Codon-Optimized NADH Oxidase Gene Expression and Gene Fusion with Glycerol Dehydrogenase for Bienzyme System with Cofactor Regeneration.

NADH oxidases (NOXs) play an important role in maintaining balance of NAD+/NADH by catalyzing cofactors regeneration. The expression of nox gene from Lactobacillus brevis in Escherichia coli BL21 (BL21 (DE3)) was studied. Two strategies, the high AT-content in the region adjacent to the initiation codon and codon usage of the whole gene sequence consistent with the host, obtained the NOX activity of 59.9 U/mg and 73.3 U/mg (crude enzyme), with enhanced expression level of 2.0 and 2.5-folds, respectively. Purified NOX activity was 213.8 U/mg. Gene fusion of glycerol dehydrogenase (GDH) and NOX formed bifuctional multi-enzymes for bioconversion of glycerol coupled with coenzyme regeneration. Kinetic parameters of the GDH-NOX for each substrate, glycerol and NADH, were calculated as Vmax(Glycerol) 20 μM/min, Km(Glycerol) 19.4 mM, Vmax (NADH) 12.5 μM/min and Km (NADH) 51.3 μM, respectively, which indicated the potential application of GDH-NOX for quick glycerol analysis and dioxyacetone biosynthesis

The Ubiquitin–Proteasome System in Tumor Metabolism

Metabolic reprogramming, which is considered a hallmark of cancer, can maintain the homeostasis of the tumor environment and promote the proliferation, survival, and metastasis of cancer cells. For instance, increased glucose uptake and high glucose consumption, known as the “Warburg effect,” play an essential part in tumor metabolic reprogramming. In addition, fatty acids are harnessed to satisfy the increased requirement for the phospholipid components of biological membranes and energy. Moreover, the anabolism/catabolism of amino acids, such as glutamine, cystine, and serine, provides nitrogen donors for biosynthesis processes, development of the tumor inflammatory environment, and signal transduction. The ubiquitin–proteasome system (UPS) has been widely reported to be involved in various cellular biological activities. A potential role of UPS in the metabolic regulation of tumor cells has also been reported, but the specific regulatory mechanism has not been elucidated. Here, we review the role of ubiquitination and deubiquitination modification on major metabolic enzymes and important signaling pathways in tumor metabolism to inspire new strategies for the clinical treatment of cancer

Mechanistic study of manganese-substituted glycerol dehydrogenase using a kinetic and thermodynamic analysis.

Mechanistic insights regarding the activity enhancement of dehydrogenase by metal ion substitution were investigated by a simple method using a kinetic and thermodynamic analysis. By profiling the binding energy of both the substrate and product, the metal ion's role in catalysis enhancement was revealed. Glycerol dehydrogenase (GDH) from Klebsiella pneumoniae sp., which demonstrated an improvement in activity by the substitution of a zinc ion with a manganese ion, was used as a model for the mechanistic study of metal ion substitution. A kinetic model based on an ordered Bi-Bi mechanism was proposed considering the noncompetitive product inhibition of dihydroxyacetone (DHA) and the competitive product inhibition of NADH. By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation. The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively. A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone. The metal ion's role in catalysis enhancement was explicated

Effects of Processing Methods and Conditioning Temperatures on the Cassava Starch Digestibility and Growth Performance of Broilers

As an important food crop, cassava is rich in nutrients and high in starch content and is widely used in the production of industrial raw materials. However, the utilization value of cassava is limited due to the reduction of planting area and the existence of anti-nutritional factors. Therefore, we evaluated in vitro cassava starch digestibility and in vivo growth performance of broilers in a 3 × 3 factorial arrangement of treatments using three processing methods (mechanical crushing (MC), steam conditioning (SC), and puffing conditioning (PU)) and three conditioning temperatures (60, 75, and 90 °C) to screen for the optimal processing method and conditioning temperature to improve the utilization of cassava. In the in vitro cassava starch digestion study, the digestibility and digestion rate (p p p p p p p < 0.05) for broilers fed SC diets than for those fed MC diets. These results indicate that cassava starch promoted starch digestion rate by reducing amylose content and amylose/amylose under PU combined with a conditioning temperature of 60 °C, ileum digestibility of starch in broilers fed SC diets was higher than MC diets regardless of conditioning temperature, and SC diets increased AME and decreased F/G to promote growth performance of broilers

Specific activities of the opt-<i>nox</i> in the process of purification.

<p>Specific activities of the opt-<i>nox</i> in the process of purification.</p

Determination of kinetic parameters of GDH-NOX.

<p>(a) Glycerol; (b) NADH. Experiment condition: glycerol concentration (0.01, 0.125, 0.014, 0.025, 0.05M), NADH concentration (20, 40, 60, 100, 200 μM). pH 7.0, 37°C.</p

The pie chart of the distribution of PDOR’ activities and identified mutants.

<p>The pie chart of the distribution of PDOR’ activities and identified mutants.</p

The bonding model of mutated PDOR’s with NADH or 3-HPA.

<p>A: before the NADH and 3-HPA bonding of mutated PDOR; B: bonding of mutated PDOR and 3-HPA; C: bonding of mutated PDOR and NADH; D: bonding of mutated PDOR, NADH and 3-HPA.</p

10% SDS-PAGE analysis of the purification fused GDH-NOX.

<p>Line 1: protein marker; Lane 2: purified GDH-NOX with His-tag.</p