Establishment And Characterization Of Protease Producing Bacterial Collection

Protease is one of the most important industrial enzymes.But there are relatively few types of commercial proteases,which can not meet the requirement of current industrial development.The selection and breeding of strains with high protease-producing ability is therefore of great importance.In the present study,a protease-producing bacterial collection was established and its basic characteristics were also investigated,which will facilitate the further development and utilization of these microbial resources.The main findings are as follows:(1)A total of 110 fruits,vegetables and soil samples were collected from 17 different locations across the country.After sample pretreatment and dilution culture,1,544 strains were isolated from these samples.Results of molecular identification demonstrated that there were 1171 bacteria which can be assigned to 62 genera and 212 species.(2)458 protease-producing bacterial strains,belonging to 31 genera and 87 species,were then screened from the above-mentioned microbial isolates.Further analysis showed that proteases from Buttiauxella,Desulfovibrio,Rodentibacter,Glutamicibacter and Hafnia had not been reported in the literature.12,13,25 and 15 strains with protease-producing ability were screened from raisins,bananas,pakchoi,and soil samples around the meat stalls,respectively.The number of protease-producing strains from Bacillus,Staphylococcus,Curtobacterium and Serratia accounted for 62.00%,6.67%,5.78 and 6.64%of the total number of the collections,whereas the proportion of protease-producing strains from other genera was less than 3%.(3)42 strains with higher proteolytic activities were then cultured and their proteases were biochemically characterized.All of the proteases produced by these strains were found to be neutral or alkaline proteases and the same microorganism could produce different types of proteases due to different culture times.The optimum temperatures of these proteases varied greatly,and proteases from the same genera or species were quiet different.(4)In the shaking flask experiments,the activities of the protease B7649 and B8102 were 41.24 U/mL and 41.76 U/mL.These two enzymes were then purified to elctrophoretic homogeneity by ammonium sulfate precipitation,desalting and gel chromatography,with the purification fold and yiled of 16.38 and 23.54%,and 21.56 and 18.47%,respectively.The temperature and pH optima of proteases from B7649 and B8102 were 60? and 9.0,and 60? and 10.0,respectively.They were very stable at 50? and 40?,respectively.After incubated at pH 7.0-11.0 for 2 h,these two proteases still retained more than 90%of its initial activity.Co2+,Cu2+ and Fe3+ exhibited different inhibitory effects on the activities of these two proteases.Zn2+ inhibited the activity of protease from B8102,whereas it had no effect on the activity of B7649 protease.Besides,the activities of these two proteases were significantly enhanced by Ca2+.