Evaluating the effects of fibrolytic enzymes on rumen fermentation, omasal nutrient flow, and production performance in dairy cows during early lactation

This study was performed to evaluate the effects of pre-treating a barley-silage-based diet with an exogenous fibrolytic enzyme derived from Trichoderma reesei (FETR, a mixture of xylanase and cellulase) on lactation performance, omasal nutrient flow and digestibility, rumen fermentation characteristics, and rumen pH profile in Holstein dairy cows during early lactation. The dairy trial was conducted using nine Holstein dairy cows (averaging 46 ± 24 days in milk and 697 ± 69 kg body weight, six cows were fitted with a rumen cannula, and three were non-cannulated). Two groups of cows were randomly assigned to each of the dietary treatments in a crossover design: control (without FETR supplementation) and supplemented [with 0.75 mL of FETR·kg−1 dry matter (DM) of the diet based on our previous study]. The application of FETR tended to decrease the DM intake compared with control. There were no effects of FETR (P > 0. 10) on omasal nutrient flow and digestibility, rumen fermentation characteristics, and rumen pH profile. In conclusion, this study lacks evidence that the fibrolytic enzyme (at a level of 0.75 mL of FETR·kg−1 DM) can affect nutrient digestibility, ruminal fermentation, and the performance of early-lactation cows. Further study with larger animal trials are needed.

Dynamics of cocoa fermentation and its effect on quality

Several research efforts on cocoa have been focused on parameters for controlling the transformation process to guarantee homogeneity and quality of cocoa beans, the main raw material in the chocolate industry. The main changes that determine the final quality of cocoa—and also the product’s homogeneity—occur during fermentation, given the great number of factors that affect the process. This research seeks to identify the most relevant factors affecting quality in order to offer higher-quality and more homogeneous cocoa for the chocolate industry. The dynamics of the fermentation process were observed in three contrasting locations, monitoring different variables and evaluating the final quality of the cocoa. Results show that temperature and pH profile are the key factors to be monitored and controlled in order to achieve high-quality cocoa beans.

Improved xylanase Characteristics upon enzyme entrapment Isolated from Thermomyces lanuginosus C9

Xylanases from microbial sources assume basic jobs in an assortment of industrial applications as a biocatalyst, and its applications generally require immobilization on supports to upgrade their stability. Enzyme immobilization is a thrilling decision to show signs of improved strength of enzymatic procedures. In this work, two sorts of polymeric backings (agar-agar and calcium alginate) are utilized to immobilize β-1,4-xylanase from Thermomyces lanuginosus C9 by entrapment, and afterward, biochemical properties of the entangled enzymes were performed. To create immobilized catalyst beads centralization of 4% agar while mix of sodium alginate 5% and calcium chloride 0.4 M was seen as ideal. Ideal reaction time for agar and calcium alginate immobilized protein increments from 10 to 25 and 30 min, separately. The incubation temperature expanded from 70°C to 75°C for agar however stayed unaltered for calcium alginate. The pH profile of free and immobilized xylanase was generally equal in both cases. Be that as it may, both the strategies changed the active boundaries of immobilized β-1,4-xylanase rather than free protein. High sub-atomic load of xylan limits dispersion which brings down the Vmax estimation of immobilized protein while Km value expanded. In contrast with agar-agar, protein immobilized inside calcium alginate display wide thermal stability and kept up 86.6% of its underlying activity at 80°C up to 150 min. Be that as it may, biotechnological portrayal demonstrated that the catalyst reusability was the most surprising discovery, predominantly of agar-agar immobilized xylanase, which held 31% activity after 7 cycles. These outcomes prove the biotechnical and monetary advantages of immobilization which help in an assortment of industrial applications.

A Single Residue on the WPD-Loop Affects the pH Dependency of Catalysis in Protein Tyrosine Phosphatases

<p>Catalysis by protein tyrosine phosphatases (PTPs) relies on the motion of a flexible protein loop (the WPD-loop) that carries a residue acting as a general acid/base catalyst during the PTP-catalyzed reaction. The orthogonal substitutions of a non-catalytic residue in the WPD-loops of YopH and PTP1B results in shifted pH-rate profiles, from an altered kinetic p<i>K</i>_a of the nucleophilic cysteine. Compared to WT, the G352T YopH variant has a broadened pH-rate profile, similar activity at optimal pH, but significantly higher activity at low pH. Changes in the corresponding PTP1B T177G variant are more modest and in the opposite direction, with a narrowed pH profile and less activity in the most acidic range. Crystal structures of the variants show no structural perturbations, but suggest an increased preference for the WPD-loop closed conformation. Computational analysis confirms a shift in loop conformational equilibrium in favor of the closed conformation, arising from a combination of increased stability of the closed state and destabilization of the loop-open state. Simulations identify the origins of this population shift, revealing differences in the flexibility of the WPD-loop and neighboring regions. Our results demonstrate that changes to the pH dependency of catalysis by PTPs can result from small changes in amino acid composition in their WPD-loops affecting only loop dynamics and conformational equilibrium. The perturbation of kinetic p<i>K</i>_a values of catalytic residues by non-chemical processes affords a means for nature to alter an enzyme’s pH dependency by a less disruptive path than altering electrostatic networks around catalytic residues themselves. </p>

Stabilitas Digester Anaerobik Satu Tahap dalam Produksi Biogas pada Variasi Temperatur Menggunakan Reaktor Batch

Anaerobic digestion is the decomposition of organic matter by microbes into methane, carbon dioxide, and hydrogen sulfide in the absence of oxygen. This study aimed to obtain the stability of the one stage anaerobic digester in biogas production that was seen through pH and alkalinity parameters. The process was carried out by varying the temperature, which is 35 °C, 45 °C, and 55 °C with pH maintained 7 (± 0.2). Analysis of pH and alkalinity was carried out to assess the stability of reactor using samples taken from the reactor overflow. The pH profile produced was relatively stable with a pH range between 6.8 - 7.3. The resulting alkalinity is relatively stable with aalkalinity range between 3.500 – 4.500 mg/L. The volume of biogas produced at 35 °C, 45 °C, and 55 °C respectively are 2065 mL, 3830 mL, and 4570 mL with the highest concentrations of methane (CH4), Carbon dioxide (CO2) and trace Hydrogen Sulfide (H2S) at a temperature of 55 oC obtained the value of the composition of methane, carbon dioxide, and hydrogen sulfide each at 89,000 %, 11,000 %, and 0,011 %.

A Single Residue on the WPD-Loop Affects the pH Dependency of Catalysis in Protein Tyrosine Phosphatases

<p>Catalysis by protein tyrosine phosphatases (PTPs) relies on the motion of a flexible protein loop (the WPD-loop) that carries a residue acting as a general acid/base catalyst during the PTP-catalyzed reaction. The orthogonal substitutions of a non-catalytic residue in the WPD-loops of YopH and PTP1B results in shifted pH-rate profiles, from an altered kinetic p<i>K</i>_a of the nucleophilic cysteine. Compared to WT, the G352T YopH variant has a broadened pH-rate profile, similar activity at optimal pH, but significantly higher activity at low pH. Changes in the corresponding PTP1B T177G variant are more modest and in the opposite direction, with a narrowed pH profile and less activity in the most acidic range. Crystal structures of the variants show no structural perturbations, but suggest an increased preference for the WPD-loop closed conformation. Computational analysis confirms a shift in loop conformational equilibrium in favor of the closed conformation, arising from a combination of increased stability of the closed state and destabilization of the loop-open state. Simulations identify the origins of this population shift, revealing differences in the flexibility of the WPD-loop and neighboring regions. Our results demonstrate that changes to the pH dependency of catalysis by PTPs can result from small changes in amino acid composition in their WPD-loops affecting only loop dynamics and conformational equilibrium. The perturbation of kinetic p<i>K</i>_a values of catalytic residues by non-chemical processes affords a means for nature to alter an enzyme’s pH dependency by a less disruptive path than altering electrostatic networks around catalytic residues themselves. </p>

A Single Residue on the WPD-Loop Affects the pH Dependency of Catalysis in Protein Tyrosine Phosphatases

<p>Catalysis by protein tyrosine phosphatases (PTPs) relies on the motion of a flexible protein loop (the WPD-loop) that carries a residue acting as a general acid/base catalyst during the PTP-catalyzed reaction. The orthogonal substitutions of a non-catalytic residue in the WPD-loops of YopH and PTP1B results in shifted pH-rate profiles, from an altered kinetic p<i>K</i>_a of the nucleophilic cysteine. Compared to WT, the G352T YopH variant has a broadened pH-rate profile, similar activity at optimal pH, but significantly higher activity at low pH. Changes in the corresponding PTP1B T177G variant are more modest and in the opposite direction, with a narrowed pH profile and less activity in the most acidic range. Crystal structures of the variants show no structural perturbations, but suggest an increased preference for the WPD-loop closed conformation. Computational analysis confirms a shift in loop conformational equilibrium in favor of the closed conformation, arising from a combination of increased stability of the closed state and destabilization of the loop-open state. Simulations identify the origins of this population shift, revealing differences in the flexibility of the WPD-loop and neighboring regions. Our results demonstrate that changes to the pH dependency of catalysis by PTPs can result from small changes in amino acid composition in their WPD-loops affecting only loop dynamics and conformational equilibrium. The perturbation of kinetic p<i>K</i>_a values of catalytic residues by non-chemical processes affords a means for nature to alter an enzyme’s pH dependency by a less disruptive path than altering electrostatic networks around catalytic residues themselves. </p>

Expanding the Application Range of Microbial Oxidoreductases by an Alcohol Dehydrogenase from Comamonas testosteroni with a Broad Substrate Spectrum and pH Profile

Alcohol dehydrogenases catalyse the conversion of a large variety of ketone substrates to the corresponding chiral products. Due to their high regio- and stereospecificity, they are key components in a wide range of industrial applications. A novel alcohol dehydrogenase from Comamonas testosteroni (CtADH) was identified in silico, recombinantly expressed and purified, enzymatically and biochemically investigated as well as structurally characterized. These studies revealed a broad pH profile and an extended substrate spectrum with the highest activity for compounds containing halogens as substituents and a moderate activity for bulky–bulky ketones. Biotransformations with selected ketones—performed with a coupled regeneration system for the co-substrate NADPH—resulted in conversions of more than 99% with all tested substrates and with excellent enantioselectivity for the corresponding S-alcohol products. CtADH/NADPH/substrate complexes modelled on the basis of crystal structures of CtADH and its closest homologue suggested preliminary hints to rationalize the enzyme’s substrate preferences

Identification of quasi-stable water molecules near the Thr73–Lys13 catalytic diad of Bacillus sp. TB-90 urate oxidase by X-ray crystallography with controlled humidity

Urate oxidases (UOs) catalyze the cofactor-independent oxidation of uric acid, and an extensive water network in the active site has been suggested to play an essential role in the catalysis. For our present analysis of the structure and function of the water network, the crystal qualities of Bacillus sp. TB-90 urate oxidase were improved by controlled dehydration using the humid air and glue-coating method. After the dehydration, the P21212 crystals were transformed into the I222 space group, leading to an extension of the maximum resolution to 1.42 Å. The dehydration of the crystals revealed a significant change in the five-water-molecules’ binding mode in the vicinity of the catalytic diad, indicating that these molecules are quasi-stable. The pH profile analysis of log(kcat) gave two pKa values: pKa1 at 6.07 ± 0.07 and pKa2 at 7.98 ± 0.13. The site-directed mutagenesis of K13, T73 and N276 involved in the formation of the active-site water network revealed that the activities of these mutant variants were significantly reduced. These structural and kinetic data suggest that the five quasi-stable water molecules play an essential role in the catalysis of the cofactor-independent urate oxidation by reducing the energy penalty for the substrate-binding or an on–off switching for the proton-relay rectification.