The Screening Of Trypsin Inhibitor From Marine Microbes With Researching On Its Properties

Trypsin inhibitor(TI)is a member of Serpin superfamily that either prevents the trypsin enzyme from breaking down the protein or reduces the enzyme’s activity.TIs are usually proteins or polypeptides which have many bioactivities mainly from a wide variety of plants and animals.Nevertheless,these TIs may well cause immune reactions in patients due to its protein components.Microbial TIs are small molecule substances which are safer and more potential on clinic.In this article,trypsin inhibitor producing strains were selected from1500 strains of marine bacteria and actinomycetes through casein punch method.These strains were then identified by 16 S rRNA in order to analyze the genus distraction.Then strains produced trypsin inhibitor stably were selected through UV spectrophotometry and BAPNA method.The trypsin inhibitor which had the highest inhibitory activity was identified based on its molecular characterization using 16 S rRNA and physiology biochemistry experiments.Its trypsin inhibitor was separated and purified from fermentation broth by the means of ammonium sulfatefractionation precipitation,sephadex G-50 followed by DEAE-cellulose-52 ion exchange chromatography.The crude product was analyzed by HPLC,full-wavelength scanning,thin layer chromatography and paper chromatography.Its component was preliminarily identified by protein,amino acid and saccharides chromogenic reaction.Meanwhile,by drawing enzymatic reaction rate curve with or without inhibitor and calculating Vm and Km,determined the inhibition type of trypsin inhibitor.The result showed that 17 trypsin inhibitor producing strains were selected which were mainly distracted in Streptomyces sp but also found in Microbacterium sp,Ensifer sp,Bacillus sp,Pseudoalteromonas sp,Vibrio sp.8 of them produced inhibitor stably by second screening.FY57 strain which had the maximum trypsin inhibition rate up to 90%was identified as Streptomyces antibioticus and its trypsin inhibitor was a polypeptide which properly contained arginine,tyrosine and aldehyde.The inhibition type of FY57 inhibitor was anticompetitive inhibition.