Cloning And Expression Of Three Function Genes From The Extremophilic Microorganisms

The extremophilic microorganism is an important source for exploitation enzymes with special traits.To find new enzyme resources,a moderate thermostable lipase andα-amylase genes were cloned from Bacillus subtilis FS321,which was isolated from the dunghill soil in the northern of China.And aα-1,4-glucosidase gene was cloned from an alkaliphilic Bacillus firmus OF4.The three genes were finally expressed in E.coli, respectively.These results provided new gene resources for molecular evolution of these enzymes and made a foundation for further studying the relationships of structure and function of the three enzymes.The main results were as following:1.The optimum culture for lipase producing medium for strain FS321 was of M4, fermentation cycle of 48～60 hrs,culture temperature of 37℃,and oxygen-consumption of 25ml medium for each 250ml flask.The lipase showed the highest activity at the temperature of 50℃and pH9.0.To further identify the strain,the cellular fatty acids composition of FS321 was analyzed,and the 16SrDNA was cloned and sequenced.Both the cellular fatty acids compositions and the phylogenetic tree based on the 16SrDNA sequence of FS321 showed that the bacterium FS321 was a Bacillus subtilis.2.Cloning the complete open reading frame(ORF)of lipase gene from B.subtilis FS321(BSL)by PCR,the ORF of BSL was in a size of 639 bp,The recombinant expression vector of pET-28a-BSL was further constructed,and the BSL was expressed in the host of E.coli BL21(DE3).A fusion protein in a size of about 27.5 kDa observed in SDS-PAGE and the clear zones developed in the tributyrin plate showed that the BSL was functional expressed in E.coli BL21.3.Theα-amylase gene from B.subtilis FS321(BSA)was cloned using the same method as above,the complete ORF of BSA was in a size of 1980 bp.The recombinant expression vector ofpET-28a-BSA was constructed,and the BSA was expressed in E.coli BL21.A fusion protein with the size of about 76.0 kDa displayed in SDS-PAGE and the clear zones developed in the soluble starch plate demonstrated that the BSA was functional expressed in E.coli.The optimum reaction temperature of the recombinantα-amylase was of 50℃,and optimum reaction pH of 7.5. 4.The amino acid sequence corresponding to a gene in the partial sequenced genome of alkaliphilic Bacillus firmus OF4 was showed 60～76%homology to the knownα-glucosidases from other Bacillus.The gene was therefore a candidate of aα-glucosidase gene,and named as ABPG.The ABPG gene was obtained by PCR,and ligated to a expression vector of pET-28a(+),overexpressed in E.coli BL21(DE3).The His-ABPG fusion protein was obtained using the affinity chromatography and in a size of about 68.5 kDa.The maximal specific activity of the enzyme was of 164.2U/mg when the maltose as the substate.And the optimum reaction temperature of the enzyme was of 30℃,the optimal reaction pH of 7.5.The recombinant enzyme was stable when kept at pH8.0～pH10.0,4℃for 12h,retaining 56.4～73.0%of initial activity.