Scale-up fermentation of Escherichia coli for the production of recombinant endoglucanase from Clostridium thermocellum

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.

Process Evaluation of Recombinant Chitin Deacetylase Expression in E. Coli Rosetta pLysS Cells using Statistical Design of Experiments

“…A similar observation with the agitation rate has been reported by Shahzadi et al in their study wherein an increase of agitation was causing downregulation of recombinant gene expression. (Shahzadi et al 2021). The pET expression system in conjunction with the E. coli as the host organism is used for high productivity.…”

Lignocellulolytic Biocatalysts: The Main Players Involved in Multiple Biotechnological Processes for Biomass Valorization

“…Concerning the heterologous expression of lignocellulolytic enzymes, it is sought to express a functional lignocellulolytic system in order to allow non-lignocellulolytic microorganisms to hydrolyze and transform lignocellulosic biomass. The most commonly used non-lignocellulolytic microorganisms for this purpose are Zymomonas mobilis , Escherichia coli , Pichia pastoris , and Saccharomyces cerevisiae [ 277 , 368 , 369 , 370 , 371 ]. The production of recombinant lignocellulases may be the solution to limitations of high substrate cost and maintenance of the necessary conditions for these enzymes production, as well as more resistant and stable strains production and higher rates of enzyme production [ 372 , 373 , 374 , 375 ].…”

Metagenomic Discovery and Characterization of Multi-Functional and Monomodular Processive Endoglucanases as Biocatalysts

“…Various engineered E. coli cells, including an endotoxin-free E. coli strain, referred to as the ClearColi ® BL21 (DE3) as developed by Mamat et al (2015), are widely used for commercial scale of pharmacological polypeptide & enzyme product production [194]. Use of E. coli competent cells for commercial scale of industrial biomass degradation enzyme production is being promoted [195]; however, governmental regulatory body approval would be needed if resulting biomass enzymes are to be used as exogenous fibre enzyme feed supplements. Several microbial platforms have been developed and used for industrial and biorefinery based commercial enzyme production via the solid state fermentation (SSF) or submerged fermentation (SmF) types of microbial cultivation, including Trichoderma reesei and/or Trichoderma longibrachiatum by Novozymes, Genencor-Danisco-Dupont, Dyadic International, AB Vista Enzymes, Biocatalysts Limited, and the formerly Iogen Bioproducts (purchased by Novozymes in 2013); Aspergillus niger by Novozymes and Amano Enzymes Inc.; and Bacillus sp.…”