The Study On Clone Of AF-03 Amylomaltase Gene And Prokaryotic Expression

Amylomaltases are intracellular 4-α-glucanotransferases, they catalyse the transfer of a segment of anα-1,4-D-glucanto a new 4-position in an acceptor, which may be glucoseor anotherα-1,4-D-glucan. Thisreaction is a variationof the a-retaining mechanism. Several amylomaltases ofvarious sources have beenstudied in detail (Table 1). In plants the enzyme is known as a disproportionating enzyme orD-enzyme.Well-known starch-acting enzymes are a-amylases and pullulanases, which degrade it tomaltooligosaccharides and glucose.Recently, a thermostable cyclodextrin glycosyl transferase (EC 2.4.1.19)that produces circular cyclodextrins was introduced on the market.glucan-branching enzymes (E.C.2.4.1.18)and amylomaltases (E.C. 2.4.1.25), are explored for theirpotential applications, as can be judgedfrom the number of patent applications in recent years.In the rest of this communication,Since solubilization of starch is desired for such enzymic treatment,the applied enzymes need to be stableand active at temperatures above 65-70℃. A natural source forextreme thermostable and thermoactive enzymes are(hyper)thermophiles that have their optimaltemperature for growth above 60℃Application of amylomaltases is in the production of a thermoreversible starch gel that can be used asa substitute for gelatin. Thermostable amylomaltases are required,since they have to perform atgelatinization temperatures o When gelatinized potato starchwas treated with the amylomaltase fromT.thermophilus, a product free of amylase and containing amylopectin with shortened and elongated sidechains was obtained This product could be dissolved in water and formed after heating and cooling a firmgel. The gel could be dissolved again by a new heating step he thermoreversible behaviour of theamylomaltase-modified potato starch product is very similar to gelatin。Purpose of this study is clone and express Amylomaltase gene by gene engineering, which will befoundation of gene-engineering strain construction further.So main goal of this study is build a highefficient vcctor that can express the Amylomaltase in Eco li. strain..We designed a pair of primer according to Amylomaltase sequence that publish on Genbank fromThermus aquaticus AF-03 that keep in our laboratory as template, then amplified and obtained targetgene(Amylomaltase) through TD-PCR. Afterward connected Amylomaltase with T-vector, transformedEschericha coli DH5αwith the recombinant plasmid. The target gene would amplify in the receptstrain.The insertion of Amylomaltase gene into T-Vector was confirmed by restriction analysis and PCR,We knew that the cloning gene is about 1500bp. Therefore, we could express the gene expression libraryimmunization. Meantime we cut the recombinant plasmid and pET-28a with Nde I and EcoRI, obtained expressionvector. Make the vectors transform BL21, and induced with IPTG, then we gained expression protein.that57KDa。In a word, this study make a steady basis for constructing gene-enginerring Eschericha coli which canproduce plenty of enzyme preparation.