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An all-inclusive Community-Based Frequency Study Nocturia throughout Hong Kong Male Grownups.
5 for guaiacol which is at the acidic end of laccases isolated from white rot fungi. The determined KM values were low while kcat values measured at acidic conditions were comparable to those reported for other laccases from white rot fungi. While both enzymes showed a moderate decrease in activity in the presence of oxalic and citric acid FpLcc2 was activated by acetic acid up to 3.7 times. This activation effect is much more pronounced at pH 5.0 compared to pH 3.0 and could already be observed at a concentration of 1 mM acetic acid.Most of the presently known β-glucosidases are sensitive to end-product inhibition by glucose, restricting their potential use in many industrial applications. Identification of novel glucose tolerant β-glucosidase can prove a pivotal solution to eliminate end-product inhibition and enhance the overall lignocellulosic saccharification process. In this study, a novel gene encoding β-glucosidase BglNB11 of 1405bp was identified in the genome of Saccharomonospora sp. NB11 and was successfully cloned and heterologously expressed in E. coli BL21 (DE3).The presence of conserved amino acids; NEPW and TENG indicated that BglNB11 belonged to GH1 β-glucosidases. The recombinant enzyme was purified using a Ni-NTA column, with the molecular mass of 51 kDa, using SDS-PAGE analysis. BglNB11 showed optimum activity at 40 °C and pH 7 and did not require any tested co-factors for activation. The kinetic values, Km, Vmax, kcat, and kcat/Km of purified enzyme were 0.4037 mM, 5735.8 μmol/min/mg, 5042.16 s-1 and 12487.71 s-1 mM-1, respectively. The enzyme was not inhibited by glucose to a concentration of 4 M but was slightly stimulated in the presence of glucose. Molecular docking of BglNB11 with glucose suggested that the relative binding position of glucose in the active site channel might be responsible for modulating end product tolerance and stimulation. β-glucosidase from BglNB11 is an excellent enzyme with high catalytic efficiency and enhanced glucose tolerance compared to many known glucose tolerant β-glucosidases. These unique properties of BglNB11 make it a prime candidate to be utilized in many biotechnological applications.Ribose-5-phosphate isomerase A (RpiA) is of great importance in biochemistry research, however its application in biotechnology has not been fully explored. In this study the activity of RpiA from Ochrobactrum sp. CSL1 (OsRpiA) towards D-allose was engineered based on sequential and structural analyses. Strategies of alanine scanning, rational design and saturated mutagenesis were employed to create three mutant libraries. A single mutant of K124A showed a 45 % activity improvement towards D-allose. The reaction properties of the mutant were analyzed, and a shift of optimal pH and higher thermal stability at low reaction temperatures were identified. The conversion of D-allose was also improved by 40 % using K124A, and higher activities on major substrates were found in the mutant's substrate scope, implying its application potential in rare sugar preparation. Kinetics analysis revealed that Km of K124A mutant decreased by 12 % and the catalytic efficiency increased by 65 % towards D-allose. Moreover, molecular dynamics simulation illustrated the binding of substrate and K124A was more stable than that of the wild-type. Arginase inhibitor The shorter distance and more relax bond angle between the catalytic residue of K124A and D-allose explained the activity improvement in detail. This study highlights the potential of OsRpiA as a biocatalyst for rare sugar preparation, and provides distinct evidences for its catalytic mechanism.In this study, a paper-based sensor combined with visual distance-readout technique for point of-care testing (POCT) of urea was developed by urease-mediated chitosan viscosity change. A series of factors that affect the performance of the sensor were investigated, including the type of filter paper, chitosan concentration, acetic acid concentration and enzymatic reaction conditions. Under optimal conditions, the proposed method for urea determination has good linearity between 3.8-15.1 mM. The limit of quantitation is 3.8 mM. Finally, the paper-based sensor was successfully applied to the determination of urea in two diesel exhaust fluid (DEF) samples. The recoveries of urea were 91.4 % and 109.9 % in DEF-1 and DEF-2, respectively. The present study provides a novel approach, which integrates paper-based sensor and visual distance-readout technique, for monitoring urea in POCT application, especially in remote or resource-limited regions.Human milk oligosaccharides (HMOs) are lactose-based glycan molecules present in human breast milk. HMOs are essentially not present in cow's milk and hence not naturally available in infant formulas. HMOs possess several health and developmentally beneficial properties, and the sialylated HMOs are thought to play a particularly important role for infant brain development. Enzymatic transsialylation directly in cow's milk, involving enzyme catalyzed transfer of sialic acid from a sialic acid donor to an acceptor, is a novel route for producing sialylated HMOs for e.g. infant formulas. The transsialidase (EC 2.4.1.-) of Trypanosoma cruzi is linked to trypanosomatid pathogenicity, but certain hydrolytic sialidases (neuraminidases), EC 3.2.1.18, from non-pathogenic organisms, can actually catalyze transsialylation. Here, we report enzymatic production of the HMO compound 3'-sialyllactose directly in cow's milk using engineeredsialidases, Tr15 and Tr16, originating from the nonpathogenic Trypanosoma rangeli. Both Tr15 and Tr16 readily catalyzed transsialylation in milk at 5 °C-40 °C using κ(kappa)-casein glycomacropeptide (cGMP) as sialyl donor substrate. Tr15 was the most efficient as this enzyme produced 1160 mg/L (1.8 mM) 3'-sialyllactose in whole milk during 10 min of reaction at 5 °C. The activation energy values, Ea, of the enzymatic transsialylation reactions were similar in milk and in buffer solutions containing cGMP and lactose. The Ea of the Tr15 catalyzed transialylation reaction in milk was 16.5 kJ/mol, which was three times lower than the Ea of Tr16 (66 kJ/mol) and of T. cruzi transsialidase (50 kJ/mol), corroborating that Tr15 was the fastest of the three enzymes and a promising candidate for potential industrial production of 3'-sialyllactose in milk. 3'sialyllactose was stable during pasteurization (30 min. at 62.5 °C) and freeze-drying.Lactobionic acid (LBA), an aldonic acid prepared by oxidation of the free aldehyde group of lactose, has been broadly used in cosmetic, food, and pharmaceutical industries. Although Escherichia coli is unable to produce LBA naturally, a wild-type E. coli strain successfully produced LBA from lactose upon pyrroloquinoline quinone (PQQ) supplementation, indicating that E. coli contains at least one lactose-oxidizing enzyme as an apo-form. By inactivating the candidate genes in the E. coli chromosome, we found that the lactose-oxidizing enzyme of E. coli was the quinoprotein glucose dehydrogenase (GCD). To improve the LBA production ability of the E. coli strain, quinoprotein glucose dehydrogenase (GDH) from Pseudomonas taetrolens was recombinantly expressed and culture conditions such as growth temperature, initial lactose concentration, PQQ concentration, and isopropyl-β-D-1-thiogalactopyranoside induction concentration were optimized. We performed batch fermentation using a 5-L bioreactor under the optimized culture conditions determined in flask culture experiments. After batch fermentation, the LBA production titer, yield, and productivity of the recombinant E. coli strain were 200 g/L, 100 %, and 1.28 g/L/h, respectively. To the best our knowledge, this is the first report to identify the lactose-oxidizing enzyme of E. coli and to produce LBA using a recombinant E. coli strain as the production host. Because E. coli is one of the most easily genetically manipulated bacteria, our result provides the groundwork to further enhance LBA production by metabolic engineering of LBA-producing E. coli.Papain was immobilized onto Ti3C2 MXene nanosheets by physical adsorption and physical adsorption combined with covalent crosslinking with glutaraldehyde. Ti3C2 MXene nanosheets were prepared by hydrofluoric acid etching method. The resulting products were well characterized by SEM, BET, XRD, FTIR, XPS. The optimized immobilization conditions are pH 6.5, immobilization time of 20 h, immobilization temperature of 10℃, and 10 mL 2 mg mL-1 papain, the amount of papain immobilized was 156 mg g-1, the activity of the immobilized papain determined was 1701 U∙g-1. The immobilized papain exhibited enhanced pH and temperature endurances, immobilized papain also showed improved storage stability (39.25 % and 65.57 % after 20 days of storage at 4 °C). papain reusability was significantly improved after immobilization and it retained more than 50 % of its initial activity after 5 repeated cycles. Interestingly, the results of immobilized enzymes demonstrated that the immobilization of enzymes on Ti3C2 MXene is feasible. Such approach could be transferred to other support systems for anchoring enzyme.L-Gulose is a rare aldohexose to serve as a building block for anticancer drug bleomycin and nucleoside-based antivirals. However, preparative inaccessibility and high cost have hindered its pharmaceutical application. Despite a regio- and stereo-selective enzymatic synthesis of l-gulose from d-sorbitol using a variant of NAD+-dependent mannitol-1-dehydrogenase from Apium graveolens (mMDH) was explored, low efficiency and productivity caused by NADH accumulation or insufficient amount of NAD+ limited the practical utility of this process. In this study, a stable and efficient NADH oxidase from Bacillus cereus (bcNOX) was found to be more compatible with mMDH to recycle NAD+ in E. coli cells for l-gulose biosynthesis. After a systematic optimization of the whole-cell system, efficient biosynthesis of l-gulose was achieved. Starting with 70 g/L of readily available and cheap d-sorbitol resulted in a volumetric productivity of 5.5 g/L/d. This whole-cell approach enables practical, efficient and environmentally friendly biosynthesis of l-gulose and exhibits the potential of becoming a biocatalytic strategy for various enzymatic oxidative transformations.Microbial production of industrial chemicals is a sustainable approach to reduce the dependence on petroleum-based chemicals such as acids, alcohols, and amines, in which the cadaverine is a natural diamide and serves as one of the key monomers for biopolymer production. In this study, the constitutive promoter J23100 driven lysine decarboxylase (CadA) for cadaverine production was established and compared in different Escherichia coli strains. The best chassis designed as JW, expressed the highest amount of CadA by using J23100 promoter, showing stable and high copy numbers (i.e., PCN > 100) when culture in the antibiotic-free medium. JW attained a CadA activity of 167 g-DAP/g-DCW-h and had the maximum biocatalyst of 45.6 g-DCW/L in fed-batch fermentation. In addition, JW was able to convert 2.5 M L-lysine to 221 g/L cadaverine, with 86 % yield and 55.3 g/L-h productivity. The whole-cell biocatalyst could be reused over four times at an average of 97 % conversion when supplied half of fresh cells in the reaction.
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