Screening And Application Of High-Producing Collagenase Strain

Collagen is widely applied in food,medicine,cosmetics and other fields for its unique structure and function.Collagenase can specifically hydrolyze collagen into small molecule peptides,making the application of collagen wider.Marine resources are rich in fish by-product resources,in which fish skin and fish bones contain a lot of collagen.Although collagenase is an important commercial enzyme,marine-derived collagenases are rare.Compared with terrestrial-derived collagenases,marine-derived collagenases usually affect the collagen of fish skin and bone in the ocean.Therefore,this project intends to screen the collagenase-producing strains from the ocean.Taking this strain as the research object,firstly,the fermentation conditions are optimized to improve its ability to produce collagenase,and then the enzymatic properties of the collagenase produced by it is studied,finally,their production of collagenase research applications in marine collagen proteolysis.The main contents and results are as follows:(1)A strain that can produce a transparent circle on the gelatin plate is selected from the sewage sample of the aquatic product processing plant,through the physiological and biochemical tests of the strain and 16S rDNA sequencing,the strain is initially identified as Stenotrophomonas maltophilia and named YQ-1.(2)Through the single factor test and orthogonal optimization test,the optimal culture conditions for the production of collagenase by strain Stenotrophomonas maltophilia YQ-1 are obtained:peptone 20 g/L,glucose 15 g/L,gelatin 5 g/L,NaCl 30 g/L.The optimal culture pH is 8,the optimal liquid volume is 50 ml/250 ml,the optimal inoculation volume is 5%,the optimal culture temperature is 28?,and the optimal rotation speed is 200 rpm.After optimization,the collagenase activity of strain YQ-1 increase from 12.4 U/ml to 15.5 U/ml.(3)By ultrafiltration and anion exchange chromatography,the collagenase is preliminarily separated and purified,and the crude collagenase solution with less impurities is obtained with a molecular weight between 25-45 kDa.Studies on the enzymatic properties of the collagenase crude enzyme solution indicate the optimal reaction pH of the enzyme is 7.5,the optimal reaction temperature is 37?,Divalent metal ions can promote collagenase,and the enzyme maintains about 92%of the enzyme activity after holding at 40? for 120min,and maintains about 61%after holding at 50? for 120min,and no enzyme activity after holding at 60? for 30min,the collagenase is type I collagenase.(4)Through the acid hydrolysis of cod skin to prepare fish skin collagen,using the degree of hydrolysis as an indicator,the single factor enzymatic hydrolysis conditions are optimized,the optimal hydrolysis conditions are determined as follows:the optimal temperature is 37?,the optimal pH is 7.5,the optimal ionic strength is 0.4 mol/L NaCl concentration,and the optimal hydrolysis time is 8 hours.