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In Association with
Utilization of agro/industrial wastes for high-level expression of recombinant endo-mannanase by Escherichia coli and its application in oil extraction from Copra
6th World Congress on Biotechnology
Gaurav Singh Kaira, Deepesh Panwar and Mukesh Kapoor
CSIR-Central Food Technological Research Institute, India
Posters-Accepted Abstracts: J Biotechnol Biomater
Abstract
The high enzyme titers of recombinant endo-mannanases (EC 3.2.1.78) have really boosted their industrial prospects in food, feed, oil drilling, textiles and pulp sectors but the high cost associated with their production is still acting as a major deterrent. In the present work, three pronged-approach was employed to ameliorate and economize ManB-1601 (recombinant endo-mannanase, accession no: KM404299) production. Firstly, pRSETA manb-1601 construct was transformed in HIControl E.coli BL21 (DE3) cells which resulted in 2.7 fold (1821 IU/ml, 42.5 hours post-induction) increase in ManB-1601 production as compared to the previous host. The second approach focused on understanding the interplay between various nutritional and physical factors. Shifting of induction time to 5 hours resulted in up to 1.89 fold improvement in enzyme yield (2268 IU/ml) and volumetric enzyme productivity (81 IU/ml/h). Replacement of IPTG with lactose improved ManB-1601 production by 1.77 fold (4031 IU/ml). Efforts to improve the cell membrane permeability using chemical (glycine, calcium chloride) and physical [incubation temperature higher (42o C) or lower (16-25o C)] methods were found counter-productive. Increment in protein (by peptone) and carbohydrate (by glycerol) concentration to 2.0 % (w/v) and 0.25% (v/v) resulted in up to 2.08 fold (8406 IU/ml) increase in ManB-1601 titers. However, doubling (2X) or quadrupling (4X) the concentration of optimized medium proved detrimental. Under the third approach, up to 70.45% (w/v) of protein in the optimized media was successfully replaced with soluble protein from defatted flax seed meal in presence of glycerol (0.25% v/v) and lactose (1.5 mM) and resulted in 5926 IU/ml of ManB-1601. Treatment of grated copra with purified ManB-1601 (220 IU/g) resulted in 9.1% higher oil yield when compared to control. Laser scanning confocal microscopy and scanning electron micrographs studies depicted significant reduction in oil-droplet size and subtle changes in the copra morphology, respectively in enzyme treated samples. No change in FAME profiles was observed after ManB-1601 treatment.Biography
Email: mkapoor@cftri.res.in
