Directed Evolution Of Glycine Oxidase From Bacillus Cereus

Glyphosate（N-phosphonomethylglycine）,the most widely used herbicide in the world,inhibits the 5-enolpyruvate shikimate-3-phosphate synthase（EPSPs）in the upstream of the shikimic acid metabolic pathway,blocks the synthesis of aromatic amino acids in plants and leads to plant death.Compared with other herbicides,glyphosate has the advantages of low toxicity,low price,efficient and broad spectrum.Through the transgenic technology to cultivate glyphosate resistant crops,it has made great economic and environmental benefits in the world.However,the widely used resistance gene is glyphosate-sensitive EPSPS gene from the microbial.It is transferred into plants to release the inhibition of aromatic amino acid synthesis by glyphosate,but it can not eliminate the glyphosate residue.The residual problem of glyphosate in crops has attracted more and more attention.Discovering the genes which can degrade glyphosate is an inevitable choice to solve this problem.In order to obtain higher glyphosate degradation activity and lower glycine degradation activity of glycine oxidase,based on the molecular orientation evolution strategy,this project improves glycine oxidase mutant（B4S7）obtained previously.The mutants with potential application prospects have been obtained,which provide genetic resources for new glyphosate-tolerant crops and important information for the in-depth study of the structural and functional relationship of glycine oxidase.The results of the study are as fllows:（1）The random mutant library was constructed and the high-throughput screening methods established in our laboratory.Three mutants,thus B5R6,B5R18and B5R26 with the highest activity were screened from 11000 mutants.The k_m values of mutants are0.052 m M,0.051m M and 0.061 m M,respectively.B5R18 has the best activity among them.The k_m k_cat and k_cat/k_m values on glyphosate are 0.051m M,9.740 min^-1,189.393m M^-1 min^-1respectively and compared with B4S7,the affinity and catalytic efficiency of glyphosate were increased by 0.95 and 4.23 times,respectively.The k_m k_cat and k_cat/k_mvalues on glycine are 59.020 m M,6.176 min^-1,0.105 m M^-1 min^-1 respectively and compared with B4S7,the affinity of glycine sightly decreases by 0.17 times and catalytic efficiency increases by 1.43 times,respectively.B5R18 specificity constant(k_cat/K_m,glyphosateand k_cat/K_m,glycineratio)was 1811,compared to B4S7 increasing by 1.16 times.（2）Based on the rational design of protein,site-directed mutation of amino acids at position 63 of glycine oxidase is performed.Seven mutants of F63A,F63G,F63S,F63D,F63R,F63H and F63K are obtained.The results show that Amino acid changes of Phe⁶³have obvious effect on glycine oxidase activity.The affinity of F63D for glyphosate decreases about 8.3 times and no significant effect changed for glycine.The k_m value of F63A and F63G is 0.038 Mm and 0.072 m M,respectively,and the affinity was significantly increased.The k_mvalue for glycine is 22.880 m M and 88.530 m M,respectively.F63H shows that the affinity（k_m=266.500 m M）and catalytic efficiency(k_cat/k_m=0.010 m M^-1 min^-1)on glycine decreases by 4.29 and 3.53 times respectively on high glyphosate activity(k_m=0.103,k_cat/k_m=38.800).F63H has a specific constant of4084,which is 3.59 times higher than that of B4S7.F63K,F63R,F63S has no significant change for the activity of both substrate molecules.The study of mutant F63H enzymatic properties shows that the optimum temperature was changed from 60°C to 50°C and the optimum reaction p H changed from 8.5 to 8.The thermal stability and p H stability were similar to those of B4S7.Fe²⁺and Cu²⁺have a significant inhibitory effect on F63H activity.Ca²⁺,Mg²⁺and Mn²⁺have a significant activated effect on F63H activity.（3）The synergistic effect of different amino acid combinations is studied by site-directed mutagenesis and the results show that the k_m,k_cat and k_cat/k_m values for glyphosate of Q346R/F63H mutant are 0.035m M,3.270 min^-1 and 93.211m M^-1 min^-1,respectively.For glycine,k_m=80.300 m M,k_cat=2.606 min^-1,k_cat/k_m=0.033 m M^-1 min^-1.Q346R/F63H has a specific constant of 2868,which is 2.4 times higher than that of B4S7.The results of enzymatic properties show that the optimum temperature is changed from60°C to 50°C,the optimum reaction p H is 8.5 and the thermal stability and p H stability were similar to those of B4S7.Fe²⁺and Cu²⁺has a very significant inhibitory effect on Q346R/F63H activity and Mn²⁺has a strong role in the activation.（4）The molecular simulation of glycine oxidase on glyphosate and glycine was studied by Molecular Operating Environment（MOE）.The effects of D103Y and R238K on the catalytic activity were determined from their hydrophobicity and the hydrogen bond effect on the catalytic key residue Glu⁵⁵.The hydrogen bonding of Glu⁵⁵ with the substrate is affected by the interaction between Phe⁶³ and Gln¹¹⁵.The study provides a good theoretical basis for the further study of the structure and function of the enzyme.