Screening And Identification Of Protease-producing Strains,Optimization Of Fermentation Process,Enzymatic Properties And Application Of Tilapia Skin

Protease is one of the major industrial enzyme,which is widely appliedto food,medicien and cosmedic fields.Protease accounts for 60%of the global enzyme market.Aquatic products are enormous in China,but the utilization rate of aquatic processingis quite low.About 50-70%of by-product is generated during processing,which has caused a serious waste of resources.Protease convertsaquatic by-product into high-value product(bioactive peptides),which has become an important issue.A high-yield protease strain was isolated from aquaculture wastewater.The strain was identified by morphological,physiological and biochemical,and sequence analysis of 16S rDNA.The fermentation culture and process of strain was optimized by single factor.Enzymatic properties of purified proteases was explored.Ferrous chelate peptide was prepared by means of ferrous ions was chelated withhydrolyzed peptide from Tilapia skin,which increased additional value of fish skin.The results are as follows.Strain 4-2 with high protease activity was screened from aquaculture wastewater and identified as Bacillus sp.4-2.The fermentation conditions of strain 4-2 were optimized by single factor experiment.The optimal source and concentration of carbon and nitrogen were 2%of corn flour and 2.5%of gelatin.The optimal pH and liquid volume was 3.0 and 30 mL in 250 mL conical flask.The best inoculum age,inoculum size and culture temperature were 12 h,2%and 30?.The maximum protease activity reached 850 U/mL under the optimal conditions,which was 4 times higher than the control.The supernatant after 48 h-fermentation was subjected to ammonium sulfate precipitation at a final concentration of 60%,resolved and dialyzed with 20 mM phosphate buffer(pH 6.8).The crude enzyme solution was separated and purified by a cation exchange column.The purified protease is a molecular weight of about 22 kD.The enzymatic properties of the protease showed that the optimum reaction temperature was 50? with thermal stability at 40? and 50?,the optimum pH was 9.0.The results of metal ions effect on protease indicated that Mg2+,Mn2+,K+(5 mM)promoted protease activity,Cu2+,Fe2+and Ag+inhibited activity.The protease is sensitive to SDS.Tween 80 and Triton X-100 have a promoting effect on the enzyme.The enzyme endured 1.5%H2O2 and was inhibited by 30%toluene,but 30%ethanol,acetone and n-butanol inhibit the enzyme activity.Strain 4-2 fermented fish skin to prepare the active peptide,which was chelated with the ferrous ion.The optimized chelation condition was as follows,50 mg/mL ferrous sulfate,0.05 g/mL ascorbic acid,pH 6.5,and the chelating time and temperature being 40 min and 40?.The ferrous-chelated peptide was characterized by ultraviolet spectrum,infrared spectrum and fluorescence spectrum.