| Glutamate decarboxylase(GAD,EC 4.1.1.15),the key enzyme for biosynthesis of gamma-aminobutyric acid(GABA),is a pyridoxal phosphate enzyme.GABA has the physiological functions of delaying brain aging,lowering blood pressure,and calming nerves.As a functional compound,GABA has broad application prospects in the food,medical,and agricultural industries.Moreover,it can activate liver and kidney function and promotes hormone secretion.GAD distributes widely from single-cell organisms to high-level mammalian organisms.However,its content is considerably low,thus hindering the industrial-scale manufacturing.Therefore,it has become a hot research topic to synthesize GABA using GAD derived from microorganisms.In our previous study,a gene encoding GAD was cloned from a Bacillus megaterium and the recombinant gene was expressed in E.coli.However,this GAD enzyme had low activity and poor stability.In order to further improve the catalytic properties of the enzyme and satisfy the needs of biological preparation,the enzyme was molecularly modified by means of directed evolution and semi-rational design strategies.Mutants with improved enzyme activity and stability were obtained,which can provide the guidance for the production and application of GABA in practice.The main findings are shown as follows:1.Directed Evolution of glutamate decarboxylase derived from Bacillus megateriumError-prone PCR was used to introduce random mutations in the gene of interest,so as toconstruct a library of mutants.After two rounds of error-prone PCR reactions,three enzyme activity-enhancing mutants A5-3,E2-4,and E3-11 were screened from more than 13,000 mutants.The enzyme activities were 21.25±0.73 U/mL,18.52±1.90 U/mL,and 14.11+2.49 U/mL,respectively.Compared with the wild type,the enzyme activities were increased by 210%for A5-3,185%for E2-4,and 118%for E3-11.The three mutants with the highest enzyme activity screened by error-prone PCR were then used as parents for DNA shuffling.Mutant Z4 was screened from more than 4,000 mutants in the library.After purification,the specific activity reached 161.63±3.31 U/mL,which was 2.91 times higher than that of wild type.The Km value of this mutant enzyme Z4 was not significantly different from that of the wild-type enzyme,whereas the kcat value was significantly higher than that of the wild-type enzyme,indicating that the catalytic efficiency of the mutant enzyme on the substrate was increased.The results of circular dichroism analysis and three-dimensional modeling analysis showed that there were little changes in the secondary structures of the four mutants compared with the wild type.Among them,the ?-helix content of E2-4 was 43.1%,which was higher than that of the wild type.The increase ofa-helix content led to an enhancement of thermal stability.The random coils of the mutants Z4,A5-3,E2-4,and E3-11 all increased,thus enriching the flexible structure of the protein.In addition,the position 55 from Ala to Asp may enhance the ability to provide H+for enzymatic reactions.The mutation from Leu to Gln in position 34 facilitated the improvement of hydrophobic interactions and enhanced the stability of the enzyme.The position 325 from Ala to Ser in position 325 may result in an increase in the flexibility of the protein structure,which favored the binding of coenzymes,substrates and enzymes,and the release of products.The increase in H+capacity and the increase in the flexibility of the protein structure may be the main reasons for the increased catalytic efficiency of mutant enzymes.In summary,the use of directed evolution strategy can effectively improve the catalytic activity of Bacillus megaterium GAD.2.Semi-rational design of glutamate decarboxylase derived from Bacillus megateriumSite-directed saturation mutations were performed using the mutation sites of the aforementioned mutants with increased enzyme activity and key sites affecting the catalytic activity of GAD.Meanwhile,a library of mutants was established.Mutants A55D,A55E,L34Q,A325S and Q346H were screened from more than 3,000 mutants,and their enzyme activities were 232%127%,138%,115%and 63%higher than that of the wild type,respectively.The mutants A55D/L34Q,A55D/A325S,and A55D/L34Q/Q346H with further increased catalytic activity were obtained by means of the multi-site combination mutation technique.Compared with the wild type,the resulting enzyme activitiesy increased by 260%,236%,and 275%,respectively.Moreover,mutant A55D/L34Q/Q346H had a specific activity of 183.22±3.33 U/mg after purification,which was 2.29 times higher than that of the wild type.The Km of the mutant A55D/L34Q/Q346H was lower than that of the wild-type enzyme,whereas the kcat value was higher than that of the wild-type enzyme,indicating that the affinity and catalytic efficiency of the mutant enzyme to the substrate were increased.The half-life of the mutant A55D/L34Q/Q346H reached 24.19 h at 55?,which was 7 h longer than that of the wild-type enzyme.Three-dimensional structure modeling and circular dichroism(CD)results showed that the increased in hydrogen bond and the number of a-helices in mutant A55D/L34Q/Q346H can stabilize the protein conformation.The enhanced the thermal stability of the enzyme.His hadthe ability to provide H+and store H+,which increaseds the catalytic efficiency of the enzyme.The position 34 Leu located at the random curl of the N segment,and the mutation to Gln led to an increase of the hydrophobic effect,thus increaseing the enzyme stability.The 5 5-position Ala located on the first helical arm of segment N.This site was mutated to Asp and the number of hydrogen bonds increaseds.The aforementioned results showed that the use of semi-rational design strategies to molecularly modify the Bacillus megaterium GAD not only improved the enzyme activity,but also 346 position was changed from Gln to His,and His was generally conserved in the protein structure.The imidazole ring in the residue could form hydrogen bond and play a role in stabilizing the enzyme protein.3.Synthetise of ?-Aminobutyric acid by transformation method of mutant A55D/L34Q/Q346HThe best mutant A55D/L34Q/Q346H was selected to synthesize GABA.studying the influences of conversion temperature,pH,surfactant,growth factor,metal ion and substrate concentration,the conversion conditions for the preparation of GABA were determined.The results showed that when the transformation temperature was 50?,pH was 5,substrate concentration was 125 mM,PLP(coenzyme for GAD)was 0.2 mM,Mg2+and Ca2+ concentration was 5 mM,the mutant A55D/L34Q/Q346H had the highest transformation capacity.Under these conditions which was 2.56 times than the wild-type strain.was 56.47 g/g*h,the transformation efficiency... |
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