Statistical analysis of production of protease and esterase by a newly isolated Lysinibacillus fusiformis AU01: purification and application of protease in sub-culturing cell lines

Abstract

A bacterial strain producing both a protease and an esterase was isolated from the seed of Tamarindus indica . The isolate was identified as Lysinibacillus fusiformis AU01 by phylogenetic analysis of the 16S rDNA sequence. The isolate produced an extracellular protease and an intracellular esterase simultaneously under the same culture conditions. Statistical methods, i.e., Plackett-Burman followed by response surface methodology (RSM), were applied to optimize media components and culture conditions in 50 mL shake flask cultures. Culturing in shake flasks with the optimized medium resulted in a 6-fold and a 3.5-fold increase in protease and esterase production, respectively, when compared to nutrient medium. The productivity of the protease and esterase was increased to 75 U/mL and 370 U/mL when cultivated under controlled conditions in a 3-L bioreactor. The extracellular protease was purified to 34.6 fold with 38.8 % recovery and the molecular mass of the enzyme was found to be 48 kDa. The partial amino acid sequence of the protease was determined by MALDI-TOF-MS analysis. The optimum temperature and pH for protease activity were found to be pH 9.0 and 40 °C. The inhibition of purified protease by EDTA and PMSF confirmed that the enzyme belonged to the family of serine metalloproteases. The enzyme was found to be stable in the presence of some hydrophobic and hydrophilic solvents. The ability of AU01 protease to dissociate monolayer cells for sub-culturing adherent cell lines was also investigated.