CN116200374A - D-carbamoyl hydrolase mutant with improved thermal stability and application thereof in synthesis of D-amino acid - Google Patents
D-carbamoyl hydrolase mutant with improved thermal stability and application thereof in synthesis of D-amino acid Download PDFInfo
- Publication number
- CN116200374A CN116200374A CN202211589530.5A CN202211589530A CN116200374A CN 116200374 A CN116200374 A CN 116200374A CN 202211589530 A CN202211589530 A CN 202211589530A CN 116200374 A CN116200374 A CN 116200374A
- Authority
- CN
- China
- Prior art keywords
- carbamoyl
- mutant
- parent
- hydrolase mutant
- mutating
- 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
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
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
-
- 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
- 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
- C12P13/04—Alpha- or beta- amino acids
-
- 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)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01077—N-Carbamoyl-D-amino-acid hydrolase (3.5.1.77)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a D-carbamoyl hydrolase mutant with improved thermal stability and application thereof in D-amino acid synthesis. Optimal mutant S202P/E208D/R277L obtained by combined mutation is t at 40 DEG C 1/2 The value was 28.5 times M4, about 36.5h. Has catalytic efficiency equivalent to M4. M4 is catalyzed at 40 DEG CThe conversion rate was only 60.8% when 100mM N-carbamoyl-D-tryptophan was reacted for 24 hours, while S202P/E208D/R277L was able to complete 99.4% conversion at 24 hours. This shows that the mutant S202P/E208D/R277L can react at a higher temperature, and has good industrial application prospect.
Description
Technical Field
The invention relates to a D-carbamoyl hydrolase mutant with improved thermal stability and application thereof in synthesis of D-amino acid, belonging to the technical field of enzyme engineering.
Background
D-amino acid is an important unnatural amino acid, widely distributed in microorganisms, plants and animals. D-amino acids can synthesize various antibiotics, and some of them, such as amoxicillin, ampicillin, penicillin, etc., are defined as necessary drugs by the world health organization. Furthermore, D-amino acids have a wide range of roles in the food, agricultural and chemical industries.
Three main classes of biocatalysts are available for the synthesis of optically pure D-amino acids, hydrolytic enzymes, oxidoreductase enzymes and D-amino acid aminotransferase enzymes. The hydrolase is a kind of enzyme with wide range of D-amino acid preparation, the hydrolase usually takes a raceme compound as a substrate, the yield can reach 50% in theory through resolution, the theoretical yield can reach 100% by combining the racemase, and the hydrolase has the advantages of wide substrate range, high product yield, few byproducts and the like. The hydrolases reported so far for the synthesis of D-amino acids mainly include the processes of D-hydantoinase and D-carbamoyl hydrolase coupled hydantoinase, N-acyl-D-amino acid amidase, D-aminopeptidase and D-peptidase.
D-carbamoyl hydrolase (HyuC) is a class 6 (EC 3.5.1.77) belonging to the nitrilase superfamily, which hydrolyzes N-carbamoyl-D-amino acids to D-amino acids, and is commonly used to produce optically pure D-amino acids by a cascade reaction with hydantoin racemase and hydantoin enzyme. However, the thermostability of the identified wild-type enzymes and their mutants is generally low. In order to meet the requirements of industrial application, researches such as directional excavation and thermal stability analysis of D-carbamyl hydrolase with different properties, thermal stability molecular modification of the D-carbamyl hydrolase based on directional evolution and rational design and the like have been widely conducted.
Disclosure of Invention
In order to solve the technical problems, the invention provides a D-carbamoyl hydrolase mutant with improved thermal stability, which is obtained by carrying out proper mutation on the basis of the D-carbamoyl hydrolase with the amino acid sequence shown as SEQ ID NO.1 studied earlier by the inventor, so that the mutant with improved thermal stability can be widely applied to the fields of foods and medicines.
The first object of the invention is to provide a D-carbamyl hydrolase mutant with improved heat stability, wherein the D-carbamyl hydrolase mutant takes D-carbamyl hydrolase with an amino acid sequence shown as SEQ ID NO.1 as a parent, and amino acids at 138, 202, 204, 208, 277 and 284 of the D-carbamyl hydrolase mutant are mutated respectively.
Further, the D-carbamoyl hydrolase mutant is characterized in that glutamic acid Glu at 138 th site of a parent is mutated into tryptophan Trp or serine Ser; or alternatively, the first and second heat exchangers may be,
mutating serine Ser at 202 th site of parent to proline Pro; or alternatively, the first and second heat exchangers may be,
mutating serine at position 204 of the parent to aspartic acid Asp or asparagine Asn; or alternatively, the first and second heat exchangers may be,
mutating glutamic acid Glu at position 208 of the parent to aspartic acid Asp; or alternatively, the first and second heat exchangers may be,
mutating arginine Arg at 277 th site of the parent to Leu, glutamine Gln or glutamic acid Glu; or alternatively, the first and second heat exchangers may be,
the histidine His at position 284 of the parent is mutated to threonine Thr or asparagine Asn.
Further, the D-carbamoyl hydrolase mutant is characterized in that serine Ser at 202 st position of a parent is mutated into proline Pro, glutamic acid Glu at 208 th position is mutated into aspartic acid Asp, and arginine Arg at 277 th position is mutated into Leu.
It is a second object of the present invention to provide a gene encoding the D-carbamoyl hydrolase mutant.
A third object of the present invention is to provide an expression vector carrying the coding gene.
The fourth object of the present invention is to provide a genetically engineered bacterium expressing the D-carbamoyl hydrolase mutant.
Furthermore, the genetically engineered bacterium takes escherichia coli as a host.
Further, the E.coli is E.coli BL21 (DE 3).
Furthermore, the genetically engineered bacterium takes pET-28a (+) as an expression vector.
The fifth object of the invention is to provide the application of the D-carbamoyl hydrolase mutant or the genetically engineered bacterium in preparing D-amino acid.
Further, the application is to take N-carbamoyl-D-amino acid as a substrate and take the D-carbamoyl hydrolase mutant or the genetically engineered bacterium as a catalyst to catalyze and generate the D-amino acid.
Further, the catalysis condition is 38-42 ℃ and 150-250 rpm.
The beneficial effects of the invention are as follows:
the invention obtains mutants with different degrees of improved thermal stability through site-directed mutagenesis based on D-carbamoyl hydrolase (NiHyuC-M4) from Nitratireductor indicus. Wherein, the single mutant E138W, E S, S P, S D, S204N, E208D, R277L, R277Q, R277E, H284T, H284N is t at 40 DEG C 1/2 The values increased from 1.3h to 2.2h, 1.7h, 2.3h, 1.9h, 1.7h, 3.3h, 8.0h, 2.3h, 1.6h, 2.2h, 1.8h, respectively, of M4. Optimal mutant S202P/E208D/R277L obtained by combined mutation is t at 40 DEG C 1/2 The value was 28.5 times M4, about 36.5h. Has a molecular weight of M4 (288 min) -1 ·mM -1 ) Equivalent catalytic efficiency of 302min -1 ·mM -1 . M4 had only 60.8% conversion at 40℃for 24h with 100mM N-carbamoyl-D-tryptophan, whereas S202P/E208D/R277L could complete 99.4% conversion at 24 h. This shows that the mutant S202P/E208D/R277L can react at a higher temperature, and has good industrial application prospect.
Detailed Description
The present invention will be further described with reference to specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the present invention and practice it.
The following examples relate to the following media:
LB liquid medium: peptone 10 g.L -1 Yeast extract 5 g.L -1 ，NaCl 10g·L -1 。
LB solid medium: peptone 10 g.L -1 Yeast extract 5 g.L -1 ，NaCl 10g·L -1 AgarPowder 15 g.L -1 。
The protein purification buffers involved in the following examples were as follows:
binding solution a:20mM imidazole, 500mM sodium chloride, 20mM Tris,5% glycerol, pH 7.4 with HCl;
eluent B:500mM imidazole, 500mM sodium chloride, 20mM Tris,5% glycerol, and adjusted to pH 7.4 with HCl.
The detection method involved in the following examples is as follows:
detection of D-carbamoyl hydrolase enzyme activity: the reaction was performed in a 1.5mL EP tube. Taking 10 mu L of protein with the concentration of 1 mg.mL -1 To a mixture of 200. Mu.L of a 10mM substrate, N-carbamoyl-D-tryptophan in Tris-HCl (pH 8.0, 100 mM) buffer, was added, and the total reaction system was 1mL. The reaction system is placed at 30 ℃ for reaction for 10min, and after the reaction is finished, the reaction system and methanol are added according to the ratio of 1:3 to terminate the reaction. The reaction product was centrifuged through a membrane (0.22 μm) and then subjected to HPLC analysis to determine the enzyme activity. Each set of data was repeated three times.
HPLC analysis conditions: the mobile phase is KH 2 PO 4 (20 mM, pH 2.5): acetonitrile=75:25, flow rate 1ml·min -1 The chromatographic column is ZORBAX SB-C 18 The column temperature is 30 ℃, and the detection wavelength is 210nm.
Definition of enzyme activity unit: the amount of enzyme required to catalyze the conversion of N-carbamoyl-D-amino acid to 1. Mu. Mol of amino acid per minute at 30℃is one enzyme activity unit (U).
Example 1: preliminary screening of thermostable D-carbamoyl hydrolase mutant enzyme
The method comprises the following specific steps:
1. (1) construction of mutants:
the mutant primers are shown below, underlined as mutation sites:
TABLE 1 mutant primers
Taking a plasmid with the amino acid sequence shown as SEQ ID NO.1 and the mutation of the 202 rd serine of the amino acid sequence into proline (S202P) as an example, carrying out PCR amplification by taking a pET-28a (+) vector connected with the nucleotide sequence shown as SEQ ID NO.2 as a template and taking S202P-F, S202P-R as a primer to obtain the nucleotide sequence of a mutant (S202P) with the mutation of the 202 rd serine of the amino acid sequence into proline;
the PCR product containing the recombinant gene obtained in the last step is digested by DpnI to remove a template, and the digested product is transformed into competent cells of E.coli BL21 (DE 3) to obtain a transformation solution; the conversion solution was applied to a solution containing 50ng mL -1 LB solid medium of kanamycin, culturing for 12h at 37 ℃ in an inverted mode, and picking single colony until the colony contains 50 ng.mL -1 The correct mutant plasmid S202P is obtained by plasmid extraction and DNA sequencing in LB liquid medium of kanamycin, which is cultured for 8-10h at 37 ℃.
Recombinant plasmids of mutant E138W/S, S202P, S D/N, E208D, R277L/Q/E, H284T/N were constructed using the primers in Table 1 and the same method as in step (1).
And (3) PCR amplification: the reaction system is described in Table 2, with a total volume of 20. Mu.L:
TABLE 2PCR reaction System
TABLE 3PCR amplification procedure
(2) Preliminary screening of thermostable D-carbamoyl hydrolase
Taking D-carbamoyl hydrolase S202P as an example, the D-carbamoyl hydrolase mutant with improved thermostability is initially screened, and the steps are as follows:
the D-carbamoylhydrolase mutant S202P obtained by the construction in the step (1) is containedRecombinant E.coli was transferred to a strain containing a final concentration of 50 ng.mL -1 40mL LB liquid medium of kanamycin, 37 ℃,120rpm culture to OD 600 IPTG was added at 0.6-0.8 and the final concentration was 0.2mM, and the culture was induced at 16℃for 20h. The cells were collected by centrifugation at 8000rpm for 5 min. After adding an appropriate amount of Tris-HCl (100 mM pH 8.0) to each 50mL centrifuge tube for collecting the cells, sonication was performed. 1mL of the crushed solution was centrifuged at 12000rpm at 4℃for 3min, 200. Mu.L of each supernatant was transferred to 3 clean 1.5mL centrifuge tubes and placed on ice for further use.
The mutant S202P crude enzyme solution is respectively incubated at 40 ℃ and 30 ℃ for 15min, then ice-bath is carried out for 2min, and then enzyme reaction is carried out. The ratio of the specific activity measured after incubation of the crude enzyme solution at 40℃for 15min to the specific activity measured after incubation at 30℃for 15min was recorded as residual activity, whereby mutants with improved thermostability (M4 residual activity of about 82%) were initially screened.
D-carbamoyl hydrolase prescreening reaction system: using N-carbamoyl-D-tryptophan as a substrate at a final concentration of 2mM, 20. Mu.L of crude enzyme solution (M4 or mutant enzyme) was added, and the mixture was made up to 1mL with 100mM Tris-HCl buffer (pH 8.0). The reaction was carried out at 30℃for 10min and terminated by adding methanol at a ratio of 1:3.
Example 2: rescreening of thermostable D-carbamoyl hydrolase mutant enzyme
(1) Purification of D-carbamoyl hydrolase mutant enzyme
The mutant plasmid with improved thermostability obtained by screening in example 1 was transformed and cultured upside down at 37℃overnight, and single colonies were picked up to a final concentration of 50 ng.mL -1 Kanamycin was cultured in 40mL LB liquid medium at 37℃and 120rpm for 8-10h. 1mL of the bacterial liquid is taken until the final concentration is 50 ng.mL -1 100mL LB liquid medium of kanamycin, 37 ℃,120rpm culture to OD 600 IPTG was added at 0.6-0.8 and the final concentration was 0.2mM, and the culture was induced at 16℃for 16-20h. The cells were collected by centrifugation at 8000rpm at 4℃for 5 min.
An appropriate amount of the binding solution A was added to each 50mL centrifuge tube for collecting the cells, and after ultrasonic disruption, the cells were centrifuged at 8000rpm and 4℃for 30min, and then subjected to membrane filtration and purification by a nickel affinity chromatography column. And after loading is finished, eluting and collecting proteins by using imidazole eluents with different concentration gradients prepared by the binding solution A and the eluent B, wherein the elution volume of each concentration gradient is about 5-10 column volumes. After the elution is finished, performing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) verification on the collected solution, collecting the protein with higher purity according to the result of the SDS-PAGE, and performing ultrafiltration concentration to remove imidazole. And (5) quick freezing with liquid nitrogen and preserving at-80 ℃.
(2) Rescreening of thermostable D-carbamoyl hydrolase
The method for detecting the thermal stability comprises the following steps: diluting the preserved mutant pure enzyme to 1 mg.mL -1 And (3) placing a proper amount of enzyme solution into a water bath at 40 ℃ for incubation, taking the incubation at 40 ℃ for 5min as a starting point for 0min, and respectively sampling the enzyme solution after incubation for different times to determine the enzyme activity. Half-life (t) was measured on a 0min enzyme activity basis of 100% 1/2 ). Half-life (t) 1/2 ) Refers to the time required to lose half of the enzymatic activity at a given temperature. Each set of data was repeated three times.
D-carbamoyl hydrolase re-screening reaction system: N-carbamoyl-D-tryptophan as substrate at a final concentration of 2mM was added at 1 mg.multidot.mL -1 10. Mu.L of the pure enzyme (M4 or mutant enzyme) was supplemented to 1mL with 100mM Tris-HCl buffer (pH 8.0). The reaction was carried out at 30℃for 10min and terminated by adding methanol at a ratio of 1:3.
The thermal stability of the mutation is R277L, E208D, S202P, R277Q, E138W, H284T, S204D, H284N, S204N, E138S and R277E in sequence from high to low.
The same procedure as in example 1 and example 2 was followed to finally obtain a combined mutant enzyme S202P/E208D, S P/R277L, E D/R277L, S202P/E208D/R277L (Table 4) with significantly improved thermostability. Wherein S202P/E208D/R277L has the best thermal stability, t 1/2 (40 ℃) was 36.5h, 28.5 times that of M4 (1.3 h).
TABLE 4 specific Activity and half-life of thermostable mutants
Example 3: property study of thermostable D-carbamoyl hydrolase mutant enzyme
1. Optimum temperature
The M4 and mutant enzymes (S202P, E208D, R277L, S P/E208D, S P/R277L, E208D/R277L, S P/E208D/R277L) were subjected to enzymatic reactions at different temperatures (25-55 ℃), respectively. The temperature at which each enzyme showed the highest activity was defined as the optimum temperature.
As shown in Table 5, the optimum temperature of the mutant enzyme S202P, E208D, S202P/E208D was 50℃and increased by 5℃compared with M4; however, the optimum temperature of the mutant enzyme R277L, S P/R277L, E D/R277L, E208D/R277L, S P/E208D/R277L was reduced to 40℃by 5℃compared with M4.
2. Thermodynamic stability experiment
Determination of the unfolding temperature by Nano-DSC (T m ) To characterize the thermodynamic stability of M4 and mutant enzymes (S202P, E208D, R277L, S P/E208D, S P/R277L, E D/R277L, S P/E208D/R277L). The specific operation steps are as follows:
(1) Baseline scan: after degassing buffer Tris-HCl (100 mM, pH 8.0) for 10min, the sample cell and the reference cell were added, and baseline scanning was performed after removing bubbles. The operation program is set as follows: the temperature scanning range is 25-85 ℃, and the temperature rising rate is 1 ℃ and min -1 The number of repetitions was 5.
(2) Sample scanning: diluting M4 or mutant enzyme to 1 mg.mL -1 Degassing for 10min, completely sucking out buffer solution in the sample cell, adding degassed M4 or mutant enzyme, and scanning. The operation program is set as follows: the temperature scanning range is 25-85 ℃, and the temperature rising rate is 1 ℃ and min -1 。
(3) And (3) result processing: using nanoAnalyze TM The software processes the running results and uses the TwoStatescaled model to perform data fitting to obtain M4 and T of mutant enzyme m Values (table 5).
The results are shown in Table 5, although M4Th3 has significantly higher thermal stability at 40℃than M4, T m The value was only 3.6℃higher. Whereas S202P, E D and R277L have a ΔT m The values are 1.1 ℃, 1.3 ℃ and 1.3 ℃ respectively, and the sum of the three values is approximately equal to the corresponding value of M4Th3, which indicates that the three single protrusionsThe variations may have additive or synergistic effects between them.
TABLE 5 characterization of the thermostable mutants
3. Dynamic stability test
By measuring
To characterize the kinetic stability of M4 obtained in example 2 with the mutant enzyme (S202P, E208D, R277L, S202P/E208D, S P/R277L, E D/R277L, S202P/E208D/R277L). Diluting the pure enzyme to 1 mg.mL -1 Split 20 μl into PCR tubes and incubate for 15min with different temperature gradients during PCR to perform the enzymatic reaction. The temperature at which half of the relative enzyme activity is lost is +.>
The results are shown in table 5, for S202P and E208D,
the increment is only 1-2 ℃; R277L->
Is 2.2 ℃. But note that S202P, E D and R277L are all three +.>
The values accumulate almost equal to M4Th3 +.>
This may further indicate an additive or synergistic effect between the three single mutations. />
4. Kinetic parameter determination
Under the measurement condition of standard enzyme activity, D-carbamyl hydrolase with a certain concentration is measured for different substrate concentrations (0.1, 0.2, 0.4,0.6, 0.8, 1.0, 2.0, 5.0 mM) and fitting with Origin 2019 software to obtain a Miq equation curve of the reaction initiation rate versus substrate concentration, to obtain K M And V max Values.
TABLE 6 kinetic parameters
The results are shown in Table 6, although S202P/E208D/R277L is V max Value (2.97 U.mg) –1 ) Compared with M4 (3.40U.mg) –1 ) K is low, but S202P/E208D/R277L M The value is also lower. Thus, the final S202P/E208D/R277L obtained has a molecular weight equal to M4 (288 min –1 ·mM –1 ) Equivalent catalytic efficiency of 302min –1 ·mM –1 。
Example 4: evaluation of the use of thermostable D-carbamoyl hydrolase mutants in the synthesis of D-tryptophan
To further investigate the use of the thermostable D-carbamoyl hydrolase mutant S202P/E208D/R277L in D-tryptophan synthesis, the reaction of M4 with S202P/E208D/R277L on 100mM N-carbamoyl-D-tryptophan at 40℃was determined.
D-carbamoyl hydrolase (5 kU.L) –1 ) Tris-HCl buffer (pH 8.0, 100 mM) containing 100mM of N-carbamoyl-D-tryptophan was added thereto and reacted at 40℃and 200rpm for 24 hours, with a constant pH of 8.0. The total volume was 500mL.
As a result, as shown in Table 7, although the conversion rate of S202P/E208D/R277L was lower than M4 in the first 2 hours due to a slightly lower specific activity, it was noted that the conversion rate of S202P/E208D/R277L was significantly higher than M4 after that. The conversion rate of M4 is basically unchanged after 12 hours of reaction, and the conversion rate is 60.8% at 24 hours; whereas S202P/E208D/R277L reached 94.5% conversion at 12h and 99.4% conversion at 24 h. This shows that S202P/E208D/R277L has significantly better thermal stability than M4, which is beneficial to the industrial application.
TABLE 7 reaction progress of 100mM N-carbamoyl-D-tryptophan catalyzed by D-carbamoyl hydrolase
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. A D-carbamyl hydrolase mutant with improved heat stability is characterized in that the D-carbamyl hydrolase mutant takes D-carbamyl hydrolase with an amino acid sequence shown as SEQ ID NO.1 as a parent, and amino acids at 138, 202, 204, 208, 277 and 284 of the D-carbamyl hydrolase mutant are mutated respectively.
2. The D-carbamoyl hydrolase mutant according to claim 1, wherein the D-carbamoyl hydrolase mutant is obtained by mutating glutamic acid Glu at 138 th position of a parent to tryptophan Trp or serine Ser; or alternatively, the first and second heat exchangers may be,
mutating serine Ser at 202 th site of parent to proline Pro; or alternatively, the first and second heat exchangers may be,
mutating serine at position 204 of the parent to aspartic acid Asp or asparagine Asn; or alternatively, the first and second heat exchangers may be,
mutating glutamic acid Glu at position 208 of the parent to aspartic acid Asp; or alternatively, the first and second heat exchangers may be,
mutating arginine Arg at 277 th site of the parent to Leu, glutamine Gln or glutamic acid Glu; or alternatively, the first and second heat exchangers may be,
the histidine His at position 284 of the parent is mutated to threonine Thr or asparagine Asn.
3. The D-carbamoyl hydrolase mutant according to claim 1, wherein the D-carbamoyl hydrolase mutant is obtained by mutating serine Ser at position 202 of the parent to proline Pro, mutating glutamic acid Glu at position 208 to aspartic acid Asp, and mutating arginine Arg at position 277 to Leu.
4. A gene encoding a D-carbamoyl hydrolase mutant according to any of claims 1 to 3.
5. An expression vector carrying the coding gene of claim 4.
6. A genetically engineered bacterium expressing the D-carbamoyl hydrolase mutant according to any one of claims 1 to 3.
7. The genetically engineered bacterium of claim 6, wherein the genetically engineered bacterium is a host of e.
8. Use of the D-carbamoyl hydrolase mutant according to any of claims 1 to 3 or the genetically engineered bacterium according to claim 6 in the preparation of D-amino acids.
9. The use according to claim 8, wherein the use is to catalyze the production of D-amino acid using N-carbamoyl-D-amino acid as substrate and the D-carbamoyl hydrolase mutant or the genetically engineered bacterium as catalyst.
10. The use according to claim 8, wherein the catalytic conditions are 38-42 ℃, 150-250 rpm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211589530.5A CN116200374A (en) | 2022-12-09 | 2022-12-09 | D-carbamoyl hydrolase mutant with improved thermal stability and application thereof in synthesis of D-amino acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211589530.5A CN116200374A (en) | 2022-12-09 | 2022-12-09 | D-carbamoyl hydrolase mutant with improved thermal stability and application thereof in synthesis of D-amino acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116200374A true CN116200374A (en) | 2023-06-02 |
Family
ID=86506665
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211589530.5A Pending CN116200374A (en) | 2022-12-09 | 2022-12-09 | D-carbamoyl hydrolase mutant with improved thermal stability and application thereof in synthesis of D-amino acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116200374A (en) |
-
2022
- 2022-12-09 CN CN202211589530.5A patent/CN116200374A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109355276B (en) | Pullulanase mutant and application thereof | |
| CN112877307B (en) | Amino acid dehydrogenase mutant and application thereof | |
| CN113151237B (en) | Sucrose isomerase mutant with improved stability and construction method thereof | |
| CN111172142B (en) | Cephalosporin C acylase mutant with high thermal stability | |
| CN107794275B (en) | Recombinant pichia pastoris for producing (+) gamma-lactamase and construction method and application thereof | |
| CN110184259B (en) | Pullulanase mutant derived from anaerobic bacillus and application thereof | |
| CN115322981A (en) | Nitrile hydratase mutant and application thereof in preparation of amide compounds | |
| CN110699396A (en) | Method for preparing D-aromatic amino acid by cascade reaction | |
| CN114317510B (en) | Maleic acid cis-trans isomerase mutant | |
| CN112481320B (en) | Method for preparing (-) gamma-lactam with high catalytic efficiency | |
| EP1553175A1 (en) | Cephalosporin C acylase mutants | |
| US20210238576A1 (en) | L-aspartate alpha-decarboxylase Mutant and Application thereof | |
| CN116200374A (en) | D-carbamoyl hydrolase mutant with improved thermal stability and application thereof in synthesis of D-amino acid | |
| CN110804602B (en) | L-aspartic acid beta-decarboxylase mutant and application thereof | |
| CN112831532B (en) | Method for enzymatic synthesis of D-leucine | |
| CN112301014B (en) | Esterase mutant with improved thermal stability and application thereof | |
| CN114836406A (en) | Agarase mutant with improved catalytic activity and application thereof | |
| CN114736884A (en) | Cytidine monophosphate kinase mutant and gene and application thereof | |
| CN111172143B (en) | D-xylonic acid dehydratase and application thereof | |
| CN108034649B (en) | Glucose isomerase mutant and application thereof | |
| CN110452899B (en) | Glucose isomerase, mutant and application of mutant in preparation of D-fructose | |
| CN112442474B (en) | Preparation method of (-) gamma-lactam | |
| CN115505581B (en) | Aromatic amine-N acetyl transferase mutant and application thereof | |
| CN110904087B (en) | L-arabinose epimerase mutant and application thereof | |
| CN107201355B (en) | High-stereoselectivity phenylalanine deaminase mutant and application 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 |