Discovery and biochemical characterization of two hexokinases from Crassostrea gigas.

Hexokinases (HKs) play a vital role in glucose metabolism, which controls the first committed step catalyzing the production of glucose-6-phosphate from glucose. Two HKs (CGIHK1 and CGIHK2) from the Pacific oyster Crassostrea giga were cloned and characterized. CGIHK1 and CGIHK2 were recombinantly expressed in Escherichia coli and successfully purified by the Ni-NTA column. The optimum pH of the two enzymes was pH 8.0 and 8.5, respectively. The optimum temperature of the two enzymes was 42 °C and 50 °C, respectively. Both enzymes showed a clear requirement for divalent magnesium and were strongly inhibited by SDS. CGIHK1 exhibited highly strict substrate specificity to glucose, while CGIHK2 could also catalyze other 11 monosaccharide substrates. This is the first report on the in vitro biosynthesis of glucose-6-phosphate by the hexokinases from Crassostrea gigas. The facile expression and purification procedures combined with different substrate specificities make CGIHK1 and CGIHK2 candidates for the biosynthesis of glucose-6-phosphate and other sugar-phosphates.

Optimization of the UDP-Xyl biocatalytic synthesis from Crassostrea gigas by orthogonal design method.

UDP-Xyl, a nucleotide sugar involved in the biosynthesis of various glycoconjugates, is difficult to obtain and quite expensive. Biocatalysis using a one-pot multi-enzyme cascade is one of the most valuable biotransformation processes widely used in the industry. Herein, two enzymes, UDP-glucose (UDP-Glc) dehydrogenase (CGIUGD) and UDP-Xyl synthase (CGIUXS) from the Pacific oyster Crassostrea gigas, which are coupled together for the biotransformation of UDP-Xyl, were characterized. The optimum pH was determined to be pH 9.0 for CGIUGD and pH 7.5 for CGIUXS. Both enzymes showed the highest activity at 37 °C. Neither enzyme is metal ion-dependent. On this basis, a single factor and orthogonal test were applied to optimize the condition of biotransformation of UDP-Xyl from UDP-Glc. Orthogonal design L9 (33) was conducted to optimize processing variables of enzyme amount, pH, and temperature. The conversion of UDP-Xyl was selected as an analysis indicator. Optimum variables were the ratio of CGIUGD to CGIUXS of 2:5, enzymatic pH of 8.0, and temperature of 37 °C, which is confirmed by three repeated validation experiments. The UDP-Xyl conversion was 69.921% in a 1 mL reaction mixture by optimized condition for 1 h. This is the first report for the biosynthesis of UDP-Xyl from oyster enzymes.

Improving microwave-assisted hydrothermal degradation of PCDD/Fs in fly ash with added Na2HPO4 and water-washing pretreatment.

In this work, microwave-assisted hydrothermal process is applied to the PCDD/F degradation of municipal solid waste incineration (MSWI) fly ash. The effects of water-washing pretreatment and the Na2HPO4 reagent on the degradation efficiency of PCDD/Fs are investigated. The PCDD/F content in MSWI fly ash is detected by high-resolution gas chromatography-mass spectrometry (HRGC/MS). The experimental results show that the efficiency of total PCDD/F degradation increases from 60.6% to 83.3% after water-washing pretreatment, with 5 wt % Na2HPO4 added for 2 h of microwave heating at 220 °C. With more Na2HPO4 (10 wt %), the degradation efficiency of PCDD/Fs reaches 91.8%, and remarkably, the WHO-TEQ is as low as 0.255 ng g-1. Analysis of the degradation pathway of PCDD/Fs indicates that a chlorination reaction happens during the microwave-assisted hydrothermal process, as do dechlorination and destruction reactions. Water-washing effectively weakens the chlorination reaction for the decrease of chlorine in fly ash, which contributes to PCDD/F degradation. The reagent Na2HPO4 has a greater effect on the dechlorination of high-chlorinated congeners. Furthermore, the results indicate that the removal efficiency of PCDDs is higher than that of PCDFs under microwave conditions. Several linear correlations between indicative congener content and I/WHO-TEQ values are summarized. Overall, microwave-assisted hydrothermal process is a promising disposal method and should receive further study for large-scale application.

UDP-Glucose 4-Epimerase and ß-1,4-Galactosyltransferase from the Oyster Magallana gigas as Valuable Biocatalysts for the Production of Galactosylated Products.

Uridine diphosphate galactose (UDP-galactose) is a valuable building block in the enzymatic synthesis of galactose-containing glycoconjugates. UDP-glucose 4-epimerase (UGE) is an enzyme which catalyzes the reversible conversion of abundantly available UDP-glucose to UDP-galactose. Herein, we described the cloning, expression, purification, and biochemical characterization of an unstudied UGE from the oyster Magallana gigas (MgUGE). Activity tests of recombinantly expressed MgUGE, using HPLC (high-performance liquid chromatography), mass spectrometry, and photometric assays, showed an optimal temperature of 16 °C, and reasonable thermal stability up to 37 °C. No metal ions were required for enzymatic activity. The simple nickel-affinity-purification procedure makes MgUGE a valuable biocatalyst for the synthesis of UDP-galactose from UDP-glucose. The biosynthetic potential of MgUGE was further exemplified in a coupled enzymatic reaction with an oyster-derived ß-1,4-galactosyltransferase (MgGalT7), allowing the galactosylation of the model substrate para-nitrophenol xylose (pNP-xylose) using UDP-glucose as the starting material.

Discovery and Biochemical Characterization of the UDP-Xylose Biosynthesis Pathway in Sphaerobacter thermophilus.

The biosynthesis of UDP-xylose requires the stepwise oxidation/ decarboxylation of UDP-glucose, which is catalyzed by the enzymes UDPglucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS). UDPxylose biosynthesis is ubiquitous in animals and plants. However, only a few UGD and UXS isoforms of bacterial origin have thus far been biochemically characterized. Sphaerobacter thermophilus DSM 20745 is a bacterium isolated from heated sewage sludge, and therefore can be a valuable source of thermostable enzymes of biotechnological interest. However, no biochemical characterizations of any S. thermophilus enzymes have yet been reported. Herein, we describe the cloning and characterization of putative UGD (StUGD) and UXS (StUXS) isoforms from this organism. HPLC- and plate reader-based activity tests of the recombinantly expressed StUGD and StUXS showed that they are indeed active enzymes. Both StUGD and StUXS showed a temperature optimum of 70°C, and a reasonable thermal stability up to 60°C. No metal ions were required for enzymatic activities. StUGD had a higher pH optimum than StUXS. The simple purification procedures and the thermotolerance of StUGD and StUXS make them valuable biocatalysts for the synthesis of UDP-glucuronic acid and UDP-xylose at elevated temperatures. The biosynthetic potential of StUGD was further exemplified in a coupled enzymatic reaction with an UDP-glucuronosyltransferase, allowing the glucuronylation of the natural model substrate bilirubin.