Investigation of thermostable recombinant glucose isomerase production by sucrose utilizing escherichia coli

Akdağ, Burcu
The aim of this M.Sc. thesis is to investigate the production of thermostable glucose isomerase (GI; EC by metabolically engineered sucrose-utilizing Escherichia coli in the designed molasses-based production media. Throughout the experiments the cell growth, sucrose consumption, recombinant GI activity, and by-product concentrations were analyzed. For this purpose, in the first part of this thesis, pRSETA plasmid carrying the thermostable GI encoding gene from Thermus thermophilus (xylA) was isolated and pRSETA::xylA plasmid was transformed into Escherichia coli W. Thereafter, the highest GI producing strain was selected using different carbon sources; and the selected strain was named as E. coli W-26. In the second part, the production of recombinant glucose isomerase was investigated firstly, in a program using laboratory scale shake-bioreactor experiments; and then by four sets of pilot scale bioreactor experiments. The recombinant GI production capacity of E. coli W-26 was tested on different carbon sources, i.e., glucose, sucrose, and molasses, at different concentrations in shake bioreactors. Growth in 32 g L-1 molasses-based medium resulted in higher recombinant GI activity and cell concentrations than those obtained in glucose and sucrose based media. Based on these results, four set of pilot scale semi-batch bioreactor vi experiment were designed where complex substrate molasses acted as the carbon and nitrogen source. In this context, four feeding strategies were designed using pulse-or exponential- feeding strategies, and the influences on the cell growth, GI production, and by-product formations were investigated with the same pre-determined specific growth rate at μ=0.05 h-1. The highest cell concentration was obtained as 18.4 g L-1 at t=26 h and the highest recombinant GI activity was achieved as 35264.5 U L-1 at t=16 h of bioprocess in BR4 operation by two molasses pulses with (NH4)2HPO4 and antibiotic addition at t=5 h and t=8 h; shifting to semi-batch operation at t=11 h by feeding molasses based medium with an exponential volumetric feeding rate calculated using the pre-determined μ=0.05 h-1.