Rational Modification Of Thermal Stability Of Lactobacillus Brevis Glutamate Decarboxylase

Glutamate decarboxylase(GAD)can catalyze the formation of L-glutamic acid to y-aminobutyric acid(GABA),and its catalytic performance has an important impact on the efficiency of the GABA biopreparation.In order to obtain GAD with high catalytic efficiency and good thermal stability,this study takes GAD 1407 from L.brevis CGMCC 1306 as the research object,and uses two rational design methods to carry out molecular modification:First,multi-sequence alignment with thermophilic GAD was conducted to identify the amino acid residue replacement sites and types that are likely to improve the thermal stability of the enzyme.Then,the optimal modification scheme of the enzyme molecule was determined by calculating the effects of these mutations on the free energy of the protein;the second is to identify Loop regions of the enzyme molecule that are highly flexible and sensitive to high temperature,and do not affect the catalytic function of the enzyme,and then analyze the structural types of β-turns in these Loop regions,and compare the differences between their amino acid residues and the typical ones,and replace the amino acid residues inconsistent at the corresponding sites with the typical amino acid residues.The mutant enzymes designed by these two schemes were constructed by using PCR-based site-directed mutation methods,and the catalytic efficiency and thermal stability of these mutant enzymes were measured.Multiple mutant enzymes with simultaneously improved catalytic efficiency and thermal stability were obtained.The main results are as follows:1.To search the thermophilic GAD and the corresponding amino acid sequence in the database,analyze the primary structure differences between GAD 1407 and these thermophilic GADs through multiple sequence alignment,and identify the amino acid residue mutation sites that may improve the thermal stability of GAD 1407,and then use Rosetta Cartesian ddG software to calculate the effect of different mutations on protein free energy in order to identify mutations that can reduce protein free energy and improve protein structural stability.The corresponding mutant enzymes were constructed by site-directed mutagenesis,their catalytic properties were measured and compared with the wild-type enzyme.It was found that among the constructed mutant enzymes,the thermal stability and catalytic efficiency of the mutant enzyme M190Y were higher than that of the wild-type enzyme:the semi-inactivation temperature T5015 of the mutant enzyme was 63.6℃,which was higher than that of the wild-type(T2015 was 62.9℃);the half-life of the mutant enzyme at 60℃ was 34.9 min,which was 3.9 min higher than that of the wild type(31.0 min);kcat/Km value of this mutant is 2.86 s-1mM-1,which is 36%higher than the wild type(Kcat/Km is 2.10 s-1mM-1).2.To analyze the crystal structure model of GAD 1407 with software such as Chimera,B-fitter,etc.,and identify the Loop regions of enzyme molecules that are more flexible and vulnerable to high temperature while not participate in substrate catalysis.The structure types ofβ-turns in these Loop regions were analyzed using the PDBsum database.The enzyme molecule modification scheme was determined by comparing the difference between amino acid in the β-turns and corresponding typical structures.The corresponding mutant enzymes were constructed by site-directed mutagenesis and their catalytic properties were measured.It was found that the catalytic efficiency and thermal stability of the mutant enzymes L152P and M415T were improved:compared with the wild-type enzymes,their kcat/Km were increased by 202.9%and 180.9%,respectively;their half-inactivation temperature T5015 were increased by 1.2℃ and 1.0℃,respectively;their half-life at 60℃ were increased by 10.8 min and 16.2 min,respectively.3.Molecular dynamics simulation lasting for 30 ns of the seven enzymes,including the wild type and mutants of M190Y,L152P,T153S,A154C,A154G,A154D and M415T,was carried out with Gromacs software in order to investigate conformation changes and backbone motion trajectory of GAD1407 caused by anmino acid residues replacements..It was found that the L152P mutation can enhance the rigidity of the Loop6 region,which not only improved the rigidity of the α/β super secondary structure formed by the α-helix(residue 126-148)and the β-sheet(residue 158-165),but also has a positive effect on the structural stability of the entire molecule.The M415T mutation can improve the flexibility of Loop 16 located in the key catalytic region of the PLP binding domain(residue 307-312);therefore,catalytic efficiency of the mutant was improved because of the consequently increased flexibility of the active center.In general,the two rational modification methods adopted in this research have obtained the mutant enzyme with improved thermal stability and catalytic efficiency,overcoming the trade off between catalytic efficiency and thermal stability in the enzyme molecular modification.In terms of molecular modification effect,the mutant enzyme L152P obtained by the second rational modification method is significantly higher in catalytic efficiency and thermal stability than other mutants,indicating that the second rational design method has better effects.It is worthy of further research,application and improvement.