Statistical optimization of alkaline protease production by Penicillium oxalicum JML 15 and evaluation of its potential in blood stain removal

Abstract

The present study describes the production, statistical optimization and characterization of alkaline protease produced by Penicillium oxalicum JML 15, and evaluation of its application as detergent additive. In One Factor At a Time (OFAT) studies, maximum protease activity was observed on day 6 at pH 9 and 30 °C, with glucose and yeast extract as optimum carbon and nitrogen sources, respectively. The presence of Mg²⁺ facilitated enhanced enzyme activity, and 1% casein supported optimum enzyme production. Statistical optimization using Plackett–Burman design identified glucose and incubation period as two significant factors for high protease yield. Response Surface Methodology was used to maximize protease yield up to 2.4-fold (348.33 U/ml) compared to unoptimized (144.72 U/ml). The partially purified protease showed optimal activity at 50 °C and pH 10, with stability retained up to 60 °C and pH 11, indicating excellent thermal–alkaline tolerance desirable for detergent applications. Enzyme activity was significantly increased in the presence of Mn²⁺ and retained about 80% of its activity at 2.0 M NaCl, indicating considerable halotolerance. The protease remained fully active in commercial detergents such as Ariel and Tide, confirming its compatibility and stability in complex detergent formulations. In stain removal assays, the enzyme achieved complete removal of bloodstains within 10 min, demonstrating its efficiency in degrading proteinaceous stains. These characteristics highlight its strong potential as a bioadditive for detergent formulations and its applicability in industries that require alkaline- and salt-tolerant proteases.

Keywords

Optimization / Response surface methodology / Halotolerant / Thermostable / Destaining

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