Optimization of an Activity Assay of Coprinus Cinereus Peroxidase

ABSTRACT To seek a simple, rapid and sensitive Coprinus cinereus Peroxidase (CIP) activity assay, a convenient one-factor-at-a-time (OFAT) method and a response surface methodology (RSM) were used. The recombinant CIP expressed in Pichia pastoris was purified with the Ni-NTA spin column. Based on the results of catalytic efficiency (kcat/Km) analysis, 2,2'-azinobis (ethylbenzthiazoline -6-sulfonate) (ABTS) was selected as the optimal enzyme substrate. Results of the OFAT method showed that enzymatic reaction performed in 0.1 mol/L sodium acetate (pH 5.0) buffer in a 200-µl reaction mixture containing 0.5 mmol/L ABTS, 10 mmol/L hydrogen peroxide (H2O2), 49.7 ng CIP at 25°C gave an average CIP activity of 88 U/mL. The ABTS and H2O2 concentrations were then further optimized to improve the sensitivity of the assay. To do that, RSM was conducted through central composite design, and a reduced quadratic model with good fit regression equation was generated. ANOVA analysis of this model indicated that the concentrations of ABTS and H2O2 and their interaction had significant impact on the assay sensitivity. The optimal reaction mixture was determined to include an initial ABTS concentration of 0.82 mmol/L 49.7 ng CIP and 16.36 mmol/L H2O2, and the activity under this condition was determined to be 138.89 U/mL.

One Factor at A Time based optimization of protease from poultry associated Bacillus licheniformis.

Bacteria were isolated from poultry farm of Guduvanchery, Tamil Nadu, India and exhibited variable protease activity on skim milk agar plates. Of 10 bacterial isolates screened, Bacillus licheniformis strain 018 was observed as a hyperprotease producer and it was further characterized using biochemical and molecular tools. Protease production from the isolate was enhanced by optimizing the culture conditions using One Factor at A Time (OFAT) method. The bacteria exhibited its optimal enzyme activity at pH- 9.0, temperature- 35⁰C, agitation speed- 130 rpm, incubation time- 24 h, carbon source- casein and nitrogen source- yeast extract. On the other hand, the crude proteases were found to be significantly active and stable at broad range of pH (5.0-9.0) and temperature (30-60⁰C). To the best of my knowledge this is the first report on the production and enhancement of alkaline protease from poultry farm isolate using OFAT method. Stability of the enzyme at high temperature and pH can be explored for varied industrial applications.