Statistical Approach for Optimization of Physiochemical Requirements on Alkaline Protease Production from Bacillus licheniformis NCIM 2042

Abstract

The optimization of physiochemical parameters for alkaline protease production using Bacillus licheniformis NCIM 2042 were carried out by Plackett-Burman design and response surface methodology (RSM). The model was validated experimentally and the maximum protease production was found 315.28 U using optimum culture conditions. The protease was purified using ammonium sulphate (60%) precipitation technique. The HPLC analysis of dialyzed sample showed that the retention time is 1.84 min with 73.5% purity. This enzyme retained more than 92% of its initial activity after preincubation for 30 min at 37°C in the presence of 25% v/v DMSO, methanol, ethanol, ACN, 2-propanol, benzene, toluene, and hexane. In addition, partially purified enzyme showed remarkable stability for 60 min at room temperature, in the presence of anionic detergent (Tween-80 and Triton X-100), surfactant (SDS), bleaching agent (sodium perborate and hydrogen peroxide), and anti-redeposition agents (Na(2)CMC, Na(2)CO(3)). Purified enzyme containing 10% w/v PEG 4000 showed better thermal, surfactant, and local detergent stability.

Figure 1

(a) Normal plot of residual; (b) residual versus predicted plot; (c) Predicted versus Actual Response plot for protease production (U) by Bacillus licheniformis NCIM 2042.

Figure 2

Perturbation plot for the production of alkaline protease by Bacillus licheniformis NCIM 2042 as a function of inoculum percentage (A), temperature (B), pH (C), agitation (D), and incubation time (E).

Figure 3

Contour plot for protease production (U) by Bacillus licheniformis NCIM 2042 as a function of (a) inoculum percentage (A) and temperature (B), (b) temperature (B) and pH, (C), (c) pH (C) and agitation (D), and (d) agitation (D), and incubation time (E) when other variables are at zero level.

Figure 4

Effect of (a) organic solvent and (b) detergent, surfactant, bleach, and anti-redeposition on protease activity.

Figure 5

Stabilizing effect of additives on protease stability in presence of (a) high temperature, (b) surfactant, and (c) local detergent.

Figure 6

Washing performance analysis of enzyme in the presence of the commercial detergent RIN. (a) Cloth stained with blood, (b) cloth stained with blood and washed with tap water; (b) blood-stained cloth and washed with RIN; (c) blood-stained cloth and washed with RIN added with crude enzyme.