Purification and biochemical characterization of a novel detergent-stable serine alkaline protease from Bacillus safensis strain RH12

Abstract

A novel protease (SAPRH) was hyper-produced (9000 U/mL) from Bacillus safensis RH12, a newly isolated enzyme from a Tunisian offshore oil field. The enzyme was purified to homogeneity, using salt precipitation, heat-treatment and FPLC anion-exchange chromatography (Fast protein liquid chromatography). SAPRH was a monomer of molecular mass of ~28 kDa. The NH2-terminal 23 amino-acid sequence of SAPRH showed high homology with those of Bacillus-proteases. SAPRH displayed optimal activity at pH 9 and 60 °C. It was strongly inhibited by (PMSF, Phenylmethane sulfonyl fluoride) and (DFP, Diisopropylfluorophosphate), indicating that it belongs to the serine-proteases family. One of the most distinctive properties is its catalytic efficiency, which is higher than that of Alcalase 2.5 L, type DX (commercial enzyme) and SAPB from Bacillus pumilus strain CBS. Interestingly, the results of the wash performance analysis demonstrated considerably good de-staining at 40 °C for 30 min with low supplementation (500 U/mL). The sapRH gene, which encodes the serine alkaline protease SAPRH, from Bacillus safensis strain RH12, was isolated and its DNA sequence was determined. The highest sequence identity value (97%) was obtained with SAPB from B. pumilus strain CBS, with only 9 amino-acids of difference. The region, encoding SAPRH was SciForum heterologously expressed in E. coli BL21AITM cells using GATEWAYTM pDESTTM17 expression-vector.