Kinetic and thermal stability studies of rulactine,a proteolytic enzyme fromMicrococcus caseolyticus

Summary Kinetics and thermostability of Rulactine, a protease fromMicrococcus caseolyticus were investigated. Study of the enzyme activity as a function of the temperature showed an optimum peak of 45°C. The effeci of the substrate concentration on the initial velocity at various temperatures was examined, and V_max and K_M were determined using a Lineweaver-Burk reciprocal plot. The activation energy evaluation gave a value of 9500 cal/mole. Studies of additives such as polyhydric alcohols (glycerol, erythritol, xylitol and sorbitol) and disaccharides (sucrose and lactose) to Rulactine at 58°C proved that they have a stabilizing effect on Rulactine.     

Polyhydric alcohol protective effect on Rhizomucor miehei lipase deactivation enhanced by pressure and temperature treatment

The influence of polyhydric alcohols (sorbitol, xylitol, erythritol, glycerol) on the thermal stability of Rhizomucor miehei lipase has been studied at high hydrostatic pressure (up to 500 MPa). In the absence of additives, a protective effect (PE) (the ratio between the residual activities determined at 480 MPa for the enzyme in the presence or absence of polyhydric alcohols) of low-applied pressures (from 50 MPa to 350 MPa) against thermal deactivations (at 50°C and 55°C) has been noticed. In the presence of additives, a strong correlation between PE and the total hydroxyl group concentration has been obtained, for the first time, under treatments of combining denaturing temperatures and high hydrostatic pressures. This relationship does not seem to be dependent on the nature polyhydric alcohols as the same effect could be observed with 1 M sorbitol and 2 M glycerol. This PE, against thermal and high pressure combined lipase deactivation, increases with polyhydric alcohol concentrations, and when temperature increases from 25°C to 55°C.     

L-Xylo-3-hexulose,a new rare sugar produced by the action of acetic acid bacteria on galactitol,an exception to Bertrand Hudson's rule

BackgroundIn acetic acid bacteria such as Gluconobacter oxydans or Gluconobacter cerinus, pyrroloquinoline quinone (PQQ) in the periplasm serves as the redox cofactor for several membrane-bound dehydrogenases that oxidize polyhydric alcohols to rare sugars, which can be used as a healthy alternative for traditional sugars and sweeteners. These oxidation reactions obey the generally accepted Bertrand Hudson's rule, in which only the polyhydric alcohols that possess cis d-erythro hydroxyl groups can be oxidized to 2-ketoses using PQQ as a cofactor, while the polyhydric alcohols excluding cis d-erythro hydroxyl groups ruled out oxidation by PQQ-dependent membrane-bound dehydrogenases.MethodsMembrane fractions of G. oxydans were prepared and used as a cell-free catalyst to oxidize galactitol, with or without PQQ as a cofactor.ResultsIn this study, we reported an interesting oxidation reaction that the polyhydric alcohols galactitol (dulcitol), which do not possess cis d-erythro hydroxyl groups, can be oxidized by PQQ-dependent membrane-bound dehydrogenase(s) of acetic acid bacteria at the C-3 and C-5 hydroxyl groups to produce rare sugars l-xylo-3-hexulose and d-tagatose.ConclusionsThis reaction may represent an exception to Bertrand Hudson's rule.General significanceBertrand Hudson's rule is a well-known theory in polyhydric alcohols oxidation by PQQ-dependent membrane-bound dehydrogenase in acetic acid bacteria. In this study, galactitol oxidation by a PQQ-dependent membrane-bound dehydrogenase represents an exception to the Bertrand Hudson's rule. Further identification of the associated enzymes and deciphering the explicit enzymatic mechanism will prove this theory.     

Stabilities of Enzymes in Polyhydric Alcohols

To keep an enzyme stable in solution, the stabilities of the enzyme in various polyhydric alcohols were investigated, and it was found that the enzyme in 70% aqueous sorbitol solution was strikingly stable against both storage at 33°C and heating at 80°C. It seems reasonable to ascribe this stability to such high osmotic pressure of the sorbitol concentration that decreased the viable counts of microorganisms. The reason why the sorbitol is the most effective among various polyhydric alcohols is now being investigated. This stable enzyme solution will be of good advantage as food improver, medicines or so on.     

Effect of glycerol,polyethylene glycol and glutaraldehyde on stability of phenylalanine ammonia-lyase activity in yeast

Summary The polyhydric alcohols, glycerol and sorbitol, significantly increased the rate ofl-phenylalanine production from trans-cinnamic acid using whole cells ofRhodotorula rubra. Chloride ions and oxygen prevented the stimulatory effect of the polyhydric alcohols. Furthermore, the severe inhibition, of the biotransformation by high trans-cinnamic acid concentrations was alleviated in the presence of glycerol, and sorbitol. The rate of conversion could be manipulated still further, even with high trnas-cinnamic acid concentrations, by elevating the reaction pH to, 12 in the presence of polyhydric alcohol. When cells were also treated first with glutaradehyde (0.1% v/v) and then polyethylene glycol (15% v/v), although neither compound stimulated the actual rate of bioconversion, the reaction was markedly stabilised and gave a 73% yield after 28 days of continuous operation.     

Influence of glycerol and other polyhydric alcohols on the quaternary structure of an oligomeric protein

Dissociation of tetrameric l-asparaginase from Escherichia coli B was examined in the presence of urea containing one of the following polyhydric alcohols: ethylene glycol, 1,2-propanediol, 1,3-propanediol, glycerol, erythritol, arabitol, adonitol, mannitol, sorbitol, inositol, glucose, sucrose, and polyethylene glycol. Low concentrations of these compounds accelerate the rate of subunit dissociation, and, with the exception of the propanediols and polyethylene glycol, higher concentrations decrease the rate at which the oligomeric enzyme dissociates. The specific concentration at which this transition occurs is related to the length of the carbon chain of the polyhydric alcohols and to the steric configuration of the hydroxyl groups about the asymmetric carbon atoms. In addition, the rate at which the oligomeric enzyme dissociates decreases as the number of hydroxymethyl groups per molecule polyol increases and reaches a maximum with the six carbon members.Low concentrations (1% by volume) of methanol, ethanol, ethylene glycol, propylene glycol, or glycerol contained in the renaturation buffer slightly accelerate the rate of reassembly of denatured subunits. The rate at which reassociation to the tetramer occurs also increases as the number of hydroxymethyl groups per molecule of polyhydric alcohol increases.     

Kinetic Behaviour of Free Lipase and Mica-Based Immobilized Lipase Catalyzing the Synthesis of Sugar Esters

The utilization of natural mica as a biocatalyst support in kinetic investigations is first described in this study. The formation of lactose caprate from lactose sugar and capric acid, using free lipase (free-CRL) and lipase immobilized on nanoporous mica (NER-CRL) as a biocatalyst, was evaluated through a kinetic study. The apparent kinetic parameters, K _m and V _max, were determined by means of the Michaelis-Menten kinetic model. The Ping-Pong Bi-Bi mechanism with single substrate inhibition was adopted as it best explains the experimental findings. The kinetic results show lower K _m values with NER-CRL than with free-CRL, indicating the higher affinity of NER-CRL towards both substrates at the maximum reaction velocity (V _max,app>V _max). The kinetic parameters deduced from this model were used to simulate reaction rate data which were in close agreement with the experimental values.     

Stabilization method of an alkaline protease from inactivation by heat,SDS and hydrogen peroxide

An investigation was carried out to evaluate the protective effect of polyhydric alcohols, such as propylene glycol and glycerol on the inactivation of an alkaline protease by sodium dodecyl sulfate (SDS) and H₂O₂. Addition of polyols increased the stability of a Bacillus clausii I-52 alkaline protease towards not only the thermal-induced, but also the SDS and H₂O₂-induced inactivation. Among the polyols examined, the best results were obtained with propylene glycol. The half-life of the enzyme was increased by 43- and >105-fold by the addition of 10% (v/v) propylene glycol to the enzyme preparations containing 5% (w/v) SDS and 5% (v/v) H₂O₂ at 50 °C, respectively. Besides the protection effect of propylene glycol from enzyme inactivation by SDS and H₂O₂, it also improved the hydrolytic efficiency towards substrate like BSA during the protease reaction containing SDS or H₂O₂. This result suggests that propylene glycol has a significant potential as a good stabilizer of an alkaline protease preparation, which finds use as an additive in industrial applications, especially, the detergent industry.     

Thermostabilization ofCucurbita ficifolia protease in the presence of additives

Summary The effect of additives such as polyhydric alcohols, polyethylene glycol and casein on the thermostability ofCucurbita ficifolia protease was determined. Glycerol and mannitol increased the half life of the enzyme approximately 2-fold. Addition of polyethylene glycol had a positive effect on enzyme stability and its stabilizing efficiency appears to correlate with additive concentration. Casein was also shown to be effective as a protease stabilizer.     

Accumulation and partial re-consumption of polyols during citric acid fermentation by Aspergillus niger

Summary Quantitative balances have been made for sugar and oxygen uptake rates during citric acid accumulation by Aspergillus niger: during the first phase of citric acid accumulation (up to 130 h) more sugar is taken up than the production of biomass, CO₂ and citric acid account for. In contrast, during later phases of fermentation more citric acid, CO₂ and biomass are formed than sugar uptake would theoretically allow. A similar pattern is obtained for oxygen uptake, where less uptake occurs during the early phase of fermentation than needed for complete balance, and the reverse is observed during the late stage of fermentation. It could subsequently be shown that this is caused by the intermediate accumulation and partial re-consumption of a number of polyhydric alcohols (glycerol, arabitol, erythritol and mannitol) during citric acid fermentation.Dedicated to Professor H. J. Rehm on the occasion of his 60th birthday with kind regards     

Measurement of free space and sorption of large molecules by cereal roots

Abstract. Large molecular weight solutes that do not penetrate the root have been used to correct for the surface film in measurements with mannitol of the volume of the Apparent Free Space (FS) in bailey roots. The results are compared with those obtained using other correction techniques for elimination of the surface film. Large molecules seem to be adsorbed on the root surface and the kinetics of adsorption differ between the polyhydric alcohol mannitol or the polysaccharide dextran on the one hand, and the polyether polyethylene glycol (PEG-4000) on the other. The significance of this difference in kinetics is discussed in relation to the use of PEG as an osmoticum in studies on root water relations and its effect on ion uptake. Although smaller molecular weight PEG's penetrate the FS and diminish sodium uptake from 10 mol m⁻³ NaCl, more dilute solutions of mannitol and larger PEG polymers are unlikely to affect ion uptake from dilute nutrient solutions. Use of these substances along with labelled nutrients in kinetic studies of the compartmentation of ions in roots can help to distinguish between ions associated with the surface film, those in the FS and those that have crossed the cell membranes into the protoplast.     

A review of anaerobic digestion bio-kinetics

Anaerobic digestion is the key to sustainable wastewater management and bioenergy production. Kinetics plays an important role in the design of bioreactors, processes, and process scale-up in anaerobic digestion. This article focuses on a state-of-the-art literature review on the experimental kinetic studies of conventional anaerobic bioreactors and anaerobic membrane bioreactors. Various kinetic models that were used to fit the experimental data and derive the kinetic parameters are summarized and discussed in the literature. The values of the maximum specific growth rate µ_max, half saturation constant K_s, decay co-efficient k_d, sludge yield Y, and methane yield Y_CH4 from experimental studies are summarized for each model. This paper can serve as an updated comprehensive source of anaerobic bio-kinetic studies and digester design.     

Formation of Dulcitol in Aerobic Dissimilation of d-Galactose by Yeasts

During the study of aerobic dissimilation of galactose by yeasts, polyhydric products were isolated in crystalline form from the fermented broths and identified. Yeast species may be divided into two groups on basis of sugar alcohol production: type I yeasts form the same end products from galactose as from glucose; type II yeasts produce dulcitol from galactose with or without other sugar alcohols but they produce no dulcitol from glucose. Isolation of dulcitol from microorganism has not been previously described.     

Predicting kinetic constants of protein-protein interactions based on structural properties

Elucidating kinetic processes of protein–protein interactions (PPI) helps to understand how basic building blocks affect overall behavior of living systems. In this study, we used structure‐based properties to build predictive models for kinetic constants of PPI. A highly diverse PPI dataset, protein–protein kinetic interaction data and structures (PPKIDS), was built. PPKIDS contains 62 PPI with complex structures and kinetic constants measured experimentally. The influence of structural properties on kinetics of PPI was studied using 35 structure‐based features, describing different aspects of complex structures. Linear models for the prediction of kinetic constants were built by fitting with selected subsets of structure‐based features. The models gave correlation coefficients of 0.801, 0.732, and 0.770 for k_off, k_on, and K_d, respectively, in leave‐one‐out cross validations. The predictive models reported here use only protein complex structures as input and can be generally applied in PPI studies as well as systems biology modeling. Our study confirmed that different properties play different roles in the kinetic process of PPI. For example, k_on was affected by overall structural features of complexes, such as the composition of secondary structures, the change of translational and rotational entropy, and the electrostatic interaction; while k_off was determined by interfacial properties, such as number of contacted atom pairs per 100 Ų. This information provides useful hints for PPI design. Proteins 2010;79:720–734. © 2010 Wiley‐Liss, Inc.     

Microbial transformation of sucrose and glucose to erythritol

Summary Two strains of osmophilic yeast which were isolated from honey-comb, produced good yields of erythritol as a main product. These strains were identified as Trichosporonoides sp., 150-5 and 331-1.From the fermentation studies with these strains using glucose and sucrose as substrate, strain 331-1 produced more erythritol as the sole polyhydric product,with trace quantities of glycerol, than strain 150-5.     

Protective Effect of Adonitol on Lactic Acid Bacteria Subjected to Freeze-Drying

The protective effects of glycerol, adonitol, and four other related polyhydric alcohols on lactic acid bacteria subjected to freeze-drying were examined. The presence of adonitol in the suspending medium markedly protected the viabilities of the 12 stains tested. Dulcitol, mannitol, m-inositol, and sorbitol were found to provide little or no protection.     

Scintillation proximity assay (SPA) as a new approach to determine a ligand’s kinetic profile. A case in point for the adenosine A<Subscript>1</Subscript> receptor

Scintillation proximity assay (SPA) is a radio-isotopic technology format used to measure a wide range of biological interactions, including drug-target binding affinity studies. The assay is homogeneous in nature, as it relies on a “mix and measure” format. It does not involve a filtration step to separate bound from free ligand as is the case in a traditional receptor-binding assay. For G protein-coupled receptors (GPCRs), it has been shown that optimal binding kinetics, next to a high affinity of a ligand, can result in more desirable pharmacological profiles. However, traditional techniques to assess kinetic parameters tend to be cumbersome and laborious. We thus aimed to evaluate whether SPA can be an alternative platform for real-time receptor-binding kinetic measurements on GPCRs. To do so, we first validated the SPA technology for equilibrium binding studies on a prototypic class A GPCR, the human adenosine A₁ receptor (hA₁R). Differently to classic kinetic studies, the SPA technology allowed us to study binding kinetic processes almost real time, which is impossible in the filtration assay. To demonstrate the reliability of this technology for kinetic purposes, we performed the so-called competition association experiments. The association and dissociation rate constants (k _on and k _off) of unlabeled hA₁R ligands were reliably and quickly determined and agreed very well with the same parameters from a traditional filtration assay performed simultaneously. In conclusion, SPA is a very promising technique to determine the kinetic profile of the drug-target interaction. Its robustness and potential for high-throughput may render this technology a preferred choice for further kinetic studies.     

Enhancement of the ozonation of wine distillery wastewaters by an aerobic pretreatment

The decomposition of the organic substrate present in wine distillery wastewaters (WDW) is studied in batch reactors, by an ozonation process, by an aerobic degradation and by another ozonation of the aerobically pretreated wastewaters. In the ozonation process, the effects on the substrate removal obtained of the temperature, pH and the presence of H₂O₂ and UV radiation are established, and an approximate kinetic study is conducted which leads to the evaluation of the apparent kinetic constants for the substrate reduction. In the aerobic degradation treatment, the evolution of the substrate, biomass and total phenolic compounds are followed during the process, and a kinetic study is performed by using the Contois model, which applied to the experimental data provides the specific kinetic parameters q_max and K₁. Finally, in the ozonation of the pretreated wastewaters, the?influence of the operating variables is established, and the effect of this aerobic pretreatment on the substrate removal and kinetic constants obtained in the ozonation stage is also discussed.     

Role of low native state kinetic stability and interaction of partially unfolded states with molecular chaperones in the mitochondrial protein mistargeting associated with primary hyperoxaluria

The G170R variant of the alanine:glyoxylate aminotransferase (AGT) is the most common pathogenic allele associated to primary hyperoxaluria type I (PH1), leading to mitochondrial mistargeting when combined with the P11L and I340M polymorphisms (minor allele; AGT_LM). In this work, we have performed a comparative analysis on the conformation, unfolding energetics and interaction with molecular chaperones between AGT_wt, AGT_LM and AGT_LRM (G170R in the minor allele) proteins. Our results show that these three variants share similar conformational and functional properties as folded dimers. However, kinetic stability analyses showed a ≈1,000-fold increased unfolding rate for apo-AGT_LRM compared to apo-AGT_wt, as well as a reduced folding efficiency upon expression in Escherichia coli. Pyridoxal 5′-phosphate (PLP)-binding provided a 4–5 orders of magnitude enhancement of the kinetic stability for all variants, suggesting a role for kinetic stabilization in pyridoxine-responsive PH1. Conformational studies at mild acidic pH and moderate guanidium concentrations showed the formation of a molten-globule-like unfolding intermediate in all three variants, which do not reactivate to the native state and strongly interact with Hsc70 and Hsp90 chaperones. Additional expression analyses in a mammalian cell-free system at neutral pH showed enhanced interaction of AGT_LRM with Hsc70 and Hsp90 proteins compared to AGT_wt, suggesting kinetic trapping of the mutant by chaperones along the folding process. Overall, our results suggest that mitochondrial mistargeting of AGT_LRM may involve the presentation of AGT partially folded states to the mitochondrial import machinery by molecular chaperones, which would be facilitated by the low native state kinetic stability (partially corrected by PLP binding) and kinetic trapping during folding of the AGT_LRM variant with molecular chaperones.     

Effects of Polyols and Sugars on the Sol-Gel Transition of Gelatin

The melting temperature of gelatin gel increased with increasing concentrations of polyols and sugars added at any gelatin concentation. The enthalpy of gel melting measured by Eldridge-Ferry plots and calorimetric measurements was decreased by addition of these polyhydric compounds, indicating that the gel stabilization by them is predominantly due to the large decrease in entropy of gel melting. Circular dichroism analysis showed that the helix formation of gelatin molecules was enhanced with increasing concentrations of these additives, but there was no positive correlation between their helix-forming ability and gel-stabilizing ability. The viscosity of gelatin solution was less in these mixed solvents than in water. Based on these results, a possible stabilization mechanism of gelatin gel by polyhydric compounds was discussed, in comparison with that of native collagen, in terms of the thermodynamics of protein-solvent interactions.