ABSTRACT
A novel extracellular protease (SAPRH) was hyper-produced (9000â¯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. The purified enzyme was a monomer of molecular mass of ~28â¯kDa. The NH2-terminal 23 amino-acid sequence of SAPRH showed high homology with those of Bacillus-proteases. SAPRH displayed optimal activity at pHâ¯9 and 60⯰C. It was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), indicating that it belongs to the serine-proteases family. Moreover, SAPRH was extremely stable at a broad range of temperature and pH retaining 85% of its activity at 50⯰C and 75% at pHâ¯11. The enzyme exhibited excellent stability and compatibility with surfactants and commercial detergents, revealing 90% stability with SDS and 100% stability with Class commercial laundry detergent. One of the most distinctive properties is its catalytic efficiency, which is higher than that of Alcalase 2.5â¯L, typeDX (commercial enzyme) and SAPB from B. pumilus CBS. Interestingly, the results of the wash performance analysis demonstrated considerably good de-staining at 40⯰C for 30â¯min with low supplementation (500â¯U/mL). Accordingly, such a protease could be considered as a good detergent-additive in detergent industry.
Subject(s)
Bacillus/enzymology , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Detergents/pharmacology , Endopeptidases/isolation & purification , Endopeptidases/metabolism , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/biosynthesis , Calcium/pharmacology , Coloring Agents/metabolism , Cotton Fiber , Drug Interactions , Endopeptidases/biosynthesis , Enzyme Inhibitors/pharmacology , Enzyme Stability/drug effects , Hydrogen-Ion Concentration , Kinetics , Phylogeny , Polymers/pharmacologyABSTRACT
The sapRH gene, which encodes the serine alkaline protease SAPRH, from Bacillus safensis RH12, was isolated and its DNA sequence was determined. The deduced amino-acid sequence showed strong homology with other Bacillus proteases. The highest sequence identity value (97%) was obtained with SAPB from B. pumilus CBS, with only 9 amino-acids of difference. The region, encoding SAPRH was heterologously expressed in E. coli BL21-AI™ cells using GATEWAY™ pDEST™17 expression-vector. The recombinant (His)6-tag enzyme (His6-rSAPRH) was purified in a single affinity chromatography step and its biochemical properties were determined and compared to those of SAPRH and rSAPB. Interestingly, His6-rSAPRH showed improved thermostability compared to SAPRH and rSAPB. The molecular dynamics of SAPRH compared to SAPB revealed a more thermostable structure, thus confirming the in vitro results showing that His6-rSAPRH has a t1/2 of 120â¯min against 90 and 30â¯min for SAPRH and rSAPB, respectively, at 70⯰C and different kinetic parameters to synthetic peptides. The docking simulations data allow in getting an insight into the involvement of some key amino-acids in substrate binding and account for the selectivity. Overall, this is the first report of a sapRH gene cloned from B. safensis which can be a promising potential candidate for future applications in detergent formulations.