Substrate inhibition by the blockage of product release and its control by tunnel engineering

Substrate inhibition can be caused by substrate binding to the enzyme–product complex and can be controlled rationally by targeting enzyme access tunnels.

Substrate Inhibition by the Blockage of Product Release and Its Control by Tunnel Engineering

<div> <p>Substrate inhibition is the most common deviation from Michaelis-Menten kinetics, occurring in approximately 25% of known enzymes. It is generally attributed to the formation of an unproductive enzyme-substrate complex after the simultaneous binding of two or more substrate molecules to the active site. Here, we show that a single point mutation (L177W) in the haloalkane dehalogenase LinB causes strong substrate inhibition. Surprisingly, a global kinetic analysis suggested that this inhibition is caused by binding of the substrate to the enzyme-product complex. Molecular dynamics simulations clarified the details of this unusual mechanism of substrate inhibition: Markov state models indicated that the substrate prevents the exit of the halide product by direct blockage and/or restricting conformational flexibility. The contributions of three residues forming the possible substrate inhibition site (W140A, F143L and I211L) to the observed inhibition were studied by mutagenesis. An unusual synergy giving rise to high catalytic efficiency and reduced substrate inhibition was observed between residues L177W and I211L, which are located in different access tunnels of the protein. These results show that substrate inhibition can be caused by substrate binding to the enzyme-product complex and can be controlled rationally by targeted amino acid substitutions in enzyme access tunnels. </p> </div> <br>

Substrate Inhibition by the Blockage of Product Release and Its Control by Tunnel Engineering

<div> <p>Substrate inhibition is the most common deviation from Michaelis-Menten kinetics, occurring in approximately 25% of known enzymes. It is generally attributed to the formation of an unproductive enzyme-substrate complex after the simultaneous binding of two or more substrate molecules to the active site. Here, we show that a single point mutation (L177W) in the haloalkane dehalogenase LinB causes strong substrate inhibition. Surprisingly, a global kinetic analysis suggested that this inhibition is caused by binding of the substrate to the enzyme-product complex. Molecular dynamics simulations clarified the details of this unusual mechanism of substrate inhibition: Markov state models indicated that the substrate prevents the exit of the halide product by direct blockage and/or restricting conformational flexibility. The contributions of three residues forming the possible substrate inhibition site (W140A, F143L and I211L) to the observed inhibition were studied by mutagenesis. An unusual synergy giving rise to high catalytic efficiency and reduced substrate inhibition was observed between residues L177W and I211L, which are located in different access tunnels of the protein. These results show that substrate inhibition can be caused by substrate binding to the enzyme-product complex and can be controlled rationally by targeted amino acid substitutions in enzyme access tunnels. </p> </div> <br>

Influence of Enzyme Additives on the Rheological Properties of Digester Slurry and on Biomethane Yield

The use of enzyme additives in anaerobic digestion facilities has increased in recent years. According to the manufacturers, these additives should increase or accelerate the biogas yield and reduce the viscosity of the digester slurry. Such effects were confirmed under laboratory conditions. However, it has not yet been possible to quantify these effects in practice, partly because valid measurements on large-scale plants are expensive and challenging. In this research, a new enzyme product was tested under full-scale conditions. Two digesters were operated at identic process parameters—one digester was treated with an enzyme additive and a second digester was used as reference. A pipe viscometer was designed, constructed and calibrated and the rheological properties of the digester slurry were measured. Non-Newtonian flow behavior was modelled by using the Ostwald–de Baer law. Additionally, the specific biomethane yield of the feedstock was monitored to assess the influence of the enzyme additive on the substrate degradation efficiency. The viscosity measurements revealed a clear effect of the added enzyme product. The consistency factor K was significantly reduced after the enzyme application. There was no observable effect of enzyme application on the substrate degradation efficiency or specific biomethane yield.

174 Multi-enzyme solution improved growth performance of piglets fed a corn-SBM-DDGS based diet

A study was conducted to determine the effect of adding a multi-enzyme product to corn-SBM-DDGS-based diet on growth performance of weaned pigs. A total of 132 pigs weaned at 21 d of age (initial BW=7.5±1.5kg) were blocked by weight and housed in pens of 4 barrows (6) or gilts (5) per pen (n = 11). The diets consisted of: 1) a corn-SBM-based diet with high animal-by products (Comp); 2) a corn-SBM-based diet with 5 to 15% DDGS and reduced animal-by products (Simp); and 3) Simp with a multi-enzyme product that supplied 4,000 U of xylanase, 150 U of β-glucanase, 150 U of amylase and 3,000 U of protease/kg (Simp+ENZ). All diets were isoenergetic and met the NRC (2012) recommendations for weaned pigs. The diets were fed for 6 weeks with a 3-phase-feeding program: phase-1, 0–7 d; phase-2, 7–21 d and phase-3, 21–42 d post-wean. A Buttiauxella spp. phytase was supplemented at 2000, 1250 and 1250 FTU/kg for phase-1, 2 and 3, respectively. Pharmaceutical level of Zn was only included in phase 1 and 2 at 3000 and 2000 ppm, respectively. Performance was measured by phase and analyzed using SAS MIXED model with repeated measure. Average ADG from 0–42 d was 521, 465 and 434 g/day in pigs fed Comp, Simp+ENZ and Simp diet, respectively (P &lt; 0.05). The ADFI was 861, 765 and 778 from 0–42 d in pigs fed Comp, Simp and Simp+ENZ (P &lt; 0.05). Final BW for pigs fed Simp+ENZ was 1.36 kg heavier than those fed Simp (26.93 vs. 25.57 kg, P &lt; 0.05). Fecal score was not affected by feeding Simp diet with or without enzyme as compared to those fed Comp diets (P &gt; 0.05). In conclusion, the multi-enzyme solution significantly improved ADG when piglets were fed a diet containing low animal by-products and high DDGS.

Bioenergy Valuation of Poultry Litter by Applying an Enzyme Product for Environmental Purposes: A New Applied Technology

The increase and intensification of food production entail some potential risks to the environment due to emissions of greenhouse gases, acid rain and other pollutants. European policies are focused on environmental protection and particularly on the health and welfare of animals intended for human consumption. The transposition of the Directive of water protection (91/676/EEC) and the increase botulism cases in countries such as Ireland have necessitated the search for alternative solutions for the management of poultry manure, which has traditionally been used as a fertilizer. Research has targeted eco-friendly, techno-economical and time-effective solutions, together with a simplified large-scale operational approach. Given this scenario, this project aims to study the use of Colombian enzyme product, called Bioterre, in European farms. This product is used in that country as a stabilizer and composting accelerator of organic waste for fertilizer production. After experimental testing application the average moisture content of the poultry litter in treated sheds of the different farms, at the end of the cycle, is 27%, versus 47% in the untreated sheds. This product decreases the moisture content in the biomass increasing the Lower Heating Value (LHV). Equipment based on this technology could be used mainly in the food processing industry and their bioenergy sustainable projects.