Pullulanases of alkaline and broad pH range from a newly isolated alkalophilic Bacillus sp. S-1 and a Micrococcus sp. Y-1

Abstract

Two highly alkalophilic bacteria, and potent producers of alkaline pullulanase, were isolated from Korean soils. The two isolates, identified as Bacillus sp. S-1 and Micrococcus sp. Y-1, grow on starch under alkaline conditions and effectively secrete extracellular pullulanases. The two isolates were extremely alkalophilic since bacterial growth and enzyme production occurred at pH values ranging from pH 6.0 to 12.0 for Micrococcus sp. Y-1 and pH 6.0 to 10.0 for Bacillus sp. S-1. Both strains secrete enzymes that possess amylolytic and pullulanolytic activities. Extracellular crude enzymes of both isolates gave maltotriose as the major product formed from soluble starch and pullulan hydrolysis. Compared to other alkalophilic microbes such as Micrococcus sp. (0.57 units ml-1), Bacillus sp. KSM-1876 (0.56 units ml-1) and Bacillus No. 202-1 (1.89 units ml-1), these isolates secreted extremely high concentrations (7.0 units ml-1 for Bacillus sp. S-1 and 7.6 units ml-1 for Micrococcus sp. Y-1) of pullulanases in batch culture. The pullulanase activities from both strains were mostly found in the culture medium (85-90%). The extracellular enzymes of both bacteria were alkalophilic and moderately thermoactive; optimal activity was detected at pH 8.0-10.0 and between 50 and 60 °C. Even at pH 12.0, 65% of original Y-1 pullulanase activity and 10% of S-1 pullulanase activity remained. The two newly isolated strains had broad pH ranges and moderate thermostability for their enzyme activities. These results strongly indicate that these new bacterial isolates have potential as producers of pullulanases for use in the starch industry.