Construction Of Genetic Engineered Strain Expressing Pyrococcus Furiosus Thermostable Amylase And The Preparation Of Linear Malto Oligosaccharides

Maltooligosaccharides and their derivatives with specific length and defined structures are wildly uesd in foods and pharmaceuticals.Maltoheptaose composed of seven glucose units with modified reducing- and non-reducing-terminal glucoses could be used as the substrate for determining alpha-amylase activity,whose indexes were abnormal will indicate the occurrence of certain diseases.So the demand for maltooligosaccharides such as maltohexaose, maltoheptaose,and maltooctaose has therefore increased rapidly.However,the industrial production of these maltooligosaccharides is extremely tedious and expensive.Undoubtedly the study of how to obtain maltooligosaccharides with specific length efficiently and conveniently will have great industrial applications and huge commercial significance.The PFTA gene was amplified by PCR from P. furiosus genomic DNA and inserted into His-tag fusion expression vector pT7473.The gene pfta was cloned and expressed in Escherichia coli.The recombinant PFTA was purified with nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography and the molecular mass of the recombinant PFTA estimated by SDS-PAGE was the same as the expected.The paper has found out that PFTA could hydrolyze the cyclodextrin in vitro reaction conditions of to produce a high purity of straight chain maltooligosaccharide with specific length such as maltohexaose, maltoheptaose,and maltooctaose. Because the thermostable amylase from Pyrococcus furiosus(PFTA) can perform best at the optimum conditions of 90℃and pH5.0,the PFTA will have high application prospects and commercial development values.