Eepression And Function Exploration Of Novel Isoamylase IsoM From Corallococcus Coralloides EGB

Starch,consisting of glucose units in the way of binding by glycosidic bond,is one of the most abundant polymers in the earth.According to the difference between the glycosidic bond,starch is divided into two essential components,1,the amylase only consisting of a-1,4 glycosidc bond.2,the amylopectin,linear dextran witha-1,4 glycosidc bond connecting on the branched chain with a-1,6 glycoside bond.These make starch form branched structure.Due to its complicated structure,single kind of enzyme can hardly hydrolysis starch,so in the process of treating the starch,it usually need to be a variety of enzyme to hydrolysis starch,including debranching enzyme,etc.Isoamylase(EC 3.2.1.68)is one of the four main types of debranching enzymes,which has different degrees of hydrolysis toa-1,6 glycoside bond in glycogen amylopectin and the limit dextrin.Isoamylase has important value in starch process industry improving the conversion efficiency of the starch,which can use cooperatively with ?-amylases and glucoamylase,etc to produce glucose,maltose,malt oligosaccharide,cyclodextrin,resistant starch and so on.In this study,we isolated and purified a class of amylase named IsoM which can specially hydrolyze the ?-1,6-glycosidic bond from the extracellular supernatant of Corallococcus sp.EGB by the way of substrates absorption.The gene sequence was obtained by analysis of peptide fingerprinting and genome of Corallococcus coralloides 2259,which was identified with isoamylase.The sequence is not homologous to the ones which were functionally testified.So the gene has been heterologous expressed and studied with the properties and functions.The eukaryotic expression system of Pichia pastoris Pichia pastoris was used to realize the heterologous expression of the GS115 gene,and then made the determination of enzyme characterization and the exploration of its function.The enzyme exhibited optimum temperature at 45 ? and the a pH at 6.0.Analysis of the substrate of IsoM demonstrated that potato starch was the optimum substrate with enzyme activity 70600U/mg as measured with I2/KI method.It could not hydrolyze amylase and hardly hydrolyze pullulan showing that it belongs strictly to isoamylase family.The research was processed by selection of five factors:methanol concentration,volume,fermentation pH,induction time,inoculum amount,and then selection the three factors which affected the result more effective to make orthogonal test.the optimum fermentation condition was that a pH value at 6.0,the amount of inoculated quantity at 8%and the amount of addition methanol at 0.8%.Using the marker recovery system constructed by our laboratory previously,the molecular chaperones HAC1,YDJ1,EROl,PDI,BIP five molecular chaperones were co-expressed.The yield of the enzyme was increased by 2.1 times by co-expressing molecular chaperone HAC1.This research also explored the hydrolysis mechanism.Through observation of the hydrolyzed solid residues by SEM,found holes,cracks and fissures within the starch granules with hydrolysis process.By analysis of HPAEC,the exploration of the preference of the length of the branched chain was carried finding that ratio of the content of low DP value with high DP value decreased with the lapse of time.The above results showed that the enzyme actived within the starch granules in the way breaking down the granules from inside to outside,and eventually could not hydrolyze starch granules until they became resistant starch with regular crystal structure.At the same time,the enzyme preferred the short branched chain rather than long branched chain.Based on the good enzymatic properties of this IsoM,this paper makes a preliminary study on its application.RS and SDS have important application value in food and medicine field,this study found that IsoM is a potential candidate for the production of RS(70.9%)and of SDS(9.2%)from raw starch.Compared with commercial pullulanase(Promozyme(?)D2),the yield of RS was equal and SDS was increased by 16.5%,which means the potential value in food and pharmaceutical additives.IsoM is often combined with other amylases to hydrolysis starch in the process of actual application.In this study,IsoM improved the maltohexaose yield from starch in combination with maltohexaose producing a-amylase AmyM(KM114206)compared with commercial pullulanase(Promozyme(?)D2).The maltohexaose yield was improved by 60%compared with the transformation with AmyM alone.