PRODUCTION AND PARTIAL PURIFICATION OF KERATINASES FROM Bacillus cereus N14 AND Bacillus halotolerans L2EN1 OBTAINED FROM POULTRY FARMS OF HIMACHAL PRADESH
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Date
2020-09
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UHF,NAUNI
Abstract
ABSTRACT
To enhance the keratinolytic potential of two bacterial strains viz., Bacillus cereus N14 and Bacillus
halotolerans, isolated from poultry farms of Nahan and Sundernagar (Himachal Pradesh), respectively, different
process parameters like temperature, pH, incubation time, inoculum size, inoculum age etc. were optimized using One
Variable at a Time (OVAT) approach and Response Surface Methodology (RSM). Bacillus cereus N14 showed
maximum enzyme activity (19.5 U/mL) after 3rd day of incubation at 35°C, pH 9.0, 12.5 (%) of inoculum size using 3
days old culture and 2.5 and 2.0 per cent of maltose and yeast extract as best carbon and nitrogen source, respectively
in the presence of Mn2+ as divalent metal ion and EDTA as best media additive. Whereas, in case of Bacillus
halotolerans L2EN1, maximum enzyme activity (26.57 U/mL) was attained after 3rd day of incubation at 45°C, pH
11.0, 12.5 (%) of inoculum size using 3 days old culture and 1.5 and 2.0 per cent of sucrose and yeast extract as best
carbon source and nitrogen source, respectively in the presence of Mn2+ as divalent metal ion and EDTA as best media
additive. Central composite design (CCD) of RSM was successfully applied to investigate the effect of 5 independent
variables viz., incubation time (days), inoculums size (%), inoculum age (days), concentration of carbon and nitrogen
source (%) on keratinase production. The maximum enzyme activity (74.86 U/mL) was attained by the B. cereus N14
at 22nd run whereas, B. halotolerans showed the maximum enzyme activity (65.50 U/mL) at 24th run. Partial
purification results reveal that 30-40 per cent ammonium sulphate precipitation cut was best for maximum keratinase
production (125.3 Ug/ml) by Bacillus cereus N14. While, 90-100 per cent ammonium sulphate concentration was
found to be ideal for Bacillus halotolerans L2EN1 (22.6 Ug/ml). The molecular weights of crude keratinases from B.
cereus N14 and B. halotolerans L2EN1 were determined through SDS-PAGE. In both the strains, keratinases were
observed to be heterotetramers made up of four polypeptide chains with different molecular weight i.e 30, 37, 70, 75
kDa in B. cereus N14 while, 17, 37, 40 and 60 kDa in B. halotolerans L2EN1. To assess the ability of these
keratinases as laundry additives, their compatibility with commercial detergents namely Tide, Reshma, Speed, Ariel
and Surf excel was examined The crude keratinase from B. cereus N14, retained its maximum activity (99.33%) in
the presence of Reshma followed by Speed (94.14%), Tide (77.78%), Ariel (71.38%) and Surf excel (60.47%).
Similarly, in case of B. halotolerans L2EN1, keratinase retained its maximum activity (97.77%) in the presence of
Reshma followed by Speed (93.44%), Tide (79.93%), Ariel (70.18%) and Surf excel (67.98%) at 50°C after 1h of
incubation hence keratinases in different combinations with the commercial detergent Reshma were tried to analyze
their wash performances. In both the strains, washing with tap water at room temperature (18°C), 28, 35 and 45°C for
30, 45 and 60 min removed some amount of blood stains from the cotton cloth pieces. However, replacement of
detergent’s enzyme(s) with crude keratinases achieved complete blood stain removal under same conditions,
suggesting their suitability as potential cleaning agents (additives) in detergents for the removal of proteinaceous
(blood) stains for long washing cycles (1 h).