HALOALKALIPHILIC BACILLUS FLEXUS AW3(2): POTENTIAL FOR BIOTECHNOLOGICAL APPLICATIONS

Abstract

The traditional cultivation based methods have a great importance in research, providing
the chance in investigations of biotechnologically significant bacterial isolates under in
vitro. A total of one hundread and fourteen bacteria was isolated from the water and
sediment samples collected from the hyperalkalinesaline environment of Lonar crater. Out
of them strain AW3(2) was selected for 16S rDNA sequencing, production and, partial
characterizations of amylase on the basis of their maximum amylolytic activity. A Gram
positive bacterium was identified as Bacillus flexus by morphological, physiological and
biochemical characterisation and 16S rDNA sequencing. The bacterium grew in sodium
chloride (NaCl w/v) from 0.5 to 7% and at pH 7–12. A substantial level of extracellular
alkaline amylase was produced by Bacillus flexus AW3(2). Optimum enzyme activity was
found to be at 70°C (1.2 unit/mL), pH 10.0 (5.9 units/mL), and 4% NaCl. The amylase was
highly stable over a broad temperature from 40 to 100°C, pH 6.0-12.0, and NaCl
concentration 0.5-10% ranges, showing excellent thermostablity, and haloalkaline tolerant
nature. A Lineweaver-Burk plot indicates that enzyme has a Km of 2.94 mg/mL and a
Vmax of 90.90 mg/mL/ min. The enzyme activity has enhanced by BaCl2 (7.4
units/mL/min), indicating it was a metalloenzyme. Among the organic nitrogen sources,
optimum amylase production was found to be in presence of yeast extract. This is valuable
information for enzyme production and optimization of amylase has a bright future towards
the improvement and production of novel enzymes for entirely new areas of industrial and
biotechnological applications involving molecular enzymology. The developing novel
techniques in genetic engineering combined with better knowledge of structure and
function allow fulfillment of industrial needs and exploration of novel applications.