Cloning And Expression Of α-Amylase From The Hypertheromophilic Archaeon Sulfolobus Solfataricus P2

Thermostableα-amylase is one of the most important enzyme in industry, which iswidely used in food and pharmacy for its heat-resistant and efficient starch hydrolyzing. Atpresent mostα-amylase used in industry in large scale is from Bacillus licheniformis. Butthe optimal pH ofα-amylase from Bacillus licheniformis is 6.0, which was used inindustry in large scale. But the optimal pH of glucosidase is 4.5, which was used in thefollowing saccharification process. So before the following technics, product of starchhydrolyzation must be adjusted pH, which enhanced the producing cost and producedwaste water not easily to be treated. Accordingly finding hyperthermophilic and acidophilicα-amylase is very important.Sulfolobus solfataricus P2 is a hyperthermophilic and acidophilic archaeon living optimallyat 80℃and pH3.0-4.5. The its genomic DNAwas sequenced in 2001, which contains aα-amylase gene.α-amylase gene of Sulfolobus solfataricus P2(sspa) was got by PCR andcloned into expression vector pET29a. The recombinant plasmid pET29a- sspa wastransformed into E.coli BL21(DE3). Induced by IPTG, theα-amylase gene was expressed.But the expressedα-amylase had low activity. By analysing the domain of theα-amylasewith SMART software. It found that the functional domain of sspa is from 7 to 447 aminoacid. After exciseing part sequences, the rest sequences of sspa was cloned and expressedonce again in E.coli BL21(DE3). Theα-amylase activity was largely enhanced.The shuttle vector pP43NMK was chosen to constructed the recombinant secretingexpression plasmid pP43NMK-sspa, and expressed in B. subtilis1A771. But expressionlevel was very low, which could be due to rare codons present in sspa. After site-directedmutagenesis expression level was largely enhanced.