Research On The Recombinant Expression, Enzymatic Properties Of C.glutamicum γ-glutamyl Transpeptidase And Its Application In Glutamic Acid Fermentation

γ-glutamyl transpeptidase(GGT),which is widely distributed in living organisms,catalyzes specificity the hydrolysis of γ-glutamyl compounds to yield glutamic acid(hydrolysis).F urthermore,GGT can also catalyze the transfer of the released glutamyl group to form the new γ-glutamyl compounds(transpeptidation).Based on its dual catalytic properties,there is an excellent prospect for γ-glutamyl transpeptidase in industrial application.At present,the study of GGT is mainly focused on the synthesis of various γ-glutamyl compounds.However,the preparation of glutamic acid using the enzyme did little research.ObjectiveIn this study,the GGT of C.glutamicum ATCC 13032(CgGT),the model organism of Corynebacterium glutamicum who is major strains for fermentative production of glutamate,was used as the object of research.This study mainly discuss on the recombinant expression,enzymatic properties,structure foundation and the application in glutamic acid fermentation of CgGT.And it aimed at laid a solid theoretical foundation for the further industrialization of glutamic acid fermentation by this enzyme,as well as provided the necessary reference for the deeper step of molecular studies.Methods1.Cloning,expression and screening of CgGT:the target gene was constructed and expressed by using genetic engineering techniques.The expression of CgGT was subsequently estimated by GGT assay and SDS-PAGE analysis.2.Fermentation optimization of CgGT from recombinant C.glutamicum:The optimal fermentation conditions of recombinant bacteria were determined by measuring the enzyme activity as well as cell density at every sampling time,and through the single factor experiments as well as the orthogonal experiment.3.Study on the extraction and characterization of CgGT:the recombinant cells was lysed by sonication and high-pressure homogenization,And then,CgGT was purified by Ni-affinity chromatography.Biochemical properties of purified CgGT were investigated by performing the reaction in the standard assay mixture.4.The intrinsic mechanism of enzymatic properties of CgGT:the mechanism was analyzed by means of bioinformatics and site directed mutagenesis.5.Application of CgGT in glutamic acid fermentation:the yield of glutamic acid in different time periods was determined by SBA-40E.Results1.Cloning,expression and screening of CgGT:the gene(ggt),encoding γ-glutamyl transpeptidase of C.glutamicum,was successfully constructed under the control of the diverse promoters of Ptac、PmaIE1/and PH36 using chromosomal DNA from C.glutamicum ATCC13032 as the template by PCR.And then the recombinant plasmids pEKEx2-TC,pEKEx2-MC and pEKEx2-HC were achieved and trans formed into the original stain.The recombinant C.glutamicum PTC was successfully screened by SDS-PAGE and enzyme assay for GGT activity.The GGT activity of recombinant PTC exhibited approximately 31 times with respect to that of PP harboring the blank vector pEKEx2.2.Fermentation optimization of CgGT from recombinant C.glutamicum:to further improve the productio n of CgGT in the recombinant PTC,the culture process were investigated and optimized.The optimum culture conditions was as follows:C.glutamicum with fusion plasmid pEKEx2-TC was cultivated at 30℃,220 r/min,until the optical density at 600 nm of the culture was about 1.2 and then induced with a final concentration of 0.6 mM IPTG for 27 h.Besides,the optimal culture medium was as follows:based on the existing medium formula of our lab,0.05%Tween-80,5.0%hexadecane and 0.01%sorbitol were added to the fermentation medium.Under these conditions,the GGT activity of fermented broth reached 869 U/L,which showed a nearly 22%increase than that not optimized.3.Study on the extraction and characterization of CgGT:The recombinant CgGT was produced in the periplasm of C.glutamicum PTC,and was purified by N i-affinity chromatography utilizing the designed C-terminal hexa-histidine tag.The purified enzyme unfolded specific activities of 5.84 U/mg protein and 0.35 U/mg protein for hydrolase and transferase,respectively.The molecular profile of the recombinant protein was analyzed by SDS-PAGE under reducing conditions.And the His6-tagged CgGT was purified as a mixture,comprising three major bands of 70,48 and 22 kDa,respectively.CgGT exhibited the optimum hydrolase activities at pH 7.0,50℃.The enzyme showed broad pH stability and was relatively thermostable,retaining almost100%of its activity at 45℃ for 24 h.The cations like Mg2+,Ca2+,Mn2+,K+,and Na+strongly enhanced the enzyme activity at the concentration exceed 5 mM.In particular,when the additive concentrations of Mg2+ and Mn2+ were 100 mM and 50 mM,CgGT displayed nearly 230 and 202%initial activity,respectively.4.The intrinsic mechanism of enzymatic properties of CgGT:the biochemical properties of the enzyme showed could be easily unveiled by analyzing its structural bases in the binding pocket using the phylogenetic tree,sequence alignment,CgGT homology modelling and site-directed mutagenesis of the putative active site.5.Application of CgGT in glutamic acid fermentation:the feasibility that CgGT,who possesses high hydrolase activity,improve glutamic acid production of C.glutamicum was primarily explored.The result suggested that the glutamic acid content of recombinant PTC was significantly higher than that of the negative control PP in the fermentation process.And at 40 h,the glutamic acid yield of C.glutamicum PTC increased more than 20%compared to the control group.In addition,when some γ-glutamyl compounds,such as Gln and GSH,were added to the fermentation system,the glutamic acid yield of C.glutamicum PTC can be further improved and exhibited 5.29-fold increase with respect to that of PP.ConclusionsIn this study,we screened successfully the recombinant strain PTC where the GGT of C.glutamicum ATCC 13032 was highly expressed.The protein of CgGT,possessing high hydrolase activity,could effectively enhance the fermentation yield of glutamic acid in the original stain.This thermostability and broad pH stability of the enzyme is desirable for its industrial application.Therefore,the study will be of great synthetic and industrial interest for the fermentation of glutamic acid.