CN109486785B - Omega-transaminase mutant with improved catalytic efficiency and application thereof - Google Patents

Omega-transaminase mutant with improved catalytic efficiency and application thereof Download PDF

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CN109486785B
CN109486785B CN201811450486.3A CN201811450486A CN109486785B CN 109486785 B CN109486785 B CN 109486785B CN 201811450486 A CN201811450486 A CN 201811450486A CN 109486785 B CN109486785 B CN 109486785B
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廖祥儒
翟李欣
赖英杰
杨邵岚
蔡宇杰
管政兵
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Abstract

The invention discloses a omega-transaminase mutant with improved catalytic efficiency and application thereof, belonging to the technical field of genetic engineering and enzyme engineering. The invention obtains the omega-transaminase mutant with improved catalytic efficiency by carrying out site-directed mutagenesis on amino acid with higher factor B in the crystal structure of the omega-transaminase, namely mutating Y at the position of 32 of the omega-transaminase into L. The catalytic efficiency Kcat/Km of the mutant Y32L is improved by 21.84 times, and the maximum conversion rate of acetophenone of the mutant is improved by 7.72 times compared with that of natural enzyme when the mutant catalyzes (R) -phenylethynamine to generate the acetophenone. The mutant obtained by the invention is more suitable for industrial application than natural omega-transaminase.

Description

Omega-transaminase mutant with improved catalytic efficiency and application thereof
Technical Field
The invention relates to a omega-transaminase mutant with improved catalytic efficiency and application thereof, belonging to the technical field of genetic engineering and enzyme engineering.
Background
Transaminase (transaminase) belongs to transferase and is generally used for catalyzing amino group to be transferred from an amino donor compound to an amino acceptor compound, protein sequences of transaminase from different sources reported in the literature are compared and clustered according to differences of different variable regions, and then the transaminase is divided into 4 types according to superposition and iterative comparison of hydrophilic sites, wherein omega-transaminase belongs to a second subfamily and is generally used for preparing chiral amine and unnatural amino acid, such as β -amino acid.
In protein molecular engineering, methods commonly used at present are rational design, irrational design and semi-rational design. The main differences between the three methods are whether the molecular structure of the enzyme protein is well understood and whether calculations and predictions need to be made using bioinformatics software. The rational design has the advantages of low experimental cost, simplicity, convenience, short time and the like.
Although the omega-transaminase has great application and research value, the catalytic efficiency of the omega-transaminase screened from wild bacteria is low, and the development and application of the omega-transaminase are greatly reduced.
Disclosure of Invention
In order to solve the problems, the invention carries out heterologous expression and site-directed mutation modification on omega-transaminase (the omega-transaminase from Bacillus pumilus W3 and the application of the omega-transaminase in biological amination 201811219769.7) from Bacillus pumilus W3, improves the catalytic rate of the omega-transaminase on specific substrates, and has profound technical guidance significance on large-scale production and popularization of the omega-transaminase.
Compared with the parent omega-transaminase, the omega-transaminase mutant has better catalytic efficiency. The parent gene is consistent with a Bacillus pumilus (Bacillus pumilus W3) omega-transaminase gene (Sequence ID: MH196528), and the plasmid template used for mutation is a carrier ota3/pCold II (application number: CN201811219769.7) carrying a natural omega-transaminase coding gene.
It is a first object of the present invention to provide a ω -transaminase mutant with improved catalytic efficiency, the amino acid sequence of the mutant comprising: the amino acid sequence obtained by mutating tyrosine at position 32 to leucine on the basis of the amino acid sequence of SEQ ID NO. 1 was named Y32L.
In one embodiment, the amino acid sequence of the ω -transaminase mutant is the sequence shown in SEQ ID NO. 5.
In one embodiment, the nucleotide sequence of the ω -transaminase mutant comprises the sequence shown in SEQ ID NO. 2.
A second object of the present invention is a method for preparing said mutant, comprising the steps of:
(1) designing primers for site-directed mutagenesis, carrying out mutagenesis by taking a vector carrying a omega-transaminase coding gene as a template, and constructing a plasmid vector of a Y32L mutant;
(2) and (3) transforming the recombinant plasmid with the correct sequence into escherichia coli BL21(DE3) to obtain a recombinant bacterium, fermenting and culturing the recombinant bacterium, and obtaining fermentation supernatant fluid containing the omega-transaminase mutant.
In one embodiment, the fermentation is by culturing the recombinant bacteria to OD at 37 ℃600After that, the temperature was decreased to 15 ℃ and IPTG was added to a final concentration of 0.4mM for induction, and the mixture was centrifuged to obtain a supernatant enzyme solution after 24 hours of culture.
In one embodiment, the preparation method further comprises purifying the ω -transaminase in the fermentation supernatant using an AKTA protein purifier and a histrappf fraction 1ml nickel column.
The third purpose of the invention is to provide a recombinant plasmid vector containing the amino acid sequence of the mutant.
In one embodiment, the plasmid vector is any one of pET series, pGEX series, pCold series, or pUB.
The invention also claims a gene for coding the mutant and a gene engineering bacterium for expressing the mutant.
The invention also claims the application of the mutant, the gene for coding the mutant and the gene engineering bacteria for expressing the mutant in the catalytic synthesis of related chiral amine in the aspects of food, chemical engineering or medicament preparation, in particular the application in the preparation of medicaments.
In one embodiment, the use comprises catalyzing the transfer of an amino group from an amino donor compound to an amino acceptor compound.
The applications include the selective catalysis and chiral synthesis of chiral amines, such as R-phenylethylamine, and a variety of unnatural amino acids.
The invention has the beneficial effects that:
the omega-transaminase mutant is mutated on the basis of R type omega-transaminase derived from Bacillus pumilus, and the constructed omega-transaminase mutant Y32L has the performance of improving the catalytic efficiency. Enzyme kinetic analysis showed that K of mutant Y32L of the inventionmThe value is reduced by 92.2 percent compared with the parent natural enzyme; catalytic efficiency Kcat/KmThe improvement is 21.84 times. When (R) -phenylethynamine is catalyzed to generate acetophenone, the maximum conversion rate of the acetophenone of the mutant is improved by 7.72 times compared with that of parent natural enzyme. Therefore, the omega-aminotransferase mutant is more suitable for the application of the omega-aminotransferase in the process of catalyzing chiral amines such as (R) -phenylethylamine and the like than the parent.
Drawings
FIG. 1: a three-dimensional simulation structure of natural omega-transaminase;
FIG. 2: performing SDS-PAGE gel electrophoresis on the natural omega-transaminase and the mutant pure enzyme; wherein lane 1 represents a protein molecular weight standard, lane 2 is mutant Y32L, and lane 3 is a native ω -transaminase.
Detailed Description
Example 1: preparation and construction of omega-transaminase site-directed mutants
Omega-transaminase 1 site-directed mutant Y32L from Bacillus pumilus W3:
in the invention, a three-dimensional simulation structure of Bacillus pumilus W3 omega-transaminase (omega-BPAT) is constructed by a Swiss-Model online server by taking a crystal structure (PDB ID:5E25) of the thermophilic archaea transaminase with the highest similarity as a template (FIG. 1). Through amino acid primary sequence alignment, the similarity between the thermophilic archaea transaminase and the omega-BPAT reaches 51.21 percent, and accords with the parameters of homology modeling, so that the omega-BPAT can be considered to have a three-dimensional structure similar to the thermophilic archaea transaminase. Based on the results predicted by the software analysis, mutant Y32L was constructed using PCR-mediated site-directed mutagenesis.
The preparation method of the site-directed mutant comprises the steps of respectively designing and synthesizing primers for introducing site-directed mutation according to the sequence (the amino acid sequence is shown as SEQ ID NO: 1) of Bacillus pumilus W3 omega-transaminase, carrying out site-directed mutation on the position Y32 of the omega-transaminase, determining a DNA coding sequence, and respectively sequencing to confirm whether the coding gene of the omega-transaminase mutant is correct or not; the mutant gene is connected to a proper expression vector (any one of pET series, pGEX series, pCold series or pUB) and is introduced into escherichia coli for expression, and the corresponding omega-transaminase site-directed mutant is obtained.
PCR amplification of site-directed mutant coding gene: using PCR technology, expression vector ota3/pCold II was used as template.
The mutation primers for introducing the site-specific mutation of Y32L are (SEQ ID NO:3 and SEQ ID NO:4, respectively):
Y32L-F：5’-CCATGGGTTCTTACTTGGCGATGGGGTCTTCG-3’
Y32L-R：5’-TCGTATACAGAGATTTTTGCGTCTTCCTTC-3’
the PCR amplification program was set up as follows: first, pre-denaturation at 94 ℃ for 5 min; then 30 cycles were entered: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 5 min; finally, extension is carried out for 10min at 72 ℃, and heat preservation is carried out at 4 ℃. The PCR product was detected by electrophoresis on a 1% agarose gel.
And (3) after purifying the PCR product, adding DPn I, heating at 37 ℃ in a water bath for 1h, degrading a template, then transforming E.coli JM109, selecting a positive clone, culturing for 8-10h in an LB liquid culture medium, preserving a glycerol tube, and sequencing. The mutant (the amino acid sequence is shown as SEQ ID NO:5, the nucleotide sequence is shown as SEQ ID NO: 2) with correct sequencing is inoculated to an LB culture medium from a glycerol tube, overnight culture is carried out, plasmids are extracted, and the plasmids are transformed to express host escherichia coli BL21(DE3) competent cells, so as to obtain the recombinant strain capable of expressing the mutant Y32L.
Example 2: expression and purification of natural omega-aminotransferase and site-directed mutants thereof
Selecting a positive monoclonal transferred into an expression host escherichia coli BL21(DE3), growing for 8-10h in an LB liquid culture medium (containing 30 mug/mL ampicillin), and inoculating the seed fermentation liquid to the LB liquid culture medium (containing 30 mug/mL ampicillin) according to the inoculation amount of 5%; culturing Escherichia coli at 37 deg.C for 2 hr to OD600Around 0.6, mutant Y32L recombinant strain was added to 0.05mMInducing extracellular expression with IPTG, further culturing and fermenting at 15 deg.c for 24 hr, centrifuging the fermented liquid at 4 deg.c and 8000g for 10min to eliminate thallus, and collecting supernatant. Slowly adding 60% (NH) into the mutant fermentation supernatant under low-speed stirring by a magnetic stirrer4)2SO4Salting out was performed overnight at 4 ℃. Centrifuging at 4 deg.C and 10000g for 20min, and collecting precipitate. After re-dissolving the precipitate with 50mmol/L of citric acid-disodium hydrogen phosphate buffer solution at pH5.3, the precipitate was dialyzed overnight against 50mmol/L of citric acid-disodium hydrogen phosphate buffer solution at pH5.3, during which the dialysis buffer solution was changed 2 to 3 times, and a sample was prepared by filtration through a 0.22 μm membrane. And (3) purifying the recombinant protein by adopting an AKTAavant protein purifier, wherein the temperature in the whole purification process is controlled to be 4 ℃. Cation exchange chromatography purification step: (1) balancing: equilibrating the strong cation exchange chromatography column with 5 volumes of 50mmol/L pH5.3 citrate-disodium phosphate buffer; (2) loading: sampling the pretreated sample at the flow rate of 1 mL/min; (3) and (3) elution: eluting unadsorbed substances, foreign proteins and target proteins at the flow rate of 1.0mL/min, wherein the eluent is a 50mmol/L citric acid-disodium hydrogen phosphate buffer solution with the pH value of 5.3 and contains 1M NaCl, carrying out linear elution, the detection wavelength is 280nm, and collecting the eluent containing sucrose isomerase activity in batches; only one target protein elution peak appears in the elution process, and enzyme activity is detected subsequently and SDS-PAGE protein electrophoresis shows that the enzyme solution collected at the peak top is the purest part no matter whether the enzyme solution is a wild type or a mutant. As shown in fig. 2.
Example 3: enzyme activity analysis method
The method for measuring the activity of ω -transaminase is described in Gao, S. (Gao, S., Su, y., Zhao, l., Li, g., Zheng, g.,2017. mutation of a (R) -selective amine transferase from fusarium oxygen system. process. biochem.63, 130-136.).
An appropriate amount of cell supernatant (or purified diluted enzyme solution) was added to 500. mu.L of sodium dihydrogen phosphate/disodium hydrogen phosphate buffer (100mM, pH7.0) containing 20mM (R) - α -phenylethyylamine (or (S) - α -phenylethyylamine), 20mM sodium pyruvate, and 0.1mM pyridoxal 5' -phosphate (PLP), and the mixture was mixed, reacted at 45 ℃ for 15 minutes, and then the reaction was terminated by adding an equal amount of ethyl acetate, and the absorbance of the solution at 254nm was measured before and after the reaction.
The amount of enzyme required to catalyze 1. mu. mol of the relevant ketones in 1 minute under the above conditions is defined as one enzyme activity unit (U/ml). The process is illustrated by △ A254Calculating the enzyme activity of omega-transaminase, U/ml (△ A/min) V/rvb, △ A/min-absorbance change, V-reaction system volume (ml), r-molar extinction coefficient (cm)2/umol); v-sample size (ml); b-cuvette optical path length (cm), the above amounts can be increased or decreased proportionally.
Through determination, the crude enzyme activity of the natural enzyme is 1.1760U/mL, and the enzyme activity of the recombinant omega-transaminase is 11.4697U/mL.
Example 4: determination of kinetic parameters of omega-transaminase mutants
Kinetic parameters of ω -transaminase were determined by reference to Gao, S. (Gao, S., Su, y., Zhao, l., Li, g., Zheng, g.,2017. characteristics of a (R) -selective amine transferase from fusarium oxysporum. process. biochem.63, 130-136.).
In this example, the kinetic parameters of the natural enzyme (amino acid sequence shown in SEQ ID NO: 1) and the mutant Y32L (amino acid sequence shown in SEQ ID NO: 5) purified in example 2 at 45 ℃ were determined by adding 500. mu.L of a sodium dihydrogen phosphate/disodium hydrogen phosphate buffer (100mM, pH7.0) containing (R) - α -phenylethynamine at different concentrations (the concentration gradient is described in the above-mentioned publication), 20mM sodium pyruvate, and 0.1mM pyridoxal 5' -phosphate (PLP) to a suitable cell supernatant (or a purified diluted enzyme solution), mixing the mixture, reacting the mixture at 45 ℃ for 15min, and adding the same amount of ethyl acetate to terminate the reaction, and the results of the kinetic study are shown in Table 1.
The results show that K of mutant Y32L compared to the native enzyme (WT)mThe value is reduced by 92.2 percent, KmThe decrease in value indicates an increased affinity of the mutant for the substrate (R) - α -phenylethylamine, in addition, the catalytic constant K of Y32LcatThe improvement is 1.69 times of that of the natural enzyme. Catalytic constant K compared to the native enzymecatAnd the increased affinity for the substrate, directly led to the catalytic efficiency K of the mutant Y32Lcat/KmThe improvement is 21.84 times.
As shown in fig. 1, Y32L is far from the catalytic center and the isomerization region of ω -transaminase, and mutation may cause the structure of the region other than the catalytic center and the isomerization region to become compact, so that the substrate is not easily detached from the catalytic center, thereby possibly having a positive effect on the kinetic parameters of the enzyme.
TABLE 1 kinetic parameters of the ω -transaminase mutants
Figure BDA0001886307820000051
The conversion rate is the conversion efficiency of (R) - α -phenylethynamine into acetophenone.
Example 5 application of the mutant in the production of (R) - α -phenylethylamine
In the embodiment, (R) - α -phenylethynylamine is taken as an amino donor, sodium pyruvate is taken as an amino acceptor, and the catalytic capability of the obtained recombinase and the mutant recombinase on the enzyme is detected, so that whether the enzyme can efficiently catalyze the (R) - α -phenylethynylamine is judged, and the application of the enzyme in the industrial synthesis of chiral amines is determined.
Chiral synthesis catalysis experiment, which is to take a proper amount of purified diluted enzyme solution, add 500 μ L of sodium dihydrogen phosphate/disodium hydrogen phosphate buffer solution (100mM, pH7.0) containing 20mM (R) - α -phenylethyylamine, 20mM sodium pyruvate, 0.1mM pyridoxal 5' -phosphate (PLP), mix well, react for 15min at 45 ℃, then add the same amount of ethyl acetate to stop the reaction, centrifuge for 1min at 12,000 Xg, take the upper organic phase, filter with 0.22 μm filter membrane, and perform High Performance Liquid Chromatography (HPLC) detection to detect the product, namely acetophenone.
Detection conditions are as follows:
column: agilent C18column (250 × 4.6mm, Agilent, USA); mobile phase: acetonitrile/water (50/50, v/v); flow rate: 0.6 mL/min; detection wavelength: 254 nm.
TABLE 2
Figure BDA0001886307820000061
And (3) comparison: and (R) -phenylethyylamine is used as a substrate, and the catalytic capability of the natural omega-transaminase on the (R) -phenylethyylamine is detected under the reaction conditions.
Test samples: the catalytic ability of the omega-transaminase Y32L mutant was tested under the above reaction conditions using (R) -phenylethylylamine as a substrate.
The data show that the catalytic activity of the recombinase on an experimental group (taking the omega-transaminase Y32L mutant as a catalyst) is better than that of a control group (taking the natural omega-transaminase as a catalyst). The data show that the recombinant omega-transaminase Y32L mutant has the function of efficiently and selectively synthesizing R-chiral amine and has larger application potential (see table 3).
TABLE 3
Figure BDA0001886307820000062
Enzyme activity (U/mg): the amount of enzyme required to catalyze 1. mu. mol of the relevant ketones in 1 minute is defined as one unit of enzyme activity (U/mg).
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
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Claims (6)

1. An omega-transaminase mutant with improved catalytic efficiency is characterized in that the amino acid sequence of the omega-transaminase mutant is shown as SEQ ID NO. 5.
2. A gene encoding the mutant of claim 1.
3. A recombinant plasmid vector containing the gene of claim 2.
4. The recombinant plasmid vector according to claim 3, wherein the recombinant plasmid vector is constructed on the basis of any one of the plasmid vectors of pET series, pGEX series, pCold series, or pUB series.
5. A genetically engineered bacterium expressing the mutant of claim 1.
6. The mutant of claim 1, or the gene encoding the mutant of claim 1, or the genetically engineered bacterium expressing the mutant of claim 1, wherein the mutant is used for catalyzing (R) - α -phenylethylylamine, and the genetically engineered bacterium is used in the fields of food, chemical engineering or pharmaceutical preparation.
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