Screening Of Collagenase-producing Strain,application And Study Of Collagenase Characteristics

Collagenase can degrade the nature collagen and gelatin,which is extensively applied in pharmaceutical,fodder,especially the leather.The bacterial strain was isolated and screened from soil of long-time deposit of chromium-containing leather shavings.The strain named as TCCC11319 was classified as B.amyloliquefaciens by Biology microbiological assay,16S rDNA sequence analysis and morphology.Based on the single factor and orthogonal experiment,conditions of fermentation and medium composition obtained for producing collagenase.Results revealed that the optimum carbon source,nitrogen source was 1%of fructose and 1.5%of soy petone,respectively.The optimum inoculation concentration(OD600),pH and temperature were 0.9,8.0 and 37?,respectively.It could produce more collagenase when it was inoculated 5%of seeds,added 50 mL medium to 250 mL triangular flask and cultured at 200 r/min.The optimal collagenase producing time was 32 h.Under these conditions,the activity of collagenase was 919.72 U/mL,increasing about 81.2%than the initiation.Enzymes produced by B.amyloliquefaciens TCCC 11319 could degrade solid leather wastes,the optimum the hydrolysis process was obtained.The optimum degradation process of rawhide shavings was that the enzymatic hydrolyzing temperature was 52?,pH value was 8 and 1 g leather shavings required 1.3 g enzyme powers(Enzyme activity was 11000 U/g)to degrade.The optimum degradation process of limed hides was that the enzymatic hydrolyzing temperature was 60 ?,pH value was 11 and 1 g leather shavings required 3 g enzyme powers and 0.4 g lipase(Enzyme activity was 4000 U/g)to require.The optimum degradation process of cowhide shavings and chromium-containing leather shavings was that the enzymatic hydrolyzing temperature was 45?,pH value was 8 and 1 g leather shavings required 1 g enzyme power to degrade.B.amyloliquefaciens TCCC 11319 could produce many types of protease,in which the content of collagenase was rather low,so it was hard to purify.In order to further study on enzyme properties of collagenase,B.subtillis WB600 was used to express the collagenase.The collagenase gene(col)was amplified from B.amyloliquefaciens TCCC11319 by PCR and subsequently cloned into pWB980.The recombinant plasmid was transformed into B.subtillis WB600,yielding the recombinant strain B.subtillis WB600/pWB980-col.The gene col was successfully expressed in Bacillus subtilis WB600.SDS-PAGE analysis showed that the molecular weights of the collagenase was about 35 KDa,and its enzyme activity was 957 U/mL.The optimal reaction pH and temperature of collagenase was at pH 11 and 40?,respectively.The collagenase was stable at a range of 30-40? and pH 7-8,whice demonstrated the collagenase was a neutral enzyme.Ca2+,Mg2+ can enhance the activity of the collagenase,but the activity of the enzyme was strongly inhibited by Mn2+,Cu2+,Fe2+,especially for Cu2+.