CN109082417A - It is mutated the bacillus megaterium ALA2 cytochrome P 450 enzymes and its preparation method and application of modification - Google Patents

It is mutated the bacillus megaterium ALA2 cytochrome P 450 enzymes and its preparation method and application of modification Download PDF

Info

Publication number
CN109082417A
CN109082417A CN201810840904.3A CN201810840904A CN109082417A CN 109082417 A CN109082417 A CN 109082417A CN 201810840904 A CN201810840904 A CN 201810840904A CN 109082417 A CN109082417 A CN 109082417A
Authority
CN
China
Prior art keywords
leu
ala
glu
lys
cytochrome
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201810840904.3A
Other languages
Chinese (zh)
Inventor
李迅
王亮亮
卢敏
王飞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Forestry University
Original Assignee
Nanjing Forestry University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing Forestry University filed Critical Nanjing Forestry University
Priority to CN201810840904.3A priority Critical patent/CN109082417A/en
Publication of CN109082417A publication Critical patent/CN109082417A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12N9/0042—NADPH-cytochrome P450 reductase (1.6.2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/02—Oxidoreductases acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12Y106/02004—NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The bacillus megaterium ALA2 cytochrome P 450 enzymes and its preparation method and application of modification are mutated, amino acid sequence is as described in SEQ ID NO.1.By to P450BM‑33 catastrophe point R966L/K972L/W1046H of the directional transformation of monooxygenase gene, selection greatly increase recombinant protein to coenzyme NAD H utilization efficiency, are protoenzymes to 2.4 times of coenzyme NAD H utilization efficiency, enzyme activity reaches 11415 U/mg.

Description

It is mutated the bacillus megaterium ALA2 cytochrome P 450 enzymes and preparation method thereof of modification And application
Technical field
The invention belongs to technical field of molecular biology, are related to a kind of bacillus megaterium ALA2 cell color of mutation modification Plain P450 enzyme and its preparation method and application.
Technical background
From bacillus megaterium (Bacillus megaterium) Cytochrome P450BM-3(also known as CYP102A1) It is a kind of monooxygenase of solubility, it can in physiological conditions, the terminal hydroxyl of catalytic long-chain fatty, unsaturated lipid The epoxidation etc. of the double bond of fat acid.(Munro AW,Girvan HM,McLean KJ:Cytochrome P450--redox Partner fusion enzymes.Biochim Biophys Acta 2007,1770 (3): 345-359.) research discovery P450BM-3Monooxygenase is mainly made of two functional domains: the P450 functional domain of N-terminal and the NADPH-P450 reductase of C-terminal Functional domain.(Degtyarenko KN:Structural domains of P450-containing monooxygenase systems.Protein engineering 1995,8(8):737-747.Guengerich FP,Martin MV,Sohl CD,Cheng Q:Measurement of cytochrome P450and NADPH-cytochrome P450reductase.Nat Protoc 2009,4 (9): 1245-1251.) wherein P450 functional domain contains prosthetic heme group (heme), and NADPH-P450 reductase functional domain includes FMN, FAD and NAD binding domain, these binding domain are responsible for electronics It is transferred to the prosthetic heme group of P450 functional domain from donor NADPH, is ultimately transferred to molecular oxygen, activate molecular oxygen.Such knot Structure composition and arrangement guarantee P450BM-3It is excellent that monooxygenase only needs fatty acid substrate and reducing power NADPH that can show Catalytic activity.(Nelson DR:The cytochrome p450homepage.Hum Genomics 2009,4(1):59- 65.) although NADPH and NADH structure and physiological function are very much like, compared with NADH, NADPH is expensive, stability It is poor.(Rosell A,Valencia E,Ochoa WF,Fita I,Pares X,Farres J:Complete reversal of coenzyme specificity by concerted mutation of three consecutive residues in alcohol dehydrogenase.J Biol Chem 2003,278(42):40573-40580.).Also there is reality both at home and abroad Show the heterogenous expression of various types of cells cytochrome p 450 enzyme and the research report of application, but does not carry out the coenzyme specificities of the gene Research is modified in the mutation of change.Therefore, if its gene order can be changed by directional transformation, thus change its coenzyme specificities, But the applicability of its enzyme is expanded significantly, and use cost substantially reduces.Cytochrome P450BM-3The NADPH- of enzyme monooxygenase P450 reductase flavine binding domain and NADPH compound crystal have good resolution ratio (PDB ID:4DQL), it can With the analysis for coenzyme specificities.Network analysis, assessment are carried out using crystal structure of the DeepViewer software to its A subunit The potential polar interaction of NADPH surrounding molecules and steric hindrance, specific assessment result are shown in Fig. 1.It was found that in NADPH molecule Phosphate groupThere are a small cavity in range, one or two of alkaline amino acid residue is contained in this cavity the inside Arg966、Lys972With a pendant hydroxyl group amino acid residue Ser965.Speculate Arg966Side-chain amino group, Lys972The guanidine radicals of side chain And Ser965The hydroxyl of side chain can interact with the phosphate group in NADPH molecule, form stable hydrogen bond or ionic bond, These interactions are conducive to P450BM-3Identification of the monooxygenase to NADPH molecule, and assist NADPH molecule in complex In positioning, to make P450BM-3Monooxygenase shows specificity to NADPH.Simultaneously, in 4DQL crystal structure, It has been found that amino acid residue Trp1046It is parallel to each other with the isoalloxazine ring of FAD molecule.According to electronics in P450BM-3Single oxygenation Mobility status analysis in enzyme, Trp1046Indyl side-chain bulk it is relatively large, space is caused to the activity of FAD isoalloxazine ring Steric hindrance is unfavorable for electronics from NADPH and flows to FAD, reduces the transmission efficiency of electronics, to influence the catalytic efficiency of enzyme.
Summary of the invention
The technical issues of solution: the present invention provides a kind of bacillus megaterium ALA2 cytochrome P 450 enzymes of mutation modification And its preparation method and application, so that its coenzyme specificities be made to change, significantly improve bacillus megaterium ALA2 cell color Plain P450 enzyme is to the utilization efficiency of coenzyme NAD H, and acquisition can be using NADH as coenzyme, and effective catalysis substrate obtains the huge of oxidation product The mutation modifier of Bacterium anthracoides ALA2 cytochrome P 450 enzymes.
Technical solution: the bacillus megaterium ALA2 cytochrome P 450 enzymes of modification, amino acid sequence such as SEQ ID are mutated Described in NO.1.
Encode the gene of the bacillus megaterium ALA2 cytochrome P 450 enzymes of above-mentioned mutation modification, the nucleotide of the gene Sequence is as described in SEQ ID NO.2.
Recombinant plasmid comprising above-mentioned nucleotide sequence.
Host cell E. coli BL21 (DE3) containing above-mentioned recombinant plasmid.
The preparation method of the bacillus megaterium ALA2 cytochrome P 450 enzymes of above-mentioned mutation modification, large intestine bar described in picking Bacterium single colonie culture；When culture to OD600Reach 0.6-0.8, induces 6-8h with final concentration 0.5mM IPTG；Thalline were collected by centrifugation And it washs；With ni-sepharose purification: 1 × Binding Buffer is added into the thallus after washing, thallus, ultrasonication bacterium solution is resuspended After be centrifuged, then extracting centrifugal liquid loading, then wash pillar with 1 × Binding Buffer and remove unbonded protein is finally used Elution containing 100mM imidazoles is to get target protein.
The bacillus megaterium ALA2 cytochrome P 450 enzymes of above-mentioned mutation modification supplemented by NADH substrate for enzymatic activity obtain Obtain the application in oxidation product.
Concrete scheme content are as follows:
The preparation of analysis and directional transformation and recombinase including original gene.
1. the analysis of original gene: Cytochrome P450BM-3The NADPH-P450 reductase flavine of enzyme monooxygenase combines Domain and NADPH compound crystal have good resolution ratio (PDB ID:4DQL), it can be used for coenzyme specificities Analysis.Network analysis is carried out using crystal structure of the DeepViewer software to its A subunit, assessment NADPH surrounding molecules are potential Polar interaction and steric hindrance, specific assessment result is shown in Fig. 1.It was found that the phosphate group in NADPH moleculeRange It is interior there are a small cavity, one or two of alkaline amino acid residue Arg is contained inside this cavity966、Lys972With a side chain Hydroxy amino acid residues Ser965.Speculate Arg966Side-chain amino group, Lys972The guanidine radicals and Ser of side chain965The hydroxyl of side chain can It interacts with the phosphate group in NADPH molecule, forms stable hydrogen bond or ionic bond, these interactions are conducive to P450BM-3Identification of the monooxygenase to NADPH molecule, and positioning of the NADPH molecule in complex is assisted, to make P450BM-3Monooxygenase shows specificity to NADPH.Simultaneously, in 4DQL crystal structure, it has been found that amino acid Residue Trp1046It is parallel to each other with the isoalloxazine ring of FAD molecule.According to electronics in P450BM-3Mobility status point in monooxygenase Analysis, Trp1046Indyl side-chain bulk it is relatively large, steric hindrance is caused to the activity of FAD isoalloxazine ring, is unfavorable for electronics FAD is flowed to from NADPH, reduces the transmission efficiency of electronics, to influence the catalytic efficiency of enzyme.
2. the directional transformation of original gene: referring to the bacillus megaterium cytochrome P 450 enzymes delivered on Genebank Gene order (accession number Genbank:J04832.1), in conjunction with the sequence information of pTrc99a-cypI recombinant plasmid, design is anti- To primer (being shown in Table 1), inverse PCR is carried out by template of pTrc99a-cypl recombinant plasmid, obtains target gene.Using restricted Restriction endonuclease Dpn I specifically degrades template plasmid, to help to obtain the mutant of high-purity.PCR product T4 poly Nucleoside monophosphate kinase carries out phosphorylation, then carries out recirculation connection.The recombination mutation plasmid pTrc99A-cypt of recirculation, It is heat-shock transformed enter Escherichia coli, the transformant plasmid for extracting mutant is sequenced, so that it is determined that whether purpose site is mutated into Function.(being specifically shown in embodiment 1)
3. the preparation of recombinase: the recombinant plasmid being correctly mutated is transferred to Escherichia coli TOP10, measures after inducing expression P450BM-3Monooxygenase enzyme activity.Trimutant P450tri (R966L/K972L/W1046H) can significantly improve enzyme to coenzyme The utilization efficiency of NADH, using oleic acid as substrate, NADH is coenzyme, specific enzyme activity 0.15U/mg.(being specifically shown in embodiment 2).
The utility model has the advantages that by P450BM-3The directional transformation of monooxygenase gene, 3 catastrophe point R966L/ of selection K972L/W1046H greatly increases recombinant protein to coenzyme NAD H utilization efficiency, is protoenzyme to coenzyme NAD H utilization efficiency 2.4 times, enzyme activity reaches 11415U/mg.
Detailed description of the invention
Fig. 1 is P450BM-3Amino acid residue figure relevant to coenzyme specificities in monooxygenase；
Fig. 2 is the protein electrophoresis figure of expression of recombinant e. coli cytochrome P 450 enzymes.M is protein Marker；1st swimming Road is supernatant after recombination bacillus coli (containing pTrc99A-cypt) whole-cell protein sample centrifugation；2nd swimming lane is on recombinant protein Efflux after sample；3rd swimming lane be recombinant protein loading after washed with combination buffer after efflux；4th swimming lane is 50mM miaow Azoles eluent；5th swimming lane is 100mM imidazole elution, and main group becomes cytochrome P 450 enzymes after purification.
Fig. 3 is wild type P450BM-3The comparison of the NADH coenzyme utilization efficiency of monooxygenase and mutant enzyme.
Specific embodiment
The present invention provides a kind of method of directional transformation monooxygenase coenzyme specificities, those skilled in the art can be borrowed Reflect present disclosure, is suitably modified mutational site and mutating acid type is realized.In particular, it should be pointed out that all similar replaces Change and change apparent to those skilled in the art, they are considered as being included in the present invention.In order to make this hair Bright objects, technical solutions and advantages are more clearly understood, and with reference to the accompanying drawings and embodiments, carry out to the present invention further It is described in detail.Related technical personnel obviously can not depart from the content of present invention, in spirit and scope to methods herein and application It is modified or appropriate changes and combinations, carrys out implementation and application the technology of the present invention.Material agents used in following embodiment Deng unless otherwise specified, commercially obtaining.
Embodiment 1: the analysis and directional transformation of bacillus megaterium P450 gene
The determination in 1.1 mutational sites
The directional transformation of bacillus megaterium cytochrome P 450 enzymes gene
By SWISS-MODEL server (http://swissmodel.expasy.org/), cytochromes are submitted P450BM-3Enzyme monooxygenase amino acid sequence information, as the result is shown Cytochrome P450BM-3The knot of enzyme monooxygenase part analysis Among structure, NADPH-P450 reductase flavine binding domain and NADPH compound crystal have good resolution ratio ( PDB ID:4DQL), it can be used for the analysis of coenzyme specificities.Under the auxiliary of Swiss-pdbViewer4.03 software (Arnold K,Bordoli L,Kopp J T.The SWISS-MODEL workspace:a web-based environment for protein structure homology modelling[J].Bioinformatics,2006, 22(2):195-201.；Schwede T,Kopp J,Guex N,et al.SWISS-MODEL:An automated protein Homology-modeling server. [J] .Nucleic Acids Research, 2003,31 (13): 3381-3385.), Assess the potential polar interaction of NADPH surrounding molecules and steric hindrance, it has been found that the phosphate group in NADPH moleculeThere are a small cavity in range, one or two of alkaline amino acid residue Arg is contained in this cavity the inside966、Lys972With One pendant hydroxyl group amino acid residue Ser965.Meanwhile in 4DQL crystal structure, it has been found that amino acid residue Trp1046 It is parallel to each other with the isoalloxazine ring of FAD molecule.According to electronics in P450BM-3Mobility status analysis in monooxygenase, Trp1046's Indyl side-chain bulk is relatively large, causes steric hindrance to the activity of FAD isoalloxazine ring, is unfavorable for electronics and flows to from NADPH FAD, reduces the transmission efficiency of electronics, to influence the catalytic efficiency of enzyme.By analyzing cytochrome P 450 enzymes structure, To Ser965、Arg966、Lys972And Trp1046Several site residues carry out rite-directed mutagenesis.
The building of 1.2 mutant
1.2.1 design of primers
According to structural analysis as a result, and combine the sequence information of pTrc99a-cypI recombinant plasmid, design is following reversed PCR primer, primer are synthesized by Nanjing Si Pu King Company.
Primer 1:5'-CTGGTACCAAATCAGCCGCTGACATACGTTCAGCACGTTATGGAA-3',
Primer 2: 5'-TGAAAAAGCGGTATGAAGCG-3'；
Primer 3:5'-CATGCTGGGTAAGGATCCTCTA-3'；
Primer 3 ': 5'-GCGGCTGGGTAAGGATCCTCTA-3'；
Primer 4:5'-CACGTCTTTTGCGTATCGGC-3'；
Primer 5:5'-GCGGCGGTACCAAATCAGCCGA-3'；
Primer 6:5'-AAAAGCGGTATGAAGCGTAATG-3'；
1.2.2 the building of recombinant plasmid pTrc99A-cypt
The acquisition of the cytochrome P 450 enzymes gene cypt of the bacillus megaterium ALA2 of artificial evolution takes inverse PCR to expand Increasing technology.
Reaction system: using 0.5 μ L pTrc99A-cypI as template, for Arg966、Lys972The mutation in site, selection are drawn 2.5 μ L of object 1 (10 μM), 2.5 μ L of primer 2 (10 μM)；For Trp1046Primer 3 or 3 ' (10 μM) 2.5 μ are selected in the mutation in site L, 2.5 μ L of primer 4 (10 μM)；For Ser965、Arg966The mutation in site；Select primer 5 (10 μM) 2.5 μ L, primer 6 (10 μM) 2.5 μ μ 5 × PCR of the L Buffer of L, 4 μ L dNTP Mixture (each 2.5mM), 10,0.25 μ L Phusion polymerase add water Supply 50 μ L.Reaction condition: 98 DEG C of initial denaturations 30s, 98 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 72 DEG C of 3min, 21 circulations, 72 DEG C extend 5min, take 5 μ L reaction product electrophoresis detections.
Pcr amplification product is purified by PCR purification kit.Because Phusion polymeric enzymatic amplification segment is flat End, can be directly with T4Polynucleotide Kinase to the phosphorylation of 5'-OH terminal oligo.
Phosphorylation reaction: T4Polynucleotide Kinase 1 μ L, 10 × Buffer 1 μ L, 8 μ L of PCR product, it is overall System's totally 10 μ L (ATP of final concentration 1mM can be added in reaction system).37 DEG C, react 3-4h.
Again overnight with 16 DEG C of T4DNA ligase connections.8 μ L liquid are taken from phosphorylation system, add 1 μ L T4DNA connection Enzyme, the 1 corresponding 10 × Buffer of μ L, 10 μ L of total system.
1.2.3 the verifying of mutant plasmid
It takes 1-2 μ L connection liquid electroporated, converts the competent cell of Escherichia coli TOP10, and be coated on containing Amp's On LLB solid medium (100 μ g/mL Amp), 37 DEG C of stationary cultures.Several white colonies are selected to 5mL LLB Liquid Culture In base (containing Amp), shake culture 8h at 37 DEG C extracts recombinant plasmid and carries out sequence verification, obtains containing mutated gene cypt's Recombinant plasmid pTrc99A-cypt.
1.2.4 the expression of recombinant protein
Positive transformant is inoculated on LLB plate and is activated, 37 DEG C of culture 12h, order bacterium colony to 5mL LLB Liquid Culture In base, 37 DEG C, 200rpm shaking table culture 8-10h turns shaking flask culture, works as OD600When reaching between 0.6-0.8, IPTG is added to end Concentration is 0.5mM, and in 30 DEG C, 6-8h is induced under the conditions of 120rpm.Sample is centrifuged 5min under 12000rpm, abandons supernatant, will be big After enterobacteria is resuspended, supersonic wave wall breaking is centrifuged again, and supernatant is taken to detect enzyme activity.Enzyme activity is measured using NADPH method and NADH method. (Budde M., Applied Microbiology and Biotechnology (2004), 66 (2), 180-186) define every point Enzyme amount needed for clock consumes the NADH/NADPH of 1.0 μm of ol is 1 enzyme-activity unit (U).
Embodiment 2: the preparation of recombinase and the measurement of enzyme activity
The preparation of 2.1 recombinases
Due to containing His-tag label in recombinant plasmid pTrc99A-cypt, pass through HisBind Purification Kit (Novagen) is purified, the recombinase purified.Specific operation process:
A. the processing of sample
(1) it by washed thallus, is resuspended with 1 × Binding Buffer 8mL, supersonic wave wall breaking.
(2) after broken wall, 13,000g centrifugation 30min, taking supernatant is sample.
B. pillar is handled
(1) 1mL filler is taken to fill column.
(2) the sterile washing pillar of 3mL is used.
(3) pillar is washed with 1 × Charge Buffer of 5mL.
(4) pillar is washed with 1 × Binding Buffer of 3mL.
C. loading
(1) pillar, coutroi velocity 6 drop about per minute is added in sample.
(2) pillar is washed with 1 × Binding of 3mL Buffer, removes unbonded protein.
(3) pillar is washed with the eluent that 4mL contains 20mM imidazoles, removes foreigh protein removing.
(4) pillar is washed with the eluent of 80mM imidazoles, destination protein is eluted.
(5) pillar is washed with 1 × Strip of 4mL Buffer.
The cytochrome P 450 enzymes purified by this process, by this enzyme solution in 50mM phosphate buffer (pH 8.0) It is dialyzed overnight, removes imidazoles.Enzyme activity is surveyed with NADPH method.
2.2P450BM-3Monooxygenase enzyme activity determination
Enzyme amount needed for definition consumes the NADH of 1.0 μm of ol per minute is 1 enzyme-activity unit (U).Recombinate the measurement body of enzyme activity System is as shown in table 1:
The measurement of 1 coenzyme specificities mutant enzyme activity of table
Component It compares (μ L) Mutant (μ L)
Imidazole buffer (100mM, pH 7.4) 140 140
Oleic acid DMSO solution (50mM) 20 20
NADH(10.0mM) 10 10
Sterile water 30 20
Enzyme solution (0.466mg/mL) 0 10
It amounts to 200 200
At room temperature, A in 10min is monitored and recorded by microplate reader340Numerical value change.At room temperature, to enzyme activity determination Reaction mixture in be added 10 μ L above-mentioned NADH (10.0mM) solution starting reaction, observe and record A in 30min340Numerical value Variation.Single mutant is investigated respectively using unmutated wild-type enzyme (NADPH utilizes type) as reference using NADH for coenzyme W1046A,W1046H,S965A,R966A；Double-mutant P450di (R966L/K972L)；Trimutant P450tri (R966L/ K972L/W1046H) to the utilization efficiency of NADH.
As can be seen from Figure 3, the P450 of wild typeBM-3Single Oxygenation (utilization rate of NADH catalysis substrate cannot be made full use of It is 40% or so), mutant protein then shows different Utilization abilities to coenzyme NAD H.It is most of in these mutant Single mutant has dropped the utilization rate of NADH instead.And Trimutant P450tri (R966L/K972L/W1046H) and list are prominent Variant W1046H significantly improves P450BM-3Utilization efficiency of the monooxygenase to NADH.Relative to wild-type enzyme, single mutant W1046H significantly improves about 95% to the utilization efficiency of coenzyme NAD H, Trimutant P450tri (R966L/K972L/ W1046H about 142%) is significantly improved to the utilization efficiency of coenzyme NAD H.
The Trp of No. 1046 positions is sported into His, while reducing side-chain bulk, in turn ensures the complexity of its structure Degree.Because the side chain of His is imidazole group, there is planar heterocyclic structure, can remain unchanged with FAD isoalloxazine plane of a loop in partial zones It is parallel in domain, stablize FAD molecule.It is compared simultaneously with the huge indoles heterocycle of Trp side chain, the imidazolyl heterocycle size of His side chain is suitable In, while reduction electronics flows to FAD resistance from NADH, and FAD molecule can be stablized and fluctuated in the range of very little, thus substantially Degree improves single mutant W1046H to the utilization efficiency of NADH.
For Trimutant P450tri (R966L/K972L/W1046H), R966L/K972L is respectively by No. 966 positions The Lys for Arg and No. 972 position set sports Leu, can be with since Leu side-chain radical is hydrophobic branch's alkane group Relevant polar interaction (hydrogen bond and ionic bond) is destroyed, enzyme is caused to be remarkably decreased the recognition capability of NADPH molecule, thus The recognition capability to NADH is improved, so Trimutant P450tri can efficiently use single Oxygenation of NADH catalysis substrate.
Sequence table
<110>Nanjing Forestry University
<120>the bacillus megaterium ALA2 cytochrome P 450 enzymes and its preparation method and application of mutation modification
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1048
<212> PRT
<213>bacillus megaterium ALA2 cytochrome P 450 enzymes (Bacillus megaterium)
<400> 1
Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn
1 5 10 15
Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile
20 25 30
Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Arg Val
35 40 45
Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu
50 55 60
Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp
65 70 75 80
Phe Ala Gly Asp Gly Leu Phe Thr Ser Trp Thr His Glu Lys Asn Trp
85 90 95
Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met
100 105 110
Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln
115 120 125
Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Glu Asp
130 135 140
Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr
145 150 155 160
Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser
165 170 175
Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Leu Gln Arg Ala Asn
180 185 190
Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp
195 200 205
Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys
210 215 220
Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly
225 230 235 240
Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr
245 250 255
Gln Ile Ile Thr Phe Leu Ile Ala Gly His Glu Thr Thr Ser Gly Leu
260 265 270
Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln
275 280 285
Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser
290 295 300
Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu
305 310 315 320
Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys
325 330 335
Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu
340 345 350
Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly
355 360 365
Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala
370 375 380
Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys
385 390 395 400
Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met
405 410 415
Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp
420 425 430
Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala
435 440 445
Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu
450 455 460
Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn Thr
465 470 475 480
Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr
485 490 495
Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln
500 505 510
Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala
515 520 525
Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala
530 535 540
Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val Lys
545 550 555 560
Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr
565 570 575
Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys
580 585 590
Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp
595 600 605
Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val
610 615 620
Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser
625 630 635 640
Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala
645 650 655
Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser Lys Glu Leu
660 665 670
Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu Leu
675 680 685
Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro
690 695 700
Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu
705 710 715 720
Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala
725 730 735
His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr
740 745 750
Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala
755 760 765
Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu
770 775 780
Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met
785 790 795 800
Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser Glu
805 810 815
Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile Ser
820 825 830
Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val
835 840 845
Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala
850 855 860
Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe
865 870 875 880
Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu Thr
885 890 895
Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly
900 905 910
Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly
915 920 925
Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu
930 935 940
Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile Ile Thr Leu
945 950 955 960
His Thr Ala Phe Ser Leu Met Pro Asn Gln Pro Leu Thr Tyr Val Gln
965 970 975
His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln
980 985 990
Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Ala
995 1000 1005
Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val His Gln Val Ser
1010 1015 1020
Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg
1025 1030 1035 1040
Tyr Ala Lys Asp Val His Ala Gly
1045
<210> 2
<211> 3147
<212> DNA
<213>bacillus megaterium ALA2 cytochrome P 450 enzymes gene (Bacillus megaterium)
<400> 2
acaattaaag aaatgcctca gccaaaaaca tttggagagc ttaaaaactt accgttatta 60
aacacagata aaccgattca aacgctgatg aaaattgcag atgaattagg ggaaatcttt 120
aaatttgaag cgcctggacg tgtgacgcgc tacttatcca gtcagcgtct aattaaagaa 180
gcatgcgatg aatcccgctt cgataaaaac ttaagtcaag cgcttaaatt tgtgcgtgat 240
tttgcaggag acggtttatt tacaagctgg acgcacgaaa aaaactggaa aaaagcgcat 300
aatatcttac ttccaagctt cagtcagcaa gcgatgaaag gctatcacgc gatgatggtc 360
gatatcgccg tgcagcttat tcaaaagtgg gagcgtctaa atgcagatga gcatattgaa 420
gtaccggaag atatgacgcg cttaacgctt gatacaatcg gtctttgcgg ctttaattac 480
cgctttaaca gcttttatcg cgatcagccg cacccgttta ttacaagtat ggtacgtgca 540
ctggacgaag cgatgaacaa gctgcagcga gcaaatccag atgatcctgc ctacgatgaa 600
aataagcgtc agtttcaaga agatattaag gtgatgaacg atctagtaga taaaattatc 660
gcagatcgca aggcaagcgg tgaacaaagc gatgatttat taacgcatat gctaaacgga 720
aaagacccag aaacaggtga gccgcttgat gatgagaaca ttcgctatca aattattacg 780
ttcttaattg cgggacacga aacaacaagt ggtcttttat catttgcgct gtatttctta 840
gtgaaaaatc cacatgtatt acaaaaagca gcagcagaag cagcacgagt tttagtagat 900
cctgttccaa gctataaaca agtgaaacag cttaaatacg tcggcatggt cttaaacgaa 960
gcgctgcgct tatggccgac agctcctgca ttttctctat atgcaaaaga ggatacggtg 1020
cttggaggag aatatccgtt agaaaaaggc gatgaactga tggttctaat tcctcagctt 1080
caccgtgata aaacgatttg gggagacgat gtggaagagt tccgcccaga gcgttttgaa 1140
aatccaagcg cgattccgca gcatgcgttt aaaccgtttg gaaacggtca gcgtgcgtgt 1200
atcggtcagc agttcgctct tcatgaagca acgcttgtac ttggtatgat gctaaaacat 1260
tttgattttg aagatcatac aaattacgag atggatatta aagaaacttt aacgttaaaa 1320
cctgaaggtt ttgttgtaaa agcaaaatca aaaaaaattc cgcttggtgg cattccttca 1380
cctagcacgg aacagcctgc taaaaaagca cgcaaaaagg tagagaacgc tcataatacg 1440
ccgctgcttg tgttatacgg ttcaaatatg ggaacagctg aaggaacggc gagggattta 1500
gcagatattg cgatgagcaa aggattcgca cctcaggttg caacgcttga ttcccatgca 1560
ggaaatcttc cgcgtgaagg agctgtttta attgtaacgg cttcttataa cggacatcca 1620
cctgataacg caaagcagtt tgtcgactgg ttagaccaag cgtctgctga tgaagtaaaa 1680
ggcgttcgct actccgtatt tggatgcggc gataaaaact gggcaactac ctatcagaaa 1740
gtgcctgctt ttatcgatga aacgctagcc gctaaagggg cagaaaacat cgctgaacgc 1800
ggcgaagcag atgcaagcga cgactttgaa ggcacatacg aagaatggcg tgaacatatg 1860
tggagtgacg tagcagctta ctttaacctc gacatagaaa acagtgaaga taataaatct 1920
acgctttcac ttcaatttgt cgacagcgct gcggacatgc cgcttgcgaa aatgcacggt 1980
gctttttcgg cgaacgttgt agcaagcaaa gaactacaaa aaccaggcag tgaacgaagc 2040
acgcgtcatc ttgaaattga gcttccaaaa gaagtttctt atcaagaagg agatcattta 2100
ggtattattc cgcgcaacta tgaaggaata gtaaatcgtg taacgacaag gttcggtcta 2160
gatgcatcac agcaaatccg tttggaagct gaagaagaaa aattagcgca tttgccactc 2220
ggtaaaaccg tatcagtaga agagcttctg caatatgtgg agcttcaaga tcctgttacg 2280
cgcacacagc ttcgcgcaat ggctgctaaa acggtctgcc cgccgcataa agtggagctt 2340
gaagccttgc ttgaaaagca agcgtataaa gaaaaggtgc tggcaaaacg cttaacaatg 2400
cttgaactgc ttgaaaaata tccggcgtgt gaaatggaat tcagcgaatt tatcgctctt 2460
cttccaagca tgcgtccgcg ctattactcc atttcttcat cacctcgcgt cgatgaaaaa 2520
caagcaagca tcacggttag cgtcgtctca ggagaggcgt ggagcggata tggagaatat 2580
aaaggaattg cgtcgaacta tcttgctgag ctgcaagaag gagatacaat tacgtgcttt 2640
gtttccacac cgcagtcagg ctttacgctg ccaaaagatc ctgaaacacc gattgtcatg 2700
gtcggaccag gaacaggcgt cgcgccgttt agaggctttg tgcaggctcg caagcagtta 2760
aaagaacaag gacagtcgct tggagaagcg catttatact ttggctgccg ttcacctcat 2820
gaagactatc tgtatcaaga agagcttgaa aatgcacaaa atgaaggtgt cattacgctt 2880
cataccgctt tttcactggt accaaatcag ccgctgacat acgttcagca cgttatggaa 2940
caagacggca cgaaattgat tgaacttctt gatcaaggag cgcacttcta tatttgcgga 3000
gacggaagcc aaatggcacc tgacgttgaa gcaacgctta ttaaaagcta tgctgatgtt 3060
catgaagtaa gtgaagcaga cgctcgctta tggctgcaac agctagaaga aaagggccga 3120
tacgcaaaag acgtgcatgc tgggtaa 3147
<210> 3
<211> 45
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ctggtaccaa atcagccgct gacatacgtt cagcacgtta tggaa 45
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tgaaaaagcg gtatgaagcg 20
<210> 5
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
catgctgggt aaggatcctc ta 22
<210> 9
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gcggctgggt aaggatcctc ta 22
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
cacgtctttt gcgtatcggc 20
<210> 7
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gcggcggtac caaatcagcc ga 22
<210> 8
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
aaaagcggta tgaagcgtaa tg 22

Claims (6)

1. being mutated the bacillus megaterium ALA2 cytochrome P 450 enzymes of modification, it is characterised in that amino acid sequence such as SEQ ID Described in NO.1.
2. being mutated the gene of the bacillus megaterium ALA2 cytochrome P 450 enzymes of modification, feature described in coding claim 1 It is the nucleotide sequence of the gene as described in SEQ ID NO.2.
3. the recombinant plasmid comprising nucleotide sequence described in claim 2.
4. the host cell E. coli BL21 (DE3) containing recombinant plasmid described in claim 3.
5. being mutated the preparation method of the bacillus megaterium ALA2 cytochrome P 450 enzymes of modification, feature described in claim 1 It is that Escherichia coli described in picking claim 4 shake bacterium culture；When culture to OD600Reach 0.6-0.8, with 0.5 mM of final concentration IPTG induces 6-8 h；Thalline were collected by centrifugation and washs；With ni-sepharose purification: 1 × Binding being added into the thallus after washing Thallus is resuspended in Buffer, is centrifuged after ultrasonication bacterium solution, then extracting centrifugal liquid loading, then wash column with 1 × Binding Buffer Son removes unbonded protein, finally with the elution containing 100 mM imidazoles to get target protein.
6. being mutated the bacillus megaterium ALA2 cytochrome P 450 enzymes of modification described in claim 1 in the enzymatic supplemented by NADH Substrate obtains the application in oxidation product.
CN201810840904.3A 2018-07-27 2018-07-27 It is mutated the bacillus megaterium ALA2 cytochrome P 450 enzymes and its preparation method and application of modification Pending CN109082417A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810840904.3A CN109082417A (en) 2018-07-27 2018-07-27 It is mutated the bacillus megaterium ALA2 cytochrome P 450 enzymes and its preparation method and application of modification

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810840904.3A CN109082417A (en) 2018-07-27 2018-07-27 It is mutated the bacillus megaterium ALA2 cytochrome P 450 enzymes and its preparation method and application of modification

Publications (1)

Publication Number Publication Date
CN109082417A true CN109082417A (en) 2018-12-25

Family

ID=64830910

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810840904.3A Pending CN109082417A (en) 2018-07-27 2018-07-27 It is mutated the bacillus megaterium ALA2 cytochrome P 450 enzymes and its preparation method and application of modification

Country Status (1)

Country Link
CN (1) CN109082417A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112359027A (en) * 2021-01-13 2021-02-12 凯莱英生命科学技术(天津)有限公司 Cytochrome P450 enzyme mutant and application thereof
CN114621934A (en) * 2020-12-10 2022-06-14 中国科学院大连化学物理研究所 P450 reductase and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102399796A (en) * 2011-11-09 2012-04-04 南京林业大学 Bacillus megaterium ALA2 cytochrome P450 enzyme gene and methods of recombinant plasmid construction and enzyme purification

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102399796A (en) * 2011-11-09 2012-04-04 南京林业大学 Bacillus megaterium ALA2 cytochrome P450 enzyme gene and methods of recombinant plasmid construction and enzyme purification

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DÖHR O ET AL.: "Engineering of a functional human NADH-dependent cytochrome P450 system", 《PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES》 *
GIRVAN H M ET AL.: "Flavocytochrome P450 BM3 mutant W1046A is a NADH-dependent fatty acid hydroxylase: implications for the mechanism of electron transfer in the P450 BM3 dimer", 《ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS》 *
RYAN J D ET AL.: "Engineering cytochrome P450 enzymes for improved activity towards biomimetic 1, 4‐NADH cofactors", 《CHEMBIOCHEM》 *
WANG T ET AL.: "Cloning, Expression and Characterization of a Monooxygenase P450BM3 from Bacillus megaterium ALA2", 《ADVANCED MATERIALS RESEARCH》 *
李迅等: "巨大芽孢杆菌细胞色素 P450 酶的表达纯化及定向进化研究", 《林产化学与工业》 *
王亮亮: "基于纤维小体脚手架蛋白的多酶共展示体系构建", 《中国博士学位论文全文数据库 工程科技I辑》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114621934A (en) * 2020-12-10 2022-06-14 中国科学院大连化学物理研究所 P450 reductase and application thereof
CN114621934B (en) * 2020-12-10 2023-11-24 中国科学院大连化学物理研究所 P450 reductase and application thereof
CN112359027A (en) * 2021-01-13 2021-02-12 凯莱英生命科学技术(天津)有限公司 Cytochrome P450 enzyme mutant and application thereof
CN112359027B (en) * 2021-01-13 2021-04-13 凯莱英生命科学技术(天津)有限公司 Cytochrome P450 enzyme mutant and application thereof

Similar Documents

Publication Publication Date Title
Su et al. CotA, a multicopper oxidase from Bacillus pumilus WH4, exhibits manganese-oxidase activity
He et al. A quinone-dependent dehydrogenase and two NADPH-dependent aldo/keto reductases detoxify deoxynivalenol in wheat via epimerization in a Devosia strain
Yu et al. Molecular mechanism of nicotine degradation by a newly isolated strain, Ochrobactrum sp. strain SJY1
Knietsch et al. Construction and screening of metagenomic libraries derived from enrichment cultures: generation of a gene bank for genes conferring alcohol oxidoreductase activity on Escherichia coli
Chen et al. Identification and molecular characterization of a novel flavin-free NADPH preferred azoreductase encoded by azoB in Pigmentiphaga kullae K24
CN103497911B (en) Application of Chryseobacterium sp. and carbonyl reductase thereof in production of aprepitant chiral intermediate
Li et al. Cloning, expression, characterization and homology modeling of a novel water-forming NADH oxidase from Streptococcus mutans ATCC 25175
WO2018113476A1 (en) Genetically engineered bacterium of coexpressing cyclohexanone monooxygenase and isopropanol dehydrogenase and use thereof
Girhard et al. Cytochrome P450 reductase from Candida apicola: versatile redox partner for bacterial P450s
CN101565709B (en) 3-sterone-9Alpha-hydroxylation enzyme gene, 3-sterone-9Alpha-hydroxylation enzyme reductase gene, relevant carriers, engineering bacteria and applications thereof
CN104152505A (en) Method for transforming and preparing 4-hydroxyl-L-isoleucine by using recombinant bacterial strains
Sjöholm et al. Transcription and regulation of the bidirectional hydrogenase in the cyanobacterium Nostoc sp. strain PCC 7120
Toshchakov et al. Genomic insights into energy metabolism of Carboxydocella thermautotrophica coupling hydrogenogenic CO oxidation with the reduction of Fe (III) minerals
Kadow et al. Recombinant expression and purification of the 2, 5-diketocamphane 1, 2-monooxygenase from the camphor metabolizing Pseudomonas putida strain NCIMB 10007
Zhang et al. Cloning and characterization of a robust recombinant azoreductase from Shewanella xiamenensis BC01
CN109082417A (en) It is mutated the bacillus megaterium ALA2 cytochrome P 450 enzymes and its preparation method and application of modification
Bertsova et al. Localization-controlled specificity of FAD: threonine flavin transferases in Klebsiella pneumoniae and its implications for the mechanism of Na+-translocating NADH: quinone oxidoreductase
CN109852597A (en) A kind of beta galactosidase galRBM20_1 and its preparation method and application
Kadow et al. Functional assembly of camphor converting two-component Baeyer–Villiger monooxygenases with a flavin reductase from E. coli
CN106754776A (en) The GDH mutant that a kind of specific enzyme activity for being catalyzed xylose is improved
Zhang et al. Functional characterization of the methylarsenite‐inducible arsRM operon from Noviherbaspirillum denitrificans HC18
US8835153B2 (en) Process and genes for expression and overexpression of active [FeFe] hydrogenases
CN103695443B (en) A kind of Novel carbonyl reductase, its gene and application
Müller et al. Putative reaction mechanism of heterologously expressed octopine dehydrogenase from the great scallop, Pecten maximus (L)
CN102154336A (en) Acidophilic vulcanized bacillus thioredoxin reductase gene and recombinant protein thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20181225

RJ01 Rejection of invention patent application after publication