An alkalophilc and thermophilic Bacillus sp. BHA that produced a thermostable alkaline protease was isolated
from decaying protein substrates. The isolate was found to grow in pH range 7
- 11 with an optimum pH 9.0 and temperature up to 55℃. The activity of
alkaline protease of Bacillus sp. BHA (68.98 APU/ml) was found higher
than the standard strains of Bacillus
amyloliquefaciens MTCC 610 (8.98 APU/ml) and Bacillus subtilis MTCC 8349 (12.14 APU/ml, used in this study, and
was comparable (68.98 APU/ml, equivalent to 30.38 APU/mg) to the activity of the commercially produced standard protease procured from Novo Nordisk, Denmark (30.35 APU/mg). Hence, the
proteolytic activity produced by this isolate was further investigated in
batch and fed-batch process. Sucrose was the
best carbon source for the production of protease activity by that
isolate. Different organic nitrogen sources (casein, peptone and beef extract)
at 1% (w/v) with varying levels of sucrose (1% - 4% w/v) initially repress
enzyme synthesis. The duration and extent of repression decreased with
increased concentration of sucrose. Maximum protease activity was found in
basal medium with 4% (w/v) sucrose and 1% (w/v) yeast extract. Yeast-extract
was thought to be an inducer of enzyme synthesis. Further, the basal medium
was unique with respect to the enzyme production, as protease production was
growth associated with the peak enzyme production being detected at the
time of maximum growth. Interestingly, a rise in 34.2% (104.86 APU/ml) of protease activity was detected at incubation temperature of 50℃ and when culture filtrate was assayed at 60℃, signifying a
high temperature stability of the produced protease by this isolate. Additional
studies on the enzyme characterization were resulted in recognition of highly
significant properties of the activity towards casein at pH 9.0 and stability
at high temperature with retention of 96% the enzyme activity at 60℃. The
parametric study under feed intervals had enabled improvement in the maximum
protease activities attainable from batch cultures in excess of 21.78% and
26.32% via two feeding strategies. A small continual increase in enzyme activity
(132.46 APU/ml during 24 h - 120 h) and enhancement in protease production in
excess of 36.84% was observed by fed-batch process than the batch experiment.
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