Production, purification and characterization of polysaccharide lytic enzymes of a marine isolate, Bacillus cereus NRC-20 and their application in biofilm removal

Abstract

In screening for marine bacterial polysaccharide lytic enzymes, the most potent isolate was identified as Bacillus cereus NRC-20. This strain showed high alginase, dextrinase, pectinase and carboxymethyle-cellulase productivity when production medium was adjusted at pH 6.0 and contains (g/l): 3.0 sodium alginate, 3.0 malt extract, 30.0 jatropha cake and inoculated with 8% (v/v) of a 24 h old cell suspension and incubated at 30°C for 48 h on a rotary shaker at 200 rpm. These enzymes mixture were precipitated by 60% acetone and purified by using sephadex G-100, whereas SDS-PAGE suggested a molecular weight of approximately 17,20, 89 and 279 KDa, respectively. The optimum partially purified enzymes activity when enzyme protein concentration was 27.4 mg/ml and substrate concentration 5.0 g/l shown after 90 min of incubation at 30°C and pH 5 and stable at 60°C for 60 min. The enzymes activities were enhanced in the presence of 0.02 M Na+ and K+ions and completely inactivated by Mg+2, Cu+2,Co+2 and Pb+2. The enzymes mixture affected the matrix integrity of different microbial biofilms artificially grown on stainless steel sheets of Bacillus subtilis NRRL B-4219, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27953 and Escherichia coli ATCC 25922.