Screening Of Thermostable Protease-Producing Strains And Cloning,Expression Of Protease Gene

Hot spring is a stable and close microbial ecosystem.The high temperature of hot spring leads the microbes to adapt a unique physiological mechanism and evolve the specific genes,which not only help them to adopt to high temperature but also mark them as a potential source enzymes with unique features that have industrial significance.Among various industrial significant enzymes,Protease is of great importance due to its widespread applications.Collceted the water samples from hot springs in Tengchong,Yunan for screening enzyme production.to explore the growth conditions and enzymatic characteristics of this strain.The protease producing strain of decomposing skimmed milk was screened by using selective medium containing casein.We used conventional method to culture bacteria and the effect of temperature,pH,carbon source of the medium,the influence of the nitrogen concentration on the strains and used the Folin method to measure enzyme activity.Extract the protease solution on the optimum pH,temperature and thermal stability,pH stability studies.A protease producing strain A-2 was isolated from the solid medium containing skim milk powder and identified as Aneurinibacillus sp.by physiological-biochemical experiments and 16 S rDNA sequence analysis.Yeast,glucose,55°C and pH 7.5 were the optimum nitrogen source,carbon source,temperature,and pH,respectively,for the growth of strain A-2.In addition,the protease produced by strain optimum temperature was 60?,has good activity at pH 7~9.The strain is Bacillus thermophilus and the produced alkaline protease which has the thermal stability and pH well tolerated.Protease gene was amplified by PCR and bioinformatics software for sequence analysis,construction of pET-28a-spr prokaryotic expression recombinant plasmid,transform Trans1 clone competent cells.The recombinant protein was expressed in Ecoli BL21 though IPTG in 17? broken cell and enzyme activity was determined.The gene fragment of protease gene spr was 1206 bp,encoded a mature protein containing 400 amino acids,which was easy to protein solution,strong hydrophilicity,the molecular mass of fusion protein wan 32 kDa,protease activity was 18u/mL.Compared to the E.coli expression system,Bacillus subtilis expression system can effectively secrete recombinant proteins out of cells.Using the vector PHY-Pamyq to clone the protease gene in Trans1 competent cells and transfer it into SCK6 Bacillus subtilis for expression,and use the prepared fermentation medium for fermentation culture for 72 hours,and the enzyme activity was measured with an empty load control.Taking Tengchong hot Spring as the research object,the work will serve as groundwork to exploit the novel products including thermos-stable enzymes and other bioactive compounds with special characteristics produced by thermophiles,as well as to develop the potential biotechnological applications in various industrial fields.