CN111944778B - Glutamine transaminase mutant and encoding gene and application thereof - Google Patents

Glutamine transaminase mutant and encoding gene and application thereof Download PDF

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CN111944778B
CN111944778B CN202010817256.7A CN202010817256A CN111944778B CN 111944778 B CN111944778 B CN 111944778B CN 202010817256 A CN202010817256 A CN 202010817256A CN 111944778 B CN111944778 B CN 111944778B
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宋小平
王雅洁
王蔷
蔡晶晶
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Abstract

The invention relates to the field of genetic engineering, in particular to a transglutaminase mutant and a coding gene and application thereof. Carrying out S2P, S23V, Y24N, R215A and K294L on a wild enzyme with the amino acid sequence shown as SEQ ID NO. 1; S2P, S23V, Y24N, R215A, H289Y; or S23V, Y24N, R215A, K269D and H289Y. The specific activities of the three mutant enzymes are respectively 1.9 times, 2.4 times and 1.4 times of that of a wild enzyme MTG-WT, and the thermal stability of the mutant enzymes is superior to that of the wild enzyme.

Description

Glutamine transaminase mutant and encoding gene and application thereof
Technical Field
The invention relates to the field of genetic engineering, in particular to a transglutaminase mutant and a coding gene and application thereof.
Background
Transglutaminase (TG) can catalyze gamma-carboxamide group of glutamine residue in protein peptide chain to produce acyl transfer reaction with various acyl acceptors, and is an effective protein cross-linking agent. TG is widely distributed in human bodies, mammals, plants, microorganisms and the like, wherein Microbial Transglutaminase (MTG) has excellent enzymological properties such as low protein molecular weight, Ca2+ independence and wider substrate range, and has wide application value in industries such as food industry, biomedicine, tissue engineering, site-specific protein crosslinking and construction of homologous antibody coupling drugs. For some applications, extrusion and heat stabilization of microcapsules, including thermoplastic processing, to produce immobilized enzymes, it may be advantageous to perform the crosslinking reaction at higher temperatures and higher activity. Therefore, mutants with higher activity and thermostability are expected to be used not only in the current process, but also to expand the range of new applications.
The molecular improvement of transglutaminase mainly focuses on the mechanism research and improvement of thermal stability. The existing heat stability research on transglutaminase mainly focuses on single-point mutation, few combined mutation researches are carried out, no report is found on the research on protein degradation in recombinant MTG secreted by pichia pastoris, and no report is found on whether the enzyme activity and heat stability are influenced by reducing the degradation of secreted protein or not and what the influence mechanism is.
Disclosure of Invention
Based on the reported problems of low thermal stability of transglutaminase, easy degradation of target protein in the fermentation process and the like, the invention provides a transglutaminase mutant.
The object of the present invention is to provide a transglutaminase mutant having improved thermostability and enzyme activity.
It is still another object of the present invention to provide a gene encoding the transglutaminase mutant having improved thermostability and enzyme activity as described above.
It is still another object of the present invention to provide a recombinant vector comprising the above gene.
It is still another object of the present invention to provide a recombinant strain comprising the above gene.
It is still another object of the present invention to provide a method for preparing transglutaminase having improved thermal stability and enzyme activity.
It is still another object of the present invention to provide use of the transglutaminase mutant having improved thermal stability and enzyme activity.
According to the transglutaminase mutant with improved heat stability and enzyme activity, the mutant is obtained by performing the following site-specific mutagenesis on wild-type transglutaminase with the amino acid sequence shown as SEQ ID NO. 1:
S2P-S23V-Y24N-R215A-K294L；
S2P-S23V-Y24N-R215A-H289Y; or
S23V-Y24N-R215A-K269D-H289Y。
The amino acid sequence of the mutant Mut1 (S2P-S23V-Y24N-R215A-K294L) according to the embodiments of the present invention is shown in SEQ ID NO: 2, respectively.
The amino acid sequence of the mutant Mut2 (S2P-S23V-Y24N-R215A-H289Y) according to a specific embodiment of the invention is as shown in SEQ ID NO: 3, respectively.
The amino acid sequence of the mutant Mut3 (S23V-Y24N-R215A-K269D-H289Y) according to the embodiments of the present invention is set forth in SEQ ID NO: 4, respectively.
The present invention also provides a gene encoding the transglutaminase mutant.
The present invention also provides a recombinant vector comprising the gene encoding the transglutaminase mutant.
The present invention also provides a recombinant cell comprising the above-described gene encoding the above-described transglutaminase mutant.
The method for preparing the transglutaminase mutant according to the present invention comprises the steps of:
constructing a recombinant vector containing a gene encoding the transglutaminase mutant;
and introducing the recombinant vector into a host cell, and performing induced expression to obtain the transglutaminase with high catalytic efficiency.
According to the specific embodiment of the invention, the mutation of the sites S2, S23, Y24, K269, R215, H289, K294 and the like of mature transglutaminase MTG-WT is combined into three mutants containing five amino acid mutation sites, and the mutant recombinant vector is transformed into Pichia pastoris pPIC9K-proGS115, the expression of the mutant is induced by small shake tube, and the result shows that the activity of the tubule supernatant of the three mutants is higher than that of the wild enzyme.
And (3) carrying out 1L shake flask expression on the clone with high enzyme activity, inducing for 72h, sampling every 24h, and detecting by SDS-PAGE. The results show that the three mutant MTG-Mut (R215) with no mutation at position 215 begins to degrade within 24h of induction, and the degradation is obvious at 72h, while the mutant MTG-Mut (A215) with mutation at position 215 does not degrade within 48h of induction, and the degradation begins to slightly occur at 72h of induction. Research shows that R215 is mutated into A215, the induction time is controlled at 72h, and the degradation of secreted protein is greatly reduced. Provides fermentation technological parameters for the industrialized production of the enzyme.
The basic enzymology property determination is carried out on the protein of three mutant enzymes and wild enzymes after concentration and purification, and the determination with N-CBZ-Gln-Gly as a substrate shows that the optimum reaction temperature of the three mutants is 55 ℃. The specific activities of the three mutant enzymes are respectively 1.9 times, 2.4 times and 1.4 times of that of the wild enzyme MTG-WT. The enzyme activity of the three mutant enzymes at 60 ℃ is more than 60% of the highest enzyme activity; when the reaction temperature is 70 ℃, the enzyme activity still reaches more than 10 percent of the highest enzyme activity. The half-life period at 50 ℃ is respectively 9.4 times, 11.6 times and 9.8 times of that of the wild enzyme, and the half-life period at 60 ℃ is respectively 6.1 times, 10.2 times and 8.7 times of that of the wild enzyme. Mut2 has the highest thermal stability with a half-life of 27.6min at 60 ℃.
The invention proves that key catalytic related sites of a mature enzyme MTG are S2 and H289, and the importance of the sites on the high catalytic efficiency of the enzyme is proved, namely the S2 and H289 sites play an important role in the high catalytic efficiency of the enzyme; it was confirmed that the S23, 24N, K269, and H289 sites were involved in the thermostability of the enzyme. Meanwhile, the mutation of R215 into A215 effectively inhibits the hydrolysis of the target protein by Kex2 protease from pichia pastoris. When pichia pastoris expresses a secretory protein containing a kr (aagaga) sequence, if the sequence is positioned on the surface of the protein, the sequence can be hydrolyzed by Kex2 protease derived from pichia pastoris, so that the secretory protein is unstable in a fermentation broth. The KR amino acid sequence in the target protein is mutated, thereby avoiding the specific recognition and the cleavage of carboxyl terminal peptide bonds in the basic amino acid residue pair on the surface of the secretory protein by the Kex2 protease and solving the degradation problem in the secretory protein of the pichia pastoris.
Drawings
FIG. 1 is a schematic diagram showing amino acid mutation at position 215 of MTG-WT (R215) and MTG-MUT (A215);
FIG. 2 is an electrophoretogram of supernatants from MTG-Mut (R215) fermentation of three mutants without mutation at amino acid position 215;
FIG. 3 is an electrophoretogram of a fermentation supernatant of MTG-Mut2 (A215) with amino acid mutation at position 215;
FIG. 4 shows the effect of temperature on enzyme activity;
FIG. 5 is a graph of residual viability versus time at 50 ℃ and 60 ℃ for various periods of time.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
Materials and methods
1. Strains and plasmids: plasmid vector pPIC9k-pro，pPICZa-mtgAnd the recombinant strain pPIC9k-pro/GS115 andpro/ mtg(GS115)；
2. enzymes, antibodies and other biochemical reagents: N-carboxylbenzoyl-L-glutamine-glycine (N-CBZ-Gln-Gly) and L-glutamic acid-gamma-monohydroxyamine were purchased from Sigma-Aldrich; anti-MTG was purchased from Eurogentec. MUT Express II Fsat Mutagenesis kit V2 was purchased from Nanjing Novowed Biotech. Other reagents are domestic analytical pure reagents (all can be purchased from common biochemical reagents companies);
3. culture medium:
(1) LB culture medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0;
(2) YPD medium: 1% yeast extract, 2% peptone, 2% glucose;
(3) BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (V/V), 1.34% YNB, 0.00004% Biotin;
(4) BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1.0% methanol (V/V). ).
EXAMPLE 1 acquisition of a mutated transglutaminase gene
TABLE 1 construction of primers for mutants using artificially synthesized Mut1 gene (mtg 1) as template
Figure DEST_PATH_IMAGE001
Mut1 gene (mtg 1) Synthesized by Nanjing Kingsrei Biotech Co., Ltd,mtg 1introduction of the 5' end of the GeneXhoI site (CTCGAG) andKex2 endopeptidase recognition site (aaaaga), 3' end is introducedNotA site I (GCGGCCGC) and a 6 XHis tag sequence (ATGGTGATGGTGATGATG), the tag sequence being primarily for ease of purification of the recombinant protein. Artificially synthesizedmtg 1The gene was cloned into a pUC57 vector,mtgis 993 bp.
Mut2, Mut3 Gene (mtg 2， mtg 3) Is pUC57-mtg1 as a template, amplifying a target plasmid by using Phanta Max Super-Fidelity DNA Polymerase, wherein an amplification product of the target plasmid is subjected to amplification by using Phanta Max Super-Fidelity DNA PolymeraseDpnI digestion, direct transformation after Clon Express recombinant cyclization, and specific operation steps refer to the use instructions of a mutation kit (MUT Express II Fsat Mutagenesis kit V2). Selecting positive transformant, extracting plasmid to sequence, and obtaining mutant plasmid (pUC 57-mtg1, PUC57-mtg2, PUC57-mtg3) Transformation of competent cellsE. coli TOP10。
EXAMPLE 2 construction of recombinant vector for transglutaminase mutant
Respectively using restriction enzymesXhoI andNoti double enzyme digestion mutant plasmid and expression vector pPICza, recovering enzyme digestion product, connecting with ligase, recovering product, transferring toE. coliTOP10 competent cells, plated evenly on 50. mu.g/mL Zeocin resistant LLB plates, 37 oCAfter overnight incubation, single colonies were grown. The plasmid with correct sequencing is named pPICZ alpha-mtg1 and the other 3 are numbered in sequence. Transforming the correctly sequenced mutant recombinant vector into the expression host pPIC9k-proGS115 (expression leader PRO peptide or PRO).
EXAMPLE 3 construction of recombinant Strain of transglutaminase mutant
The three successfully constructed mutant recombinant vectors pPICZ alpha-mtg-mut are respectively usedSpeI linearization and electrotransformation into pPIC9k-pro(iii) GS115 (Productivity in advance), obtaining recombinant Yeast Strain GS115 (pro/mtg-mut）。
Selecting the strain GS115 containing the recombinant plasmid (pro/mtg-mut), inoculating in a culture tube of 4mL BMGY medium, and shake culturing at 28 ℃ and 200rpm for 48 h; the culture broth was then centrifuged at 3000g for 5min, the supernatant was discarded, and the pellet was resuspended in 4mL BMMY medium containing 1.0% methanol and placed at 25 ℃ for induction culture at 200rpm for 48 h. And finally taking the supernatant to detect the enzyme activity. The results of the mini-shake tube induced expression show that the activity of the tubule supernatant of all three mutants is higher than that of the wild enzyme.
Selecting GS115 with highest enzyme activity in the supernatant (1)pro/mtg-mut) 60. mu.L of the strain solution into 5 mL YPD liquid medium, shaking the flask overnight, transferring all the strain solution into 50mL BMGY (pH = 6.0) shaking flask, culturing the strain solution overnight at 28 ℃ at 200 r/min, and measuring OD after 24h600When OD is reached600When the concentration reached 6, the cells were collected by centrifugation at 3000g for 5min, and after resuspension of the cells with 200 mL of BMMY (methanol 1%, pH = 6.0), expression was induced, and the initial OD was determined600 And (4) induction temperature of 25 ℃ for 72h, adding methanol every 24h until the final concentration is 1% (V/V), and taking supernatant to detect enzyme activity. And (5) after the fermentation is finished, purifying the recombinant transglutaminase mutant enzyme and the wild enzyme. The results show that the three mutants MTG-Mut (R215) without mutation at position 215 begin to degrade after 24h induction, and the degradation is obvious after 72h (as shown in FIG. 2). While the mutant MTG-Mut with the 215-site mutation (A215) has no degradation within 48h of induction, and a small amount of degradation begins to appear after 72h of induction (as shown in FIG. 3). Research shows that R215 is mutated into A215, the induction time is controlled at 72h, and the degradation of secreted protein is greatly reduced. Provides fermentation technological parameters for the industrialized production of the enzyme.
Example 4 analysis of expression Activity of recombinant transglutaminase mutant and wild type
To compare the enzymatic activities of mutant MTG and wild-type MTG, the enzymatic activity of MTG was determined by hydroxamic acid colorimetry using N-benzylcarbonyl-L-glutamine-glycine (N-CBZ-Gln-Gly, Sigma) as a substrate. Colorimetric determination of M by hydroxamic acidTG enzyme activity, 1 unit of enzyme activity defined as: 37oEnzyme amount (U/mL) used by MTG to catalyze substrate (alpha-N-CBZ-Gln-Gly, Sigma) to generate 1 μmoL L-glutamic acid-monohydroxyaminoic acid per minute at C. The purified recombinant transglutaminase mutant enzyme of example 3 and the wild-type enzyme were subjected to enzymatic property examination.
1. The optimal temperature measurement method of the recombinant transglutaminase mutant and the wild type comprises the following steps:
and (3) detecting the optimal reaction temperature: the optimum temperature of the MTG and the mutant thereof is determined by determining the enzyme activities of the MTG at 20-70 ℃, determining the enzyme activities of the MTG at 20, 30, 37, 50, 60 and 70 ℃ respectively, determining a narrower optimum reaction temperature range, and then determining the optimum reaction temperature by taking 5 ℃ as a gradient. The activity determination method is carried out according to the method, the final concentration of the substrate N-CBZ-Gln-Gly is 30mmol/L, and 20mmol/L Tris buffer solution is adjusted to the pH value of 6.0 at the respective temperature. Temperature was plotted against viability to obtain a temperature-viability curve (FIG. 4). The results show that the enzyme activity change conditions of the three mutants of Mut1, Mut2 and Mut3 at 20-70 ℃ are basically consistent, the optimal reaction temperature is 55 ℃, and the optimal reaction temperature of the wild MTG is 50 ℃.
2. The heat stability and half-life period detection method of the recombinant transglutaminase mutant and the wild type comprises the following steps:
and (3) measuring the thermal stability: diluting the purified MTG and the mutant thereof to the same concentration, respectively preserving the temperature in a water bath at 50 ℃ and 60 ℃ for different times, then placing the MTG and the mutant on ice for cooling for 10min, and determining the residual activity of the enzyme according to the method. The residual activity was plotted as a function of time at 50 ℃ and 60 ℃ for different periods of time (FIG. 5). The result shows that after all mutant enzymes are treated for 5 hours at 50 ℃, the enzyme activity is more than 50 percent of the highest enzyme activity; and after the wild type is treated for 1h at 50 ℃, the enzyme activity is reduced to 50 percent of the highest enzyme activity. All mutant enzymes are treated for 10min at 60 ℃, and the enzyme activity still reaches more than 60 percent of the highest enzyme activity; and the wild type is treated at 60 ℃ for 10min, and the enzyme activity cannot reach 10% of the highest enzyme activity. The law of the percentage decrease of the residual enzyme activity at 50 ℃ and 60 ℃ is as follows: mut2 < Mut3 < Mut1 < WT. The above results show that all mutants have different degrees of increased thermostability compared to WT, Mut2 being the most thermostable, Mut3 times.
Half-life detection (t 1/2): according to the results of FIG. 5, an Ln (residual activity) -time relationship graph is prepared and linear fitting is carried out, and the obtained slope is the first-order inactivation constant of the enzyme at the temperature (k d). Half-life of enzyme at 50 ℃ and 60 ℃ ((t 1/2) By the formulat 1/2 = Ln2/k dThe results were obtained (Table 2).
TABLE 2 comparison of the enzymatic Properties of the mutant and of the wild enzyme
Figure 572201DEST_PATH_IMAGE003
Sequence listing
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Pro Ala Pro Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile
225 230 235 240
Pro Arg Ser Pro Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp Tyr
245 250 255
Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp Asp Thr Val Trp
260 265 270
Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala Met
275 280 285
Tyr Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Glu Gly Tyr Ser Asp
290 295 300
Phe Asp Arg Gly Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn
305 310 315 320
Thr Ala Pro Asp Lys Val Lys Gln Gly Trp Pro
325 330

Claims (6)

1. A transglutaminase mutant with improved heat stability and enzyme activity, which is obtained by performing the following site-directed mutagenesis on a wild-type transglutaminase having an amino acid sequence shown in SEQ ID NO. 1:
S2P-S23V-Y24N-R215A-K294L；
S2P-S23V-Y24N-R215A-H289Y; or
S23V-Y24N-R215A-K269D-H289Y。
2. A transglutaminase gene encoding the transglutaminase mutant of claim 1, wherein the transglutaminase mutant has improved thermostability and enzyme activity.
3. A recombinant vector comprising the transglutaminase gene according to claim 2.
4. A recombinant cell comprising the transglutaminase gene according to claim 2.
5. The use of the transglutaminase mutant having improved thermostability and enzyme activity according to claim 1 as a protein crosslinking agent.
6. A method for producing transglutaminase having improved thermal stability and enzyme activity, comprising the steps of:
constructing a recombinant vector comprising the transglutaminase gene according to claim 2;
and introducing the recombinant vector into a host cell, performing induced expression, and separating and purifying to obtain the transglutaminase with improved thermal stability and enzyme activity.
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CN112553176B (en) * 2020-12-29 2022-04-29 江南大学 Glutamine transaminase with improved thermal stability
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CN118043463A (en) * 2021-10-07 2024-05-14 天野酶制品株式会社 Modified transglutaminase

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