achieved that the double mutant strain of R35E/R36V, R35D/R36V and R35D/R36Q tri-；With R35E/R36V as template, use primer 9 and 10, it is thus achieved that R35E/R36V/Y76V tri-mutant strain.

Embodiment 2 : the expression of mutant enzyme, preliminary purification

The mutants which had obtained in embodiment 1 is cultivated in 2 L LB fluid mediums, cultivates to OD prior to 37 DEG C₆₀₀After about 0.8, the isopropyl-beta D-thio galactopyranoside (IPTG) adding final concentration 0.5 mM carries out abduction delivering, and inducing temperature is 25 DEG C, and induction time is 20 hours.After abduction delivering terminates, it is centrifuged 5 minutes in 5000 rpm and collects thalline, utilize buffer A (20 mM Tris-Cl pH 8.0,50 mM sodium chloride) resuspended and wash thalline, and recentrifuge thalline.Follow-up all purification experiment are all carried out at 4 DEG C, and all buffer are all first cooled to 4 DEG C in advance.By the thalline after washing with the 100 resuspended thalline of mL buffer A, high-pressure homogenization crushes, and 14000 rpm are centrifuged 30 minutes and remove broken precipitation, the Ni-NTA chromatographic column that supernatant loading buffer A is equilibrated, and with buffer B (20 mM Tris-Cl pH 8.0,50mM imidazoles, 500 mM sodium chloride) remove foreign protein, with buffer C(20 mM Tris-Cl pH 8.0,250mM imidazoles, 500 mM sodium chloride) elute destination protein.By destination protein to buffer D(20 mM Tris-Cl pH 8.0,50 mM sodium chloride) dialyse, to remove high concentration imidazoles and sodium chloride.Accompanying drawing 1 is muton protein electrophoresis collection of illustrative plates after purification.

Embodiment 3 : the vitality test of mutant

The vigor of muton utilizes SPECTRAMAXM2e (MD, USA) microplate reader is measured, survey live body system used is as follows: the final concentration of each composition is respectively as follows: 20 mM substrate pyruvate, 200 mM substrate ammonium chloride, 1 mM coenzyme NAD H(or NADPH), the appropriate pure enzyme of StDapdh mutant, surveying buffer of living is 100 mM sodium carbonate/bicarbonate buffer solution pH 9.0, final volume 200 L.Substrate and protein sample are all initially charged in 96 orifice plates and balance 10 minutes in 30 ° of C, then are added to coenzyme NAD H initial action, measure enzyme activity by measuring the minimizing of absorbance at 340 nm (NADH is 6.22 mM at 340 nm molar extinction coefficients^-1∙cm^-1), enzyme activity unit is defined as enzyme amount during catalytic reaction needed for consumption 1 mol coenzyme NAD H per minute, table 2 is wild-type enzyme and the muton enzyme Rate activity to NADH and NADPH, as can be seen from the table, the coenzyme Preference of all mutants all overturns as NAD (H) dependent form from the NADP (H) of wild-type enzyme.

Table 2 Wild-type enzyme and mutant pair NADH And NADPH Specific enzyme activity

Claims (9)

1. five kinds of NAD(H) dependent form amino acid dehydrogenase mutant, can be as biocatalyzer.

2. include: utilize derive from thermophilic symbiosis bacillus (Symbiobacterium thermophilum) NADP(H) dependent form amino acid dehydrogenase StDapdh as template, carry out sudden change by Protocols in Molecular Biology and obtain NAD(H) the amino acid dehydrogenase mutant of dependent form.

3. biocatalyzer as claimed in claim 1meso-diaminopimelate dehydrogenase mutant, its protein specificity is, mutant template be thermophilic symbiosis bacillus (Symbiobacterium thermophilum)meso-diaminopimelate dehydrogenase or be not less than the protein of 80% with its amino acid sequence similarity.

4. biocatalyzer as claimed in claim 1meso-diaminopimelate dehydrogenase mutant, its protein sequence is characterised by that the arginine (R) that tetraploid rice is equivalent to StDapdh the 35th replaces with glutamic acid (E), i.e. R35E.

5. biocatalyzer as claimed in claim 1meso-diaminopimelate dehydrogenase mutant, its protein sequence is characterised by that the arginine (R) that tetraploid rice is equivalent to StDapdh the 35th replaces with glutamic acid (E)；The arginine (R) that tetraploid rice is equivalent to StDapdh the 36th replaces with valine (V), i.e. R35E/R36V.

6. biocatalyzer as claimed in claim 1meso-diaminopimelate dehydrogenase mutant, its protein sequence is characterised by that the arginine (R) that tetraploid rice is equivalent to StDapdh the 35th replaces with aspartic acid (D)；The arginine (R) that tetraploid rice is equivalent to StDapdh the 36th replaces with valine (V), i.e. R35D/R36V.

7. biocatalyzer as claimed in claim 1meso-diaminopimelate dehydrogenase mutant, its protein sequence is characterised by that the arginine (R) that tetraploid rice is equivalent to StDapdh the 35th replaces with aspartic acid (D)；The arginine (R) that tetraploid rice is equivalent to StDapdh the 36th replaces with glutamine (Q), i.e. R35D/R36Q.

8. biocatalyzer as claimed in claim 1meso-diaminopimelate dehydrogenase mutant, its protein sequence is characterised by that the arginine (R) that tetraploid rice is equivalent to StDapdh the 35th replaces with glutamic acid (E)；The arginine (R) that tetraploid rice is equivalent to StDapdh the 36th replaces with valine (V)；The tyrosine (Y) that tetraploid rice is equivalent to StDapdh the 76th replaces with valine (V), i.e. R35E/R36V/Y76V.

9. biocatalyzer as claimed in claim 1meso-diaminopimelate dehydrogenase mutant, its coenzyme Preference is from nicotinamide-adenine dinucleotide phosphate (NADPH or NADP⁺) become nicotinamide adenine dinucleotide (NADH or NAD⁺).