Cloning, expression and characterization of a novel alkaline serine protease gene from native Iranian Bacillus sp.; a producer of protease for use in livestock

Abstract

The use of proteases in the last decade has been welcomed in livestock and poultry industries and has led to significant results such as improved feed conversion ratio, weight gain and increased growth performance. In the present study, isolation and identification of a novel alkaline protease from Iranian Bacillus species was performed in order to use in livestock feed. After primary isolation of bacteria from soil samples of rice fields and early detection of bacterial genus, the zymogram plate was performed for evaluation of production extracellular proteases. Of the 11 strains producing protease, one strain that produced more enzymes was selected to continue the work. Characterization of alkaline protease was done using specific enzyme assays. To confirm the genus of isolates as well as to identify the species close to, molecular analysis of 16S rRNA gene sequence was done. After that, bioinformatics analysis carried out in NCBI database for searching bacterial alkaline proteases gene sequences. The primer designed based on gene homology of close species for extraction of alkaline protease gene. The results showed that the enzyme extract had the highest activity at pH 9.0 and 50 °C. The 16S rRNA gene sequence was submitted for the strain called Bacillus sp. RAM on the NCBI site. According to the results of the phylogenetic tree, the bacterium was belonged to Bacillus genus and Bacillus sp. RAM was close to Thuringiensis C405. The isolated alkaline protease gene successfully cloned in pET28a and transferred to the expression host E.coli BL21. The expression of the protease gene was evaluated by SDS-PAGE electrophoresis. The induced recombinant cells expressed the protease and revealed molecular weight band of about 38 kDa. According to the enzyme properties, this alkaline protease can useful for application in animal industry.