Optimization of media for improved production of recombinant T1 lipase using local substrates

Thermostable T1 lipase carries a lot of potential in industrial applications such as in diesel production and detergent formulation. However, the usage of laboratory media can cost a fortune when used at commercial scale (adding up to the final cost value of the enzyme). In order to create a cheaper enzyme product a new medium formulation from cheaper sources and readily available is crucial. This study was designed to formulate new medium and to develop an efficient large scale bioprocess strategie for thermostable T1 lipase from recombinant E. coli BL21. Different carbon and nitrogen sources from agro and industrial waste were screened. The compositions of the medium were optimized using response surface methodology (RSM). Isopropyl β-D-1-thiogalactopyranoside (IPTG) and lactose capability as inducer were also studied. The kinetics of T1 lipase production by recombinant E. coli were evaluated using Monod and Luedeking-Piret equations. The effects of dissolved oxygen tension (DOT) level on growth of recombinant E. coli and T1 lipase production were investigated in batch fermentation using 7.5 L stirred tank bioreactor. Fed-batch fermentation for T1 lipase production was initially developed in 7.5 L stirred tank bioreactor and then scaled up to 30 L. A newly formulated medium for production of T1 lipase was formulated using 5th grade molasses and fish processing waste as carbon and nitrogen sources. The medium consisted of molasses (2 g/L), fish waste (12%), NaCl (5 g/L), MgSO4 (0.5 g/L) and KH2PO4 (1 g/L). Through centre composite design (CCD), medium compositions using IPTG as an inducer showed higher T1 lipase production in predicted (172.89 U/mL) and actual run (164.37 U/mL) compared to lactose as an inducer in predicted (123.47 U/mL) and actual run (120.34 U/mL). Both R2 values calculated using RSM showed a good fit and the proposed models for T1 lipase production by recombinant E. coli were sufficient to describe the processes. T1 lipase production was found to be a growth associated process and 30% showed the optimal level of DOT for production of T1 lipase. The constant feed rate for fed-batch fermentation at 160 mL/h using 50% lactose as feeding medium was found to be optimal for production of T1 lipase (260.10 U/mL) and recombinant E. coli growth (51.30 g/L). The fermentation employing recombinant E. coli for T1 lipase production was successfully scaled-up to 30 L stirred tank bioreactor using a constant DOT approach, with DOT level controlled at 30% saturation. 50% of cost reduction was successfully achieved in production of T1 lipase when using new formulated medium and so far, this is the first report of using molasses and fish waste in the medium formulation. The information and findings obtained from this study are very useful in designing and in the preparation of standard operating procedure (SOP) for production of T1 lipase by recombinant E. coli at pilot plant and at industrial scale.

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