High Yield Expression Of A Novel Maltohexaose-forming A-amylase AmyM And Characterization Of A Novel Sacharrifying A-amylase AmyC From Corallococcus Coralloides Egb

a-Amylases(E.C.3.2.1.1)are starch degrading enzymes which can hydrolyze the internal a-1,4-glycosidic bonds in oligosaccharides with the retention of a-anomeric configuration in the products.a-Amylases can be produced by plants,animals or microbial,where they play a dominant role in carbohydrate metabolism.a_Amylases are one of the most important industrial enzymes with a wide variety of applications ranging from conversion of starch to sugar syrups,textile industry,and detergent to the use for pharmaceutical industry.In this study,two novel a-amylases AmyM and AmyC were successfully cloned and expressed.The enzymatic assay of the recombinant AmyM expressed by P.pastoris GS115 revealed that this amylase can well maintain the stability with high concentration of KCl,NaCl,or ethanol.The product was assayed by the HPLC,finding that maltohexaose is the main product covering 54%of the hydrolysis.Also the medium and conditions for fermentation were optimized,with the protein expression of 223 mg/L induced by 0.5%methanol for 60h in a 50 L fermenter.Considering these characters,the recombinant AmyM may meet the demand for industrial uses.The recombinant AmyC(rAmyC)was purified and biochemically characterized.rAmyC hydrolyzes various starch and maltooligosaccharides larger than G4 to glucose to maltotetraose without production of other oligosaccharides,indicating that it is a special saccharifying ?-amylase.AmyC possesses weak but significant a-1,6-bond cleaving activity and transglycosylation activity.The time course of the reaction with maltotriose as a substrate evidently showed the formation of oligosaccharide of DP4 by glycosyl transfer.The substrate specificities and end products analysis showed that the hydrolytic pattern of rAmyC is unique.rAmyC exhibited maximum hydrolysis activity at 50 ? in 50 mM sodium phosphate buffer(pH 6.0)with a specific activity of 180 U/mg.These results indicate that AmyC is a novel multifunctional sacharrifying a-amylase possessing distinct characteristics.