Identification of Potential Aldose Reductase Inhibitors Using Convolutional Neural Network-Based in Silico Screening.

Aldose reductase (ALR2) is a notable enzyme of the polyol pathway responsible for aggravating diabetic neuropathy complications. The first step begins when it catalyzes the reduction of glucose to sorbitol with NADPH as a coenzyme. Elevated concentrations of sorbitol damage the tissues, leading to complications like neuropathy. Though considerable effort has been pushed toward the successful discovery of potent inhibitors, its discovery still remains an elusive task. To this end, we present a 3D convolutional neural network (3D-CNN) based ALR2 inhibitor classification technique by dealing with snapshots of images captured from 3D chemical structures with multiple rotations as input data. The CNN-based architecture was trained on the 360 sets of image data along each axis and further prediction on the Maybridge library by each of the models. Subjecting the retrieved hits to molecular docking leads to the identification of the top 10 molecules with high binding affinity. The hits displayed a better blood-brain barrier penetration (BBB) score (90% with more than four scores) as compared to standard inhibitors (38%), reflecting the superior BBB penetrating efficiency of the hits. Followed by molecular docking, the biological evaluation spotlighted five compounds as promising ALR2 inhibitors and can be considered as a likely prospect for further structural optimization with medicinal chemistry efforts to improve their inhibition efficacy and consolidate them as new ALR2 antagonists in the future. In addition, the study also demonstrated the usefulness of scaffold analysis of the molecules as a method for investigating the significance of structurally diverse compounds in data-driven studies. For reproducibility and accessibility purposes, all of the source codes used in our study are publicly available.

Identification and functional characterization of a novel aldo-keto reductase from Aloe vera.

MAIN CONCLUSION: The present investigation profoundly asserted the catalytic potential of plant-based aldo-ketoreductase, postulating its role in polyketide biosynthesis and providing new insights for tailored biosynthesis of vital plant polyketides for therapeutics. Plants hold great potential as a future source of innovative biocatalysts, expanding the possibilities within chemical reactions and generating a variety of benefits. The aldo-keto reductase (AKR) superfamily includes a huge collection of NAD(P)H-dependent oxidoreductases that carry out a variety of redox reactions essential for biosynthesis, detoxification, and intermediary metabolism. The present study involved the isolation, cloning, and purification of a novel aldo-ketoreductase (AvAKR) from the leaves of Aloe vera (Aloe barbadensis Miller) by heterologous gene expression in Escherichia coli based on the unigene sequences of putative ketoreductase and cDNA library screening by oligonucleotide hybridization. The in-silico structural analysis, phylogenetic relationship, and molecular modeling were outranged to approach the novelty of the sequence. Additionally, agroinfiltration of the candidate gene tagged with a green fluorescent protein (GFP) was employed for transient expression in the Nicotiana benthamiana to evaluate the sub-cellular localization of the candidate gene. The AvAKR preferred cytoplasmic localization and shared similarities with the known plant AKRs, keeping the majority of the conserved active-site residues in the AKR superfamily enzymes. The enzyme facilitated the NADPH-dependent reduction of various carbonyl substrates, including benzaldehyde and sugars, proclaiming a broad spectrum range. Our study successfully isolated and characterized a novel aldo-ketoreductase (AvAKR) from Aloe vera, highlighting its versatile NADPH-dependent carbonyl reduction proficiency therewith showcasing its potential as a versatile biocatalyst in diverse redox reactions.

Crystal structures and kinetic studies of a laboratory evolved aldehyde reductase explain the dramatic shift of its new substrate specificity.

The Fe2+-dependent E. coli enzyme FucO catalyzes the reversible interconversion of short-chain (S)-lactaldehyde and (S)-1,2-propanediol, using NADH and NAD+ as cofactors, respectively. Laboratory-directed evolution experiments have been carried out previously using phenylacetaldehyde as the substrate for screening catalytic activity with bulky substrates, which are very poorly reduced by wild-type FucO. These experiments identified the N151G/L259V double mutant (dubbed DA1472) as the most active variant with this substrate via a two-step evolutionary pathway, in which each step consisted of one point mutation. Here the crystal structures of DA1472 and its parent D93 (L259V) are reported, showing that these amino acid substitutions provide more space in the active site, though they do not cause changes in the main-chain conformation. The catalytic activity of DA1472 with the physiological substrate (S)-lactaldehyde and a series of substituted phenylacetaldehyde derivatives were systematically quantified and compared with that of wild-type as well as with the corresponding point-mutation variants (N151G and L259V). There is a 9000-fold increase in activity, when expressed as kcat/KM values, for DA1472 compared with wild-type FucO for the phenylacetaldehyde substrate. The crystal structure of DA1472 complexed with a non-reactive analog of this substrate (3,4-dimethoxyphenylacetamide) suggests the mode of binding of the bulky group of the new substrate. These combined structure-function studies therefore explain the dramatic increase in catalytic activity of the DA1472 variant for bulky aldehyde substrates. The structure comparisons also suggest why the active site in which Fe2+ is replaced by Zn2+ is not able to support catalysis.

Synthesis and characterization of novel acyl hydrazones derived from vanillin as potential aldose reductase inhibitors.

In the polyol pathway, aldose reductase (AR) catalyzes the formation of sorbitol from glucose. In order to detoxify some dangerous aldehydes, AR is essential. However, due to the effects of the active polyol pathway, AR overexpression in the hyperglycemic state leads to microvascular and macrovascular diabetic problems. As a result, AR inhibition has been recognized as a potential treatment for issues linked to diabetes and has been studied by numerous researchers worldwide. In the present study, a series of acyl hydrazones were obtained from the reaction of vanillin derivatized with acyl groups and phenolic Mannich bases with hydrazides containing pharmacological groups such as morpholine, piperazine, and tetrahydroisoquinoline. The resulting 21 novel acyl hydrazone compounds were investigated as an inhibitor of the AR enzyme. All the novel acyl hydrazones derived from vanillin demonstrated activity in nanomolar levels as AR inhibitors with IC50 and KI values in the range of 94.21 ± 2.33 to 430.00 ± 2.33 nM and 49.22 ± 3.64 to 897.20 ± 43.63 nM, respectively. Compounds 11c and 10b against AR enzyme activity were identified as highly potent inhibitors and showed 17.38 and 10.78-fold more effectiveness than standard drug epalrestat. The synthesized molecules' absorption, distribution, metabolism, and excretion (ADME) effects were also assessed. The probable-binding mechanisms of these inhibitors against AR were investigated using molecular-docking simulations.

N-substituted phthalazine sulfonamide derivatives as non-classical aldose reductase inhibitors.

Aldose reductase (AR, AKR1B1; EC 1.1.1.21) is an aldo-keto reductase that has been widely investigated as an enzyme crucially involved in the pathogenesis of several chronic complications, including nephropathy, neuropathy, retinopathy, and cataracts associated with diabetes mellitus. Although sulfonamides have been reported to possess many other biological activities, in continuation of our interest in designing and discovering potent inhibitors of AR, herein, we have evaluated the AR inhibitory potential of N-substituted phthalazine sulfonamide derivatives 5a-l. The biological studies revealed that all the derivatives show excellent activity against AR, with KI constants ranging from 67.73 to 495.20 nM. Among these agents, 4-(6-nitro-1,4-dioxo-1,2,3,4-tetrahydrophthalazine-2-carbonyl)benzenesulfonamide (5e) and 1,4-dioxo-3-(4-sulfamoylbenzoyl)-1,2,3,4-tetrahydrophthalazine-6-carboxylic acid (5f) showed prominent inhibitory activity with KI values of 67.73 and 148.20 nM, respectively, vs AR and were found to be more potent than epalrestat (KI  = 852.50 nM), the only AR inhibitor currently used in the therapy. Moreover, molecular docking studies were also performed to rationalize binding site interactions of these sulfonamides (5a-l) with the target enzyme AR. According to ADME-Tox, predicts were also determined that these derivatives be ARIs displaying suitable drug-like properties. The sulfonamides identified in this study may be used to develop lead therapeutic agents inhibiting diabetic complications.

In Search of Differential Inhibitors of Aldose Reductase.

Aldose reductase, classified within the aldo-keto reductase family as AKR1B1, is an NADPH dependent enzyme that catalyzes the reduction of hydrophilic as well as hydrophobic aldehydes. AKR1B1 is the first enzyme of the so-called polyol pathway that allows the conversion of glucose into sorbitol, which in turn is oxidized to fructose by sorbitol dehydrogenase. The activation of the polyol pathway in hyperglycemic conditions is generally accepted as the event that is responsible for a series of long-term complications of diabetes such as retinopathy, cataract, nephropathy and neuropathy. The role of AKR1B1 in the onset of diabetic complications has made this enzyme the target for the development of molecules capable of inhibiting its activity. Virtually all synthesized compounds have so far failed as drugs for the treatment of diabetic complications. This failure may be partly due to the ability of AKR1B1 to reduce alkenals and alkanals, produced in oxidative stress conditions, thus acting as a detoxifying agent. In recent years we have proposed an alternative approach to the inhibition of AKR1B1, suggesting the possibility of a differential inhibition of the enzyme through molecules able to preferentially inhibit the reduction of either hydrophilic or hydrophobic substrates. The rationale and examples of this new generation of aldose reductase differential inhibitors (ARDIs) are presented.

Structural and Biochemical Analysis of the Furan Aldehyde Reductase YugJ from Bacillus subtilis.

NAD(H)/NADP(H)-dependent aldehyde/alcohol oxidoreductase (AAOR) participates in a wide range of physiologically important cellular processes by reducing aldehydes or oxidizing alcohols. Among AAOR substrates, furan aldehyde is highly toxic to microorganisms. To counteract the toxic effect of furan aldehyde, some bacteria have evolved AAOR that converts furan aldehyde into a less toxic alcohol. Based on biochemical and structural analyses, we identified Bacillus subtilis YugJ as an atypical AAOR that reduces furan aldehyde. YugJ displayed high substrate specificity toward 5-hydroxymethylfurfural (HMF), a furan aldehyde, in an NADPH- and Ni2+-dependent manner. YugJ folds into a two-domain structure consisting of a Rossmann-like domain and an α-helical domain. YugJ interacts with NADP and Ni2+ using the interdomain cleft of YugJ. A comparative analysis of three YugJ structures indicated that NADP(H) binding plays a key role in modulating the interdomain dynamics of YugJ. Noticeably, a nitrate ion was found in proximity to the nicotinamide ring of NADP in the YugJ structure, and the HMF-reducing activity of YugJ was inhibited by nitrate, providing insights into the substrate-binding mode of YugJ. These findings contribute to the characterization of the YugJ-mediated furan aldehyde reduction mechanism and to the rational design of improved furan aldehyde reductases for the biofuel industry.

Bombyx mori-derived aldo-keto reductase AKR2E8 detoxifies aldehydes present in mulberry leaves.

Lepidopterans are agricultural pests. Since the silkworm is a model for lepidopterans, analysis of the enzymes produced by silkworms is of great interest for developing methods of pest control. The aldo-keto reductase (AKR) superfamily catalyzes the reduction of aldehydes by converting a carbonyl group to an alcohol group. Here, we characterized a new AKR present in the silkworm Bombyx mori, which has been designated as AKR2E8. Amino acid sequence and phylogenetic analyses showed that AKR2E8 is similar to human AKR1B1 and AKR1B10. Three amino acid residues in the active site were identical among AKR2E8, AKR1B1, and AKR1B10. Recombinant AKR2E8 overexpressed in Escherichia coli used nicotinamide adenine dinucleotide phosphate as a coenzyme to reduce the aldehydes present in mulberry (Morus alba) leaves. AKR2E8 was found to reduce benzaldehyde, hexanal, heptanal, nonanal, trans-2-nonenal, and citral. No nicotinamide adenine dinucleotide-dependent activity was detected. Akr2e8 mRNA was detected in the testes, ovaries, and fat body; the highest expression was found in the midgut. The substrate specificity and highest observed expression of AKR2E8 in the midgut suggests that AKR2E8 may play a major role in aldehyde detoxification in silkworms. The findings of this study may assist in the development of pest control methods for controlling the population of lepidopterans, such as silkworms, that damage crops.

Role of Cys-298 in specific recognition of glutathione by aldose reductase.

Aldose reductase (AR) is an NADPH-dependent oxidoreductase that is well-studied for its role in Diabetes Mellitus. Glutathione conjugated aldehydes are efficiently catalysed by AR. We have employed molecular dynamics simulations to investigate the dynamics of a glutathione analog, γ-glutamyl-S-(1,2-di-carboxyethyl)-cysteinyl-glycine (DCEG), into the binding pocket of AR. Study revealed that backbone nitrogens of Ala-299 and Leu-300 form a tiny pocket gated by thiol group of Cys-298. The glycine moiety of DCEG was able to displace the thiol group of Cys-298 to make hydrogen bond interactions with backbone of Ala-299, Leu-300, and Leu-301. This study provides the details of the dynamic interactions of DCEG in the binding pocket of AR, and shall aid in the design/discovery of differential inhibitors against AR.Communicated by Ramaswamy H. Sarma.

Synthesis, biological evaluation, and in silico study of novel library sulfonates containing quinazolin-4(3H)-one derivatives as potential aldose reductase inhibitors.

A series of novel sulfonates containing quinazolin-4(3H)-one ring derivatives was designed to inhibit aldose reductase (ALR2, EC 1.1.1.21). Novel quinazolinone derivatives (1-21) were synthesized from the reaction of sulfonated aldehydes with 3-amino-2-alkylquinazolin-4(3H)-ones in glacial acetic acid with good yields (85%-94%). The structures of the novel molecules were characterized using IR, 1 H-NMR, 13 C-NMR, and HRMS. All the novel quinazolinones (1-21) demonstrated nanomolar levels of inhibitory activity against ALR2 (KI s are in the range of 101.50-2066.00 nM). Besides, 4-[(2-isopropyl-4-oxoquinazolin-3[4H]-ylimino)methyl]phenyl benzenesulfonate (15) showed higher inhibitor activity inhibited ALR2 up to 7.7-fold compared to epalrestat, a standard inhibitor. Binding interactions between ALR2 and quinazolinones have been investigated using Schrödinger Small-Molecule Drug Discovery Suite 2021-1, reported possible inhibitor-ALR2 interactions. Both in vitro and in silico study results suggest that these quinazolin-4(3H)-one ring derivatives (1-21) require further molecular modification to improve their drug nominee potency as an ALR2 inhibitor.

Which Properties Allow Ligands to Open and Bind to the Transient Binding Pocket of Human Aldose Reductase?

The transient specificity pocket of aldose reductase only opens in response to specific ligands. This pocket may offer an advantage for the development of novel, more selective ligands for proteins with similar topology that lack such an adaptive pocket. Our aim was to elucidate which properties allow an inhibitor to bind in the specificity pocket. A series of inhibitors that share the same parent scaffold but differ in their attached aromatic substituents were screened using ITC and X-ray crystallography for their ability to occupy the pocket. Additionally, we investigated the electrostatic potentials and charge distribution across the attached terminal aromatic groups with respect to their potential to bind to the transient pocket of the enzyme using ESP calculations. These methods allowed us to confirm the previously established hypothesis that an electron-deficient aromatic group is an important prerequisite for opening and occupying the specificity pocket. We also demonstrated from our crystal structures that a pH shift between 5 and 8 does not affect the binding position of the ligand in the specificity pocket. This allows for a comparison between thermodynamic and crystallographic data collected at different pH values.

Multi-Targeted Molecular Docking, Pharmacokinetics, and Drug-Likeness Evaluation of Okra-Derived Ligand Abscisic Acid Targeting Signaling Proteins Involved in the Development of Diabetes.

Diabetes mellitus is a global threat affecting millions of people of different age groups. In recent years, the development of naturally derived anti-diabetic agents has gained popularity. Okra is a common vegetable containing important bioactive components such as abscisic acid (ABA). ABA, a phytohormone, has been shown to elicit potent anti-diabetic effects in mouse models. Keeping its anti-diabetic potential in mind, in silico study was performed to explore its role in inhibiting proteins relevant to diabetes mellitus- 11ß-hydroxysteroid dehydrogenase (11ß-HSD1), aldose reductase, glucokinase, glutamine-fructose-6-phosphate amidotransferase (GFAT), peroxisome proliferator-activated receptor-gamma (PPAR-gamma), and Sirtuin family of NAD(+)-dependent protein deacetylases 6 (SIRT6). A comparative study of the ABA-protein docked complex with already known inhibitors of these proteins relevant to diabetes was compared to explore the inhibitory potential. Calculation of molecular binding energy (ΔG), inhibition constant (pKi), and prediction of pharmacokinetics and pharmacodynamics properties were performed. The molecular docking investigation of ABA with 11-HSD1, GFAT, PPAR-gamma, and SIRT6 revealed considerably low binding energy (ΔG from -8.1 to -7.3 Kcal/mol) and predicted inhibition constant (pKi from 6.01 to 5.21 µM). The ADMET study revealed that ABA is a promising drug candidate without any hazardous effect following all current drug-likeness guidelines such as Lipinski, Ghose, Veber, Egan, and Muegge.

Identification of a new class of potent aldose reductase inhibitors: Design, microwave-assisted synthesis, in vitro and in silico evaluation of 2-pyrazolines.

Aldose reductase (AR) acts as a multi-disease target for the design and development of therapeutic agents for the management of diabetic complications as well as non-diabetic diseases. In the search for potent AR inhibitors, the microwave-assisted synthesis of twenty new compounds with a 1,3-diaryl-5-(4-fluorophenyl)-2-pyrazoline moiety as a common fragment in their structure (1-20) was carried out efficiently. Compounds 1-20 were subjected to in vitro studies, which were conducted to assess their AR inhibitory effects and cytotoxicity towards L929 mouse fibroblast (normal) cells. Among these compounds, 1-(3-bromophenyl)-3-(4-piperidinophenyl)-5-(4-fluorophenyl)-2-pyrazoline (20) was identified as the most promising AR inhibitor with an IC50 value of 0.160 ± 0.005 µM exerting competitive inhibition with a Ki value of 0.019 ± 0.001 µM as compared to epalrestat (IC50 = 0.279 ± 0.001 µM; Ki = 0.801 ± 0.023 µM) and quercetin (IC50 = 4.120 ± 0.123 µM; Ki = 6.082 ± 0.272 µM). Compound 20 displayed cytotoxicity towards L929 cells with an IC50 value of 18.75 ± 1.06 µM highlighting its safety as an AR inhibitor. Molecular docking studies suggested that π-π stacking interactions occurred between the m-bromophenyl moiety of compound 20 and Trp21. Based on in silico pharmacokinetic studies, compound 20 was found to possess favorable oral bioavailability and drug-like properties. It can be concluded that compound 20 is a potential orally bioavailable AR inhibitor for the management of diabetic complications as well as non-diabetic diseases.

Non-carboxylic acid inhibitors of aldose reductase based on N-substituted thiazolidinedione derivatives.

In search of dually active PPAR-modulators/aldose reductase (ALR2) inhibitors, 16 benzylidene thiazolidinedione derivatives, previously reported as partial PPARγ agonists, together with additional 18 structural congeners, were studied for aldose reductase inhibitory activity. While no compounds had dual property, our efforts led to the identification of promising inhibitors of ALR2. Eight compounds (11, 15-16, 20-24, 30) from the library of 33 compounds were identified as potent and selective inhibitors of ALR2. Compound 21 was the most effective and selective inhibitor with an IC50 value of 0.95 ± 0.11 and 13.52 ± 0.81 µM against ALR2 and aldehyde reductase (ALR1) enzymes, respectively. Molecular docking and dynamics studies were performed to understand inhibitor-enzyme interactions at the molecular level that determine the potency and selectivity. Compound 21 was further subjected to in silico and in vitro studies to evaluate the pharmacokinetic profile. Being less acidic (pKa = 9.8), the compound might have a superior plasma membrane permeability and reach the cytosolic ALR2. This fact together with excellent drug-likeness criteria points to improved bioavailability compared to the clinically used compound Epalrestat. The designed compounds represent a novel group of non-carboxylate inhibitors of aldose reductase with an improved physicochemical profile.

Evaluation of the role of the DNA surface for enhancing the activity of scaffolded enzymes.

The catalytic enhancements of enzymes loaded on DNA nanostructures have been attributed to the characteristics provided by highly negative charges on the surface of the DNA scaffold, such as the modulation of the local pH near enzymes. In this study, two types of enzymes with optimal activity at pH 6 and 8 equally displayed significant catalytic enhancements on the DNA scaffold surface. By using a ratiometric pH indicator, a lower local pH shift of 0.8 was observed near the DNA scaffold surface. The postulated local pH change near the DNA scaffold surface is unlikely to play a general role in enhancing the activity of the scaffolded enzymes.

Assessing the In Vitro Inhibitory Effects on Key Enzymes Linked to Type-2 Diabetes and Obesity and Protein Glycation by Phenolic Compounds of Lauraceae Plant Species Endemic to the Laurisilva Forest.

Methanolic leaf extracts of four Lauraceae species endemic to Laurisilva forest (Apollonias barbujana, Laurus novocanariensis, Ocotea foetens and Persea indica) were investigated for the first time for their potential to inhibit key enzymes linked to type-2 diabetes (α-amylase, α-glucosidase, aldose reductase) and obesity (pancreatic lipase), and protein glycation. Lauraceae extracts revealed significant inhibitory activities in all assays, altough with different ability between species. In general, P. indica showed the most promissing results. In the protein glycation assay, all analysed extracts displayed a stronger effect than a reference compound: aminoguanidine (AMG). The in vitro anti-diabetic, anti-obesity and anti-glycation activities of analysed extracts showed correlation with their flavonols and flavan-3-ols (in particular, proanthocyanins) contents. These Lauraceae species have the capacity to assist in adjuvant therapy of type-2 diabetes and associated complications, through modulation of the activity of key metabolic enzymes and prevention of advanced glycation end-products (AGEs) formation.

Dynamic Preference for NADP/H Cofactor Binding/Release in E. coli YqhD Oxidoreductase.

YqhD, an E. coli alcohol/aldehyde oxidoreductase, is an enzyme able to produce valuable bio-renewable fuels and fine chemicals from a broad range of starting materials. Herein, we report the first computational solution-phase structure-dynamics analysis of YqhD, shedding light on the effect of oxidized and reduced NADP/H cofactor binding on the conformational dynamics of the biocatalyst using molecular dynamics (MD) simulations. The cofactor oxidation states mainly influence the interdomain cleft region conformations of the YqhD monomers, involved in intricate cofactor binding and release. The ensemble of NADPH-bound monomers has a narrower average interdomain space resulting in more hydrogen bonds and rigid cofactor binding. NADP-bound YqhD fluctuates between open and closed conformations, while it was observed that NADPH-bound YqhD had slower opening/closing dynamics of the cofactor-binding cleft. In the light of enzyme kinetics and structural data, simulation findings have led us to postulate that the frequently sampled open conformation of the cofactor binding cleft with NADP leads to the more facile release of NADP while increased closed conformation sampling during NADPH binding enhances cofactor binding affinity and the aldehyde reductase activity of the enzyme.

Screening and characterization of aldose reductase inhibitors from Traditional Chinese medicine based on ultrafiltration-liquid chromatography mass spectrometry and in silico molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenqi Jiangtang granule (SJG) is an ancient Chinese herbal formula used for treatment of Diabetes mellitus and its complications. AIM OF THE STUDY: To establish an integrated approach for discovery of effective Aldose reductase inhibitors (ARIs) from SJG. MATERIALS AND METHODS: An integrated approach combining ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS) with in silico molecular docking was established for development of ARIs. AR enzyme was separated from the rabbit's crystalline lens. The inhibitory activities of these compounds were detected by UV spectrophotometry with DL-glyceraldehyde as a substrate. Furthermore, molecular docking was used to understand the binding mechanism of these screened compounds interacting with AR. RESULTS: After optimization of AR reaction system and ultrafiltration incubation system, 17 active ingredients were screened from SJG by UF-LC-MS technique. Among these potential AR inhibitors, ginsenoside Rd exhibited the strongest activity with IC50 value of 45.77 µM. Three of them, calycosin, gomisin J and schisandrin A were demonstrated to be potential inhibitors for the first time, with IC50 at 447.34 µM, 181.73 µM, and 429.00 µM, respectively. Most of the active compounds exhibited competitive inhibition against AR. The docking scores of saponins were higher than that of lignans, which was consistent with the verification results. CONCLUSION: The results indicated that TCM formula with clinical efficacy was indeed hopeful source for screening active ingredients, and the combination of UF-LC-MS and in silico molecular docking was a universal and promising approach for development of effective enzyme inhibitors.

Simultaneous Optimization of Activity and Stability of Xylose Reductase from D. nepalensis NCYC 3413 Using Statistical Experimental Design.

BACKGROUND: Physical parameters like pH and temperature play a major role in the design of an industrial enzymatic process. Enzyme stability and activity are greatly influenced by these parameters; hence optimization and control of these parameters becomes a key point in determining the economic feasibility of the process. OBJECTIVE: This study was taken up with the objective to optimize physical parameters for maximum stability and activity of xylose reductase from D. nepalensis NCYC 3413 through separate and simultaneous optimization studies and comparison thereof. METHODS: Effects of pH and temperature on the activity and stability of xylose reductase from Debaryomyces nepalensis NCYC 3413 were investigated by enzyme assays and independent variables were optimised using surface response methodology. Enzyme activity and stability were optimised separately and concurrently to decipher the appropriate conditions. RESULTS: Optimized conditions of pH and temperature for xylose reductase activity were determined to be 7.1 and 27 °C respectively, with predicted responses of specific activity (72.3 U/mg) and half-life time (566 min). The experimental values (specific activity 50.2 U/mg, half-life time 818 min) were on par with predicted values indicating the significance of the model. CONCLUSION: Simultaneous optimization of xylose reductase activity and stability using statistical methods is effective as compared to optimisation of the parameters separately.

Michaelis-Menten Kinetics Measurements of Aldo-Keto Reductases for Various Substrates in Murine Tissue.

Aldo-keto reductases (AKRs) are responsible for the detoxification of harmful aldehydes. Due to the large number of isotypes, the physiological relevance of AKRs cannot be obtained using mRNA or protein quantification, but only through the use of enzymatic assays to demonstrate functionality. Here, we present a fast and simple protocol to determine the important Michaelis-Menten kinetics of AKRs, which includes various aldehyde substrates of interest such as 4-hydroxynonenal, methylglyoxal, and malondialdehyde. For complete details on the use and execution of this protocol, please refer to Morgenstern et al. (2017) and Schumacher et al. (2018).