Mapping substrate selectivity of lipases from thermophilic fungi

New methods were adapted to screen, fast and easily, the lipase specificity (topo- or enantio-selectivity) on crude extracellular extracts from thermophilic fungi. Substrate acyl chain length specificity was tested using p-nitrophenyl esters and vinyl esters by the detection of released p-nitrophenolate anions in the first case and protonation of p-nitrophenolate anions (color diminution) in the second case. Enantioselectivity was tested using either the direct reaction rates on individual enantiomers of glycidyl butyrate or on competition between these enantiomers and resorufin esters (-butyrate or -acetate). Among a library of 44 thermophilic fungi, 10 strains were pre-selected (based on their capabilities to produce constitutively extracellular lipases) for further lipase specificity studies. The above methods were applied to lipases from these pre-selected fungi and also to other several lipases preparations from bacterial, fungal and mammalian origin. Remarkably, the method on competition allowed the accurate determination of the enantiomeric ratio (E), since experimental data fitted correctly with the E determined by classical chemical methods. Consequently, these methods can be applicable for screening selectivity in a high number of lipases or esterases from wild isolates or variants generated by directed evolution, using directly in the test, the substrate (i.e. esters) that will be worked out in a given process.     

New enzymes with potential for PET surface modification

This work describes newly isolated organisms and their potential to modify the surface of polyethylene terephthalate (PET). Out of the different screening processes, four bacterial and five fungal strains were isolated. A PET model substrate was synthesized (bis (benzoyloxyethyl) terephthalate) and used in the screening process, mimicking the polymer in its crucial properties and having the advantage of defined hydrolysis products. On this model substrate, extracellular enzyme preparations from the isolated microorganisms showed a maximum activity of 8.54 nkat/L. All enzyme preparations showed esterase activity on p-nitrophenyl-acetate while no activity was found on p-nitrophenyl decanoate or p-nitrophenyl palmitate. Increased hydrophilicity of PET fabrics after enzyme treatment was found based on rising height and water dissipation measurements.     

Morphological, biochemical and kinetic properties of lipase from Candida rugosa immobilized in zirconium phosphate

Zirconium phosphate (ZrP), a low-cost inorganic material with well-defined physicochemical properties, was successfully used as support for immobilizing Candida rugosa lipase by covalent bonding. The immobilized derivative showed high catalytic activity in both aqueous and non-aqueous media. Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy measurements demonstrated that the ZrP fulfilled the morphological requirements for use as a matrix for immobilizing lipases. The free and immobilized lipases were compared in terms of pH, temperature and thermal stability. The immobilized lipase had a higher pH optimum (7.5) and higher optimum temperature (50°C) than the free lipase. Immobilization also increased the thermal stability. The hydrolysis of p-nitrophenyl palmitate (pNPP) by immobilized lipase, examined at 37°C, followed Michaelis-Menten kinetics. Values for K_m=1.18 µM and V_max=325Umg^-1 indicated that the immobilized system was subject to mass transfer limitations. The immobilized derivative was also tested under repetitive reaction batches in both ester hydrolysis and synthesis.     

Purification and properties of lipase from a Bacillus strain for catalytic resolution of (R)-Naproxen

A Bacillus strain was screened for asymmetric resolution of (R)-Naproxen. The optical purity (ee (%)) of (R)-Naproxen was found to be 86.47% and conversion rate was 40–50% in bacterial cells PBS reaction system. The dissolved lipase was clarified from the Bacillus bacterial cells by centrifugation and loaded on a phenyl-Sepharose CL-4B column. After purification by a single hydrophobic chromatography, the activity of lipase was approximately 43 times higher than the crude one. The hydrolytic activity of lipase using Naproxen ethyl ester and p-nitrophenyl acetate (p-NPA) as substrate remained essentially constant during the purification procedure. A Bacillus strain with stereochemical selectivity was obtained.     

Substrate selectivity of imidazole-appended dimethyl-β-cyclodextrin

The substrate selectivity of the hydrolysis reaction by imidazole-appended dimethyl-β-cyclodextrin (2) was studied using different kinds of p-nitrophenyl esters of amino acids. The tendency for the hydrolysis reaction of the amino acid ester by 2 reflected a difference in K_m rather than in k_cat. 2 had the ability to undergo a stereo-selective hydrolysis reaction.     

The expression in E. coli of a polymeric gene coding for an esterase mimic catalyzing the hydrolysis of p-nitrophenyl esters

We have prepared a hybrid protein consisting of seven esterase units, Glu-Ala-His-Ala-Ser-Phe-Phe-Phe, fused to the N-terminal of galactokinase (E. coli). The structural gene for this bifunctional protein was obtained by cloning a polymer made up of three chemically synthesized oligonucleotides to which the galactokinase gene was fused in frame. The hybrid protein was purified to homogeneity with the aid of the galactokinase moiety and showed an M_r of 51 000-53 000. The preparation could catalyze the hydrolysis of p-nitrophenyl esters and, due to the inbuilt hydrophobic spacers, Phe-Phe-Phe, improved catalysis of more hydrophobic substrates was obtained.     

A novel esterase from a psychrotrophic bacterium, Acinetobacter sp. strain no. 6, that belongs to the amidase signature family

A novel esterase that belongs to the amidase signature family was found in a psychrotrophic bacterium, Acinetobacter sp. strain no. 6, isolated from Siberian soil. The gene coding for the esterase, named EstA8, was cloned, and an open reading frame of 1488 bp corresponding to 496 amino acid residues was identified. EstA8 showed 30% sequence identity with 6-aminohexanoate-cyclic-dimer hydrolases from Pseudomonas sp. strain NK87 and Flavobacterium sp. strain K172, which degrade a by-product of the nylon-6 industry. EstA8 was overproduced in Escherichia coli JM109 under the control of the lac promoter of pUC118 and purified. Consistent with the fact that the source microorganism is cold-adapted, the enzyme was unstable at moderate temperatures. It lost 75% of its original activity by incubation at 40 °C for 30 min. Despite its structural similarity to 6-aminohexanoate-cyclic-dimer hydrolase, 6-aminohexanoate cyclic dimer did not serve as the substrate. EstA8 is a member of the amidase signature family, but its esterase activity toward p-nitrophenyl esters, such as p-nitrophenyl acetate, was much higher than its amidase activity toward p-nitroanilides, such as p-nitroacetanilide.     

Water activity dependence of lipase catalysis in organic media explains successful transesterification reactions

The water activity dependence of lipase kinetics in organic media was evaluated using lipases from Rhizopus oryzae and Candida rugosa immobilised on polypropene EP-100. The conversion studied was the transesterification of ethyl decanoate to hexyl decanoate with hydrolysis to decanoic acid as competing reaction. The reactions were carried out at controlled water activity in diisopropyl ether. Substrate inhibition was observed at hexanol concentrations of 100 mM or higher. The Rhizopus lipase expressed the highest activity and the best selectivity for transesterification at the lowest water activity (a_w=0.06). The Candida lipase expressed the highest transesterification/hydrolysis ratio at a_w=0.11 and the highest total activity at a_w=0.53. Several glycosidases previously tested under conditions similar to those used here expressed both maximal total activity and the best selectivity at water activities close to 1.0. The water activity dependence of the lipases is thus fundamentally different from that of glycosidases and it is a major part of the reason why lipases are more suited for transferase-type reactions than the glycosidases.     

The Comparative Discriminating Abilities of Lipases in Different Media and Their Application in Fatty Acid Enrichment

The generally held belief that the selectivity of lipase can be changed by changing the media from aqueous to non-aqueous was tested by monitoring the rates of hydrolysis, ester synthesis and transesterification with a range of fatty acid mono-esters. Although the absolute rates of reaction varied, hydrolysis was by far the most rapid of the three, the relative rates for the fatty acids used were similar in all three reaction types. The selectivity of the five enzymes used appeared to remain unchanged irrespective of the type of reaction, i.e. hydrolysis of p-nitrophenyl esters, direct ester synthesis with butanol and fatty acid or transesterification with butyl butyrate and fatty acid, and could not be changed by changing water activity. This principle was applied to screen for suitable lipases which could be used to increase the gamma linolenic acid content of a fatty acid mixture. Enzymes could be selected by measuring the rate of hydrolysis of a range of P-nitrophenyl esters.     

Lipase catalysed synthesis of sebacic and phthalic esters

The enzymatic synthesis and hydrolysis of alkyl sebacates and o-, m-, p-phthalates were studied. Biosyntheses were conducted through alcoholysis of dimethyl phthalates and dimethyl sebacate with 2-ethylhexanol and 3,5,5-trimethylhexanol in a solvent-free medium, using lipases from Candida antarctica (Novozym 435), Rhizomucor miehei (Lipozyme IM) and Porcine pancreas (PPL). It was found that the synthesis and hydrolysis of sebacic acid esters were characterised by a satisfactory rate, however, by low enantioselectivity. The yield of synthesis of di-3,5,5-trimethylhexyl sebacate catalysed by Novozym 435 at 50 °C was 84%, after 20 h of reaction. The degree of conversion, 62.9% after 350 h, was obtained for alcoholysis reaction of dimethyl m-phthalate with 3,5,5-trimethylhexanol. For the enzymes used, no activity was detected at all on both the synthesis and hydrolysis of di-2-ethylhexyl o-phthalate and di-3,5,5-trimethylhexyl o-phthalate.     

Enzymatic transesterifications of carbonates in water-restricted environments

We investigated the ability of several hydrolases to catalyze reactions with an abiotic water-insoluble substrate, carbonic acid diphenyl ester, also known as diphenyl carbonate (DPC). In single-phase water/organic systems, turnover numbers (TN) of greater than 2 x 10(4) min(-1)have been achieved for the hydrolysis of DPC. The K(m) values for the hydrolytic reaction were measured to be 200muM and 330muM for Candida cylindracea lipase and Porcine liver esterase, respectively. In addition to hydrolysis, we observed transesterification of carbonates with a wide variety of alcohol and phenol species. Transesterifications of DPC with bifunctional alcohols resulted in the synthesis of polycarbonates. We investigated the stability and transesterification activity of these enzymes in several water-restricted environments to limit competing hydrolysis reactions. We find that, with the removal of water, hydrolysis is reduced more than four orders of magnitude while transesterification is diminished only 10-fold (turnover numbers of 600 min(-1) in water-miscible systems to 60 min(-1) in water-restricted environments with pure Candida lipase). Stability of the Candida lipase in these water-restricted environments (half-life of longer than 3 days) is much greater than in water/organic single phase systems (5 h in 20% methanol). In addition, the Candida lipase displayed enantiomeric selectivity in transesterifications of DPC with racemic 2-butanol (greater than 80% ee).     

Biochemical properties and potential applications of an organic solvent-tolerant lipase isolated from Serratia marcescens ECU1010

An extracellular lipase was purified to homogeneity with a purification factor of 5.5-fold from a bacterial strain Serratia marcescens ECU1010. The purified lipase is a dimer with two homologous subunits, of which the molecular mass is 65 kDa, and the pI is 4.2. The pH and temperature optima were shown to be pH 8.0 and 45 °C, respectively. Among p-nitrophenyl esters of fatty acids with varied chain length, the lipase showed the maximum activity on p-nitrophenyl myristate (C₁₄). The lipase was activated by some surfactants such as Gum Arabic, polyvinyl alcohol (PVA) and Pg350me, but not by Ca²⁺. The enzyme displayed pretty high stability in many water miscible and immiscible solvents. This is a unique property of the enzyme which makes it extremely suitable for chemo-enzymatic applications in non-aqueous phase organic synthesis including enantiomeric resolution. Several typical chiral compounds were tested for kinetic resolution with this lipase, consequently giving excellent enantioselectivities (E = 83 >100) for glycidyl butyrate (GB), 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one acetate (HMPCA), naproxen methyl ester (NME) and trans-3-(4′-methoxyphenyl) glycidic acid methyl ester (MPGM).     

Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis

Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (≈90 mmol L(-1)). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex(?) 100 L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1)).     

Enzymatic synthesis of structured phenolic lipids by incorporation of selected phenolic acids into triolein

The synthesis of structured phenolic lipids by lipase-catalyzed transesterification of selected phenolic acids, including p-hydroxyphenyl acetic, p-coumaric, sinapic, ferulic and 3,4-dihydroxybenzoic acids, with triolein was investigated. The highest enzymatic activity (248 nmol esterified phenolic acid/g solid enzyme/min) and bioconversion (62%) was obtained for the transesterification of p-hydroxyphenyl acetic acid with triolein. In addition, the transesterification of p-coumaric with triolein resulted in a higher enzymatic activity (87 nmol esterified phenolic acid/g solid enzyme/min) and bioconversion (46%) than those obtained for the transesterfication of ferulic and sinapic acids. The results also showed that using p-hydroxyphenyl acetic, p-coumaric and ferulic acids as substrate, the maximum bioconversion of phenolic monoacylglycerols was close to that of phenolic diacylglycerols. Although p-coumaric acid had very low radical scavenging activity (2%) compared to that of ferulic acid (62%), the p-coumaroylated lipids demonstrated a higher scavenging potency (16%) than that of the feruloylated one (10%).     

Integrated enzymatic production of specific structured lipid and phytosterol ester compositions

Mixtures of specific structured lipids and phytosterol esters, valuable food components, were synthesized by an enzymatic one-pot process in organic-solvent-free medium starting from a mixture of phytosterol, caprylic acid and sunflower oil. Nine biocatalysts, seven commercially available lipases and two air-dried solid state (SSF) fermentation preparations of Aspergillus oryzae NRRL 6270 (AoSSF) and Aspergillus sojae NRRL 6271 (AsSSF), were screened for lipase activity in the transesterification reactions of sunflower oil with caprylic acid and for sterol esterase activity in the direct esterification of phytosterols with free fatty acids. The best process variant using a sequence of sterol esterase (AoSSF)-catalyzed esterification reaction of the free fatty acids and phytosterols, followed by water removal in vacuum and lipase-catalyzed transesterification with immobilized lipase from Rhizomucor miehei (Lipozyme) resulted in 92.1% conversion to phytosterol esters and 44.1% conversion to triacylglycerols containing two caprylic esters.     

Lipases have similar water activity profiles in different reactions

The shape of the profiles of enzyme activity versus water activity for four different lipases were independent of the reaction used to determine the activity. The profile for each lipase (Rhizopus arrhizus, Pseudomonas sp., Candida rugosa and Lipozyme) in esterification, hydrolysis and transesterifications profiles were the same. In transesterification the yield was unaffected by the water activity but the hydrolysis yield increased with increasing aW .     

Polar organic solvent added to an aqueous solution changes hydrolytic property of lipase

For developing further uses of lipase as a biocatalyst, its hydrolytic activity toward some esters was investigated in a miscible solution composed of a buffer and a polar organic solvent. Twenty percent dimethylformamide, 35% dimethylsulfoxide, 15% 1,4-dioxane, 15% dimethoxyethane, and 2% diethoxyethane promoted the hydrolysis by a lipase from Rhizomucor miehei toward some hydrophobic substrates, 4-methylumbelliferyl oleate, 4-methylumbelliferyl palmitate, and monoolein. While hydrolysis by this lipase toward the substrates with a relatively weak hydrophobicity (4-metylumbelliferyl heptanoate and 4-methylumbelliferyl nanoate) was suppressed by these solvents. A fluorometric analysis showed that the polar organic solvent in the buffer induced some conformational change around a tryptophan residue of R. miehei lipase. In addition to the influence of the miscible solvent on the solubility of the substrates, the conformational change of the protein induced by the miscible solvent would also affect the reactive properties of the lipase. Adding a polar organic solvent to an aqueous solution will be an efficient method for changing hydrolytic performance of lipases.     

Comparison of esterification and transesterification of fructose by porcine pancreas lipase immobilized on different supports

The syntheses of fructose butyrate with five different immobilized porcine pancreas lipases were studied in acetonitrile with butyric acid and tributyrin as acyl donors. The hydrolytic activities of the enzymes were measured during the syntheses. The transesterification yield was the highest with the Celite-adsorbed enzyme: 57% (1.42 g fructose butyrate l–1), and this form exhibited the highest degree of hydrolytic activity preservation (57%) after operation for 20 days.     

Hydrolysis of methyloleate using immobilized lipase from Chromobacterium viscosum

Various lipases were screened for their hydrolytic efficiency towards methyloleate. Lipase from Chromobacterium viscosum gave highest hydrolysis efficiency of 92% in 24?h. Different cation exchange resins were screened to immobilize lipase from Chromobacterium viscosum. A weakly acidic macroreticular type resin, IRC-50 having carboxyl end group functionality gave highest activity yield of 18.8%. Strongly acidic cation exchange resins with sulphonic functionality and macroreticular type did not give much activity yield when compared to weakly acidic non macroreticular type resins. It was observed that end group functionality and structure of the matrices plays an important role in obtaining highest activity yield. For a specific water concentration, the hydrolysis ratio reached 85% in less than 7?h when the substrate to enzyme ratio was 4. As the ratio is increased above 4, the availability of water at the interface has become a limitation for obtaining maximum hydrolysis.     

A hexaimino cryptand containing naphthyl groups; structural characterization of mononuclear complexes and catalysis in hydrolysis of p-nitrophenyl phosphate

Mononuclear metal complexes of a hexaimino cryptand containing three naphthyl groups have been prepared and the structure of the copper(I) complex was determined by X-ray diffraction. The complexes show acceleration of hydrolysis of p-nitrophenyl phosphate ester.