Expression, purification, and characterization of recombinant nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum

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• 作者：Iddar ; Abdelghani ; Valverde ; Federico ; Serrano ; Aurelio ; Soukri ; Abdelaziz
• 关键词：Clostridium acetobutylicum ; Nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) ; Expression ; Purification
• 刊名：Protein Expression and Purification
• 出版年：2002
• 出版时间：August, 2002
• 年：2002
• 卷：25
• 期：3
• 页码：519-526
• 全文大小：241 K

文摘

Clostridium acetobutylicum gapN was cloned and expressed in Escherichia coli BL-21. The IPTG-induced nonphosphorylating NADP-dependent GAPDH (GAPN) has been purified about 34-fold from E. coli cells and its physical and kinetic properties were investigated. The purification method consisted of a rapid and straightforward procedure involving anion-exchange and hydroxyapatite chromatographies. The purified protein is an homotetrameric of 204kDa exhibiting absolute specificity for NADP. Chromatofocusing analysis showed the presence of only one acidic GAPN isoform with an acid isoelectric point of 4.2. The optimum pH of purified enzyme was 8.2. Studies on the effect of assay temperature on enzyme activity revealed an optimal value of about 65°C with activation energy of 18KJmol⁻¹. The apparent K_m values for NADP and d-glyceraldehyde-3-phophate (d-G3P) or dl-G3P were estimated to be 0.200±0.05 and 0.545±0.1mM, respectively. No inhibition was observed with L-D3P. The V_max of the purified protein was estimated to be 78.8Umg⁻¹. The Cl. acetobutylicum GAPN was markedly inhibited by sulfhydryl-modifying reagent iodoacetamide, these results suggest the participation of essential sulfhydryl groups in the catalytic activity.