Molecular Engineering Of Glutamate Decarboxylase To Improve Its Thermostability And Expression Level

Glutamate decarboxylase(GAD)is a key enzyme in the biosynthesis of y-aminobutyric acid(GABA).The thermostability and expression level of GAD are critical factors limiting its large-scale application.Therefore,in this study,molecular engineering of GAD 1407 from Lactobacillus brevis was carried out to improve its thermostability and expression level.First,the amino acid residues that may contribute to the high thermostability were identified through multiple-sequence alignment,and were replaced to enhance the thermostability as well as catalytic efficiency.Then,the coevolutionary network related to the critical site was analyzed,of which one amino acid residue was mutated to investigate its influence on the stability and activity.In addition,the effect of N-terminus of mRNA on the expression of GAD was studied by synonymous codon substitution,and a strategy for high-level expression of recombinant GAD in Escherichia coli was developed.The main results are listed as follows:(1)A series of residues in GAD 1407 that may affect thermostability were identified by sequence alignment using ClustalW method and five mutants were constructed by site-directed mutagenesis.After screening,a mutant H322R with both improved thermostability and specific activity was found.The semi-inactivation temperature(T5010)and half-life(t1/2)at 60℃ of H322R were 65.44℃and 102.17 min,respectively,which were 4.14℃ higher and 73.4 min longer than those of the GAD wild-type(WT),respectively.Moreover,the specific activity of H322R was increased by 20%compared with that of WT.Molecular dynamics simulation results showed that the mutation enhanced the overall rigidity of the PLP binding domain and increased flexibility of the catalyzed key region loop(residues 308-312),which may be the main reason for the improvement of the thermostability and specific activity.Through conformation analysis of the local region of H322R,it was found that the introduction of arginine at the H322 site enhanced the polar interactions between the surrounding residues,which made their spatial arrangement closer and formed new hydrogen bonds and stable salt bridges.Thereby,the mutant H322R exhibited a higher thermostability and catalytic activity owing to the optimized overall structure.(2)The residues M316 and E99 were inferred in the coevolutionary subnetwork to have interactions with H322 by the EVcouplings sever using plmDCA method.Based on the mutant H322R,site-directed saturation mutagenesis library was constructed at the position 316.Four mutants M316C-H322R、M316V-H322R、M316I-H322R、M316L-H322R were screened with varying degrees of reduced activity and thermostability fallen in between WT and H322R.It proved that the 316 position played an important role in the thermostability of GAD 1407,and methionine at this position may bring a benefit for the thermostability.MD simulation revealed that the reduced flexibility of the catalytic region(308-312)and the reduced rigidity of overall structure were the main reasons for the decreased catalytic activity and thermostability of the double-site mutants,respectively.(3)The secondary structure of GAD N-terminal mRNA and the corresponding free energy were analyzed by DNAMAN software.A series of mutants with distinct free energy of secondary structure were constructed by introducing synonymous mutations.It was found that the total GAD expression levels of G27A,G27T,A12G/T18A and T18A/G27T were significantly increased.The best mutant G27A exhibited more than 67%increased recombinant expression activity compared to WT.The above results indicated that reducing the free energy of the N-terminal mRNA secondary structure can effectively improve the expression level of GAD.