Effect of pH on the Stability of Hexokinase and Glucose 6-Phosphate Dehydrogenase

Abstract

Hexokinase (HK) and glucose 6-phosphate dehydrogenase (G6PDH) are important enzymes used in biochemical studies and in analytical methods. The stability of the enzymes can be affected by several variables, pH being one of them. The effect of pH on the stability of HK and G6PDH was evaluated in this work. Baker's yeast cells were suspended in 50 mM Tris-HCl buffer (pH 7.5) containing 5.0 mM MgCl2, and submitted to disruption by agitation with glass beads and in the presence of protease inhibitors. The cell-free extract was obtained by centrifugation (2880g; 10 min), followed by dilution into the buffers: 0.1 M acetate-acetic acid (pH: 4.0, 4.5, 5.0, or 5.5), 0.1 M phosphate buffer (pH: 6.0, 6.5, or 7.0), and 0.1 M Tris-HCl buffer (pH: 7.5, 8.0, 8.5, 9.0 or 9.5). The residual activity of HK and G6PDH, expressed as micromol of NADPH formed per min, were measured through a period of buffer-enzyme contact from 0 to 51 h at 4 degrees C. It was observed that up to 4 h both enzymes were stable in all buffers used. However, after 51 h HK was stable at pH 6.0 and 7.5, whereas G6PDH was stable at pH 7.0, 9.5, and between 4.5 and 5.5.

Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production

“…The glucose-6-phosphate dehydrogenase assay was performed according to Souza et al (2002). Briefly, cells were mechanically disrupted with glass beads, and the cell extracts were added to the reaction buffer (100 mM Tris–HCl pH=7.5) containing 3 mM MgCl 2 , 0.2 mM NADP + and 4 mM glucose-6-phosphate.…”

Process Integration for the Disruption of Candida guilliermondii Cultivated in Rice Straw Hydrolysate and Recovery of Glucose-6-Phosphate Dehydrogenase by Aqueous Two-Phase Systems

“…In general, it is desirable that partition coefficients of total proteins and target enzyme present different values, which could suggest that purification was reached. K value above 1.0 denotes that proteins and enzyme are preferably distributed to the upper phase [24]. As can be seen in Table 1, estimated values of K to G6PD (K E ) from clarified homogenate, were lower than those of proteins (K P ), although both were favorably transferred to the salt-enriched phase (bottom phase), leading to a decrease in partition coefficient.…”

“…The development of a purification procedure requires the variation of factors to get the optimum intracellular protein recovery, namely polymer concentration, polymer molar mass, pH, and temperature system. Other authors analyzed stability of G6PD, and their results allowed setting the parameters temperature and pH system in 25°C and 7.5, respectively [5,24]. Therefore, we used a 2 2 central composite design to identify which ATPS composition (independent variables such as M PEG and TLL) is able to maximize G6PD purification factor and yield in the bottom phase (PF B , Y B ) of integrated process (Table 2).…”

Purificação da enzima glicose-6-fosfato desidrogenase por processo de extração líquido-líquido em sistemas aquosos bifásicos integrado ao rompimento celular de Candida guilliermondii

“…No entanto, a purificação dessa enzima requer como etapa inicial, o rompimento das células para a sua liberação. Dentre as diversas técnicas empregadas destacam-se: o rompimento mecânico com esferas de vidro (ABRAHÃO-NETO et al, 1997;RICCI-SILVA et al, 2000;OLIVEIRA et al, 2001;SOUZA et al, 2002) (MORELLI et al, 1978;LEVY, 1979). Yüregir et.…”

“…Os valores dos volumes das fases, das atividades enzimáticas e dos teores de proteínas totais nas fases obtidas foram utilizados para avaliar a eficiência do processo de SDFA. Esse estudo foi realizado a temperatura de 25°C em banho-maria, pois foi mostrado em trabalho anterior que durante 8 horas a essa temperatura e pH 7,5, na presença de inibidores de proteases, não há desnaturação da G6PD(SOUZA et al, 2002). foram retiradas para análises posteriores.…”