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.
Aldose-Ketose Isomerases ; biosynthesis ; Arabidopsis ; enzymology ; Arabidopsis Proteins ; biosynthesis ; Cloning, Molecular ; Escherichia coli ; metabolism ; Metals ; Pentosephosphates ; Recombinant Proteins ; biosynthesis

L-Arabinose isomerase (L-AI) is an intracellular enzyme that catalyzes the reversible isomerization of D-galactose and D-tagatose. Given the widespread use of D-tagatose in the food industry, food-grade microorganisms and the derivation of L-AI for the production of D-tagatose is gaining increased attention. In the current study, food-grade strains from different foods that can convert D-galactose to D-tagatose were screened. According to physiological, biochemical, and 16S rDNA gene analyses, the selected strain was found to share 99% identity with Pediococcus pentosaceus, and was named as Pediococcus pentosaceus PC-5. The araA gene encoding L-AI from Pediococcus pentosaceus PC-5 was cloned and overexpressed in E. coli BL21. The yield of D-tagatose using D-galactose as the substrate catalyzed by the crude enzyme in the presence of Mn2+ was found to be 33% at 40 degrees C.
Aldose-Ketose Isomerases ; biosynthesis ; genetics ; Biotransformation ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Galactose ; metabolism ; Genetic Vectors ; genetics ; Hexoses ; metabolism ; Pediococcus ; classification ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; genetics

With the development of low-carbon economy and renewable resource, fermentation of the pentose sugar xylose to produce ethanol becomes a very hot topic. The recombinant Saccharomyces cerevisiae can be constructed by expressing heterologous xylose isomerase (XI). Because Thermus thermophilus XI (TthXI) does not need cofactor, it has been developed for establishing the utilization pathway of xylose in S. cerevisiae. In this article, we reviewed the progress on xylose isomerase. We first introduced the primary properties, sequence and structure characters of xylose isomerase, and discussed its thermostability. The molecular modification of xylose isomerase, including of substrate specificity and thermostability were discussed in detail. Meanwhile, combined with our own research, we also discussed how to improve the xylose isomerase activity at room temperature. Finally, we suggested perspectives of xylose isomerase.
Aldose-Ketose Isomerases ; chemistry ; genetics ; metabolism ; Catalysis ; Enzyme Stability ; Hot Temperature ; Recombinant Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Substrate Specificity

Acanthamoeba cysts are resistant to unfavorable physiological conditions and various disinfectants. Acanthamoeba cysts have 2 walls containing various sugar moieties, and in particular, one third of the inner wall is composed of cellulose. In this study, it has been shown that down-regulation of cellulose synthase by small interfering RNA (siRNA) significantly inhibits the formation of mature Acanthamoeba castellanii cysts. Calcofluor white staining and transmission electron microscopy revealed that siRNA transfected amoeba failed to form an inner wall during encystation and thus are likely to be more vulnerable. In addition, the expression of xylose isomerase, which is involved in cyst wall formation, was not altered in cellulose synthase down-regulated amoeba, indicating that cellulose synthase is a crucial factor for inner wall formation by Acanthamoeba during encystation.
Acanthamoeba castellanii/*enzymology/genetics/metabolism ; Aldose-Ketose Isomerases/*biosynthesis ; Amebiasis/*pathology ; Benzenesulfonates ; Cell Wall/chemistry/genetics/*metabolism ; Cellulose/biosynthesis ; Down-Regulation ; Encephalitis/parasitology ; Glucosyltransferases/*biosynthesis/genetics ; Keratitis/parasitology ; Microscopy, Electron, Transmission ; RNA Interference ; RNA, Small Interfering

Our previous research indicated that the Streptomyces pactum Act12 (Act12) had a certain promotional effect on tanshinone accumulation and up-regulated the expression of genes 3-hydroxy-3-methyglutaryl-CoA reductase (HMGR) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) in Salvia miltiorrhiza hairy roots. This study focuses on the roles of reactive oxygen species in S. pactum Act12-induced tanshinone production in S. miltiorrhiza hairy roots. The 4% Act12, 4% Act12 + CAT and 4% Act12 + SOD were added to S. miltiorrhiza hairy root and subcultured for 21 days, the dry weight, contents of reactive oxygen species, contents of tanshinones and expression of HMGR and DXR were determined at different harvest-time. The generation of reactive oxygen species (ROS) in S. miltiorrhiza hairy roots was triggered by 4% Act12 treatment. The relative expressions of genes HMGR and DXR in 4% Act12 treatment were 32.4 and 4.8-fold higher than those in the control. And the total tanshinone in the hairy roots was 10.2 times higher than that of the control. The CAT and SOD could significantly inhibit the ROS accumulation and relative expressions of genes HMGR and DXR in 4% Act12 treatment, which induced the total tanshinone content was decreased by 74.6% comparing with the 4% Act12 treatment. ROS mediated Act12-induced tanshinone production. The Act12 may be via the ROS signal channel to activate the tanshinone biosynthesis pathways. Thereby the tanshinon content in hairy roots was increased.
Aldose-Ketose Isomerases ; genetics ; metabolism ; Diterpenes, Abietane ; biosynthesis ; Plant Proteins ; genetics ; metabolism ; Plant Roots ; enzymology ; genetics ; metabolism ; microbiology ; Reactive Oxygen Species ; metabolism ; Salvia miltiorrhiza ; enzymology ; genetics ; metabolism ; microbiology ; Secondary Metabolism ; Streptomyces ; physiology