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 pPICZ&alpha / -A expression vector. Thereafter, this pPICZ&alpha / -A::xylA vector was cloned into AOX1 locus in P. pastoris genome and expressed under alcohol oxidase promoter which is induced by methanol. After constructing the recombinant P. pastoris strains, the best producing strain was selected according to the specific enzyme activity assay and SDS-PAGE analyses in batch shaker-bioreactor experiments. The selected recombinant P. pastoris clone carrying xylA gene in its genome was named as eP20. Using recombinant P. pastoris eP20 clone, effects of salt and sorbitol concentration on the cell growth and recombinant GI activity were investigated. The data obtained from the experiments showed that the maximum cell and GI activity values were obtained in production medium that contained 30 g L-1 sorbitol, 4.35 g L-1 ammonium sulphate, 0.1 M potassium phosphate buffer (pH 6.0), 14.9 g L-1 MgSO4&bull / 7H2O, 1.17 g L-1 CaSO4 &bull / 2H2O, 1 ml L-1 chloramphenicol and 4.35 ml L-1 PTM1 / where, the maximum biomass and recombinant GI activity were calculated , respectively, as 6.3 g L-1 and 3.21 U L-1. Moreover, the research program related with the effect of initial sorbitol concentration shows that optimum initial sorbitol concentration, CS0 is 50 g L-1 that resulted a cell concentration and recombinant GI activity which are 7.32 g L-1 and 3.6 U L-1, respectively. In the second part of the M.Sc. of the study, a pilot scale bioreactor experiment in a working volume of 1 L was performed in controlled bioreactor system. The variations in the cell growth, recombinant GI activity, AOX activity, total protease activity and organic acid concentrations throughout the fermentation were analyzed whereas the specific growth rates, yield coefficients and specific consumption rates were also calculated. The results showed that a pH and oxygen controlled operation enabled an important increase in recombinant GI activity. In this context, recombinant GI activity was increased as 56.1-fold and resulted in 202.8 U L-1 at t=12 whereas the maximum biomass concentration was obtained as 85.2 g L-1 at t=36. In this study, an active thermostable recombinant GI enzyme was produced extracellularly by a yeast cell, i.e. recombinant P. pastoris, for the first time.

Structural biology is a branch of science related to biochemistry, biophysics, and molecular biology that deals with the molecular structures of biological macromolecules, in particular nucleic acids and proteins. Structure-guided drug design uses three-dimensional knowledge of protein structures to design small molecules which block the action of specific proteins. When crystals of theses macromolecules and their complexes can be obtained, their crystal structures can be determined by using isomorphous differences between a native structure and a derivative structure. This allows crystallographers to determine the coordinates of a small number of heavy atoms which provide initial phases for macromolecules. The advent of synchrotron radiation allowed determination of a heavy atom substructure by use of anomalous differences using either multiple wavelengths (MAD) or a single wavelength (SAD); the latter has become the most common phasing method in crystallography and is the method used in this study. The use of SeMet has been by far the most successful method employed in SAD. However, in some cases production of SeMet proteins is not possible thus necessitating additional options, for example, xenon. Noble gases such as xenon may be used in SAD experiments by binding to various, non-specific sites. Advances in noble gas pressurization systems like the Hampton Research Xenon Chamber have greatly eased the production of noble gas derivatives, xenon itself being a prime candidate with a very strong anomalous signal when compared to lighter noble gases like krypton and argon. Investigation of the phasing properties of xenon was carried out on test proteins hen egg white lysozyme (HEWL), thermolysin, glucose isomerase, and thaumatin II. Phases were successfully determined for all four proteins including thaumatin II which did not bind xenon but was successful due to the anomalous signal from 17 native sulfurs. The three remaining proteins showed varying occupancies and numbers of sites including xenon sites in thermolysin and glucose isomerase which have not been observed previously. This document will serve as a guide for the preparation of xenon derivative crystals and provides a strategy for the collection and processing of data from xenon derivatives.

In this study, the effects of bioprocess parameters on glucose isomerase roduction by Bacillus thermoantarcticus (DSMZ 9572) were investigated. For his purpose, firstly, in laboratory scale shake-bioreactors, a defined medium was designed in terms of its carbon and nitrogen sources, to achieve the highest glucose isomerase activity. Optimum concentrations of medium components were determined by a statistical approach, namely Response Surface Methodology (RSM). RSM defined the relationship between the response, glucose isomerase activity and the independent variables, medium components. The highest glucose isomerase volumetric activity was obtained as 1630 U L-1 in the optimized medium containing 10.64 kg m-3 xylan, 5.66 kg m-3 yeast extract, 5.92 kg m-3 (NH4)2SO4, 0.25 kg m-3 MgSO4.7H2O, 0.001 kg m-3 FeSO4.7H2O, 0.001 kg m-3 ZnSO4.7H2O , 0.000075 kg m-3 MnSO4. H2O, and 0.00001 kg m-3 CuSO4.5H2O at conditions: pH0= 6.0, T=55&deg / C, N=200 min-1. Then by using the designed medium, pH and oxygen transfer conditions of the bioprocess were investigated in 3.0 dm3 pilot scale bioreactor. The effect of pH was analyzed at pH=6 uncontrolled and controlled operations with the following conditions: air inlet rate of Q0/VR=0.5 vvm and agitation rate of N=500 min-1. The effects of oxygen transfer parameters were examined, at pH=6 controlled condition, at an air inlet rate of Q0/VR=0.5 vvm and the agitation rates of N=300,500,750 min-1. The variations in glucose isomerase activity, cell, amino acid and organic acid concentrations with the cell cultivation time, specific cell growth rate, the oxygen uptake rate, the liquid phase coefficient by using the dynamic method, maintenance coefficient for oxygen and yield coefficients were determined. The highest glucose isomerase volumetric activity and cell concentration, 1838 U L-1 and 2.26 kg m-3 at the 24th of the cell cultivation were attained at 0.5 vvm, 500 min 1 and pH=6 uncontrolled operation.

ABSTRACT Covalent Immobilization of Glucose Isomerase on Poly (2-hydroxyethyl methacrylate) Particles Yildiz, Hakan &Uuml / mit M.S., Department of Chemistry Supervisor: Prof. Dr. Nesrin Hasirci July 2004, 54 pages In this study, poly (2-hydroxyethyl methacrylate), P(HEMA), particles were prepared by suspension polymerization of the monomer 2-hydroxyethyl methacrylate with addition of ethylene glycol dimethyacrylate, EGDMA, as cross linker. Glucose isomerase, GI, enzyme was covalently immobilized on the prepared P(HEMA) particles after activation of the particles with cyanuric chloride. The activities of the free and immobilized enzymes were measured with Ethanol-Carbazole method. The immobilization of GI on P(HEMA) particles promoted enzyme stability and as a result, the enzyme became more stable to temperature, storage, and reuse. For maximum substrate conversion, optimum temperature was determined as 70 oC for free GI and this value shifted to 60 oC for immobilized enzyme. Optimum pH for maximum substrate conversion was found to be 7.0 for free GI and 8.0 for immobilized GI. The change of enzyme activity with substrate concentration were determined to calculate Km and Vmax values of the free and immobilized enzymes. Km values were found to be 1.7x10-2 mol/L and 3.1x10-1 mol/L while Vmax values were 1.01x10-4 mol/L.min, 1.65x10-3 mol/L.min for free and immobilized GI, respectively. Reuse capability of immobilized GI on P(HEMA) particles was measured and compared with commercial GI. Both systems retained 80 % of their original activities after 40th use, within 6 days. The change of enzyme activities upon storage were detected at certain time intervals for the samples stored in buffer solution at 4 oC. Immobilized enzyme was retained 60% of its original activitiy in 60 days of storage at 4 oC. Immobilized GI and commercial GI both retained 90% of their activities under continuous flow after 180 mL of substrate solution passed through the column.

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&minus / 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.