Optimization of fermentation strategy for enhanced production of thermostable xylanase by recombinant Escherichia Coli

• Main Author: Kandiyil, Subeesh Kunhi
• Format: Thesis
• Language: English
• Published: 2019
• Subjects: TP Chemical technology
• Online Access: http://eprints.utm.my/id/eprint/87115/1/SubeeshKunhiKandiyilPSChE2019.pdf; http://eprints.utm.my/id/eprint/87115/; http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:131508

Xylan is the second most abundant polysaccharide in plant cell wall which is hydrolyzed by the group of enzymes called hemicellulase. ß -1, 4 endo xylanase is considered as the most important among the xylanase enzymes, due to its wide industrial applications. Escherichia coli BL21 with a plasmid vector pET-22b (+) carrying xylanase coding gene, which was isolated from the extremely thermophilic bacterium called Thermotoga neapolitana, was used in the current study to enhance xylanase production. In phase 1 of this study, using the statistical approach called response surface methodology, the optimum media composition for enhanced xylanase production was successfully identified. Up to 800 IU mL-1 xylanase activity was observed in optimized media, which is around 3 folds higher compared to the activity achieved in unoptimized medium. In phase 2, optimization of lactose-based induction strategy was carried out to enhance the xylanase production. As a result of this induction optimization, the intracellular xylanase production was enhanced up to 2600 IU mL-1. In phase 3, as a part of process scale up, the study was focused on developing suitable fed-batch fermentation conditions, by optimizing nutrients and inducer feeding strategy. With the optimized fed batch fermentation conditions in 16 L stirred tank bioreactor, the xylanase activity was enhanced up to 11000 IU mL-1, which is 4 to 5 folds higher compared to activity reported in previous studies. During physicochemical characterization in phase 4 of the current study, the optimum temperature and pH of xylanse enzyme was found to be 80°C and 6.5, respectively. Among the metal ions and chelating agents tested, zinc sulfate and ethylenediaminetetraacetic acid were found to have the highest inhibitory effect on xylanase enzyme in this study.