CN111944778A - Glutamine transaminase mutant and encoding gene and application thereof - Google Patents
Glutamine transaminase mutant and encoding gene and application thereof Download PDFInfo
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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 wild enzyme MTG-WT, and the thermal stability is superior to that of the wild enzyme.
Description
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 the generation of gamma-carboxamide group of glutamine residue in protein peptide chain and various acyl acceptorsThe acyl transfer reaction is an effective protein cross-linking agent. TG is widely distributed in human body, mammal, plant and microorganism, etc., wherein Microbial Transglutaminase (MTG) has low protein molecular weight and is not Ca2+Dependence, wide substrate range and other excellent enzymological properties, and has wide application value in food industry, biomedicine, tissue engineering, site-specific protein crosslinking, homogeneous antibody coupling medicine construction and other industries. 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。
SEQ ID NO：1：
DSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP
The amino acid sequence of the mutant Mut1(S2P-S23V-Y24N-R215A-K294L) according to a specific embodiment of the invention is shown in SEQ ID NO: 2, respectively.
SEQ ID NO: 2 is shown in
DPDDRVTPPAEPLDRMPDPYRPVNGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKAKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESLFRNWSEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP
The amino acid sequence of the mutant Mut2(S2P-S23V-Y24N-R215A-H289Y) according to the embodiments of the present invention is set forth in SEQ ID NO: 3, respectively.
SEQ ID NO：3：
DPDDRVTPPAEPLDRMPDPYRPVNGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKAKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMYVYESKFRNWSEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP
Mutant Mut3 according to embodiments of the invention
(S23V-Y24N-R215A-K269D-H289Y) has the amino acid sequence shown in SEQ ID NO: 4, respectively.
DSDDRVTPPAEPLDRMPDPYRPVNGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKAKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADDTVWTHGNHYHAPNGSLGAMYVYESKFRNWSEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP
The present invention also provides a gene encoding the transglutaminase mutant.
The present invention also provides a recombinant vector comprising the above gene encoding the above 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, three mutants containing five amino acid mutation sites are combined by mutating sites S2, S23, Y24, K269, R215, H289, K294 and the like of mature transglutaminase MTG-WT, the mutant recombinant vector is transformed into Pichia pastoris pPIC9K-pro/GS115, and the expression is induced by shaking a small tube, and the results show that the activity of the tubular supernatants 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 is measured after the protein of three mutant enzymes and wild enzyme is concentrated and purified, and the optimum reaction temperature of the three mutants is 55 ℃ when the N-CBZ-Gln-Gly is used as a substrate. 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 time at 50 ℃ is respectively 9.4 times, 11.6 times and 9.8 times of that of the wild enzyme, and the half-life time 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. For pichia pastoris to express a secreted protein containing the kr (aagaga) sequence, if the sequence is located on the protein surface, it may be hydrolyzed by Kex2 protease derived from pichia pastoris, resulting in instability of the secreted protein in the 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 fermentation supernatant of three mutant MTG-Mut (R215) 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 vectors pPIC9k-pro, pPICZa-mtg and recombinant strains pPIC9k-pro/GS115 and pro/mtg (GS 115).
2. Enzymes, antibodies and other biochemical reagents: N-carboxybenzoyl-L-glutamine-glycine (N-CBZ-Gln-Gly) and L-glutamic acid-gamma-monohydroxyamine acid were purchased from Sigma-Aldrich; anti-MTG was purchased from Eurogentec. MUT Express II Fsat Mutagenesis kit V2 was purchased from Nanjing Novowed Biotech. The others are domestic analytical pure reagents (all can be purchased from common biochemical reagents).
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 (mtg1) as template
Mut1 gene (mtg)1) Synthesized by Nanjing Kingsrei Biotech Co., Ltd., mtg1The 5 'end of the gene is introduced with Xho I site (CTCGAG) and Kex2 endopeptidase recognition site (aaaaga), the 3' end is introduced with NotI site (GCGGCCGC) and 6 XHis tag sequence (ATGGTGATGGTGATGATG), and the tag sequence is mainly convenient for purifying recombinant protein. Artificial synthesis mtg1The gene was cloned into pUC57 vector, mtg was 993 bp.
Mut2, Mut3 gene (mtg)2，mtg3) Using pUC57-mtg1 as template
Max Super-Fidelity DNApolymerase amplifies target plasmid, the target plasmid amplification product is subjected to DpnI digestion,
The transformation was carried out directly after the recombinant cyclization, and the specific procedures were carried out according to the instructions of the mutation kit (MUT Express II Fsat Mutagenesis kit V2). Positive transformants were selected, plasmid sequencing was extracted, and correctly sequenced mutant plasmids (pUC57-mtg1, pUC57-mtg2, pUC57-mtg3) were transformed into competent cells e.
EXAMPLE 2 construction of recombinant vector for transglutaminase mutant
Restriction enzymes Xho I and NotI are respectively used for double enzyme digestion of mutant plasmids and expression vectors pPICza, enzyme digestion products are recovered through glue, the products are recovered through ligase ligation, E.coli TOP10 competent cells are transferred to be evenly coated on an LLB plate containing 50 mu g/mL Zeocin resistance, and the single clone is grown through overnight culture at 37 ℃. The correctly sequenced plasmid was designated pPICZ α -mtg1, the remaining 3 were numbered sequentially. The correctly sequenced mutant recombinant vector was transformed to express host pPIC9k-PRO/GS115 (expression leader PRO peptide or PRO).
EXAMPLE 3 construction of recombinant Strain of transglutaminase mutant
The successfully constructed three mutant recombinant vectors pPICZ alpha-mtg-mut were linearized with Spe I and transformed into pPIC9k-pro/GS115 (made competent in advance) to obtain recombinant yeast strain GS115 (pro/mtg-mut).
Selecting a strain GS115(pro/mtg-mut) containing the recombinant plasmid, inoculating the strain into a culture tube of 4mL BMGY medium, and carrying out shake culture 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 60 μ L of GS115(pro/mtg-mut) bacterial liquid with highest supernatant enzyme activity to 5mLYPD liquid culture medium, shaking the flask overnight for culture, transferring all the obtained liquid into 50mL BMGY (pH 6.0) shaking flask, culturing at 28 deg.C for overnight at 200r/min, measuring OD 24h later600When OD is reached600When the pH reached 6, the cells were collected by centrifugation at 3000g for 5min, and after resuspension of the cells in 200mL of BMMY (methanol 1%, pH 6.0), expression was induced, and the initial OD was determined600And (3) 1.0-1.5, inducing at 25 ℃ for 72h, supplementing methanol every 24h until the final concentration is 1% (V/V), and taking supernatant to detect the 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) is not degraded within 48h of induction, and the small degradation begins to appear at 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. The enzyme activity of MTG is determined by a hydroxamic acid colorimetric method, and the enzyme activity of 1 unit is defined as: amount of enzyme (U/mL) used by MTG to catalyze the production of 1. mu. moL L-glutamic acid-monohydroxyaminoic acid (hydroxamic acid) from substrate (. alpha. -N-CBZ-Gln-Gly, Sigma) per minute at 37 ℃.
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 MTG and its mutant is determined by measuring the MTG enzyme activities at 20, 30, 37, 50, 60 and 70 ℃ within the range of 20-70 ℃ respectively, determining a narrower optimum reaction temperature range, and then determining the optimum reaction temperature by taking 5 ℃ as a gradient. The activity determination was carried out as described above, with a final concentration of 30mmol/L substrate N-CBZ-Gln-Gly and a pH of 20mmol/L buffer adjusted to 6.0 at the respective temperatures. 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 are basically consistent at 20-70 ℃, 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 1 hour 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; while the wild type is treated at 60 ℃ for 10min, the enzyme activity can not reach 10% of the highest enzyme activity. The percentage decrease rule 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 fig. 5And (3) drawing an Ln (residual activity) -time relation graph and performing linear fitting to obtain a slope, namely the first-order inactivation constant (k) of the enzyme at the temperatured). Half-lives (t) of the enzymes at 50 ℃ and 60 ℃1/2) From the formula t1/2＝Ln2/kdThe results were obtained (Table 2).
TABLE 2 comparison of the enzymatic Properties of the mutant and of the wild enzyme
Sequence listing
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Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala
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Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Asp Ser Asp Asp Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met
1 5 10 15
Pro Asp Pro Tyr Arg Pro Val Asn Gly Arg Ala Glu Thr Val Val Asn
20 25 30
Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg
35 40 45
Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys
50 55 60
Val Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu
65 70 75 80
Ala Phe Ala Ser Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys Asn
85 90 95
Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val
100 105 110
Ala Lys Glu Ser Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg Glu
115 120 125
Val Ala Ser Val Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu Ser
130 135 140
Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala
145 150 155 160
Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn
165 170 175
Thr Pro Ser Phe Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser Arg
180 185 190
Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg
195 200 205
Ser Ser Ser Ala Asp Lys Ala Lys Tyr Gly Asp Pro Asp Ala Phe Arg
210 215 220
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 (7)
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. Use of the transglutaminase mutant with improved thermostability and enzyme activity according to claim 1.
6. Use of the transglutaminase gene according to claim 2.
7. A method for preparing transglutaminase with improved thermal stability and enzyme activity comprises the following steps:
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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| CN112553176A (en) * | 2020-12-29 | 2021-03-26 | 江南大学 | Glutamine transaminase with improved thermal stability |
| CN112553177A (en) * | 2020-12-29 | 2021-03-26 | 江南大学 | Glutamine transaminase variant with improved heat stability |
| WO2023058765A1 (en) * | 2021-10-07 | 2023-04-13 | 天野エンザイム株式会社 | Modified transglutaminase |
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| CN112553177B (en) * | 2020-12-29 | 2022-04-01 | 江南大学 | Glutamine transaminase variant with improved heat stability |
| CN112553176B (en) * | 2020-12-29 | 2022-04-29 | 江南大学 | Glutamine transaminase with improved thermal stability |
| WO2023058765A1 (en) * | 2021-10-07 | 2023-04-13 | 天野エンザイム株式会社 | Modified transglutaminase |
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