CN106148310A - A kind of nitrilase mutants and the application in prepared by nicotinic acid thereof - Google Patents

A kind of nitrilase mutants and the application in prepared by nicotinic acid thereof Download PDF

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CN106148310A
CN106148310A CN201610659707.2A CN201610659707A CN106148310A CN 106148310 A CN106148310 A CN 106148310A CN 201610659707 A CN201610659707 A CN 201610659707A CN 106148310 A CN106148310 A CN 106148310A
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ala
leu
nitrilase
glu
gly
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CN106148310B (en
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姜岷
李�禾
董维亮
马江锋
章文明
信丰学
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Nanjing Tech University
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    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/05—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in nitriles (3.5.5)
    • C12Y305/05001—Nitrilase (3.5.5.1)

Abstract

The invention discloses a kind of nitrilase mutants and the application in prepared by nicotinic acid thereof, described mutant is the 168th phenylalanine of aminoacid sequence shown in sequence SEQ ID NO. 2 to be sported valine and 192 mutant serines are phenylalanine.With the recombination engineering containing nitrilase mutants encoding gene after abduction delivering obtain wet thallus for enzyme source, with 3 cyanopyridines as reaction substrate, being constituted reaction system with the PBS of pH=7.0 for reaction medium, water-bath at 40 DEG C, reaction terminates to obtain nicotinic acid.

Description

A kind of nitrilase mutants and the application in prepared by nicotinic acid thereof
Technical field
The invention belongs to molecular biology and biological technical field, relate generally to a kind of nitrilase mutants and at cigarette Application in acid synthesis.
Background technology
Nicotinic acid, has another name called nicotinic acid, chemical entitled pyridone-3-carboxylic acid, and its chemical constitution is simple, stable in physicochemical property, with nicotinoyl Amine is collectively known as vitamin B3 or vitamin PP, at food, medical and industrial have important application.Lead at medicine Territory, nicotinic acid can be directly as dermatosis and similar avitaminotic active drugs such as preventing and treating pellagra, it may also be used for control Treat the disease such as atherosclerosis and peripheral nerve vasospasm；Nicotinic acid can be as medicine intermediate synthesis phthalein amine and acyl class medicine Thing.At field of food, nicotinic acid is commonly used for the additive of milk product, cake, Semen Maydis powder etc..Nicotinic acid can also adding at meat product As colour former in work, additionally they can be as antistaling agent, deodorant and color preserving agent etc..The topmost application of nicotinic acid is exactly As feed additive, in the U.S., nicotinic acid and nicotiamide are as deputy feedstuff vitamin substances.Additionally, nicotinic acid also may be used It is applied in daily chemical products do dyestuff or antioxidant etc..
At present, nicotinic acid industrial process mainly have potassium permanganate oxidation method, nitric acid oxidation method, nitric acid-sulfuric acid oxidizing process, Ammonia oxidation etc..Its shortcoming responds to be needed to carry out energy consumption height under high temperature, and course of reaction is complicated, and environmental pollution is serious.Cause This, seeking the nicotinic acid mode of production of high effective green environmentally friendly is one of major issue of facing at present.
At present, utilizing microbial enzyme method to convert acquisition nicotinic acid and come into one's own, existing many researcheres are constantly excavated to be had The microorganism of nitrilase activity also is applied to prepare nicotinic acid.The conversion of resting cells nicotinonitrile of R.rhodochrousJ1 Generating nicotinic acid, its conversion ratio can reach 100%, uses the mode of batch feeding to add after substrate converts 26h continuously, the product of nicotinic acid Amount reaches 172g L-1.Immobilization B.pallidus Dac521 cell transformation nicotinonitrile obtains nicotinic acid, uses pillar reaction Device, is continuously injected into 100mmol L-1Substrate, obtaining transformation efficiency at 50 DEG C and 60 DEG C respectively is 104mg (substrate) h-1·g (cell)-1With 208mg (substrate) h-1G (cell)-1.Sharma etc., in the batch feeding of 1L system converts, utilize 4.2g The resting cell of globule nocardia (N.globerula) NHB-2 is for converting the nicotinonitrile of common 1M, the 3-cyanogen of 98.6% Yl pyridines is converted into nicotinic acid, and its productivity is 3.21g (nicotinic acid) h-1G (cell)-1。
Summary of the invention
The invention provides a kind of method that nicotinic acid prepared by nitrilase of recombinating, and improve this by site-directed mutagenesis technique The nitrilase catalytic capability to nicotinonitrile a, it is thus achieved that plant height produces the bacterial strain of nicotinic acid.
An object of the present invention is to provide a kind of nitrilase mutants, and described mutant is by sequence SEQ ID 168th phenylalanine of aminoacid sequence shown in NO.2 sports valine and 192 mutant serines are phenylalanine, institute Stating described variant amino acid sequence as shown in SEQ ID NO.4, nucleotide sequence is shown in SEQ ID NO.3.
The two of the purpose of the present invention are to provide the recombiant plasmid that above-mentioned nitrilase mutants builds.
The three of the purpose of the present invention are to provide the Escherichia coli recombinant strain containing above-mentioned recombiant plasmid.
The four of the purpose of the present invention are to provide the application in producing nicotinic acid of the described nitrilase mutants.
Above-mentioned application is particularly as follows: with the recombination engineering containing nitrilase mutants encoding gene after abduction delivering The wet thallus obtained is enzyme source, with nicotinonitrile as reaction substrate, is constituted with the PBS of pH=7.0 for reaction medium Reaction system, water-bath at 40 DEG C, reaction terminates to obtain reactant liquor.The consumption of reaction thalline is 10g/L, described substrate Concentration is 100mmol/L.
Wet thallus preparation method of the present invention is as follows: be inoculated into by the engineering bacteria containing nitrilase mutants gene containing eventually Concentration be 0.1g/L card receive mycin 5ml LB liquid medium in, 37 DEG C, 200rpm/min shakes overnight incubation, takes culture fluid Be inoculated into 1% volume ratio receive containing 0.1g/L card mycin fresh LB fluid medium in.37 DEG C, 200rpm/min shakes training Support to OD600The IPTG of final concentration of 0.1mmol/L is added when=0.4～0.8,25 DEG C, 200rpm/min inducing culture 16h, will At fermentation liquid and 4 DEG C, 8000rpm/min is centrifuged 10min, collects thalline.
The beneficial effects of the present invention is: (1) present invention provides a kind of nitrilase encoded by nitrilase gene, its Under the proper conditions, can effectively produce nicotinic acid, a step has expanded the microbial resources that nicotinic acid produces.(2) mutant strain obtains more High enzyme's reaction speeding.Converting 100mM substrate under cellular level, original bacteria needs 30min, and saltant type nitrilase converts Time shortens to 10min, and the speed of the corresponding nicotinic acid converted is respectively 42.1mg/g/min, 123mg/g/min.
Accompanying drawing explanation
Fig. 1 is restructuring E. coli BL21pET28a-nitA and mutant E.coli BL21pET28a- The albumin glue electrophoresis schematic diagram of nitA-C2.
Fig. 2 nitrilase enzyme activity determination standard curve.
Fig. 3 is restructuring E. coli BL21pET28a-nitA, E.coli BL21pET28a-nitA-C2 catalysis Prepare the reaction of nicotinic acid
Detailed description of the invention
Technical scheme is described further by the present invention below in conjunction with specific embodiment, but should not be understood For limitation of the present invention:
Embodiment one, the structure of archetype nitrilase recombinant bacterium E.coli BL21pET28a-nitA
With the genes of SEQ ID NO.1 (NCBI GenBank No.DQ444267, Jin Sirui company synthesis) of synthesis it is Template, according to gene order and the coli expression carrier pET-28a of SEQ ID NO.1 (+) polyclone restriction enzyme site set Meter primer.
Forward primer: P1:5 '-GGTGCCGCGCGGCAGCCATATGGTTTCGTATAACAGCA-3 '.
Downstream primer: P2:5 '-GCGGCCGCAAGCTTGTCGACCTTTGCTGGGACCGGTTCT-3 '.
PCR reaction condition is:
The reaction condition of PCR is: 99 DEG C of Gai Wen；94 DEG C of denaturations 10min；94 DEG C of degeneration 45s, 50 DEG C of annealing 45s, 72 prolong Stretch 2min, totally 32 circulations.PCR primer uses the agarose gel of 0.8% to verify, verifies that correct rear cutout glue uses Biomiga company DNA gel reclaims test kit and reclaims fragment.
Bacillus coli DH 5 alpha (buying in ATCC DSMZ) containing pET-28a plasmid is connect test tube, adds card Receiving mycin, shaking table 37 DEG C, 200rpm, shaken cultivation is overnight.Use plasmid extraction kit (village alliance is biological) to extract plasmid, use Nde I and Sal I carries out double digestion, and at 37 DEG C, enzyme action is overnight, and enzyme action system is as follows:
The linearizing carrier of double digestion and PCR primer carry out one-step cloning, are attached, it is thus achieved that recombiant plasmid.
Recombiant plasmid converts with competent E. coli BL21 (DE3), is applied to containing kanamycin LB solid medium on, cultivate about 8h.
Embodiment two, the structure of mutant bacteria
With the genes of SEQ ID NO.1 of the synthesis in embodiment one as template, according to the gene order of SEQ ID NO.1 with The nucleotide sequence design primer in mutational site.
Using containing genes of interest fragment recombiant plasmid pET-28a (+)-Nit is as template, according to Overlap extension PCR Template is expanded by method, and Method And Principle is with embodiment one.Whole amplification procedure is undertaken in two steps.First primer it is separately added into P1 and F168V-F, expands template with P2 and F168V-R, it is thus achieved that two segment DNA sequences.Then two sections of sequences are added into In one PCR system, add primer P1 and P2, proceed PCR amplification, it is thus achieved that the F168V mutant DNA sequence of total length.? According to the method described above 192 amino acids are suddenlyd change on the basis of 168 mutants, it is thus achieved that double sudden changes of F168V-S192F Body NitA-C2.
Mutant plasmid builds and colibacillary conversion reference example one.
Embodiment three, the expression of nitrilase gene
Picking individual colonies accesses in 5ml LB liquid medium test tube, and mycin received by the culture medium card containing 0.1g/L.37 DEG C, 200rpm, shaken cultivation is overnight.Test tube liquid seeds is transferred to fresh containing with the inoculum concentration of volume ratio 3% by next day In the LB fluid medium of the kanamycin of 0.1g/L, cultivate to thalline OD for 37 DEG C600When being about about 0.4-0.8, to above-mentioned LB In fluid medium add derivant IPTG (final concentration of 0.1mM), 25 DEG C induction 20h, then by fermentation liquid at 4 DEG C, 8000rpm is centrifuged 10min, collects wet thallus, washs thalline 2 times with the PBS of pH=7.0, collects thalline.
Embodiment four, nitrilase enzyme activity determination method
Enzyme activity determination is set up according to Berthelot colorimetry.
1, the preparation of developer:
(1) A liquid: be dissolved in distilled water by 6g NaOH, is settled to 100ml after adding 3ml sodium hypochlorite.
(2) B liquid: 30mg sodium nitroprusside and 6g phenol are dissolved in 100ml distilled water and keep in Dark Place.
2, the foundation of standard curve
(the NH of preparation 10mmol/L4)2SO4Titer, prepares reaction system, 37 DEG C of shaking table vibration colour developings according to following table 30min, surveys light absorption value under 630nm, draws it, such as Fig. 2.
3. the detection of testing sample: take 10ml centrifuge tube and add 2ml H2O, 1ml A liquid, 1ml B liquid, 20 μ l treat test sample Product, mixing is placed in 37 DEG C of shaking tables vibration colour developing 30min, detects A630, bring suction into according to the equation of linear regression of standard curve Light value tries to achieve NH4 +Amount, calculate the amount of the nicotinic acid of conversion further.
Nicotinic acid prepared by embodiment five, utilization restructuring nitrilase
Using the thalline collected in embodiment three is enzyme source, is added in reaction system (total system 1ml, substrate 3-cyanogen The PBS of yl pyridines 100mM, pH=7.0), water-bath 10min sampling at 40 DEG C, add 100 μ l 2M HCl and terminate Reaction.Extract reaction solution color developing detection product assay.Result is as shown in Figure 3.
From above-mentioned experimental result, nitrilase gene is transformed into the restructuring large intestine that escherichia coli obtain by the present invention Bacillus has good nitrilase ability, directly can carry out living things catalysis with the somatic cells containing enzyme for enzyme source or convert anti- Should, nicotinonitrile conversion can be prepared nicotinic acid by this nitrilase.And mutant NitA-C2 relatively archetype NitA has more High reactivity.Primitiveness recombinant bacterium E.coli BL21pET28a-nitA and E.coli under these conditions BL21pET28a-nitA-C2 is 0.342mmol/g/ min and 1mmol/g/min in the reaction rate of 10min.Corresponding conversion The amount of nicotinic acid be 42.1mg/g/min, 123mg/g/min.
Sequence table
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Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu
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Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu Ala Glu Asp Ala Glu
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Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu Gln Ile Leu Leu Ala Lys
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Ala Gly Ala Asp Pro Val Gly His Tyr Ser Arg Pro Asp Val Leu Ser
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Claims (8)

1. a nitrilase mutants, it is characterised in that described mutant is by aminoacid sequence shown in sequence SEQ ID NO. 2 168th phenylalanine of row sports valine and 192 mutant serines are phenylalanine.
Nitrilase mutants the most according to claim 1, it is characterised in that described variant amino acid sequence such as SEQ Shown in ID NO.4.
3. the recombiant plasmid built by nitrilase mutants described in claim 1 or 2.
4. contain the Escherichia coli recombinant strain of recombiant plasmid described in claim 3.
5. the application in producing nicotinic acid of the nitrilase mutants described in claim 1 or 2.
Apply the most as claimed in claim 5, it is characterised in that described application is: with containing nitrilase mutants encoding gene Recombination engineering after abduction delivering obtain wet thallus be enzyme source, with nicotinonitrile as reaction substrate, with pH=7.0 PBS be reaction medium constitute reaction system, water-bath at 40 DEG C, reaction terminate obtain reactant liquor.
Apply the most as claimed in claim 6, it is characterised in that described bacterium source consumption is 10 g/L, described reaction substrate Concentration is 100 mmol/L.
Apply the most as claimed in claim 6, it is characterised in that the preparation method of described wet thallus is as follows: nitrilase will be contained The engineering bacteria of mutant gene be inoculated into receive containing final concentration of 0.1 g/L card mycin 5ml LB liquid medium in, 37 DEG C, 200 Rpm/min shakes overnight incubation, take culture fluid with 1% volume ratio be inoculated into containing 0.1 g/L card receive mycin fresh LB liquid training Support in base；37 DEG C, 200 rpm/min concussions are cultivated to OD600The IPTG of final concentration of 0.1 mmol/L is added when=0.4 ~ 0.8, 25 DEG C, 200 rpm/min inducing culture 16h, 8000 rpm/min at fermentation liquid and 4 DEG C are centrifuged 10 min, collect thalline.
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Cited By (7)

* Cited by examiner, † Cited by third party
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CN106636044A (en) * 2017-03-07 2017-05-10 东莞东阳光药物研发有限公司 Nitrilase mutant as well as coding gene and application thereof
CN108384739A (en) * 2018-01-10 2018-08-10 上海晶诺生物科技有限公司 A kind of integrated recombinant Mycobacterium smegmatis and its construction method of production niacin
CN111172140A (en) * 2020-01-21 2020-05-19 浙江工业大学 Nitrilase mutant and application thereof in preparation of anti-epileptic drug intermediate
CN111321132A (en) * 2020-02-18 2020-06-23 浙江工业大学 Nitrilase mutant with improved reaction specificity and application thereof
CN111690675A (en) * 2019-08-01 2020-09-22 安徽瑞邦生物科技有限公司 Recombinant bacterium for expressing nitrile hydratase mutant and preparation method and application thereof
CN114317506A (en) * 2022-01-13 2022-04-12 兄弟科技股份有限公司 Nitrilase, engineering bacteria constructed by nitrilase and application of nitrilase in green synthesis of nicotinic acid
WO2022073331A1 (en) * 2020-10-09 2022-04-14 浙江工业大学 Nitrilase mutant and use thereof in catalytic synthesis of 2-chloronicotinic acid

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CN106636044A (en) * 2017-03-07 2017-05-10 东莞东阳光药物研发有限公司 Nitrilase mutant as well as coding gene and application thereof
CN106636044B (en) * 2017-03-07 2019-10-18 东莞东阳光药物研发有限公司 Nitrilase mutants and its encoding gene and application
CN108384739A (en) * 2018-01-10 2018-08-10 上海晶诺生物科技有限公司 A kind of integrated recombinant Mycobacterium smegmatis and its construction method of production niacin
CN108384739B (en) * 2018-01-10 2020-03-27 上海晶诺生物科技有限公司 Integrated recombinant mycobacterium smegmatis producing nicotinic acid and construction method thereof
CN111690675A (en) * 2019-08-01 2020-09-22 安徽瑞邦生物科技有限公司 Recombinant bacterium for expressing nitrile hydratase mutant and preparation method and application thereof
CN111172140A (en) * 2020-01-21 2020-05-19 浙江工业大学 Nitrilase mutant and application thereof in preparation of anti-epileptic drug intermediate
CN111172140B (en) * 2020-01-21 2022-04-19 浙江工业大学 Nitrilase mutant and application thereof in preparation of anti-epileptic drug intermediate
CN111321132A (en) * 2020-02-18 2020-06-23 浙江工业大学 Nitrilase mutant with improved reaction specificity and application thereof
CN111321132B (en) * 2020-02-18 2021-08-24 浙江工业大学 Nitrilase mutant with improved reaction specificity and application thereof
WO2022073331A1 (en) * 2020-10-09 2022-04-14 浙江工业大学 Nitrilase mutant and use thereof in catalytic synthesis of 2-chloronicotinic acid
CN114317506A (en) * 2022-01-13 2022-04-12 兄弟科技股份有限公司 Nitrilase, engineering bacteria constructed by nitrilase and application of nitrilase in green synthesis of nicotinic acid

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