Identification And Application Of The Amylosucrase From Cellulomonas Carboniz T26

Amyloscurase is a glucosyltransferase which belongs to glycoside hydrolase family(glycoside hydrolase,GH)13.It could catalyze the formation of ?-(1,4)-glucan when using sucrose as a sole substrate.The catalytic reactions of amylosucrase include hydrolysis reaction,polymerization reaction,and transglucosylation reaction.Amylosucrase is a versatile enzyme,it could transfer the glucosyl residues to other effective acceptors,such as,saligenin,catechin,hydroquinone,etc.Because of the transglucosylation reaction,the products appear a better property.In this study,we use the genetic engineering means to get the gene segment of amylosucrase from a gram-positive bacterium,Cellulomonas carboniz T26,then sub-clone it into pET-22b(+)vector and get the pET-CC-AS plasmid.The recombinant plasmid is transformed into E.coli BL21(DE3)for expression,and after all the processes including enlage cultivation,inducible expression and purification,we get the recombinant enzyme,CC-AS.SDS-PAGE is used to check the enzyme and a single band is found around 70 kDa,which is same as the theoretical value.We try to use sucrose as sole substrate and find CC-AS could catalyze the formation of ?-(1,4)-glucan under the proper condition.We also find that CC-AS possesses a good transglucosylation property,which implies a bright application prospect.The optimum pH and temperature of transglucosylation reaction are pH 7.0 and 40°C,which is different from hydrolysis reaction.The enzyme displays a poor themostability at 45°C and 50°C,but a good themostability at 40°C.In this study,all the metal irons inhibit the activity of the enzyme.We try to use iodine binding,fourier-transform infrared spectroscopy(FT-IR)analysis and nuclear magnetic resonance spectroscopy(NMR)analysis to identify the structure of the reaction product and confirm that the product is the pure ?-(1,4)-glucan.HPAEC is used to figure out the ratio of hydylysis reaction,polymerization reaction and isomerization reaction.We find the advantage of CC-AS in polymerization reaction after calculation.Then we also find that a lower temperature is helpful for the elongation of the ?-glucan polysaccharide.We use sucrose as donor,hydroquinone as acceptor,CC-AS as catalyst to produce ?-arbutin.The molar production yield of the reaction is 40%~44.7% when performing at pH 7.0 and 40°C.We find that the influence of L-ascorbic acid is limited when the concentration of hydroquinone is low.We also find that the best Donor/Acceptor is 4:1.Some evidences show that higher hydroquinone may lead to a lower molar production of ?-arbutin.In fact,CC-AS possesses a wonderful transglucosylation ability which could also performs an outstanding transglucosylation activity at a higher concentration.We indicate that when the reaction system contains 70 ?g/m L(1.26 U/m L)CC-AS,a lower concentration of substrate and is performed at pH 7.0,40°C for 2 h,we could get a desired yield.