A Comparative study for the production of recombinant intracellular glucose isomerase by escherichia coli and pichia pastoris

Date

2014

Author

Yaman, Sena

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In this M.Sc. study, intracellular thermostable glucose isomerase (EC 5.3.1.5) production capacities of two metabolically engineered microorganisms, Escherichia coli and Pichia pastoris, were investigated. In this context, to construct intracellular glucose isomerase (GI) producing recombinant P. pastoris, the cells were transfected with pPICZ-A expression vector containing GI coding gene (xylA) of Thermus thermophilus. After the confirmation of the transfection, effects of co-carbon source sorbitol were investigated. CS0=30 g L-1 initial sorbitol concentration was found as optimal condition in terms of the cell growth and GI activity. The highest cell concentration and volumetric activity were attained as CX= 4.2 g L-1 (t=36h) and vi A=792.9 U L-1 (t=24h), respectively; whereas the highest activity was obtained as A=545 U L-1 (24h) using methanol as the sole carbon source. In the second part of the study, the research focused on GI production using Escherichia coli BL21 (DE3) pLysS carrying pRSETA::xylAint. Laboratory scale air- filtered shake bioreactor experiments were designed to determine the effects of carbon sources on the cell growth of E. coli. The highest cell concentration was obtained in CM0=30 g L-1 hydrolyzed molasses-based medium where the GI activity was A=2312 U L-1 being 3.17-fold higher than that of P. pastoris. Based on the revealed data, feeding strategy development experiments were carried out using E. coli. Effects of pulse and continuous feeding of molasses on GI production, the cell and by-product formations were investigated by four sets of pilot scale bioreactor experiments. The highest cell concentration was obtained as CX=17.9 g L-1 at t=28h with a pre-determined exponential feeding calculated for the specific growth rate of 0=0.1 h-1. In terms of GI activity, the most prospering strategy was PM-0.05. In this strategy, one molasses pulse given at t=7h and continuous feeding of molasses started at t=10h with a pre-determined exponential feeding rate of 0=0.05 h-1. The highest volumetric GI activity was obtained as A=29050 U L-1 at t=26h of the bioprocess with YX/S=0.20 g g-1 overall specific cell yield on subsrate and 48.8 g total substrate consumption.

Subject Keywords

Isomerases., Escherichia coli., Pichia pastoris., Sucrose.

URI

http://etd.lib.metu.edu.tr/upload/12616786/index.pdf
https://hdl.handle.net/11511/23269

Collections

Graduate School of Natural and Applied Sciences, Thesis