Secretory Expression In Escherichia Coli And Molecular Modification Of 1,4-?-glucan Branching Enzyme

1,4-?-Glucan branching enzyme(1,4-?-glucan branching enzyme,GBE,EC 2.4.1.18)catalyzes the formation of ?-1,6-branch points in starch by hydrolyzing ?-1,4-glucosidic linkages and then synthesizing ?-1,6-glucosidic linkages.So far,there have been many reports on the cloing and expression of different GBE genes in E.coli.However,it is highly desirable to construct the extracellular secretion system of GBE.Moreover,specific activity has been considered as a critical factor determining the feasibility for industrial applications.Besides,the relationship between structure and function is still unclear.The confirmation of the active center of GBE is important for the enhancement of enzymatic activity through site-directed mutagenesis.In previous study,the plasmid pET-20b(+)containg the gene encoding GBE from G.thermoglucosidans STB02 was constructed without pe1 B signal.This study aimed to achieve the secretory expresseion of plasmid pET-20b(+)/gbe in E.coli.The reaction mechanism of GBE was analyzed.Then site-directed mutagenesis was used to determine the function of Ala310 in GBE.On the basis,the mutants of different amino acid residues at position 349,which was located on the active center,were constructed and obtained.The main results are attached as follows:(1)The extracellular secretion of GBE in E.coli was verified in different fermentation conditions,which included four typical culture medium,fermentation temperature,fermentation time,initial pH,IPTG concentration.The optimized conditions for extracellular production of the GBE were as follows: TB culture medium,initial pH 7.5,induced by 0.005 mM IPTG at 30°C for 48 h.To the best of our knowledge,this is the first report on the secretory expression of GBE in E.coli.(2)The mechanism of secreted GBE exported from E.coli cells in the absence of signal sequences was analyzed.The secretion of GBE was related to growth of E.coli.The GBE was observed in extracellular,periplasmic,soluble intracellular,insoluble inclusion bodies.This demonstrated that GBE achieved movement across the periplasm and two membranes.The cell morphology of E.coli was observed using SEM and TEM,which meant that the secretion of GBE to extracelluar medium did not rely on the cells autolysis.The N-terminal sequence of the purified GBE showed the sequnence of NH2-SVVP,rather than MKYLL(pe1B).By comparison,the plasmid pET-20b(+)/gbe-pe1 B had an obviously different secretion process.Thus,the pe1 B signal had been cut off when the plasmid pET-20b(+)/gbe was constructed.When the plasmid changed into pET-24a(+)/gbe,pET-22b(+)/gbe,the secretion of GBE remained unchanged.Mutations at conserved residues(D352N?E452Q?D420N)had lost enzymatic activity,but the level of protein expression was equivalent to that of wild-type GBE.The results showed that enzymatic activity had no effect on the secretion of GBE.Last,Nd1-10 reduced the extracellular production of GBE and decreased the enzymatic activity,which meant the secretion of GBE was related with N-terminal residues.(3)The action mode of GBE was analyzed.Then,the substrate specificity of GBE was analyzed in the optimum reaction conditioin.GBE had strong preference for amylopectin from potato starch.The hydrolyzing activity and branching activity were determined at intervals during incubation.The results showed that both types of activity took effects simutanenously,in which the branching activity played a leading role.The chain transfer pattern of GBE was analyzed by HPAEC-PAD.The minimum chain length required by GBE was 20 residues.GBE preferentially transfered chains of DP 7~17.The cyclic-products were verified by MALDI-TOF MS.(4)The active center of GBE was verified.In the GBE from G.thermoglucosidans STB02,alanine 310(Ala310)was located in conserved region II.An analysis of the amino acid sequence showed that Ala310 was highly conserved in the prokaryotic GBE subfamily.Site-directed mutagenesis was used to determine the function of Ala310 in GBE.Replacement of Ala310 with glycine,aspartate,asparagine,isoleucine,glutamate,or glutamine resulted in mutant enzymes with less than 10% to 25% of wild-type activity when amylopectin or amylose was used as substrate.HPAEC-PAD showed that A310 G mutant had no effect on the transfer pattern,but the branching activity had been repressed to a large extent.Kinetic analysis also showed that mutations of Ala310 had an effect on the Km value that changed the preferred substrate from amylopectin to amylose.These results showed that Ala310 was important for the catalytic activity and substrate specificity of the GBE.(5)Based on an alignment of the amino acid sequences of the branching enzymes from a variety of bacterias,the residue 349(G.thermoglucosidans STB02 numbering)in region III was generally methionine in bacteria with higher identity,while it was threonine or serine in bacteria with lower identity.Four mutants(M349T,M349 S,M349H,M349Y)were constructed by site-directed mutagenesis and characterized.The M349 T and M349 S mutations showed 24.5%,21.1% increases in the activity compared to that of wild-type GBE,respectively.In addition,the M349 T and M349 S mutants displayed 24.2%,17.6% enhancements in the ?-1,6-glycosidic linkage ratio of potato starch samples,respectively.These results suggested that enhancing the contact with conserved residue Asn337 and maintaining the polarity of the environment enhanced enzyme activity.