A novel,two consecutive enzyme synthesis of feruloylated monoacyl- and diacyl-glycerols in a solvent-free system

Feruloylated mono- and di-acylglycerols were synthesized in a two step reaction: ethyl ferulate was first transesterified with glycerol and then this was esterified with oleic acid. The yield of the combined feruloylated mono- and di-acylglycerols in the second reaction reached 96% when esterification of 0.3 g transesterification products with 2.22 g oleic acid was catalyzed with 0.25 g Candida antarctica lipase at 60°C under a vaccum of 10 mmHg for 1.33 h.     

Continuous synthesis of alkyl ferulate by immobilized Candida antarctica lipase at high temperature

1-Pentyl, 1-hexyl and 1-heptyl ferulates were continuously synthesized at 60–90°C using a reactor system in which a column packed with ferulic acid powders and another column packed with immobilized Candida antarctica lipase particles were connected in series. Conversions greater than 0.9 were achieved for the synthesis of the 1-hexyl and 1-heptyl ferulates at 90°C. The system could be stably operated for the 1-heptyl ferulate synthesis at 90°C for at least two weeks.     

Lipase-catalyzed preparation of mono- and diesters of ferulic acid

Lipophilic and stable derivatives of ferulic acid are required to improve its efficacy in fatty foods and to optimize its use in cosmetic and pharmaceutical preparations. We report an improved synthesis of ferulic acid monoesters (ethyl ferulate and lauryl ferulate) using immobilized lipase from Candida antarctica B (CALB) in diisopropyl ether (DIPE). Maximum yields were 89% and 85% in 200 h for ethyl and lauryl ferulate, respectively. Ethyl ferulate was further acylated with vinyl esters to form ferulate diesters. 4-Acetoxy-ethyl ferulate was obtained with the immobilized lipase from Alcaligenes sp. (QLG) with 59% yield in 72 h, whereas 4-dodecanoyloxy-ethyl ferulate (a new compound) was synthesized with 52% yield in 72 h using CALB. DIPE was the best solvent for the transesterifications. Finally, the anti-inflammatory activity of the synthesized derivatives was evaluated in vitro; the compounds bearing a dodecyl chain showed improved anti-inflammatory activity compared with short-chain esters.     

Synthesis of ethyl ferulate in organic medium using celite-immobilized lipase

In the present work we have evaluated synthesis of ethyl ferulate by the esterification reaction of ferulic acid and ethanol catalyzed by a commercial lipase (Steapsin) immobilized onto celite-545 in a short period of 6 h in DMSO. The immobilized lipase was treated with cross-linking agent glutaraldehyde (1%; v/v). The optimum synthesis of ethyl ferulate was recorded at 45 °C, pH 8.5 and 1:1 ratio of ethanol and ferulic acid. Co²⁺, Ba²⁺and Pb²⁺ ions enhanced the synthesis of ethyl ferulate Hg²⁺, Cd³⁺and NH⁴⁺ ions had mild inhibitory effect. The celite-bound lipase produced 68 mM of ethyl ferulate under optimized reaction conditions.     

Inhibition of hepatic lipase by m-aminophenylboronate application of phenylboronate affinity chromatography for purification of human postheparin plasma lipases

Phenylboronates are competitive inhibitors of serine hydrolases including lipases. We studied the effect of m-aminophenylboronate on triglyceride-hydrolyzing activity of hepatic lipase (EC 3.1.1.3). m-Aminophenylbo ronate inhibited hepatic lipase activity with a K₁ value of 55 μM. Furthermore, m-aminophenylboronate protected hepatic lipase activity from inhibition by di-isopropyl fluorophosphate, an irreversible active site inhibitor of serine hydrolases. Inhibition of hepatic lipase activity by m-aminophenylboronate was pH-dependent. The inhibition was maximal at pH 7.5, while at pH 10 it was almost non-existent. These data were used to develop a purification procedure for postheparin plasma hepatic lipase and lipoprotein lipase. The method is a combination of m-aminophenylboronate and heparin-Sepharose affinity chromatographies. Hepatic lipase was purified to homogeneity as analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of purified hepatic lipase was 5.46 mmol free fatty acids h⁻¹ mg⁻¹ protein with a total purification factor of 14 400 and a final recovery of approximately 20%. The recovery of hepatic lipase activity in m-aminophenylboronate affinity chromatography step was 95%. The purified lipoprotein lipase was a homogeneous protein with a specific activity of 8.27 mmol free fatty acids h⁻¹ mg⁻¹ The purification factor was 23 400 and the final recovery approximately 20%. The recovery of lipoprotein lipase activity in the m-aminophenylboronate affinity chromatography step was 87%. The phenylboronate affinity chromatography step can be used for purification of serine hydrolases which interact with boronates.     

Isolation and characterization of Acinetobacter sp. ES-1 excreting a lipase with high enantioselectivity for (S)-ketoprofen ethyl ester

A new Acinetobacter sp. ES-1, grown on triolein, tryptone and Triton X-100, excreted a lipase that hydrolyzed 10m M (R,S)-ketoprofen ethyl ester into (S)-ketoprofen. The crude lipase had an activity of 10Uml^-1 and, at 30°C and pH7 over 48h, gave a conversion yield of 35% with an enantiomeric excess for the product 96%.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

The Putative Lipase,AF1763, from <Emphasis Type="Italic">Archaeoglobus fulgidus</Emphasis>is a Carboxylesterase with a Very High pH Optimum

The open reading frame AF1763, annotated as a putative lipase gene (lipA) of the hyperthermophilic archaeon, Archaeoglobus fulgidus DSM 4304, was cloned and over-expressed in E. coli. A sequence analysis of LipA and the investigation of a truncated enzyme implied a special function of the C-terminal part of LipA. The substrate spectrum of the enzyme suggested that LipA is a carboxylesterase rather than a canonical lipase. The enzyme showed optimal activity at 70 °C and between pH 10 and 11, which is among the most alkaline pH range detected for hydrolases.     

The effect of kinetin on stomata of the grass Anthephora pubescens Nees

Transpiration from excised leaves of Anthephra pubescens Nees was enhanced by 1 and 10 mmol m^-3 kinetin. Stomatal opening in isolated epidermal strips of A. pubescens under CO₂-free air and in the absence of K⁺ was enhanced by 10 mmol m^-3 kinetin.Abbreviations ABA abscisic acid     

An inducible,intracellular, alkalophilic lipase inAspergillus flavipes grown on triacylglycerols

An intracellular lipase was induced inAspergillus flavipes grown on various triacylglycerols at pH 6.0 and at 30°C, with maximum activity with sunflower oil. The lipase had an optimum pH for activity of 8.8 and retained 30% of its activity at pH 10.0. It had an optimum temperature for activity, measured over 30 min, of 45°C. It was completely inactivated at 60°C within 10 min.     

Gene Cloning,Expression, and Characterization of the <Emphasis Type="Italic">Geobacillus Thermoleovorans</Emphasis> CCR11 Thermoalkaliphilic Lipase

The gene for a Geobacillus thermoleovorans CCR11 thermostable lipase was recovered by PCR and cloned. Four genetic constructions were designed and successfully expressed in E. coli: (i) the lipase structural gene (lipCCR11) in the PinPoint Xa vector; (ii) the lipase structural gene (lipACCR11) in the pET-28a(+) vector; (iii) the lipase structural gene minus the signal peptide (lipMatCCR11) in the pET-3b vector; and (iv) the lipase structural gene plus its own promoter (lipProCCR11) in the pGEM-T cloning vector. The lipase gene sequence analysis showed an open reading frame of 1,212 nucleotides coding for a mature lipase of 382 residues (40 kDa) plus a 22 residues signal peptide. Expression under T7 and T7lac promoter resulted in a 40- and 36-fold increase in lipolytic activity with respect to the original strain lipase. All recombinant lipases showed an optimal activity at pH 9.0, but variations were found in the temperature for maximum activity and the substrate specificity among them and when compared with the parental strain lipase, especially in the recombinant lipases that contained fusion tags. Therefore, it is important to find the appropriate expression system able to attain a high concentration of the recombinant lipase without compromising the proper folding of the protein.     

<Emphasis Type="Italic">Candida rugosa</Emphasis> lipase-catalyzed polyurethane degradation in aqueous medium

Candida rugosa lipase (EC 3.1.1.3) was used to degrade commercially-available solid poly(ester)urethane (Impranil) in an aqueous medium under different temperature, pH, enzyme and substrate concentrations. A mathematical model was developed and applied to represent the degradation kinetics of the solid polyurethane. Reaction optima were found to be pH 7 and 35°C. Diethylene glycol, a degradation byproduct, generation rate was measured to be 0.12 mg/l min and the activation energy was calculated as 9.121 kcal/gmol K. This information will be useful in developing bioreactors for practical applications to manage polyurethane wastes using lipase.     

Coprecipitation of Pseudomonas fluorescens Lipase with Hydrophobic Compounds As an Approach to Its Immobilization for Catalysis in Nonaqueous Media

The precipitation of N-cetylamine, N-cetylacetamide, hexadecane-1,2-diol, cetyl alcohol, and poly(butyl metacrylate) in acetone–water media in the presence of the lipase from Pseudomonas fluorescens was found to be accompanied by the coprecipitation of the enzyme. Within the lyophilized coprecipitates, the lipase exhibits a high catalytic activity and enantioselectivity in the reaction of (1RS)-phenylethanol acetylation with vinyl acetate in t-butyl methyl ether. In order of increasing lipase activity, the coprecipitates can be arranged in the series: cetyl alcohol, poly(butyl metacrylate), hexadecane-1,2-diol, N-cetylamine, and N-cetylacetamide, with the activity 2.5- to 19-fold exceeding the activity of the native enzyme. Immobilization of the lipase on solid supports, such as Celite 545 (physical sorption) and Eupergit C250L (covalent binding), in the presence of hexadecane-1,2-diol was found to increase the esterifying activity of the enzyme.     

Application of central composite rotatable design to lipase catalysed synthesis of m-cresyl acetate

Esterification of m-cresol with acetic acid using porcine pancreas lipase (PPL) was investigated by response surface methodology (RSM). A central composite rotatable design (CCRD) involving 32 experiments of five variables at five levels was employed to analyse the esterification behaviour. The effect of five variables studied namely, m-cresol concentrations (0.005–0.025 mol), enzyme/substrate ratios (0.18–1.22 activity units/millimol, AU/mmol), incubation periods (6–54 h), pH (4–8) and buffer volumes (0–0.2 ml) was useful in arriving at an optimum ester yield. The methodology projected conditions for higher yields up to 6.0 mmol. Validation experiments carried out under these predicated conditions showed good correspondence between experimental and predicted yields. CCRD treatment clearly showed the inhibitory nature of m-cresol in the esterification process. The reaction required the presence of buffer for better conversions and a minimum amount of 0.1 ml buffer was found necessary for this reaction. Buffer pH values around 6.0 and below appeared to favour better esterification than those at pH values in the range 6.0–8.0. However an optimum condition for maximum yield was: incubation period: 54 h; buffer volume: 0.2 ml; pH: 8; E/S ratio: 0.83 AU/mmol; m-cresol: 0.02 mol; predicted yield: 6.0 mmol; experimental yield: 6.4 mmol.     

Fatty acid preference of mycelium-bound lipase from a locally isolated strain of <Emphasis Type="Italic">Geotrichum candidum</Emphasis>

Mycelium-bound lipase (MBL) was prepared using a strain of Geotrichum candidum isolated from local soil. At the time of maximum lipase activity (54 h), the mycelia to which the lipase was bound were harvested by filtration and centrifugation. Dry MBL was prepared by lyophilizing the mycelia obtained. The yield of MBL was 3.66 g/l with a protein content of 44.11 mg/g. The lipase activity and specific lipase activity were 22.59 and 510 U/g protein, respectively. The moisture content of the MBL was 3.85%. The activity of free (extracellular) lipase in the culture supernatant (after removal of mycelia) was less than 0.2 U/ml. The MBL showed selectivity for oleic acid over palmitic acid during hydrolysis of palm olein, indicating that the lipase from G. candidum displayed high substrate selectivity for unsaturated fatty acid containing a cis-9 double bond, even in crude form. This unique specificity of MBL could be a direct, simple and inexpensive way in the fats and oil industry for the selective hydrolysis or transesterification of cis-9 fatty acid residues in natural triacylglycerols.     

Syntrophococcus sucromutans sp. nov. gen. nov. uses carbohydrates as electron donors and formate,methoxymonobenzenoids or Methanobrevibacter as electron acceptor systems

Most probable number (MPN) estimates indicated that a mean of 4.3×10⁷ and 5×10⁶ bacteria per ml of rumen fluid from a predominantly alfalfa hay-fed steer demethoxylated ferulate and syringate, respectively. After further enrichment from an MPN tube of the highest dilution showing demethoxylation of syringate, strain S195 was isolated using roll tubes with syringate as an added energy source. S195 was an anaerobic, Gram-negative, nonmotile coccus, 1 to 1.3 m in diameter, and was unique in using various carbohydrates as electron donor with acetate as the sole organic product. One of the following electron acceptor systems allowed growth (organic products in parentheses): Methanobrevibacter simithii (CH₄), formate (acetate), 3,4,5-trimethoxybenzoate and syringate (acetate and gallate), vanillate (acetate and protocatechuate), vanillin (acetate, protocatechuic aldehyde and protocatechuate), ferulate (acetate, caffeate and hydrocaffeate), caffeate (hydrocaffeate). Strain S195 required 30% (v/v) rumen fluid in the medium for good growth. S195 was placed in a new genus and species, Syntrophococcus sucromutans, of the family Veillonellaceae.Abbreviations G+C Guanine plus cytosine - MPN most probable number - OD optical density     

Development of recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica

To expand the industrial applications of Candida antarctica lipase B (CALB), we developed Aspergillus oryzae whole-cell biocatalyst expressing the lipase-encoding gene from C. antarctica. A. oryzae niaD300, which was derived from the wild type strain RIB40, was used as the host strain. The CALB gene was isolated from C. antarctica CBS6678 and expression plasmids were constructed with and without secretion signal peptide. The lipase gene was expressed under the control of improved glaA and pNo-8142 promoters of plasmids pNGA142 and pNAN8142, respectively. The Southern blot analysis demonstrated the successful integration of the CALB gene in the genome of A. oryzae. To determine the role of signal peptide, the expression plasmids were constructed with homologous and heterologous secretion signal sequences of triacylglycerol lipase gene (tglA) from A. oryzae and lipase B (CALB) from C. antarctica, respectively. The C-terminal FLAG tag does not alter the catalytic properties of the lipase enzyme and Western blotting analysis using anti-FLAG antibodies demonstrated the presence of cell wall and membrane bound lipase responsible for the biocatalytic activity of the whole-cell biocatalyst. The resultant recombinant A. oryzae was immobilized within biomass support particles (BSPs) made of polyurethane foam (PUF) and the BSPs were successfully used for the hydrolysis of para-nitrophenol butyrate (p-NPB) and for the optical resolution of (RS)-1-phenyl ethanol by enantioselective transesterification with vinyl acetate as acyl donor.     

Spectrophotometric assay for online measurement of the activity of lipase immobilised on micro-magnetic particles

A spectrophotometric assay has been adapted to directly measure the activity of enzymes immobilised on insoluble magnetic particles. Three different types of lipases (Candida antarctica lipase A and B and Thermocatenulatus lanuginosus lipase) were immobilised on two types of magnetic beads. The activity of the resulting immobilised lipase preparations was measured directly in the reaction solution by using a modified p-nitrophenol ester assay using a spectrophotometer. Removal of the solid particles was not necessary prior to spectrophotometric measurement, thus allowing reliable kinetic measurements to be made rapidly. The method was effective for a wide range of magnetic bead concentrations (0.01–0.2 mg ml⁻¹). In all cases the assay could determine the bead-related specific enzyme activity. The assay was validated by comparing with a pH-stat method using p-nitrophenol palmitate as the substrate with an excellent correlation between the two methods. The utility of the spectrophotometric assay was demonstrated by applying it to identify the best combination of lipase type, activation chemistry and magnetic particle. Epoxy activation of poly vinyl alcohol-coated magnetic particles prior to immobilisation of commercial C. antarctica lipase A gave the best preparation.     

Characterization and optimization of extracellular alkaline lipase production by Alcaligenes sp. Using stearic acid as carbon source

The aim of this study was to improve the production of an extracellular alkaline lipase from Alcaligenes sp. (ATCC 31371) by optimization of the culture medium, for economic production of biodiesel from waste vegetable oil. A number of carbon sources including different types of starch, sugar, sugar alcohol, organic acids, and surfactants were investigated. Polyoxyethylene (20) sorbitan tristearate, whose side chain is stearic acid, was the most effective carbon source for lipase production. Box-Behnken experimental design was used for three factors (soy protein, sodium nitrate, and stearic acid) and the optimal composition for maximum lipase production (1.7-fold enhancement) was established as soy protein 4.07%, sodium nitrate 0.17%, and stearic acid 0.28% at 28°C with an agitation rate of 220 rpm for 24 h. The enzyme was purified to homogeneity and the recovery of the lipase activity was 7.8% with a 30-fold purification. The estimated molecular size of the protein determined by SDS-PAGE was 33 kDa. The optimum pH and temperature of the purified lipase was 8.5 and 40°C, respectively. The purified enzyme was stable in the pH range of 6.0 and 9.5 and in the temperature range of 20 and 50°C.     

Enhancement of apparent thermostability of lipase from Rhizopus sp. by the treatment with a microbial transglutaminase

Crude lipase from Rhizopus sp. was moderately stable against heat treatment at 45 °C. However, after incubation for 1 h at 25 °C with Streptoverticillium transglutaminase (MTG), the half-life of crude lipase in the heat treatment was increased more than 10-fold compared to that of untreated one. The result can be ascribed by the MTGase-mediated crosslinking of contaminating proteins that affect the apparent thermostability of lipase in the crude sample.