Enzymatic Characterization Of A Exo-?-1,4-glucosidase And Substrate Specificity Of Endogenous Protease In Model Expression Hosts

This study consists of two parts:1.Enzymatic characterization of a exo-?-1,4-glucosidase gene;2.Substrate specificity of endogenous protease in model expression systems.1.Starch,which possess both?1,4?-?-and?1,6?-?-linkages,is divided into amylose and amylopectin.Besides,amylopectin contain more?1,6?-?-linkages.In order to improve the efficiency of hydrolyzation of starch,it is of great industrial value to separate a strain,Bacillus pseudofirmus 703,which highly product alkaliphilic amylase with?1,4?-?-and?1,6?-?-linkages hydrolytic activities simultaneously.Bacillus pseudofirmus OF4,the homologous strain of Bacillus pseudofirmus 703,has been whole-genome sequenced.In the present study,we designed the primers according to the genomic sequence of Bacillus pseudofirmus OF4 and cloned an exo-?-1,4-glucosidase gene,named amy112.Sequence analysis of Amy112 revealed that it belongs to glycoside hydrolase family 13,which presented 65%and 64%identities of the alpha-glucosidase Gsj from Geobacillus sp.HTA-462 and exo-alpha-1,4-glucosidase from G.stearothermophilus,respectively.The gene amy112 was expressed in E.coli followed by purification through Ni affinity resin,desalting and anion exchange chromatography.SDS-PAGE detection showed a 64 KDa band,which consistent with theoretical value.Biochemical analysis demonstrated that Amy112 exhibited a higher activity toward amylopectin compared to soluble starch with the optimal temperature and pH of 40?and pH 7.0,respectively.Amy112 retained more than 95%residual activity after 30 min incubation at 40oC and retained more than 60%residual activity between pH6.2-8.6.Li⁺,Ca²⁺and Mg²⁺didn't affect the activity of Amy112,but K⁺significantly improved the enzyme activity of Amy112.Comparably,some divalent heavy metal ions(Zn²⁺,Cd²⁺,Co²⁺,Hg²⁺and Cu²⁺)inhibited the enzyme activity.Although no transglycosylation activity was detected through LC-MS analysis,Amy112 was identified to hydrolyze?-1,4-glucosidic linkages in maltose and maltotriose,as well as?-1,6-glucosidic linkages in isomaltose,indicating its exo-?-1,4-glucosidase and oligo-l,6-glucosidase activities.Amy112could hydrolyze pNPG at K_m of 2.99 mM and V_max of 66.62 mmol/min.The template with highest identity derived from?-glucosidase GSJ from Geobacillus sp.HTA-462.The 3D structure of Amy112 was simulated by Swiss-Model program with the 3D structure of GSJ?PDB number:2ZE0?.Biochemical assay suggested that Glu257 together with Asp200 and Asp327 forming a catalytic triad of DED in Amy112.Mutation of Glu257 to Ala in Amy112nearly abolished its enzymatic activity,indicating that Glu257 is one of the active sites of Amy112.Amy112,which possessed exo-?-1,4-glucosidase and oligo-l,6-glucosidase activities,would have great potential applications in starch-debranching industries.In future,we will improve its enzyme activity.2.It always happens that heterologous expression of recommbinant protein encounters low expression or no expression,which is partly due to the degradation by the endogenous proteases in the host cells.Particularly in eukarotic cells,post-translational modifications including proteolysis,phosphorylation,and glycosylation,usually cause the incorrect folding and inactivation of target protein.Therefore,the analysis of substrate specificities of endogenous protease in common expression systems is of great significance.In our studies here,the substrate specificities of endogenous proteases in three model expression systems,including E.coli,B.subtillis,and P.pastrois,were analyzed from the view of single cell level point.We found that specific substrate sequences could be cleavage by endogenous proteases,such as EspP in E.coli,aprE/nprE in B.subtitis,and Prb1 in P.pastrois GS115,respectively,through bioinformatics analysis and literature search.In order to screen more potential protease,we firstly expressed the substrate sequences of these three proteases displayed Saccharomyces cerevisiae surface.The fermentation supernatant of three model strain were mixed with Saccharomyces cerevisiae cells displayed different substrate sequence of protease for cleavage reaction.Then,screen model was constructed using flow cytometry to detect whether these protease cut relevant substrate sequences.The results showed that the fermentation supernatant of E.coli Rosetta?DE3?could effectively cleave the corresponding substrate sequence of EspP,while E.coli BL21?DE3?could not effectively cleave the corresponding substrate sequence of EspP.Meanwhile,the fermentation supernatant of B.subtilis could cleave the corresponding substrate sequence of aprE/nprE.The fermentation supernatant of of GS115 strain could effectively cleave the corresponding substrate sequence of Prb1.Based on these results,the substrate specificities of endogenous proteases in three model expression systems will be mapped by way of analyzing the substrate mutant library of protease from the perspective of the whole level in the future studies.Our reseach on the substrate specificities of endogenous proteases will provide prediction for the efficient expression of recombinant protein and new data supporting the identification of unknown functional proteases.