Abstract
Endoglucanase (EC 3.2.1.4) catalysing the hydrolysis of β-1.4-glycosidic linkage of cellulose molecules is an enzyme of tremendous industrial importance. The present study describes a response surface methodology based predicted model to deduce a set of fermentation conditions for optimum growth and activity of recombinant endoglucanase in E. coli BL21 (DE3). Numerous significant parameters including fermentation media composition, temperature (Celsius), pH and agitation rate (rpm) were analysed systemically by employing central composite design. This effort reports highly efficient recombinant endoglucanase overproduction (6.9 gl−1 of biomass) with 30% expression by E. coli in modified M9NG media incubated at 37 °C and pH 7 agitated at 200 rpm. Addition of 3 mM glucose and 24 mM glycerol in the M9NG media has shown positive effect on the enzyme yield and activity. The CMCase activity experimentally estimated was found to be 1185 U/mg with the optimized parameters. The outcomes of both the responses by the predicted quadratic model were found in consensus with the obtained values. Our results well depicted the favourable conditions to further scale-up the volumetric yield of other relevant recombinant enzymes and proteins.
Highlights
Endoglucanase (EC 3.2.1.4) catalysing the hydrolysis of β-1.4-glycosidic linkage of cellulose molecules is an enzyme of tremendous industrial importance
Significant fermentation parameters including fermentation temperature, pH and agitation rate were optimized by varying the levels of these factors using central composite design for achieving higher cell growth with maximum CMCase activity
The present study describes our experiments for the scaling-up of fermentation conditions to improve the yield and quality of endoglucanase by exploiting various influencing factors
Summary
Endoglucanase (EC 3.2.1.4) catalysing the hydrolysis of β-1.4-glycosidic linkage of cellulose molecules is an enzyme of tremendous industrial importance. Numerous significant parameters including fermentation media composition, temperature (Celsius), pH and agitation rate (rpm) were analysed systemically by employing central composite design. This effort reports highly efficient recombinant endoglucanase overproduction (6.9 gl−1 of biomass) with 30% expression by E. coli in modified M9NG media incubated at 37 °C and pH 7 agitated at 200 rpm. Lactose being metabolized as a carbon source by the cells for optimal growth, results in maximum biomass but becomes inadequate for the expression of recombinant proteins, costs at prolonged induction time. Optimization of growth media, supplementing with enriched nutrients, and fermentation crucial parameters will help to develop cost-effective media to achieve higher productivity with reduced induction time for the industrial benefits
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