Expression,Enzymatic Properties And Fermentation Optimization Of Collagenase In Bacillus Subtilis

Collagenase is a collagen hydrolase that gently hydrolyzes the three-dimensional structure of collagen without damaging other protein structures.The peptides produced by the enzymatic degradation of collagen have been shown to be biologically active and have certain research value.In this paper,the gene encoding collagenase col in the Bacillus velezensis G341 strain was synthesized and inserted into the p P43NMK vector to obtain the recombinant plasmid p P43NMK-col.The recombinant plasmid p P43NMK-col was introduced into Bacillus subtilis WB600 to obtain the recombinant Bacillus subtillis WB600/P43NMK-col.In this paper,the successfully constructed collagenase-producing recombinant strain was fermented and cultured.The fermentation broth of the recombinant strain was used to study the purification and enzymatic properties of collagenase,and at the same time,it was optimized from several aspects such as medium composition and culture conditions,and the purpose of improving the yield and industrial application of collagenase was achieved by constructing engineering bacteria producing single collagenase.The main conclusions are as follows:（1）Heterologous expression of collagenase col.The highly expressed collagenase target gene was screened from the NCBI database,the collagenase col target gene was obtained by synthesis,and the recombinant p P43NMK-col was constructed in Escherichia coli using shuttle plasmid p P43NMK,and successfully transferred to Bacillus subtillis WB600 for heterologous expression.After ammonium sulfate precipitation,ultrafiltration concentration and Ni-NTA affinity purification,the fermentation supernatant was subjected to SDS-PAGE to obtain a single collagenase band.The purified collagenase activity was 667.9 U/m L.（2）Study on the enzymatic properties of collagenase col.Preliminary studies have shown that recombinase exhibits optimal activity at p H 9.0 and 50°C.The catalytic efficiency of recombinant collagenase is inhibited by Fe³⁺and Cu²⁺,but promoted by Co²⁺,Ca²⁺,Zn²⁺and Mg²⁺.（3）Media composition optimization.Determine the optimal medium composition by single factor method,including carbon source,nitrogen source and metal ion optimization.The optimal medium was 15 g/L fructose,36 g/L yeast powder and peptone mixture（2:1）,and 10 g/L Ca²⁺.（4）Optimization of enzyme production conditions in fermentation.The effects of initial p H,speed,temperature and inoculation amount on enzyme production under fermentation conditions were explored.The optimal conditions for its growth were that the maximum extracellular activity of recombinant collagenase reached 2746.7 U/m L at p H 7.0,temperature 35°C,speed 260 rpm and inoculation amount of 11%,which was 2.4times that before optimization.