Optimization of Conditions for the Production of Recombinant Cellulase by using E.COLI BL21 Codon Plus in Fermenter

Maliha Nawaz^1*, S.Zafar¹, Sameen Khan Shervani², Sidra Ray¹, Ummul Buneen¹, Arzoo Rubab¹, Maria Kanwal¹ , Qandeel-e-Arsh¹, M. Faizan Khurram¹ and Syed Naeem Sajid^1*

¹Centre of Agricultural Biochemistry& Biotechnology (CABB), University of Agriculture, Faisalabad, Pakistan

²Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan

Corresponding Author E-mail: snomi432@gmail.com

DOI : http://dx.doi.org/10.13005/bbra/2822

ABSTRACT: Cellulose is the richest plant biomass on earth and is an unbranched polymer composed of D-glucose residues joined by β-1, 4-D-glycosidic bonds. The utmost abundant carbohydrates in nature are considered to be cellulases and hemicellulases. Cellulases are inducible enzymes that catalyze the hydrolysis of a β-1, 4-glycosidic bond to release the glucose units in a cellulose molecule. Thermophilic cellulases are relatively expensive and a very significant industrial enzyme. In this study, the recombinant plasmid pET22b (+) containing the cellulase encoding gene was transformed in E.coli BL21 codon plus. A Shake flask fermentation study was performed using modified M9NG media. Lactose and IPTG were used as an inducer. After SDS-PAGE analysis, the predicted molecular weight of a protein was 62kDA Batch culture fermentation was performed using LB and modified M9NG media. Lactose was used as the cheapest inducer. Under optimized fermentation conditions, the enzyme displayed maximum activity at 37^oC and pH 7. The specific activity of the enzyme was 70U/ml. The production of the recombinant enzyme was enhanced approximately 6 times in E.coli BL21 as compared to wild type strain. The expression level of the recombinant cellulase was round about 30%-40%. Molecular cloning of the cellulase encoding genes resulted in the maximum production of the cheapest enzymes that can be used for industrial purposes.  

KEYWORDS: Cellulases; M9NG; IPTG; Heteropolymers; Glucomannan; Arabinoxylan; Galactoglucomannans

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