Bioprocess development for thermostable glucose isomerase production

Angardi, Vahideh
In this study, process development for glucose isomerase (GI) was aimed. In this context, firstly, thermostable xyl genes, PCR amplified from Thermus thermophilus and Pyrococcus furiosus cells, were recombined to the E.coli BL21 (DE3) and P.pastoris strains, respectively. But significant increase in the term of GI activity compared with wild type cells only detected in recombinant E.coli strain so this strain was selected for further experiments. Then, the effect of different natural and artificial inducers on the production of rGI under control of LacUV5 promoter was investigated in laboratory-scale bioreactors. Lactose was shown to be more efficient in the term of operon induction for long time bioprocesses. Thereafter, in order to increase thermostable rGI production rate, to achieve high cell density culture of E.coli BL21 (DE3) pLysS pRSETA::xylT as well as to evade acetate accumulation, the effect of exponential feeding strategy of carbon source on the production of thermostable GI enzyme, cell concentration and acetate formation by recombinant E.coli BL21 (DE3) pLysS was investigated at four sets of fed-batch bioreactor experiments at three different predetermined specific growth rates 0.1 h-1 (M-0.1), 0.15 h-1 (M-0.15), 0.2 h-1 (M-0.2) and a glucose based exponential feeding at specific growth rate of 0.15 h-1(G-0.15) were performed by recombinant E.coli BL21 (DE3) pLysS cells. The highest biomass was obtained in M-0.15 condition as 9.6 kg m−3 at t=32 h and the highest rGI activity was achieved in M-0.1 operation as A=16399 U L-1 at t=32 h of bioprocess. Moreover, peptide ligand with specific affinity toward histidin-tag peptide was selected by phage display technology. Isothermal titration calorimetry and surface plasmon resonance analyses were carried out to determine peptide-peptide interaction properties.


A conducting polymer and a calixarene derivative A novel surface design for glucose detection
Gökoğlan, Ceren; Söylemez, Saniye; Kesik, Melis; Ünay, Hande; Sayın, Serkan; Çırpan, Ali; Yıldız, Hüseyin Bekir; Toppare, Levent Kamil (null; 2016-07-17)
In this study, a novel amperometric glucose biosensor based on a conducting polymer and a calixarene was developed. Conducting polymer of (2‐(2‐oc‐tyldodecyl)‐4,7‐di(selenoph‐2‐yl)‐2H‐benzo[d][1,2,3]triazole)) (SBTz) was used as the immobilization matrix for biomolecule deposition to achieve an effective surface design to detect glucose. After successful deposition of SBTz on graphite electrode, a newly synthesized calixarene and gold nanoparticle (AuNP) mixture were used for improving biosensor character...
Metabological engineering of Pichia Pastoris for extracellular thermostable glucose isomerase production
Ata, Özge; Çalık, Pınar; Özdamar, Tunçer H.; Department of Biotechnology (2012)
The aim of this study is to develop a metabolically engineered P. pastoris strain for production of an active extracellular thermostable glucose isomerase (GI) enzyme by using genetic engineering techniques. For this purpose, research program was performed in two sub-programs. In the first sub-program, xylA gene from Thermus thermophilus was amplified and inserted into pPICZα-A expression vector. Thereafter, this pPICZα-A::xylA vector was cloned into AOX1 locus in P. pastoris genome and expressed under alco...
Immobilization of glucose isomerase in surface-modified alginate gel beads
In this study, glucose isomerase enzyme was entrapped into modified and nonmodified calcium alginate gel beads. Various characteristics of free and immobilized enzymes such as the optimum pH, temperature and dependence of activity on storage and operational stability were evaluated. The optimum pH and temperature of free and immobilized glucose isomerase were found to be the same values as 7.5 and 60C, respectively. For free and immobilized enzymes, kinetic parameters were calculated as 1.79 x 10(-2) and 8....
A novel architecture based on a conducting polymer and calixarene derivative: its synthesis and biosensor construction
GOKOGLAN, Tugba Ceren; SOYLEMEZ, Saniye; KESIK, Melis; UNAY, Hande; Sayin, Serkan; Yildiz, Huseyin Bekir; Çırpan, Ali; Toppare, Levent Kamil (2015-01-01)
In this study, a novel amperometric glucose biosensor based on a selenium comprising conducting polymer and calixarene was developed. Firstly, poly(2-(2-octyldodecyl)-4,7-di(selenoph-2-yl)-2H-benzo[d][1,2,3]-triazole), poly((SBTz)) was electrodeposited onto a graphite electrode by an electropolymerization technique. Then, a newly synthesized calixarene and gold nanoparticle (AuNP) mixture was used for the improvement of biosensor characteristics. GOx, as a model enzyme was immobilized on the modified electr...
GURSEL, I; Hasırcı, Vasıf Nejat (1992-01-01)
In this study, matrix entrapment of the enzyme glucose oxidase was achieved through gamma-irradiation of monomers N-vinyl pyrrolidone, 2-hydroxyethyl methacrylate and their mixture. To test the effect of radiation on entrapment efficiency, retention of activities and properties of the system, duration and temperature were varied. The reusability of the resultant products was tested. It was generally found that inclusion of the hydrophilic monomer N-vinyl pyrrolidone into the matrix increased the water conte...
Citation Formats
V. Angardi, “Bioprocess development for thermostable glucose isomerase production,” Ph.D. - Doctoral Program, Middle East Technical University, 2011.