Expression, purification and characterization of arabinose-5-phosphate isomerase from Arabidopsis thaliana / 生物工程学报

Arabinose-5-phosphate isomerase (KdsD) is the first key limiting enzyme in the biosynthesis of 3-deoxy-D-manno-octulosonate (KDO). KdsD gene was cloned into prokaryotic expression vector pET-HTT by seamless DNA cloning method and the amount of soluble recombinant protein was expressed in a soluble form in E. coli BL21 (DE3) after induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The target protein was separated and purified by Ni-NTA affinity chromatography and size exclusion chromatography, and its purity was more than 85%. Size exclusion chromatography showed that KdsD protein existed in three forms: polymers, dimmers, and monomers in water solution, different from microbial KdsD enzyme with the four polymers in water solution. Further, the purified protein was identified through Western blotting and MALDI-TOF MASS technology. The results of activity assay showed that the optimum pH and temperature of AtKdsD isomerase activities were 8.0 and 37 ℃, respectively. The enzyme was activated by metal protease inhibitor EDTA (5 mmol/L) and inhibited by some metal ions at lower concentration, especially with Co²⁺ and Cd²⁺ metal ion. Furthermore, when D-arabinose-5-phosphate (A5P) was used as substrate, Km and Vmax of AtKdsD values were 0.16 mmol/L, 0.18 mmol/L·min. The affinity of AtKdsD was higher than KdsD in E. coli combined with substrate. Above results have laid a foundation for the KdsD protein structure and function for its potential industrial application.

Radiorespirometric determination of contribution of pentose phosphate cycle to metabolism of glucose in dermacentor andersoni

The contribution of both pentose phosphate cycle [PPC] and glycolytic-citric acid cycle [GCAC] to metabolism of glucose by different physiological states of Dermacentor andersoni female was evaluated. The radiorespirometric method was used after injection of glucose 3,4-[14]C [G[3,4]] glucose-1-[14]C [G[1]] and glucose-6-[14]C [G[6]] into the hemocoel of separate females of different physiological states and following by the expired [14]CO[2], was trapped for 7 hrs. at 1 hr. intervals. The contribution of PPC to glucose metabolism is 23, 50 and 84% in the unfed, unmated-partly fed, and ovipositing females respectively. Accordingly, the GCAC contribution is 77, 50 and 16% in the same physiological states respectively. The greatly increasing trend in PPC activity from the unfed to the ovipositing state is attributed to increasing need to requisite two-carbon unit intermediates for other metabolic processes. The metabolic products of PPC used in biosynthetic processes associated with feeding and oogenesis is a reliable reason for the active operation of this metabolic pathway

Glycolytic enzymes in juvenile and adult Angiostrongylus cantonensis.

In extracts of adult Angiostrongylus cantonensis, the activities of enzymes including glucokinase, phosphoglucoisomerase, phosphofructokinase, aldolase, triosepho sphate isomerase, glyceraldehydephosphate dehydrogenase, phosphoglycerokinase, phosphoglyceromutase, enolase, pyruvate kinase, lactate dehydrogenase, pyruvate decarboxylase, alcohol dehydrogenase, glucose 6-phosphate dehydrogenase, glycerophosphate dehydrogenase and pyruvate dehydrogenase complex were demonstrated. The present of significant activity of glycerophosphate dehydrogenase and glucose 6-phosphate dehydrogenase may indicate the possibility of an operative of alpha-glycerophosphate and pentose phosphate pathway.