Identification Of A Protease Producing Strain,Enzymatic Property And Heterologous Expression

Protease is a kind of enzyme which is widely used in the manufacturing industry of food,detergent and medicine.A protease-producing strain named CF-02 was isolated by casein nutrient medium,subsequently its enzymatic properties and identification were tested in this paper.Additionally,enzyme operation conditions were optimized,and eukaryotic expression plasmid carrying this protease gene was also constructed.The results are listed below:?1?Strain CF-02 showed perfect ability of protease production and was identified as Bacillus subtilis by physiological-chemical test and 16S rDN A sequencing.The optimal period of protease production appeared at 36 h,the late stationary phase of growth period in fermentation medium.The optimal formula of medium shake culture was 4%soybean meal,1%corn meal,0.41%glucose,0.94%yeast extract,0.15%CaC l₂,0.3%NaCl.Its optimal p H and temperature for enzyme production was 7.0 and 20?respectively.?2?Enzyme properties analysis shows that the optimum reaction pH and temperature was pH 8.0 and 55?,respectively,which was regarded as mesophilic and neutral protease.Enzyme activity decreased sharply when the reaction buffer pH value was higher than 9.0 or lower than 5.0,or the reaction temperature higher than 60?,indicated that the protease was susceptible to high temperature,acidity and alkalinity.2.5mmol/L of Ca²⁺,Mn²⁺and Cu²⁺could enhance 10%of the protease activity,while Fe²⁺and Zn²⁺showed inhibition effect on protease activity,decreased approximate 7%of the protease activity;Mg²⁺,K⁺and Na⁺have little significant effect on its activity.?3?Separation and purification of crude enzyme was conducted by ammonium sulfate and DEAE-Sepharose FF anion exchange chromatography,and the purified protease was tested by SDS-PAGE.MS analysis showed that the protease is amino peptidase and similar with Bacillus subtilis Yto P protein sequence,which can select protein and amino-acid residue at N-terminate resection.SDS-PAGE revealed that the molecular size of the protease produced by CF-02 was about 34kDa.?4?The result is consistent with YtoP protein molecular weight.Based on the design of primers of protease ytop gene,target gene were obtained by PCR,the taeget gene was named as yyqx.Then the yyqx gene and the plasmid vector pPIC9K were digested by restriction endonucleases SnaB?and Not?.The target gene was fused with plasmid vector to constructed plasmid pPIC9K-yyqx,the plasmid was linearized and transformed into Pichia pastoris GS115 with the aim to cultivate and preserve the transformant.