Lipase Type Research Articles

Latest Trends in Lipase-Catalyzed Synthesis of Ester Carbohydrate Surfactants: From Key Parameters to Opportunities and Future Development.

Carbohydrate-based surfactants are amphiphilic compounds containing hydrophilic moieties linked to hydrophobic aglycones. More specifically, carbohydrate esters are biosourced and biocompatible surfactants derived from inexpensive renewable raw materials (sugars and fatty acids). Their unique properties allow them to be used in various areas, such as the cosmetic, food, and medicine industries. These multi-applications have created a worldwide market for biobased surfactants and consequently expectations for their production. Biobased surfactants can be obtained from various processes, such as chemical synthesis or microorganism culture and surfactant purification. In accordance with the need for more sustainable and greener processes, the synthesis of these molecules by enzymatic pathways is an opportunity. This work presents a state-of-the-art lipase action mode, with a focus on the active sites of these proteins, and then on four essential parameters for optimizing the reaction: type of lipase, reaction medium, temperature, and ratio of substrates. Finally, this review discusses the latest trends and recent developments, showing the unlimited potential for optimization of such enzymatic syntheses.

Polyurethane degradation by extracellular urethanase producing bacterial isolate Moraxella catarrhalis strain BMPPS3

Plastic waste has become a global issue and a threat to the ecosystem. The present study isolated polyurethane (PU) degrading bacterial species from soil dumped with plastic wastes. Four bacterial isolates, RS1, RS6, RS9 and RS13 were obtained and their ability to degrade PU in a synthetic medium with PU as a sole source of carbon was assessed individually. After thirty days of incubation, the highest PU weight loss of 67.36 ± 0.32% was recorded in the medium containing RS13 isolate. The results of FTIR revealed the occurrence of carbonyl peaks. The putative isolate RS13 confirmed with the genus Moraxella according to 16S rRNA gene sequencing and the isolate was specified as Moraxella catarrhalis strain BMPPS3. The restriction analysis of Moraxella catarrhalis strain BMPPS3 revealed that the GCAT content to 51% and 49% correspondingly. Moraxella catarrhalis strain BMPPS3 was able to colonize on PU surface and form a biofilm as revealed by SEM investigation. Fatty acids and alkanes were found to be the degradation products by GC-MS analysis. The presence of these metabolites facilitated the growth of strain RS13 and suggested that ester hydrolysis products had been mineralized into CO2 and H2O. Extracellular biosurfactant synthesis has also been found in Moraxella catarrhalis strain BMPPS13 inoculated with synthetic media and mineral salt media containing PU and glucose as carbon sources, respectively with a significant level of cell-surface hydrophobicity (32%). The production and activity of extracellular esterase showed consistent increase from day 1–15 which peaked (1.029 mM/min/mg) on day 24 significantly at P < 0.001. Crude biosurfactants were lipopeptide-based, according to the characteristic investigation. According to this study findings, Moraxella catarrhalis produces biosurfactants of the esterase, urethanase and lipase (lipopeptide) types when carbon source PU is present.

Differential effect of nine cinnamic acid derivatives on the biocatalytic activity of Candida antarctica lipase type B

Naturally occurring cinnamic acid derivatives are a broad family of aromatic carboxylic acids with bioactive properties. Among the cinnamic acid derivatives, for instance, are ferulic acid and caffeic acid, which have been widely studied for their antioxidant and anti-inflammatory properties. These active ingredients are mostly poorly soluble in water, which greatly limits their bioavailability. To increase the bioavailability of these acids, green esterification protocols can be developed exploiting lipases. In particular, this article reports the process optimization for the enzymatic esterification of nine cinnamic acid derivatives with erythritol, a polyol highly soluble in water. The study explores how the different substituents present on the aromatic ring of the cinnamic acid derivatives affect the catalytic capacity of the Candida antarctica lipase type B. The study, conducted through both molecular docking and experimental evidence, shows how hydroxyl groups on the aromatic ring can strongly limit the conversion of the acids to the corresponding esters. At the same time, the degree of unsaturation of the derivative also influences the favorable poses in the active site of the lipase. The best results (yields over 95 %) were obtained with 10 g/L of lipase, a temperature of 90 °C, molar ratio (acid/alcohol) of 3:1, for 72 h of reaction time.

Process Parameters Influence Product Yield and Kinetic Parameters in Lipase Catalysis

AbstractEffects of process parameters like enzyme concentration, concentration and type of substrate, pH, temperature, speed of agitation, and pressure on lipase catalysis are reviewed. The enzyme concentration controls its interfacial presence and consequently the rate of reaction. A change in substrate concentration alters lipase kinetics. Substrate‐lipase interaction varies with substrate type and pH. Water concentration and agitation affect the extent of interfacial area. Temperature impacts the rate and thermal denaturation of enzymes. Statistical optimization can solve the problem of controlling a variable's effect by other variables. Immobilization support and nonionic surfactant altered the significance of enzyme concentration. The lipase type controlled the impact of concentrations of enzyme, substrate, and water. The water content was important during lipase‐catalyzed hydrolysis and esterification. The mode of agitation influenced the significance of enzyme concentration and temperature. Time had a remarkable impact during hydrolysis. Temperature, substrate type, and chain length notably controlled kinetic parameters. This work paves the way for similar studies on other enzymes.

Enzymatic synthesis of novel pyrrole esters and their thermal stability

In the present work a simple enzymatic approach (Novozym 435) for transesterification to synthesize pyrrole esters was reported. To generate the best reaction conditions, which resulted in the optimum yield of 92%, the effects of lipase type, solvent, lipase load, molecular sieves, substrate molar ratio of esters to alcohol, reaction temperature, reaction duration, and speed of agitation were evaluated. The range of alcohols was assessed under optimal circumstances. The spectrum observations conclusively demonstrated that the compounds could be generated with high yield under the circumstances utilized for synthesis. The odor characteristics of the pyrrolyl esters obtained were examined by gas chromatography–mass spectrometry-olfactometry (GC–MS–O). Among them, compounds of benzhydryl 1H-pyrrole-2-carboxylate (3j), butyl 1H-pyrrole-2-carboxylate (3k) and pentyl 1H-pyrrole-2-carboxylate (3l) present sweet and acid aroma. In addition, the thermal degradation process was further studied using the Py–GC/MS (pyrolysis–gas chromatography/mass spectrometry), TG (thermogravimetry), and DSC (differential scanning calorimeter) techniques. The outcomes of the Py–GC/MS, TG, and DSC techniques show that they have excellent thermal stability.

Engineered aldoxime dehydratase to enable the chemoenzymatic conversion of benzyl amines to aromatic nitriles

A chemoenzymatic strategy has been implemented to synthesize nitriles from benzyl amines under mild conditions. Aldoxime dehydratase (Oxd) plays a decisive role to convert aldoximes into corresponding nitriles. However, natural Oxds commonly exhibit extremely low catalytic capacity toward benzaldehyde oximes. Here, we engineered the OxdF1 from Pseudomonas putida F1 to enhance its catalytic efficiency toward benzaldehyde oximes by a semi-rational design strategy. The protein structure-based CAVER analysis indicates that M29, A147, F306, and L318 are located adjacent to the substrate tunnel entrance of OxdF1, which were responsible for the transportation of substrate into the active site. After two rounds of mutagenesis, the maximum activities of the mutants L318F and L318F/F306Y were 2.6 and 2.8 U/mg respectively, which were significantly higher than the wild OxdF1 of 0.7 U/mg. Meanwhile, the lipase type B from Candida antarctica was functionally expressed in Escherichia coli cells to selectively oxidize benzyl amines to aldoximes using urea-hydrogen peroxide adduct (UHP) as an oxidant in ethyl acetate. To merge the oxidation and dehydration reactions, a reductive extraction solution was added to remove the residue UHP, which is critical to eliminate its inhibition on the Oxd activity. Consequently, nine benzyl amines were efficiently converted into corresponding nitriles by the chemoenzymatic sequence.

Lipase-catalyzed ring-opening polymerization of natural compound-based cyclic monomers.

The need for sustainable and environment-friendly materials has led to growing interest in the development of biodegradable polymers based on natural compounds. However, metal-based catalysts used in the polymerization process may cause concerns about the toxicity of the resultant polymers. Therefore, polymers derived from natural compounds and synthesized through the use of green catalysts are highly desirable. Lipase-catalyzed ring-opening polymerization (ROP) of biocompound-based cyclic monomers has emerged as a promising and green strategy for the design and synthesis of such polymers. In this review, we summarize reports on the use of ROP catalyzed by lipase for cyclic monomers derived from natural compounds, including bile acid- and porphyrin-based macrocycles, carbonate-based macrocycles, lactones, and cyclic anhydrides, with an emphasis on ring-closure reactions for the synthesis of cyclic monomers, the types of lipases for the ROP and the choice of reaction conditions (temperature, solvent, reaction time, etc.). Moreover, the current challenges and perspectives for the choice and reusability of lipases, ring-closure versus ring-opening reactions, monomer design, and potential applications are discussed.

Lipid metabolism in sarcopenia.

Sarcopenia is an age-related disease associated with loss of muscle mass and strength. This geriatric syndrome predisposes elderly individuals to a disability, falls, fractures, and death. Fat infiltration in muscle is one of the hallmarks of sarcopenia and aging. Alterations in fatty acid (FA) metabolism are evident in aging, type 2 diabetes, and obesity, with the accumulation of lipids inside muscle cells contributing to muscle insulin resistance and ceramide accumulation. These lipids include diacylglycerol, lipid droplets, intramyocellular lipids, intramuscular triglycerides, and polyunsaturated fatty acids (PUFAs). In this review, we examine the regulation of lipid metabolism in skeletal muscle, including lipid metabolization and storage, intervention, and the types of lipases expressed in skeletal muscle responsible for the breakdown of adipose triglyceride fats. In addition, we address the role of FAs in sarcopenia and the potential benefits of PUFAs.

Synthesis, Molecular Docking and Molecular Dynamics Simulation Studies of Some Pyridazinone Derivatives as Lipase Inhibitors

Human health and illness are dependent on lipases, which play a key role in maintaining cell integrity, storing fat for energy and serving as signaling molecules. In this study, 4 compounds that carry 6-phenylpyridazin-3(2H)-one main nucleus, which can be effective as lipase inhibitors, were synthesized and their structures were elucidated. The biological activity of synthesized compounds was evaluated via the porcine pancreatic lipase type II (PLL) inhibitor assay. Orlistat, a lipase inhibitor, was used as a positive control. Compound 8d was found to be the most effective compound, with an IC50 value of 32.66±2.8265 (μg/mL). In addition, molecular docking and molecular dynamics simulations studies were carried out to examine the interactions of the compounds with the target in detail. The results obtained as a result of these in silico studies were found to be compatible with the lipase inhibition effects of the compounds. It was observed that the compounds may have potential lipase inhibitory effects as a result of the substitutions of the 3-(6-oxo-3-phenylpyridazin-1(6H)-yl)propanehydrazide structure.

Development of a Tailored Sol-Gel Immobilized Biocatalyst for Sustainable Synthesis of the Food Aroma Ester n-Amyl Caproate in Continuous Solventless System.

This study reports the synthesis of a hybrid sol-gel material, based on organically modified silanes (ORMOSILs) with epoxy functional groups, and its application in the stabilization of lipase type B from Candida antarctica (CalB) through sol-gel entrapment. The key immobilization parameters in the sol-gel entrapment of lipase using epoxysilanes were optimized by the design of numerous experiments, demonstrating that glycidoxypropyl-trimethoxysilane can allow the formation of a matrix with excellent properties in view of the biocatalytic esterifications catalyzed by this lipase, at an enzyme loading of 25 g/mol of silane. The characterization of the immobilized biocatalyst and the correlation of its catalytic efficiency with the morphological and physicochemical properties of the sol-gel matrix was accomplished through scanning electron microscopy (SEM), fluorescence microscopy (FM), as well as thermogravimetric and differential thermal analysis (TGA/DTA). The operational and thermal stability of lipase were increased as a result of immobilization, with the entrapped lipase retaining 99% activity after 10 successive reaction cycles in the batch solventless synthesis of n-amyl caproate. A possible correlation of optimal productivity and yield was attempted for this immobilized lipase via the continuous flow synthesis of n-amyl caproate in a solventless system. The robustness and excellent biocatalytic efficiency of the optimized biocatalyst provide a promising solution for the synthesis of food-grade flavor esters, even at larger scales.

Towards an alcohol-free process for the production of palm phytonutrients via enzymatic hydrolysis of crude palm oil using liquid lipases.

Crude palm oil (CPO) is rich with phytonutrients such as carotenoids and tocols which possesses many health benefits. The aim of this research was to develop a methanol-free process to produce palm phytonutrients via enzymatic hydrolysis. In this work, triacylglycerol was hydrolyzed into free fatty acids (FFAs) using three different types of liquid lipases derived from Aspergillus oryzae (ET 2.0), Aspergillus niger (Habio) and Candida antartica (CALB). ET 2.0 was found to be the best enzyme for hydrolysis. Under the optimum condition, the FFA content achievable was 790 g kg-1 after 24 h of reaction with 1:1 water-to-oil mass ratio at 50 °C and stirring speed of 9 × g. Furthermore, with the addition of 2g kg-1 ascorbic acid, it was found that 98% of carotenoids and 96% of tocols could be retained after hydrolysis. This work shows that enzymatic hydrolysis, which is inherently safer, cleaner and sustainable is feasible to replace the conventional methanolysis for the production of palm phytonutrients. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Biomolecular Chemical Simulations on Enantioselectivity and Reactivity of Lipase Enzymes to Azulene Derivatives

Biomolecular chemical simulations have recently become a useful research method in the fields of organic chemistry and bioscience. In the last few years, we have been focusing on the biomolecular computational simulation on lipase enzyme and ligand complexes to predict the enantioselectivity and reactivity of lipases toward non-natural organic compounds. In this paper, we describe the molecular simulations including molecular dynamics (MD) and fragment molecular orbital (FMO) calculations for the complexes of Candida antarctica lipase type A (CALA) and trifluoromethylazulene alcohol derivatives. From the MD calculations, we found that the fast-reacting enantiomer of esters with high enantioselectivity stays in the vicinity of the active site of CALA, while the slow-reacting enantiomer leaves the active site of CALA. On the other hand, both ( R)- and ( S)-enantiomers of ester with low ensntioselectivity were found to keep near to near the active site of CALA. Further, for the esters that do not react with lipase enzyme, we found that both ( R)- and ( S)-enantiomers move away from the active site of lipase enzyme. From the FMO calculations, we found that each fast-reacting enantiomer of esters with high enantioselectivity strongly interacts with certain particular amino acid residues in CALA containing Asp95, while both ( R)- and ( S)-enantiomers of ester with low enantioselectivity interact with same amino acid residues in CALA including Asp95. These results suggest that it is possible to predict not only the enantioselectivity but also the reactivity of CALA and to identify the amino acid residues important to the enzymatic reaction. Therefore, we consider that our computational simulations would be a useful method for predicting and understanding the reactivity and the enantioselectivity of lipase-catalyzed biotransformations.

Expanding Access to Optically Active Non-Steroidal Anti-Inflammatory Drugs via Lipase-Catalyzed KR of Racemic Acids Using Trialkyl Orthoesters as Irreversible Alkoxy Group Donors

Studies into the enzymatic kinetic resolution (EKR) of 2-arylpropanoic acids (‘profens’), as the active pharmaceutical ingredients (APIs) of blockbuster non-steroidal anti-inflammatory drugs (NSAIDs), by using various trialkyl orthoesters as irreversible alkoxy group donors in organic media, were performed. The enzymatic reactions of target substrates were optimized using several different immobilized preparations of lipase type B from the yeast Candida antarctica (CAL-B). The influence of crucial parameters, including the type of enzyme and alkoxy agent, as well as the nature of the organic co-solvent and time of the process on the conversion and enantioselectivity of the enzymatic kinetic resolution, is described. The optimal EKR procedure for the racemic profens consisted of a Novozym 435-STREM lipase preparation suspended in a mixture of 3 equiv of trimethyl or triethyl orthoacetate as alkoxy donor and toluene or n-hexane as co-solvent, depending on the employed racemic NSAIDs. The reported biocatalytic system provided optically active products with moderate-to-good enantioselectivity upon esterification lasting for 7–48 h, with most promising results in terms of enantiomeric purity of the pharmacologically active enantiomers of title APIs obtained on the analytical scale for: (S)-flurbiprofen (97% ee), (S)-ibuprofen (91% ee), (S)-ketoprofen (69% ee), and (S)-naproxen (63% ee), respectively. In turn, the employment of optimal conditions on a preparative-scale enabled us to obtain the (S)-enantiomers of: flurbiprofen in 28% yield and 97% ee, ibuprofen in 45% yield and 56% ee, (S)-ketoprofen in 23% yield and 69% ee, and naproxen in 42% yield and 57% ee, respectively. The devised method turned out to be inefficient toward racemic etodolac regardless of the lipase and alkoxy group donor used, proving that it is unsuitable for carboxylic acids possessing tertiary chiral centers.

Efficacy of Enriched Medium-chain Fatty Acids in Virgin Coconut Oil in Their Antibacterial Activity against Food Pathogens

Free fatty acids (FFA) have been previously shown to be an effective bactericidal agent. In this study, the FFA mixture was attempted to isolate from virgin coconut oil (VCO) using lipases and was fractionated to obtain the enriched medium-chain fatty acids (MCFA) to compare its antibacterial efficacy against food pathogens. Three types of lipases – namely, lipozyme TL 100L, Candida rugosa lipase (CRL), and porcine pancreas lipase (PPL) – were used to compare their efficacy in VCO hydrolysis, in which CRL was found to be the most efficient lipase for the process of VCO hydrolysis at the water: buffer ratio of 1:5, lipase concentration of 5310 U/g, pH 7, and temperature of 40 °C. The vacuum-distillation process was successfully found to fractionate the initial FFA into three fractions: Fraction I containing MCFA (C8–C12) plus Fraction II (C12–C14) and Fraction III containing LCFA (C14–C20). Among these fractions, Fraction I was observed to be the most effective anti-agent against four out of five food pathogens (negative result against Salmonella typhimurium), followed by Fraction II and initial FFA. Antimicrobial activities of study fractions were found effective on both Gram-positive strains and Gram-negative strains.

Combination of Biocatalysis and Sonochemistry in the Ethyl Oleate Production

Objectives:Fatty acid alkyl esters (FAAEs) are a family of natural neutral lipids and can be produced cleanly and sustainably by esterification of free fatty acids (FFAs) with short chain alcohol using enzymatic catalysts. In this study, the use of lipases was evaluated in enzymatic esterification of oleic acid with ethanol using the combination of biocatalysis and sonochemistry (ultrasound) in the absence of co-solvents.Methods:Reaction parameters, such as type of lipase, amount of enzyme, reaction time, alcohol hydration level and enzyme turnover were evaluated for the enzymatic synthesis of ethyl oleate.Results:C. antarctica lipase provided yields above 95% in less than 10 h with 10% (w/w) of lipase. The use of hydrous ethanol (5% of water) showed a slight drop in yield but remained above 90% of ethyl oleate production. C. antarctica lipase showed no loss efficiency even after 10 reaction cycles.Conclusion:The combination of biocatalysis and ultrasound radiation provided a high yield, showing that the combination of these tools may be a good choice for the enzymatic synthesis of ethyl oleate.

Phytochemical and biological characterization of dry outer scales extract from Tropea red onion (Allium cepa L. var. Tropea)–A promising inhibitor of pancreatic lipase

BackgroundAllium cepa L. var. Tropea is typically cultivated in Calabria (Italy) and it is certified as “Cipolla Rossa di Tropea Calabria-PGI” (Tropea red onion). The use of clinically available anti-obesity drugs such as Orlistat is being gradually dismissed due to their side-effects and this has encouraged the search for alternative inhibitors of intestinal lipases such as phytochemicals showing less side-effects. In this study we aimed to evaluate for the first time the anti-obesity potential of the hydroalcoholic extract from the dry outer scales of Tropea red onion by the assesment of its capacity to inhibit pancreatic lipase. Its possible mechanism of action was also studied with planar lipid membranes (PLMs) surrogate of intestinal membranes. MethodsSpecialized metabolites in the extract were determined by GC–MS, HPLC-DAD, HPLC-UV-DAD and HPLC-HRMS analyses. Inhibition of pancreatic lipase was studied in vitro against crude lipase Type II from porcine pancreas. PLMs used in the electrophysiology measurements were made up of DOPS:DOPE:POPC. ResultsThe extract contained quercetin-4′-O-glucoside, quercetin and quercetin-3,4′-O-diglucoside as the most abundant phenolics. Among apolar constituents, γ-sitosterol, linoleic and stearic acids were dominant. The lipase inhibitory effect of the extract had an IC50 value equal to 0.77±0.03 mg/mL (positive control, IC50 = 0.018 mg/mL). The electrophysiological study demonstrated that the extract is able to incorporate into PLMs and to form transient channel-like events ConclusionsTaken altogether, the results allow us to suggest that the hydroalcoholic extract from the dry outer scales of Tropea red onion could prevent lipid ester hydrolysis and it has a protective effect against phospholipase as found for interfacially active compounds.

Evaluation of the Impact of Esterases and Lipases from the Circulatory System against Substrates of Different Lipophilicity.

Esterases and lipases can process amphiphilic esters used as drugs and prodrugs and impact their pharmacokinetics and biodistribution. These hydrolases can also process ester components of drug delivery systems (DDSs), thus triggering DDSs destabilization with premature cargo release. In this study we tested and optimized assays that allowed us to quantify and compare individual esterase contributions to the degradation of substrates of increased lipophilicity and to establish limitations in terms of substrates that can be processed by a specific esterase/lipase. We have studied the impact of carbonic anhydrase; phospholipases A1, A2, C and D; lipoprotein lipase; and standard lipase on the hydrolysis of 4-nitrophenyl acetate, 4-nitrophenyl palmitate, DGGR and POPC liposomes, drawing structure–property relationships. We found that the enzymatic activity of these proteins was highly dependent on the lipophilicity of the substrate used to assess them, as expected. The activity observed for classical esterases was diminished when lipophilicity of the substrate increased, while activity observed for lipases generally increased, following the interfacial activation model, and was highly dependent on the type of lipase and its structure. The assays developed allowed us to determine the most sensitive methods for quantifying enzymatic activity against substrates of particular types and lipophilicity.

Assessment on the oil accumulation by knockdown of triacylglycerol lipase in the oleaginous diatom Fistulifera solaris

Microalgae are promising producers of biofuel due to higher accumulation of triacylglycerol (TAG). However, further improvement of the lipid metabolism is critical for feasible application of microalgae in industrial production of biofuel. Suppression of lipid degradation pathways is a promising way to remarkably increase the lipid production in model diatoms. In this study, we established an antisense-based knockdown (KD) technique in the marine oleaginous diatom, Fistulifera solaris. This species has a capability to accumulate high content of lipids. Tgl1 KD showed positive impact on cell growth and lipid accumulation in conventional culture in f/2 medium, resulting in higher oil contents compared to wild type strain. However, these impacts of Tgl1 KD were slight when the cells were subjected to the two-stage growth system. The Tgl1 KD resulted in slight change of fatty acid composition; increasing in C14:0, C16:0 and C16:1, and decreasing in C20:5. This study indicates that, although Tgl1 played a certain role in lipid degradation in F. solaris, suppression of only a single type of TAG lipase was not significantly effective to improve the lipid production. Comprehensive understanding of the lipid catabolism in this microalga is essential to further improve the lipid production.

Study of enzymatic production of biodiesel using vegetable oils and commercial ethanol

Biodiesel is a combination of alkyl fatty acid esters generated by a catalyst and supported by acid, basic, or enzymatic processes from vegetable oils to short-chain alcohols such as methanol or ethanol. However, the high costs for raw materials utilising vegetable oil of food grade have made it commercially impracticable to produce this biofuel. Research has therefore expanded with residual oil indicating that biomass from household and industrial waste has been technically feasible. The outcome of this investigation coincides with the enzyme production study of biodiesel utilising residual oil and ethanol, according to this principle. In esterification of commercial oleic acid, the behaviour of commercial immobilised lipase from CAL-B was examined and the variables affecting the process were investigated. Based on the results presented and discussed in this paper, the use of immobilized Candida antarctica lipase type B (CAL-B) for biodiesel production is more viable when using acidic substrates, since the best results were achieved with such raw materials and with a reaction rate comparable to esterification with an acid catalyst. The results of this work showed that the enzymatic esterification of commercial oleic acid with ethanol provided a conversion of 87.3% within 60 minutes of reaction, at a temperature of 30 °C, in a stoichiometric proportion and without adsorption of water. Both biocatalysts showed good stability, which produced over 80% conversion and 60 minutes of reaction and could be repeated without substantial loss of activity for at least 10 consecutive occasions

Optimization of Spectrophotometric and Fluorometric Assays Using Alternative Substrates for the High-Throughput Screening of Lipase Activity

The effects of reaction conditions on the spectrophotometric and fluorometric assays using alternative substrates (p-nitrophenyl palmitate and 4-methylumbelliferyl oleate) were investigated to optimize them for the high-throughput screening of lipase activity from agricultural products. Four model lipases from Chromobacterium viscosum, Pseudomonas fluorescens, Sus scrofa pancreas, and wheat germ (Triticum aestivum) were allowed to hydrolyze the alternative substrates at different substrate concentrations (1–5 mM), operating pH (5.0–8.0), and operating temperatures (25–55°C). The results show that both the spectrophotometric and fluorometric assays worked well at the standard reaction conditions (pH 7.0 and 30°C) for finding a typical lipase, although pH conditions should be considered to detect the catalytic activity of lipases, which are applicable to more acidic or alkaline pH circumstances. To validate the optimized conditions, the high-throughput screening of lipase activity was conducted using 17 domestic agricultural products. A pileus of Pleurotus eryngii showed the highest activity in both the spectrophotometric (633.42 μU/mg) and fluorometric (101.77 μU/mg) assays. The results of this research provide practical information for the high-throughput screening of lipases using alternative substrates on microplates.