Commercial Lipase Preparations Research Articles

Extending the computational and experimental analysis of lipase active site selectivity.

Molecular docking is an important computational analysis widely used to predict the interaction of enzymes with several starting materials for developing new valuable products from several starting materials, including oils and fats. In the present study, molecular docking was used as an efficient in silico screening tool to select biocatalysts with the highest catalytic performance in butyl esters production in a solvent-free system, an eco-friendly approach, via direct esterification of free fatty acids from Licuri oil with butanol. For such purpose, three commercial lipase preparations were used to perform molecular docking studies such as Burkholderia cepacia (BCL), Porcine pancreatic (PPL), and Candida rugosa (CRL). Concurrently, the results obtained in BCL and CRL are the most efficient in the esterification process due to their higher preference for catalyzing the esterification of lauric acid, the main fatty acid found in the licuri oil composition. Meanwhile, PPL was the least efficient because it preferentially interacts with minor fatty acids. Molecular docking with the experimental results indicated the better performance in the synthesis of esters was BCL. In conclusion, experimental results analysis shows higher enzymatic productivity in esterification reactions of 1294.83μmol/h.mg, while the CRL and PPL demonstrated the lowest performance (189.87μmol / h.mg and 23.96μmol / h.mg, respectively). Thus, molecular docking and experimental results indicate that BCL is a more efficient lipase to produce fatty acids and esters from licuri oil with a high content of lauric acid. In addition, this study also demonstrates the application of molecular docking as an important tool for lipase screening to achieve more sustainable production of butyl esters with a view synthesis of biolubricants.

Lipase‐mediated plant oil hydrolysis—Toward a quantitative glycerol recovery for the synthesis of pure allyl alcohol and acrylonitrile

AbstractPlant based triacylglycerides are a sustainable feedstock for the chemical, bioenergy, food and cosmetic sector. While fatty acids conversion has been intensively studied, processes for glycerol valorization have been scarce. In contrast to chemical hydrolysis of plant triacylglycerides enzymatic hydrolysis methods provide a cleaner glycerol stream. This study focuses on the selection of a commercial lipase capable of quantitative hydrolysis of rapeseed‐ and high oleic sunflower oils. Enzymatic process using only water as the reaction medium allows simplified recovery of pure glycerol. Among the six commercial lipase preparations tested, Candida rugosa lipase was identified as the most effective biocatalyst. Catalytic behavior in buffer and pure water was equivalent. Glycerol generated using a washed lipase was recovered by just three purification steps. The FTIR spectrum of the recovered glycerol was equivalent to pure glycerol standard. Over the entire recovery process, 82%–88% of the theoretical glycerol yield could be obtained. Purified glycerol was further didehydroxylated to allyl alcohol by a formic acid mediated open distillation process. The enriched allyl alcohol had a quality, which allows to use it for the synthesis of bio‐based acrylonitrile.

Tuning Immobilized Commercial Lipase Preparations Features by Simple Treatment with Metallic Phosphate Salts.

Four commercial immobilized lipases biocatalysts have been submitted to modifications with different metal (zinc, cobalt or copper) phosphates to check the effects of this modification on enzyme features. The lipase preparations were Lipozyme®TL (TLL-IM) (lipase from Thermomyces lanuginose), Lipozyme®435 (L435) (lipase B from Candida antarctica), Lipozyme®RM (RML-IM), and LipuraSelect (LS-IM) (both from lipase from Rhizomucor miehei). The modifications greatly altered enzyme specificity, increasing the activity versus some substrates (e.g., TLL-IM modified with zinc phosphate in hydrolysis of triacetin) while decreasing the activity versus other substrates (the same preparation in activity versus R- or S- methyl mandelate). Enantiospecificity was also drastically altered after these modifications, e.g., LS-IM increased the activity versus the R isomer while decreasing the activity versus the S isomer when treated with copper phosphate. Regarding the enzyme stability, it was significantly improved using octyl-agarose-lipases. Using all these commercial biocatalysts, no significant positive effects were found; in fact, a decrease in enzyme stability was usually detected. The results point towards the possibility of a battery of biocatalysts, including many different metal phosphates and immobilization protocols, being a good opportunity to tune enzyme features, increasing the possibilities of having biocatalysts that may be suitable for a specific process.

Structures of two novel crystal forms of Aspergillus oryzae alpha amylase (taka-amylase)

The structures of Aspergillus oryzae α-amylase were determined in a tetragonal crystal, having one molecule as asymmetric unit, and a monoclinic crystal with two molecules as asymmetric unit. Both crystal forms were obtained from trace contaminants of an old commercial lipase preparation. Structures were determined and refined to 1.65Å and 1.43Å resolution respectively. The latter crystal has a non-crystallographic (NCS) twofold axis within the asymmetric unit. Glycosylation at Asn197 is evident, and in the tetragonal crystal can be seen to include three, partially disordered sugar residues following the initial N-acetyl glucosamine (NAG). Superposition of the tetragonal crystal model on the α-amylases from Bacillus subtilis (PDB:1BAG), pig pancreas (PDB:3L2L), and barley (PDB:1AMY), show a high degree of coincidence, particularly for the (β/α)8-barrel domains, and especially within the active site. Using this structural agreement between amylases, we extrapolated the binding model of a six residue, limit dextrin found in pig pancreas α-amylase to the A.oryzae enzyme model, which predicts substrate interacting amino acid residues.

Application of an immobilized Rhizopus oryzae lipase to batch and continuous ester synthesis with a mixture of a lauric acid and fusel oil

Five commercial lipase preparations immobilized on epoxy-polysiloxane- hydroxyethylcellulose were tested as potential biocatalysts to mediate the esterification reaction of isoamyl alcohol with lauric acid. Among them, the immobilized lipase from Rhizopus oryzae was the most active biocatalyst, attained high ester yield (81.26%) in 24 h, being selected for subsequent tests using fusel oil as a source of isoamyl alcohol under batch and continuous runs. Under batch conditions, no adverse effect was found when pure isoamyl alcohol was replaced by fusel oil. However, under continuous runs, the selected biocatalyst was found to have limited performance (half-life time = 173 h) and the average ester productivity was only 277.8 ± 19.3 μmol g−1 min−1. These results can be credited to the high affinity of the hybrid matrix for the water formed as a by-product, modifying the substrate interaction with the essential water around the enzyme limiting the substrate migration to the solid phase and thereby reducing the enzyme activity. By replacing the hybrid support for a higher hydrophobic matrix such as commercial copolymer styrene-divinylbenzene it was possible to attain a stable operation, achieving an average ester productivity of 794.43 ± 66.04 μmol g−1 min−1 and increasing the half-life time to 285 h. Further productivity incremental (1598.68 ± 135.79 μmol g−1 min−1) was also attained by using higher substrate loading. This process shows to be an attractive alternative to bulk chemical routes to satisfy increasing commercial demands for isoamyl laurate an essential emollient in the cosmetic industry.

Incorporation of capric acid in pumpkin seed oil by sn-1,3 regioselective lipase-catalyzed acidolysis

Structured lipids (SLs) are novel triacylglycerols obtained by changing the native fatty acid (FA) profiles or by the incorporation of a new desired FA in the acylglycerol backbone. These modified fats present important medical and functional properties for food applications. This work aimed to synthetize a MLM-type SL, which consists of triacylglycerols containing a medium-chain FA (M) at sn-1,3 positions and a long-chain FA (L) at sn-2 position, by acidolysis of pumpkin seed oil with capric acid, catalyzed by a commercial lipase preparation from Thermomyces lanuginosa (Lipozyme TL IM). Reactions were performed at 45 °C, in solvent-free media, at 1:2 molar ratio (pumpkin seed oil:capric acid) and a fixed amount of immobilized lipase of 5%, 10%, 15% or 20%. Incorporations of C10:0 increased with time up to 31 h (29.9 ± 0.7 mol-%) when 5% lipase load was used. Significant differences were only observed between the results obtained with 5 and 20% of biocatalyst load. The subsequent experiment was carried out with 5% lipase load, at 45 °C, 1:2 molar ratio and in the presence of n-hexane. The results showed slightly higher incorporation yields in the presence of solvent, namely at 48 h-reaction (34.7 ± 1.0 mol-%). However, since the structured lipids are to be used in food products, together with environmental and economic concerns, solvent-free systems are preferred. In this study, the synthesis of a MLM-type SL from pumpkin seed oil for food uses was well succeeded.

Lipase-Catalyzed Synthesis of Structured Lipids at Laboratory Scale.

In this chapter, some examples of laboratory protocols to produce functional structured lipids, namely, human milk fat substitutes, dietetic triacylglycerols, and interesterified fat blends with improved nutritional and rheological properties, catalyzed either by immobilized commercial or noncommercial lipase preparations, are presented. In addition to batch synthesis, the continuous production in packed- or fluidized-bed bioreactors is addressed, as well as the evaluation of operational stability of the biocatalysts used (either in batch reuses or in continuous mode).

Enzymatic tailoring of oleuropein from Olea europaea leaves and product identification by HRMS/MS spectrometry

Oleuropein, a bioactive compound found in all parts of olive tree, especially in leaves and branches, presents numerous health promoting properties that increase research and market interest the last few years. In addition, oleuropein degradation products, such as hydroxytyrosol, elenolic acid, and the aglycones also exhibit biological activities with different properties compared to the starting compound. Under this view, a commercial lipase preparation Lipolase 100L and a thermophilic β-glucosidase from Myceliophthora thermophila were used for the regioselective hydrolysis of oleuropein towards the production of the corresponding biologically active compounds. The enzymatic degradation products of oleuropein, such as hydroxytyrosol, elenolic acid and its glucoside, and oleuropein aglycones were identified by LC-HRMS/MS and NMR spectroscopy. The latter, was found as a mix of diastereomers of the monoaldehydic form of oleuropein aglycone, identified as (5S, 8R, 9S)-, (5S, 8S, 9S)- and (5S, 8R, 9R). The high substrate specificity exhibited by both lipase and β-glucosidase allows the successful tailoring of oleuropein towards the production of different biologically active compounds with significant potential in the cosmeceutical and food industry.

Síntese enzimática do caprilato de isopentila utilizando óleo fúsel como matéria-prima

Four commercial lipase preparations immobilized by covalent attachment on epoxy-polysiloxane-β-cyclodextrin were tested as potential biocatalysts to mediate the esterification reaction of isopentanol with caprylic acid in solvent-free medium. Among them, the immobilized lipase from Rhizopus oryzae was the most active biocatalyst, attained ester conversion higher than 80% in 24 h, being selected for subsequent tests using fusel oil as source of isopentanol. An experimental design was performed and the analysis of the results revealed that the formation of the isopentyl caprylate was strongly influenced by the variable molar ratio at 95% of confidence level. The proposed mathematical model allowed predicting that the excess of caprylic acid (molar ratio fusel oil to acid of 1:1.5) and reaction temperature of 45 oC favor the reaction to attain high ester conversion. Sensory evaluation of the flavors profile in relation to all the components involved in the reaction (caprylic acid, fusel oil and esters) showed that the formed esters had distinct aromas from the starting materials, confirming that the changes in flavors occurred before and after esterification reactions.

Selection of Lipases for the Synthesis of Biodiesel from Jatropha Oil and the Potential of Microwave Irradiation to Enhance the Reaction Rate.

The present study deals with the enzymatic synthesis of biodiesel by transesterification of Jatropha oil (Jatropha curcas L.) with ethanol in a solvent-free system. Seven commercial lipase preparations immobilized by covalent attachment on epoxy-polysiloxane-polyvinyl alcohol composite (epoxy-SiO2-PVA) were tested as biocatalysts. Among them, immobilized lipases from Pseudomonas fluorescens (lipase AK) and Burkholderia cepacia (lipase PS) were the most active biocatalysts in biodiesel synthesis, reaching ethyl ester yields (FAEE) of 91.1 and 98.3% at 72 h of reaction, respectively. The latter biocatalyst exhibited similar performance compared to Novozym® 435. Purified biodiesel was characterized by different techniques. Transesterification reaction carried out under microwave irradiation exhibited higher yield and productivity than conventional heating. The operational stability of immobilized lipase PS was determined in repeated batch runs under conventional and microwave heating systems, revealing half-life times of 430.4 h and 23.5 h, respectively.

Methyl Oleate Production in a Supported Sol–Gel Immobilized Lipase Packed Bed Reactor

A supported immobilized lipase was investigated as a suitable biocatalyst for the transesterification of triolein to methyl oleate in a packed bed reactor (PBR). The packing material for the bioreactor was comprised of lipase immobilized in sol–gel and supported on Celite (CSG). A kinetic- and mass transfer-based model was developed, incorporating a novel efficiency correlation to account for the effect of glycerol on the immobilized enzyme. Comparing the CSG PBR performance to that using a commercial lipase preparation (Novozym 435 (N435)), higher product conversion was achievable using CSG over a shorter time period, indicating the superior performance of CSG for enzymatic transesterification. A rate equation accounting for alcohol inhibition was used to compare the kinetics of the CSG and N435 PBRs. It was found that CSG exhibited a higher maximum reaction rate and was less prone to inhibition by methanol relative to N435. The resulting model showed good agreement with the experimental data, and CSG exhibited good stability without loss of activity over a five day period. This biocatalyst shows significant potential for the efficient production of biodiesel, and the mechanistic model developed has broader applications for similar enzyme and reaction systems.

Comparison of the performance of commercial immobilized lipases in the synthesis of different flavor esters

Abstract In this work, it is compared the performance of three commercial lipase preparations (Novozym 435, Lipozyme TL-IM, and Lipozyme RM-IM) in the synthesis of flavor esters obtained by esterification of acetic, propionic, and butyric acids using ethanol, isopropyl alcohol, butanol, or pentanol. A comprehensive comparison was performed verifying activities of these three enzyme preparations versus the different couples of substrates, checking the obtained yields. In general, the longer the acid chain, the higher the reaction yields. Novozym 435 was the most efficient enzyme in most cases, and only Lipozyme RM-IM offered better results than Novozym 435 in the production of ethyl butyrate. Reactions with butyric acid showed the highest conversion rates using all biocatalysts. Using optimal substrates, the reactions catalyzed by the three enzymes were optimized using the response surface methodology, and the catalytic performance of the biocatalysts in repeated batches was assessed. After optimization, yields higher than 90% were obtained for all three enzymes, but Lipozyme TL-IM needed four-times more biocatalyst content than the other two preparations. Novozym 435 kept over 80% of its activity when reused in 9 successive batches, whereas Lipozyme RM-IM can be reused 5 times and Lipozyme TL-IM only 3 times. In general, Novozym 435 showed to be more suitable for these reactions than the other two enzyme preparations.

Enabling Industrial Biocatalytic Processes by Application of Silicone‐Coated Enzyme Preparations

AbstractComposite particles of the commercial lipase preparation NZ435 and silicone (silcoat‐NZ435) have recently been described as promising biocatalysts for synthetic use. In this study, their actual potential for enhanced performance in industrial applications was evaluated, focusing on scenarios where carrier disintegration and catalyst leaching constitute key limitations. All three investigated model reactions, the syntheses of myristyl myristate, poly(ethylene glycol) 400‐coconut fatty acid monoester and ethylene oxide and propylene oxide copolymer (EO/PO)‐oleic acid diester, were considerably improved in terms of the maximal number of reaction cycles performed with the same batch of catalyst, and consequently in terms of the obtainable product amount. The total turnover numbers (TTN) increased by a factor up to 50, making the realization of this type of reactions in an industrial process more feasible. The utility of silcoat‐NZ435 for stereoselective transformations was demonstrated with the enantioselective acylation of 1‐phenylethanol with vinyl butyrate, in which full retention of the excellent stereoselectivity of native NZ435 was observed. Moreover, it was demonstrated for the first time that the methodology by which silcoat‐catalysts are obtained can be successfully transferred to alternative carriers and enzymes (e.g., protease, esterase and laccase), opening a broad field for the implementation and advancement of biocatalysis in industrial processes.

Synthesis activity-based zymography for detection of lipases and esterases

A new zymography method for lipases and esterases was developed on the basis of the esterification reaction between fatty acids and alcohols. The enzymes were separated by SDS-PAGE and native PAGE. The gel was washed and then incubated in an aqueous solution containing fatty acids (oleic acid 18:1 or caprylic acid 8:0) and dodecanol. Synthesis was visualized by in situ precipitation of water-insoluble and non-diffusible fatty acid esters, such as dodecyl oleate and dodecyl octanoate. The synthesis activity-based zymography was confirmed with different enzyme samples, including commercial lipase preparations, purified recombinant lipase and cutinase, and crude culture supernatants of lipolytic enzyme-producing soil bacteria.

Lipase of Aspergillus niger NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate

Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80g/l of isoamyl acetate within 96h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5g/l) and conversion (73.5%) were obtained within 24h using Novozyme 435 under these conditions.

Enzymatic hydrolysis of structurally diverse phthalic acid esters by porcine and bovine pancreatic cholesterol esterases

A weak hydrolyzing activity against bis (2-ethylhexyl) phthalate (DEHP) was discovered in a commercial crude lipase (EC 3.1.1.3) preparation from porcine pancreas. DEHP was hydrolyzed to mono (2-ethylhexyl) phthalate (MEHP) not by a pancreatic lipase but by a cholesterol esterase (CEase, EC 3.1.1.13), a trace contaminant in the crude lipase preparation. Enzymatic hydrolysis of phthalic acid esters (PAEs), suspected to be endocrine-disrupting chemicals, was investigated using CEases from two species of mammals and a microorganism. Eight structurally diverse PAEs, namely diethyl phthalate (DEP), di- n-propyl phthalate (DPrP), di- n-butyl phthalate (DBP), di- n-pentyl phthalate (DPeP), di- n-hexyl phthalate (DHP), DEHP, n-butyl benzyl phthalate (BBP), and dicyclohexyl phthalate (DCHP), were hydrolyzed to their corresponding monoesters by both porcine and bovine pancreatic CEases, while a microbial CEase from Pseudomonas sp. had no hydrolyzing activity against these PAEs. The hydrolysis experiments with bovine pancreatic CEase (50 U) indicated complete hydrolysis of every PAE (5 μmole) except for BBP and DCHP within 15 min; BBP and DCHP were hydrolyzed within 30 min and 6 h, respectively. The rates of PAE hydrolysis could be affected by the bulkiness of alkyl side chains in the PAEs. This study provides important evidence that mammalian pancreatic CEases, such as those from porcine and bovine sources, are potential enzymes for nonspecific degradation of structurally diverse PAEs.

Purification and characterization of exo-β- d-glucosaminidase from commercial lipase

A chitosanolytic enzyme without lipolytic activity was purified to apparent homogeneity from a commercial lipase preparation by using a combination of DEAE-Sepharose CL-6B exchange chromatography, Phenyl-Sepharose CL-4B hydrophobic interaction chromatography, and Sephacryl S-200 gel filtration chromatography, and the purified enzyme was characterized. The molecular mass of the homodimeric protein was about 130 kDa. The optimum action temperature and pH of the enzyme on chitosan were 60 °C and 4.6–4.8, respectively, and the enzyme was stable at temperatures lower than 60 °C and at pH 4–9. The purified enzyme exhibited the highest activity toward chitosan which was 73–82% deacetylated. TLC analysis shows that the purified enzyme released glucosamine residues successively from the substrates and final hydrolysis products of both chitosan tetramer and pentamer were glucosamine, indicating that the enzyme exhibited exo-β- d-glucosaminidase activity.

Enantiomer selective acylation of racemic alcohols by lipases in continuous-flow bioreactors

Continuous-flow mode enantiomer selective acetylations of racemic 1-phenylethanol, 1-cyclohexylethanol, and 1-phenylpropan-2-ol ( rac- 1a– c, respectively) with vinyl acetate were performed in small stainless steel packed-bed reactors filled with different commercial lipase preparations. In several lipase-filled columns, highly enantiomer selective ( E > 100) kinetic resolutions of these alcohols were achieved. In most cases, comparison of the continuous-flow and batch mode (shake flask) biotransformations indicated similar enantiomer selectivities ( E) but higher productivities (specific reaction rate: r) in the corresponding continuous-flow reaction. The effect of temperature (0–60 °C) and pressure (1–120 bar) on the continuous-flow acetylation of racemic 1-phenylpropan-2-ol was investigated in an immobilized Candida antarctica lipase B (CaLB) filled reactor. Pressure had no significant effect on r and E. Expectedly, a monotonous increase of specific reaction rate ( r) was observed within this temperature range. Most surprisingly, the enantiomer selectivity had a maximum ( E ∼ 25, at 20 °C) and a minimum ( E ∼ 7, at 50 °C). The continuous-flow reactions in CaLB-filled columns were successfully applied for preparative scale kinetic resolutions of rac- 1a– c.

A potential high-throughput method for the determination of lipase activity by potentiometric flow injection titrations

Potentiometric FIA titrations were performed to determine enzyme activities of lipase type B from Candida antarctica, CAL-B. Two substrates, triacetin and tributyrin were hydrolyzed in phosphate buffer solutions, and the concentration change of the base component of the buffer was titrated in a carrier solution containing hydrochloric acid and potassium chloride. The system was calibrated with butyric acid and acetic acid, respectively. FIA titration peaks were evaluated with respect to peak height and peak area. Butyric acid and acetic acid could be titrated in the buffer solution from 3 × 10 −3 mol L −1 to 0.1 mol L −1. The detection limit of enzyme activity was determined to be 0.07 U mL −1 (15 min reaction time) and the minimum activity was calculated to be 0.035 units corresponding to 35 nmol min −1. The specific activities of lipase B for the hydrolysis of tributyrin and triacetin were determined as 16 ± 2 U mg −1 and 2 ± 0.2 U mg −1 (per mg commercial lipase preparation), respectively.

Dipeptide synthesis in biphasic medium: evaluating the use of commercial porcine pancreatic lipase preparations and the involvement of contaminant proteases

Dipeptide syntheses starting from Ac-L-Tyr-OEt or Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys or Thr) and glycine amide in biphasic reaction media were achieved using two commercially available porcine pancreatic lipase (PPL) preparations (crude (cPPL) and purified PPL (pPPL)). Under the mild conditions employed, α-chymotrypsin, a pancreatic protease that also presents esterase activity, catalyzed Ac-L-Tyr-Gly-NH2 synthesis with high productivity. Product hydrolysis also occurred in most of the syntheses studied. Polyacrylamide gel electrophoresis, enzymatic assays employing specific chromogenic substrates and size-exclusion chromatography revealed that cPPL and pPPL contain contaminant proteases and, therefore, exhibit esterase and amidase activities. Overall, these data indicate that those contaminants may be the main catalysts of peptide bond synthesis when Nα-blocked-L-amino acid esters and the commercial PPL preparations are used. On the other hand, such data do not contest the possibility of using such enzyme preparations as an inexpensive source of catalysts for dipeptide synthesis under soft conditions.