Enzymatic conversion is very important to produce functional rare sugars, but the conversion rate of single enzymes is generally low. To increase the conversion rate, a dual-enzyme coupled reaction system was developed. Dual-enzyme coupled reaction system was constructed using D-psicose-3-epimerase (DPE) and L-rhamnose isomerase (L-RhI), and used to convert D-fructose to D-psicose and D-allose. The ratio of DPE and L-RhI was 1:10 (W/W), and the concentration of DPE was 0.05 mg/mL. The optimum temperature was 60 degrees C and pH was 9.0. When the concentration of D-fructose was 2%, the reaction reached its equilibrium after 10 h, and the yield of D-psicose and D-allose was 5.12 and 2.04 g/L, respectively. Using the dual-enzymes coupled system developed in the current study, we could obtain sugar syrup containing functional rare sugar from fructose-rich raw material, such as high fructose corn syrup.
Aldose-Ketose Isomerases ; metabolism ; Carbohydrate Epimerases ; metabolism ; Fructose ; chemistry ; Glucose ; chemistry ; Hydrogen-Ion Concentration ; Temperature

Rare sugar is a kind of important low-energy monosaccharide that is rarely found in nature and difficult to synthesize chemically. D-allose, a six-carbon aldose, is an important rare sugar with unique physiological functions. It is radical scavenging active and can inhibit cancer cell proliferation. To obtain D-allose, the microorganisms deriving D-psicose 3-epimerase (DPE) and L-rhamnose isomerase (L-RhI) have drawn intense attention. In this paper, DPE from Clostridium cellulolyticum H10 was cloned and expressed in Bacillus subtilis, and L-RhI from Bacillus subtilis 168 was cloned and expressed in Escherichia coli BL21 (DE3). The obtained crude DPE and L-RhI were then purified through a HisTrap HP affinity chromatography column and an anion-exchange chromatography column. The purified DPE and L-RhI were employed for the production of rare sugars at last, in which DPE catalyzed D-fructose into D-psicose while L-RhI converted D-psicose into D-allose. The conversion of D-fructose into D-psicose by DPE was 27.34%, and the conversion of D-psicose into D-allose was 34.64%.
Aldose-Ketose Isomerases ; metabolism ; Bacillus subtilis ; enzymology ; Carbohydrate Epimerases ; metabolism ; Clostridium cellulolyticum ; enzymology ; Escherichia coli ; metabolism ; Fructose ; metabolism ; Glucose ; metabolism

Using the large scale sequencing, a novel human cDNA of 1,645 bp was screened from the cDNA library of human fetal brain. The cDNA contains an ORF encoding a 296-aa protein with a calculated molecular weight of 34.0 KD. Compared with that of current sequence databases, the putative protein was found to have 36% homology with protein-disulfide isomerase (PDI). So this cDNA was named PDI like (PDI-L) gene. Multiple tissue Northern blot analysis shows that PDI-L cDNA is expressed in heart, brain, liver, kidneys and so on. The ORF fragment of PDI-L cDNA was inserted into reconstructed pBV220 expression vehicle and its predicted expressive protein was obtained.
Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Gene Library ; Humans ; Protein Disulfide-Isomerases ; biosynthesis ; genetics

Taxol is one of the most potent anti-cancer agents, which is extracted from the plants of Taxus species. Isopentenyl diphosphate isomerase (IPI) catalyzes the reversible transformation between IPP and DMAPP, both of which are the general 5-carbon precursors for taxol biosynthesis. In the present study, a new gene encoding IPI was cloned from Taxus media (namely TmIPI with the GenBank Accession Number KP970677) for the first time. The full-length cDNA of TmIPI was 1 232 bps encoding a polypeptide with 233 amino acids, in which the conserved domain Nudix was found. Bioinformatic analysis indicated that the sequence of TmIPI was highly similar to those of other plant IPI proteins, and the phylogenetic analysis showed that there were two clades of plant IPI proteins, including IPIs of angiosperm plants and IPIs of gymnosperm plants. TmIPI belonged to the clade of gymnosperm plant IPIs, and this was consistent with the fact that Taxus media is a plant species of gymnosperm. Southern blotting analysis demonstrated that there was a gene family of IPI in Taxus media. Finally, functional verification was applied to identify the function of TmIPI. The results showed that biosynthesis of β-carotenoid was enhanced by overexpressing TmIPI in the engineered E. coli strain, and this suggested that TmIPI might be a key gene involved in isoprenoid/terpenoid biosynthesis.
Amino Acid Sequence ; Carbon-Carbon Double Bond Isomerases ; genetics ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; Paclitaxel ; biosynthesis ; Phylogeny ; Plant Proteins ; genetics ; Taxus ; enzymology ; genetics

The rhizome of Alpinia officinarum is a widely used Chinese herbal medicine. The essential oil in A. officinarum rhizome is mainly composed of 1, 8-cineole and other monoterpenes, as the major bioactive ingredients. In plants, monoterpenes are synthesized through the methylerythritol phosphate (MEP) pathway in the plastids, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is an enzyme catalyzing a committed step of the MEP pathway. In the present study, the full-length cDNA encoding DXR was cloned from the rhizome of A. officinarum, using homology-based RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The new cDNA was designated as AoDXR and submitted to GenBank to be assigned with an accession number HQ874658. The full-length cDNA of AoDXR was 1 670 bp containing a 1 419 bp open reading frame encoding a polypeptide of 472 amino acids with a calculated molecular mass of 51.48 kDa and an isoelectric point of 6.15. Bioinformatic analyses revealed that AoDXR showed extensive homology with DXRs from other plant species and contained a conserved plastids transit peptide, a Pro-rich region and two highly conserved NADPH-binding motifs in its N-terminal region characterized by all plant DXRs. The phylogenetic analysis revealed that AoDXR belonged to angiosperm DXRs. The structural modeling of AoDXR showed that AoDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that AoDXR expressed strongly in leaves, weak in rhizomes of A. officinarum. Exogenous methyl jasmonate (MeJA) could enhance the expression of AoDXR and the production of 1, 8-cineole in A. officinarum rhizomes. The cloning and characterization of AoDXR will be helpful to reveal the molecular regulation mechanism of monoterpene biosynthesis in A. officinarum and provides a candidate gene for metabolic engineering in improving the medicinal quality of A. officinarum rhizome.
Aldose-Ketose Isomerases ; genetics ; Alpinia ; chemistry ; enzymology ; genetics ; Amino Acid Sequence ; DNA, Complementary ; genetics ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Plant ; Molecular Sequence Data ; Monoterpenes ; metabolism ; Phylogeny

Disulfide bond formation protein A, DsbA, is one of the important proteins located in E. coli periplasm, which is a foldase facilitating the folding of nascent secreted proteins, especially for those with many pairs of disulfide bonds. The crystal structure and phylogenetic analysis of DsbA and DsbA-mediated protein folding, alternatively in vivo and in vitro, are summarized. Both the extremely low pKa of Cys30 , about 3.5, and the destabilizing effect of the active site disulfide contribute to its strong oxidizing power. The Cys30 is also considered as the most important residue closely related to its activity using site-directed mutagenesis methodology. DsbA could effectively assist proteins folding, both in vivo coexpressed with the target protein, and in vitro replenished as foldases. Moreover, DsbA also has the chaperone-like activity in the assistant refolding of genetically engineered inclusion bodies.
Amino Acid Sequence ; Disulfides ; chemistry ; metabolism ; Escherichia coli ; enzymology ; genetics ; Escherichia coli Proteins ; chemistry ; classification ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Phylogeny ; Protein Disulfide-Isomerases ; chemistry ; classification ; metabolism ; Protein Folding ; Protein Structure, Tertiary ; Sequence Homology, Amino Acid

Disulfide bond-forming (Dsb) protein is a bacterial periplasmic protein that is essential for the correct folding and disulfide bond formation of secreted or cell wallassociated proteins. DsbA introduces disulfide bonds into folding proteins, and is re-oxidized through interaction with its redox partner DsbB. Mycobacterium tuberculosis, a Gram-positive bacterium, expresses a DsbA-like protein ( Rv2969c), an extracellular protein that has its Nterminus anchored in the cell membrane. Since Rv2969c is an essential gene, crucial for disulfide bond formation, research of DsbA may provide a target of a new class of anti-bacterial drugs for treatment of M.tuberculosis infection. In the present work, the crystal structures of the extracellular region of Rv2969c (Mtb DsbA) were determined in both its reduced and oxidized states. The overall structure of Mtb DsbA can be divided into two domains: a classical thioredoxin-like domain with a typical CXXC active site, and an α-helical domain. It largely resembles its Escherichia coli homologue EcDsbA, however, it possesses a truncated binding groove; in addition, its active site is surrounded by an acidic, rather than hydrophobic surface. In our oxidoreductase activity assay, Mtb DsbA exhibited a different substrate specificity when compared to EcDsbA. Moreover, structural analysis revealed a second disulfide bond in Mtb DsbA, which is rare in the previously reported DsbA structures, and is assumed to contribute to the overall stability of Mtb DsbA. To investigate the disulphide formation pathway in M.tuberculosis, we modeled Mtb Vitamin K epoxide reductase (Mtb VKOR), a binding partner of Mtb DsbA, to Mtb DsbA.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; metabolism ; Catalytic Domain ; Crystallography, X-Ray ; Disulfides ; chemistry ; Escherichia coli ; metabolism ; Escherichia coli Proteins ; chemistry ; metabolism ; Molecular Docking Simulation ; Molecular Sequence Data ; Mycobacterium tuberculosis ; metabolism ; Oxidation-Reduction ; Protein Disulfide-Isomerases ; chemistry ; metabolism ; Protein Folding ; Protein Structure, Tertiary ; Sequence Alignment ; Static Electricity

BACKGROUND: Topoisomerase II (TOPO II) is an enzyme that separates intertwined chromosomes during DNA synthesis by transiently breaking and joining DNA strands. The level of TOP II is one of the determinants of cellular sensitivity to anti-tumor drugs in non-Hodgkin's lymphoma patients. The alpha form of TOPO II has been recently used as a marker of cellular proliferation. High levels of TOPO IIalpha are expressed in aggressive and proliferative tumors. METHODS: This study was designed to evaluate the relationship between TOPO IIalpha expression and clinicopathological parameters including age, gender, the serum LDH level, the serum beta2-microglobulin level and stage, or expressions, of Ki-67, p53 and p27, in non-Hodgkin's lymphoma. We analyzed forty-one biopsied tissue specimens from patients with non-Hodgkin's lymphoma. RESULTS: The expression of TOPO IIalpha increased with the clinical stage and it was correlated with Ki-67 and p53 expressions. However, TOPO IIalpha expression did not have any significant correlation with age, gender, the serum LDH level, the serum 2-microglobulin level and the p27 expression. CONCLUSIONS: TOPO IIalpha expression is a useful marker of cellular proliferation and it may serve as a prognostic factor of a tumor's progression and aggressiveness in non-Hodgkin's lymphomas.
Cell Proliferation ; DNA Topoisomerases, Type I* ; DNA Topoisomerases, Type II ; DNA* ; Humans ; Ki-67 Antigen ; Lymphoma, Non-Hodgkin*

Taxa-4(5),11(12)-diene is the precursor for paclitaxel biosynthesis. The diterpenoid paclitaxel (marketed as Taxol), a plant secondary metabolite isolated from yew, is an effective drug widely used in the treatment of numerous cancers. However, further application of taxol has been restricted due to its low yield in plants and the difficulties in extraction. To increase the intact isoprene flux, we constructed the fusion gene plasmid pBgGGTS and individual cassette plasmid pBgGGgTS to enhance the expression levels of geranylgeranyl diphosphate synthase gene (ggpps) and a taxadiene synthase gene (ts) in Coprinopsis cinerea. These two plasmids were separately transformed into C. cinerea LT2 strain, resulting in several putative transformants. Putative transformants were determined by PCR technique, indicating that 5 out of 13 putative transformants transformed by pBgGGTS and 6 out of 13 putative transformants transformed by pBgGGgTS, respectively. Additionally, the Southern blotting analysis of these 10 transformants confirmed that both ggpps and ts gene were stably integrated into the genome of C. cinerea. Crude extracts from each of the transformants were analyzed. There is no difference in the mycelium extracts among the wild-type LT2 and two types of transformants. However, analysis of culture filtrates indicated that an additional GC peak was found at the retention time of 16.762 min which was absent in the wild type control. The mass fragmentation pattern of this peak had the same diagnostic ions with taxa-4(5),11(12)-diene. According to peak area, the amounts of taxa-4(5),11(12)-diene in each fermented broth were 44 ng/L (transformed with pBgGGgTS) and 30 ng/L (transformed with pBgGGTS), respectively. In conclusion, co-expression of the ggpps and ts gene could increase the taxadiene production in C. cinerea.
Agaricales ; metabolism ; Alkenes ; metabolism ; Diterpenes ; metabolism ; Farnesyltranstransferase ; genetics ; metabolism ; Genetic Engineering ; Isomerases ; genetics ; metabolism ; Paclitaxel ; Plasmids

The shuttle expression vectors pHZGI1 and pHZGI2 were successfully constructed by inserting structural genes of GI containing single mutated site G138P and double mutated site G138P-G247D into E. coli-Streptomyces shuttle vector pHZ-1272, respectively. Then they were transformed into S. lividans TK54 strain by protoplast transformation. SDS-PAGE indicated that two shuttle vectors in TK54 strain expressed obviously specific bands at 42.5 kD after inducted by 2 micrograms/mL thiostrepton. Optical densitometric scan showed that the content of the mutant enzymes GIG138P and GIG138P-G247D were about 19% and 22% of dissoluble proteins, respectively. Western blotting farther proved that GIG138P and GIG138P-G247D were expressed in S. lividans TK54.
Aldose-Ketose Isomerases ; analysis ; genetics ; Blotting, Western ; Genetic Vectors ; Mutation ; Streptomyces ; genetics