CN104498466B - nitrile hydratase and its application - Google Patents
nitrile hydratase and its application Download PDFInfo
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- CN104498466B CN104498466B CN201410758150.9A CN201410758150A CN104498466B CN 104498466 B CN104498466 B CN 104498466B CN 201410758150 A CN201410758150 A CN 201410758150A CN 104498466 B CN104498466 B CN 104498466B
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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
- C12N9/88—Lyases (4.)
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- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/01—Hydro-lyases (4.2.1)
- C12Y402/01084—Nitrile hydratase (4.2.1.84)
Abstract
The invention discloses a kind of nitrile hydratase and its application, the nitrile hydratase is made up of α subunits and β subunits, and as shown in SEQ ID NO.6, the amino acid sequence of β subunits is as shown in SEQ ID NO.7 for the amino acid sequence of the α subunits.The present invention is cloned into nitrile hydratase gene from special Salmonella DSM 12804 (Bordetella petrii DSM 12804) is won, is successfully obtained after the gene expression with expression quantity high and high activity and substrate spectrum is wide, tool chiral selectivity nitrile hydratase.Existing nitrile hydratase typically has vigor higher to aliphatic nitrile compound, very low to the general vigor of aromatic nitrile compound.Nitrile hydratase of the present invention has catalysis activity higher to aromatic nitrile compound, especially 2 isopropyl p-chlorobenzyl cyanide.
Description
Technical field
The present invention relates to genetic engineering field, more particularly to a kind of nitrile hydratase and its application.
Background technology
Nitrile is a kind of important compound, is widespread in nature.The method of nitrile hydrolysis mainly has chemical hydrolysis
And biotransformation method, wherein, biotransformation method relates generally to nitrile hydratase, amidase and nitrilase.Nitrile hydratase is with sulphur
Atom and cysteine sulfinic acid residue can be catalyzed the enzyme of nitrile hydrolysis for a class of absolute center, can be catalyzed nitrile hydration generation acyl
Amine, amidase further by acid amides catalyzing hydrolysis into carboxylic acid compound, and nitrilase can be directly catalyzed nitrile generation Carboxylation
Compound.Nitriles substance can be converted into by acid amides, acid, amino-compound, ester, alcohol, enamine etc. by the enzymatic reaction of these enzymes
Value wider array of some compounds of higher, range of application.Therefore, the living things catalysis hydrolysis of nitrile is in fine chemical product, doctor
The aspects such as medicine intermediate, the synthesis of chipal compounds and environmental improvement are with a wide range of applications.
The nitrile hydratase of microorganism is applied as biocatalyst and possesses huge potentiality in organic synthesis technique, is had
The irreplaceable superiority of chemical method, such as：Reaction condition is gentle, environment-friendly, low cost, with region and three-dimensional selection
Property, so as to promote development and exploitation of the nitrile hydratase in commercial Application.The distribution of nitrile hydratase production microorganism is quite wide
It is general, such as Rhodococcus sp, pseudomonad, Nocard's bacillus, Selective medium, Bacillus alcaligenes, corynebacteria, the nitrile water of separate sources
Synthase often has different characteristics.Wherein, the microorganism of some nitrile hydratase productions has been used for acrylamide, niacinamide, 2- ammonia
In the production of base -2,3- amide dimethyl butyrates etc..Patent document such as Patent No. US007405064B2 discloses a kind of testis
Ketone comamonas 5-MGAM-4D；The patent document of Patent No. ZL88106735 discloses a kind of rhodococcus rhodochrous J-1；
The patent document of Patent No. ZL86100062 discloses a kind of Rhodococcus sp S-6, oxidation arthrobacterium and microbacterium flavum；The patent No.
For the patent document of ZL99106291.4 discloses a kind of thermophilic Selective medium JCM3095；Patent No. ZL03115536.7
Patent document disclose a kind of propionic acid bar bacterium；The patent document of Patent No. ZL103834600 discloses one plant of stench vacation
Monad, these wild strains are all used in the production of acrylamide.
Acrylamide is widely used in papermaking, decoration and oil exploitation etc. as a kind of important basic chemical industry raw material
Aspect.The acrylamide annual production produced with bioanalysis has reached more than 600,000 tons.In China, third is synthesized with nitrile hydratase
Although the biotechnology of acrylamide starts late, but have developed rapidly, and statistics shows within 2012, and the third of China's bioanalysis production
Acrylamide accounts for the 52% of whole acrylamide yield, and increases year by year.The patent of Patent No. ZL01822031.2 discloses one
If serial microorganism includes bacillus, Micrococcus, brevibacterium, pseudomonas, Cattell Pseudomonas and Rhod
Microorganism catalytic production niacinamide under the conditions of reaction temperature is for 10 DEG C.Niacinamide belongs to B family vitamin, in vivo with DPN
And the form of DPN I participates in organism metabolism, medicine is widely used in, on food additives and Feed Manufacturing.Patent No.
The patent of ZL201210239547.8 discloses one plant of wild strain celebrating sheng, a reed pipe wind instrument Rhodococcus sp CCTCC No：M2010050 is used for catalytic water
Close and prepare 2- amino -2,3- amide dimethyl butyrates, it is the intermediate of imidazolone type superelevation effective herbicide.
Although being widely used using the technique of wild strain industrialized production amides compound,
The enzyme activity stability of the nitrile hydratase of above-mentioned technique generally existing wild strain is poor, and the tolerance to substrate and product is not high, with
And interval metaplasia is produced, the problems such as product quality is not sufficiently stable.Additionally, also there is acid amides in addition to nitrile hydratase in wild strain
Enzyme and nitrilase, it can cause the accumulation of accessory substance carboxylic acid compound, so as to reduce the yield of amides compound
And purity, while also increasing the difficulty and production cost for isolating and purifying.With the fast development of molecular biology, using gene weight
Group technology, constructs the novel gene engineered strain with nitrile hydratase activity and carries out protein engineering transformation, and being expected to can be
Solve the problems, such as actual production in varying degrees, such as improve the expression of enzyme, the heat endurance of enhancing enzyme and to substrate and
The tolerance of product, the generation of blocking side reaction；In addition, the strong adaptability of genetic engineering bacterium, fermentation period are short, it is advantageously implemented
Large-scale culture and industrialized production, it helps coordinate production and separation costs of reduction enzyme etc. with follow-up new technology.
The content of the invention
The invention provides the nitrile hydratase that a kind of with R- isomers stereoselectivities, vigor is high, substrate spectrum is wide.
A kind of nitrile hydratase, is made up of α subunits and β subunits, the amino acid sequence such as SEQ ID NO.6 institutes of the α subunits
Show, the amino acid sequence of β subunits is as shown in SEQ ID NO.7.
Encode the gene of coding for alpha subunit of the gene of the nitrile hydratase comprising base sequence as shown in SEQ ID NO.1
With the gene of the coding β subunits as shown in SEQ ID NO.2.The base sequence is derived from and wins special Salmonella DSM 12804
(Bordetella petrii DSM 12804), by 1429 base compositions, from 5 ' end, the 1st encodes nitrile water to 762 bit bases
Synthase α subunits；The 773rd to 1429 bit base encoding nitrile hydratase β subunits, base sequence such as SEQ ID NO.3 institutes is held from 5 '
Show.
The gene of described nitrile hydratase is also comprising the base sequence of encoding nitrile hydratase activation.Described nitrile hydratase
The base sequence of son is activated as shown in SEQ ID NO.4, the activation subbase is because of the high efficient expression for nitrile hydratase gene to pass
It is important.
In order to improve expression activity, activation is connected with after described nitrile hydratase gene (SEQ ID NO.3) is modified
Gene (SEQ ID NO.4), obtains the base sequence as shown in SEQ ID NO.5.
The invention provides a kind of nitrile hydratase activation, amino acid sequence is as shown in SEQ ID NO.8.
Present invention also offers a kind of expression cassette of the gene for encoding the nitrile hydratase, recombinant vector and transformant.
Carrier of the present invention can be pET-30a (+), pET-21a (+), pET-22b (+), pET-28a (+),
PETDuet-1, pACYCDuet-1, pCDFDuet-1 and RSFDuet-1, but it is not limited only to these described carriers.Preferably
By PCR expand it is modified after nitrile hydratase gene be connected on expression vector pET-30a (+), formed recombination expression carry
Body pET-30a (+)-NHaseP.
Host cell of the present invention is Escherichia coli, preferably E.coli BL21 (DE3) bacterial strain.Will be above-mentioned heavy
Group expression vector pET-30a (+)-NHaseP Transformed E .coli BL21 (DE3) competent cell, wherein containing pET-30a (+)-
The bacterial strain of NHaseP recombinant plasmids, as genetically engineered E.coli BL21 (DE3)/pET-30a (+)-NHaseP.This bacterial strain
Can under conventional IPTG inductive conditions high efficient expression nitrile hydratase albumen.Genetically engineered E.coli BL21 (DE3)/
The Liquid Culture based formulas of pET-30a (+)-NHaseP are：Peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, pH
It is 7.0.The temperature of culturing engineering bacterium is 35~40 DEG C, and rotating speed is 180~220rpm.
Invention further provides a kind of described nitrile hydratase answering in catalysis nitrile compound hydration reaction generation acid amides
With.
Shown in described nitrile compound such as formula (I) or (II)：
In formula (I)：
X is OH, H, NH2Or alkyl；
R ' is H or with the 1-3 alkyl of C atoms；
R is H or optionally H by NH2Substitution with the 1-12 alkyl of C atoms；
In formula (II)：
R " is the unsaturation ring of monokaryon or double-core, and it has 6-12 C atom, and its optionally H by 1 or 2 Cl, Br
Or F substitutions.
It is further preferred that in formula (I)：
X is OH, H, NH2Or with 1-3 C atom alkyl；
R ' is H or with the 1-3 alkyl of C atoms；
R is H or optionally H by NH2Substitution with the 1-6 alkyl of C atoms；
In formula (II)：
R " is the unsaturation ring of monokaryon or double-core, and it has a 6-12 C atom, and its optionally H by the Cl of 1 or 2,
Br or F replaces.
Preferably, described nitrile compound is acrylonitrile, nicotinonitrile, 2- isopropyls p-chlorobenzyl cyanide and Alpha-Methyl
One kind in benzene acetonitrile.It is further preferred that described nitrile compound is 2- isopropyl p-chlorobenzyl cyanides.
Existing nitrile hydratase typically has vigor higher to aliphatic nitrile compound, to the general vigor of aromatic nitrile compound
It is very low.Nitrile hydratase of the present invention has higher urging to aromatic nitrile compound, especially 2- isopropyls p-chlorobenzyl cyanide
Change vigor.
The present invention from win special Salmonella DSM 12804 (Bordetella petrii DSM 12804) be cloned into nitrile hydration
Enzyme gene, successfully obtains with expression quantity high and high activity after the gene expression and substrate spectrum is wide, tool chiral selectivity nitrile
Hydrase.
Brief description of the drawings
Fig. 1 be nitrile hydratase gene of the present invention and activation subbase because PCR expand electrophoretogram；
M：Nucleic acid Marker, 1~3：Nitrile hydratase gene and activation subbase because pcr amplification product.
Fig. 2 is the collection of illustrative plates of recombinant plasmid pET-30a (+)-NHaseP of the present invention.
Fig. 3 is for after genetically engineered E.coli BL21 (DE3) of the present invention/pET-30a (+)-NHaseP induced expressions
SDS-PAGE；
M：Low-molecular-weight standard protein；
1：The broken cytosol of pET-30a (+) empty plasmid control；
2：The broken cytosol of thalline after genetically engineered E.coli BL21 (DE3)/pET-30a (+)-NHaseP inductions；
3：The broken born of the same parents' supernatant of thalline after genetically engineered E.coli BL21 (DE3)/pET-30a (+)-NHaseP inductions；
4：The broken born of the same parents' precipitation of thalline, arrow after genetically engineered E.coli BL21 (DE3)/pET-30a (+)-NHaseP inductions
Indicate respectively the position of nitrile hydratase α subunits and β subunits.
Fig. 4 is the structure schematic diagram of expression plasmid pET-30a (+)-NHaseP of the present invention.
Fig. 5 is the efficient liquid phase detection figure that present invention restructuring nitrile hydratase is catalyzed racemic 2- isopropyl p-chlorobenzyl cyanides
Spectrum.
Specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited only to
This.
Materials and methods in embodiment are：
Experimental technique in the present invention is conventional method unless otherwise instructed, for details, reference can be made to the volume such as J. Pehanorm Brookers
's《Molecular Cloning:A Laboratory guide》.
Restriction enzyme EcoRI, HindIII and T4DNA ligase used in the embodiment of the present invention is purchased from
TaKaRa, precious bioengineering (Dalian) Co., Ltd；Genome extracts kit, plasmid extraction kit, the examination of DNA recovery purifyings
Agent box is purchased from Axygen Hangzhou Co., Ltd；E.coli DH5 α, E.coli BL21 (DE3), plasmid pET-30a (+), pET-
21a (+), pET-22b (+) etc. are purchased from Novagen companies；DNA marker, FastPfu archaeal dna polymerase, low-molecular-weight standard
Albumen, agarose electrophoresis reagent are purchased from Beijing Quanshijin Biotechnology Co., Ltd；Primer synthesizes with sequence work by upper
Hai Shenggong biotechnologies Co., Ltd completes.Above reagent application method refers to catalogue.It is rich that the present invention is used
Special Salmonella DSM 12804 (Bordetellapetrii DSM 12804) is obtained purchased from German microorganism and cell culture preservation
The heart (DSM), preserving number is DSM 12804.
Embodiment 1
First, nitrile water is cloned from special Salmonella DSM 12804 (Bordetella petrii DSM 12804) genome is won
Synthase gene and activation subbase because
According to winning the special genomic dna sequences of Salmonella DSM 12804 (GenBank accession number：AM902716.1) design is drawn
Thing NP-F (sense primer) and NP-R (anti-sense primer).
NP-F sequences：5’-CCGGAATTCATGCTCGAAGTTCTTTGCATGG-3’
NP-R sequences：5’-TTCCCAAGCTTGTTAAGCCATTGCGGCAACG-3’
Restriction enzyme site EcoRI, HindIII are separately added into the primer of upstream and downstream (shown in underscore).To win
The special genomic DNAs of Salmonella DSM 12804 are template, and NP-F and NP-R enters performing PCR amplification, PCR reaction systems and reaction for primer
Condition is as follows：
PCR amplification system：
PCR amplification conditions：
1) predegeneration：95℃5min；
2) it is denatured：98℃10s；Annealing：56℃15s；Extend：72℃100s；Circulate 30 times altogether；
3) extend：72℃10min；
4) 4 DEG C of preservation 2.0h.
After PCR amplifications terminate, amplified production is detected with 1.0% agarose gel electrophoresis, as a result shows that amplified production is
Single band, size is 1800bp or so (as shown in Figure 1).Amplified production is purified back with DNA recovery purifying kits
Receive, specific steps are with reference to purification kit specification.
2nd, the structure of expression vector and engineering bacteria
Expression vector pET-30a (+) and pcr amplification product are carried out double with restriction enzyme EcoRI and HindIII respectively
Digestion.Digestion products are carried out with purifying with DNA purification kits after the completion of digestion to reclaim to remove restriction enzyme and digestion
The nucleotides small fragment for getting off.Pcr amplification product after double digestion is connected to corresponding otch with T4DNA ligases
Expression vector pET-30a (+) on, linked system is as shown in table 1 below：
Table 1pET-30a (+)-NHaseP recombinant expression plasmid linked systems
After each reagent in above-mentioned linked system is mixed, it is put in 16 DEG C of metal baths and connects 12h.By enzyme connect product thing
In conversion to E.coli DH5a competent cells, apply flat board, choose single bacterium colony LB Liquid Cultures, the sun that the identification of PCR methods is successfully constructed
Property transformant, and the correctness of insetion sequence is verified by sequencing company.Recombinant expression carrier is transferred to expressive host again
In E.coli BL21 (DE3), the recon of conversion is verified with PCR methods, errorless genetic engineering bacterium is E.coli after checking
BL21(DE3)/pET-30a(+)-NHaseP。
It is prepared by the 3rd, expression and purifying of restructuring nitrile hydratase
Genetically engineered E.coli BL21 (DE3)/pET-30a (+)-NHaseP for building is seeded to containing 50 μ g/ml
In the 5mL LB fluid nutrient mediums of Kan, 37 DEG C of concussion and cultivate 12h.Take 1.5mL nutrient solutions and be forwarded to 50mL equally containing 50 μ g/ml
In the fresh LB fluid nutrient mediums of Kan, 37 DEG C of concussion and cultivates to OD600When reaching 0.8 or so, addition IPTG to its concentration is
0.5mM, 20h is induced at 20 DEG C.The SDS-PAGE of engineering bacteria induced expression result is as shown in Figure 3.
After culture terminates, thalline is collected by centrifugation, abandons supernatant, bacterium is washed with the buffer solution of 50mM Tris-HCl (pH 7.0)
Body is twice.Thalline is resuspended in Tris-HCl (50mM, pH 7.5,20mM imidazoles, 0.3M NaCl, 5mM dithiothreitol (DTT)) afterwards
In buffer solution, ultrasonication bacteria suspension, centrifugation removal precipitation, the supernatant for obtaining is the crude enzyme liquid containing restructuring nitrile hydratase.With
0.45 μm of membrane filtration crude enzyme liquid, because restructuring nitrile hydratase contains histidine-tagged, then carries out nickel ion affinity chromatograph
Purifying, obtains the pure enzyme protein of restructuring nitrile hydratase.
The genetic engineering bacterium of embodiment 2 catalysis acrylonitrile generation acrylamide
Enzyme activity unit is defined as：At reaction conditions, catalytic substrate reaction per minute produces 1 μm of enzyme amount of ol products.
The zymotic fluid of engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseP of the structure of 25ml embodiments 1 is taken,
10000rpm, 10min are collected by centrifugation thalline, then with the resuspended collection of buffer solution of 250ml 50mM Tris-HCl (pH 8.5)
Thalline, the enzyme activity of resuspended enzyme liquid is 1700U/ml.To 1.0ml acrylonitrile is added in re-suspension liquid, carry out being hydrated instead at 20 DEG C
Should, react 120min.Afterwards with the acrylonitrile in gas chromatography detection reaction system, acrylamide and acrylic acid content.
The conversion ratio of substrate is more than 99%, and yield is more than 92%, without the acrylic acid generation of discovery in reaction system.
The genetic engineering bacterium of embodiment 3 catalysis nicotinonitrile generation niacinamide
The zymotic fluid of engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseP of the structure of 25ml embodiments 1 is taken,
10000rpm, 10min are collected by centrifugation thalline, then with the buffer solution of 250ml 50mM Tris-HCl (pH 6.0) to collection
Thalline carries out resuspended.To 5.0g nicotinonitriles are added in re-suspension liquid, hydration reaction is carried out at 37 DEG C, react 120min.It
Nicotinonitrile, the content of niacinamide in reaction system are detected with high performance liquid chromatography afterwards.Substrate conversion efficiency is more than
99%, yield is more than 89%.
The genetic engineering bacterium of embodiment 4 catalysis Alpha-Methyl benzene acetonitrile generation Alpha-Methyl phenyl acetamide
The zymotic fluid of engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseP of the structure of 25ml embodiments 1 is taken,
10000rpm, 10min are collected by centrifugation thalline, then with the buffer solution of 250ml 50mM Tris-HCl (pH 7.5) to collection
Thalline carries out resuspended.To 1.0g Alpha-Methyl benzene acetonitriles are added in re-suspension liquid, hydration reaction is carried out at 25 DEG C, react 120min.
Alpha-Methyl benzene acetonitrile, the Alpha-Methyl phenyl acetamide in reaction system are detected with high performance liquid chromatography (chiral chromatographic column) afterwards
Content.Result finds that engineering bacteria, when racemic ' alpha '-methylbenzeneacetonitrile is catalyzed, shows R- isomers stereoselectivities, converts
Rate is 49%, and yield is 87%, and product e.e. values are 58%.
The genetic engineering bacterium of embodiment 5 catalysis 2- isopropyls p-chlorobenzyl cyanide generates 2- isopropyls to chlorobenzene acetamide
The zymotic fluid of engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseP of the structure of 25ml embodiments 1 is taken,
10000rpm, 10min are collected by centrifugation thalline, then with the buffer solution of 250ml 50mM Tris-HCl (pH 7.0) to collection
Thalline carries out resuspended.To 0.5g 2- isopropyl p-chlorobenzyl cyanides are added in re-suspension liquid, hydration reaction is carried out at 35 DEG C, reacted
120min.Detected with high performance liquid chromatography (chiral chromatographic column) afterwards 2- isopropyls p-chlorobenzyl cyanide in reaction system,
Content of the 2- isopropyls to chlorobenzene acetamide.Result find engineering bacteria be catalyzed racemic 2- isopropyl p-chlorobenzyl cyanides when, table
Reveal R- isomers stereoselectivities, liquid phase testing result as shown in figure 5, conversion ratio be 49%, yield is 90%, product e.e.
Be worth is 65%.
Comparative example 1
Fallon, R.d. (Applied Microbiology and Biotechnology, 47,156-161,1997) etc.
2- (4- chlorphenyls) -3- first is catalyzed with pseudomonas putida NRRL-18668 (Pseudomonasputida NRRL-18668)
The hydration reaction of base butyronitrile.It was found that the nitrile hydratase that the wild mushroom contains can be catalyzed the hydration of 2- (4- chlorphenyls) -3- methylbutyronitriles
Generation 2- (4- chlorphenyls) -3- methylbutyryl amine, and (S) type stereoselectivity is shown, enzyme activity is 7.1 × 10-3μmol/
Min/mg wet cells.Its optimal reactive temperature is 30 DEG C, and when temperature is more than 30 DEG C, zymoprotein starts rapid deactivation.It is of the invention
During engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseP catalysis 2- (4- chlorphenyls) -3- methylbutyronitriles, with (R)
Type stereoselectivity, its enzyme activity reaches 25.4 × 10-3μm ol/min/mg wet cells.Optimal reactive temperature is 35 DEG C, at 35 DEG C
Following zymoprotein shows fairly good stability.
Comparative example 2
Shun-Ichi Masutomo (Bioscience Biotechnology and Biochemistry, 59 (4),
720-722,1995) etc. 2- (4- chlorphenyls) -3- methyl is catalyzed with pseudomonad B21C9 (pseudomonas sp.B21C9)
The hydration reaction of butyronitrile.It was found that the nitrile hydratase that the wild mushroom contains can be catalyzed the hydration life of 2- (4- chlorphenyls) -3- methylbutyronitriles
Into 2- (4- chlorphenyls) -3- methylbutyryl amine, and certain (S) type stereoselectivity is shown, but its activity is relatively low, only
1.8×10-4μm ol/min/mg stem cells.Engineering bacteria E.coli BL21 (DE3) of the invention/pET-30a (+)-NHaseP is urged
When changing 2- (4- chlorphenyls) -3- methylbutyronitriles, with (R) type stereoselectivity, its enzyme activity reaches 7.8 × 10-2μmol/min/
Mg stem cells.
Claims (1)
1. application of a kind of nitrile hydratase in catalysis nitrile compound hydration reaction generation acid amides, it is characterised in that；
The nitrile hydratase is made up of α subunits and β subunits, and the amino acid sequence of the α subunits is as shown in SEQ ID NO.6, and β is sub-
The amino acid sequence of base is as shown in SEQ ID NO.7；
The nitrile compound is nicotinonitrile, R types 2- isopropyls p-chlorobenzyl cyanide or R type Alpha-Methyl benzene acetonitriles.
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| CN104862296B (en) * | 2015-04-22 | 2017-12-12 | 江南大学 | A kind of specific enzyme activity and stability-enhanced pattern of fusion nitrile hydratase |
| CN107881163A (en) * | 2017-09-29 | 2018-04-06 | 浙江大学 | A kind of heat-resisting nitrile hydratase, engineering bacteria and its application in catalysis nitrile compound hydration reaction generation acid amides |
| CN107815446B (en) * | 2017-10-16 | 2018-09-28 | 浙江大学 | A kind of fermentation process in high density of recombination nitrile hydratase Recombinant organism |
| CN113444714B (en) * | 2021-07-29 | 2022-06-21 | 浙江大学杭州国际科创中心 | Beta subunit mutant nitrile hydratase mutant and application thereof |
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