The Study Of Thermostableα-Amylase From Pyrococcus Furiosus

α-Amylases are among the most important commercial enzymes, having wide applications in starch-processing, brewing, alcohol production, textile, and other industries. It has long been the research hotspot to develop new thermostableα-amylases. The extracellularα-amylase from Pyrococcus furiosus (PFA) displays a temperature optimum of 100°C and retains above 80% of maximal activity at pH 4.5 without calcium salt addition. These predominant properties of PFA make this enzyme a potential candidate for industrial application.This research is mainly focused on cloning,expression and purification of the PFA.The PFA gene was amplified by PCR from P. furiosus genomic DNA and inserted into His-tag fusion expression vector pT7473. The fusion protein of His-PFA was mainly expressed in the form of inclusion bodies. The inclusion body protein was solubilized with 8M urea and purified by Ni-NTA chromatography.Since His-tag destablized the enzyme at high temperature, the expression vector pT7473-PFA without His-tag was constructed. The gene was highly expressed in Escherichia coli BL21-Codon Plus (DE3)-RIL. The recombinant α-amylase was also mainly expressed in the form of insoluble inclusion bodies. Solubilization and renaturation of the recombinant PFA was investigated and an improved purification method was established. The solubilization of the inclusion bodies was achieved by 90°C treatment for 3 min in Britton-Robinson buffer at pH 10.5. The solubilized PFA was then diluted and subsequently purified by Phenyl-Sepharose chromatography. By this simple, economical and efficient method, the yield of active enzyme was greatly increased compared with the works previous reported,and the purifed protein showed similar thermal stability and specific activity to the results reported before. This study might be beneficial for the future works on the large-scale productions of thermostableα-amylases with industrial potentials.