Catalytic Properties of Lipase Entrapped as Lysates of Recombinant Strain-Producer r<Emphasis Type="Italic">Escherichia coli</Emphasis>/lip into Nanocarbon-in-Silica Composites in the Bioconversion of Triglycerides and Fatty Acids

Composite multi-component biocatalysts were prepared by entrapping lysates of a recombinant rE. coli/lip strain producing Thermomyces lanuginosus lipase into composite nanocarbon-containing matrices based on a SiO₂ xerogel. The dependence of the lipase activity and operational stability on the type of the carbon component (nanotubes or nanospheres of different diameters) was studied in the bioconversion of triglycerides (hydrolysis and interesterification), as well as in the esterification of saturated fatty acids—namely, butyric (C4:0), capric (C10:0), and stearic (C18:0) acids—with isoamyl alcohol. It was shown that the biocatalytic properties were determined by both the texture parameters of the nanostructured carbon included and the type of enzymatic reaction performed. Biocatalysts without a nanocarbon component had the highest operational stability in the batch process of interesterification of sunflower oil with ethyl acetate; the half-life time was found to be 720 h at 40°C. Biocatalysts containing carbon nanotubes of ~21 nm in diameter were five to six times more active in the batch esterification process than biocatalysts without a nanocarbon component. Biocatalysts containing carbon nanotubes catalyzed the synthesis of esters in a binary organic solvent (hexane and diethyl ether) without a loss of activity for more than 500 h at 40°C.     

Lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> 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 120 rpm. 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 80 g/l of isoamyl acetate within 96 h 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 (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.     

Green synthesis of isoamyl acetate via silica immobilized novel thermophilic lipase from <Emphasis Type="Italic">Bacillus aerius</Emphasis>

Isoamyl acetate, a pear or banana flavor, is widely used in food, beverage, cosmetic, and pharmaceutical industries. In the present work, lipase from Bacillus aerius was immobilized on silica gel matrix in the presence of a cross-linking agent, glutaraldehyde, and its efficiency in synthesizing isoamyl acetate using esterification reaction was studied. The esterification of acetic acid and isoamyl alcohol by silica-bound lipase was studied as a function of time and temperatures. The incubation time of 10 h, temperature of 55°C, substrate molar ratio 1: 1, and the amount of lipase as 1% were found to be optimal for the esterification reaction. The bound lipase catalyzed the esterification of acetic acid by isoamyl alcohol with the yield of about 68% under the optimized reaction conditions. The product was identified as isoamyl acetate using gas-liquid chromatography, nuclear magnetic resonance, and Fourier transform IR spectroscopy analysis by the presence of an ester group at the wavenumber of 1720.5 cm^–1.     

Biocatalytic methanolysis activities of cross-linked protein-coated microcrystalline lipase toward esterification/transesterification of relevant palm products

Biocatalysis by immobilized lipase is an efficient alternative process for conversion of crude vegetable oil with high free fatty acid content to biodiesel, which is the limit of the conventional alkaline-catalyzed reaction. In this study, influences of solid-state organic and inorganic buffer core matrices with different pK_a on catalytic performance of cross-linked protein coated microcrystalline biocatalysts prepared from Thermomyces lanuginosus lipase (CL-PCMC-LIP) toward esterification of palmitic acid (PA), transesterification of refined palm oil (RPO), and co-ester/transesterification of crude palm oil (CPO) to fatty acid methyl ester (FAME) was studied. Glycine, CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), and TAPS ([(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid) were shown to be potent core matrices for these reactions. The optimal reaction contained 4:1 [methanol]/[fatty acid] molar equivalence ratio with 20% (w/w) CL-PCMC-LIP on glycine in the presence of tert-butanol as a co-solvent. Deactivation effect of glycerol on the biocatalyst reactive surface was shown by FTIR, which could be alleviated by increasing co-solvent content. The maximal FAME yields from PA, RPO, and CPO reached 97.6, 94.9, and 95.5%, respectively on a molar basis under the optimum conditions after incubation at 50 °C for 6 h. The biocatalyst retained >80% activity after recycling in five consecutive batches. The work demonstrates the potential of CL-PCMC-LIP on one-step conversion of inexpensive crude fatty acid-rich feedstock to biodiesel.     

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.     

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)).     

Immobilization of porcine pancreatic lipase on poly-hydroxybutyrate particles for the production of ethyl esters from macaw palm oils and pineapple flavor

Poly-hydroxybutyrate particles (PHB) were used as support to immobilize porcine pancreatic lipase (PPL). The biocatalysts prepared were tested in the synthesis of pineapple flavor by esterification of butanol and butyric acid in heptane medium, and in the synthesis of ethyl esters by transesterification of macaw palm pulp (MPPO) and macaw palm kernel (MPKO) oils with ethanol in solvent-free systems. The effect of protein loading on the biocatalyst activity was assessed in olive oil hydrolysis. Maximum hydrolytic activity of 292.8 ± 8.60 IU/g was observed. Langmuir isotherm model was applicable to the adsorption of PPL on PHB particles. Maximum immobilized protein amount was 24.3 ± 1.70 mg/g. The optimal pH and temperature in hydrolysis reaction for the immobilized PPL were at pH 8.5 and 50 °C, while for the crude PPL extract were at pH 8.0 and 45 °C. Immobilized PPL exhibited full hydrolytic activity after 2 h of incubation in non-polar solvents. In esterification reaction, optimal conversion was around 93% after 2 h of reaction. After six esterification cycles, the biocatalyst retained 63% of its initial activity. The biocatalyst prepared attained transesterification yield of 50% after 48 h of reaction for MPKO and 35% after 96 h of reaction for MPPO.     

脂解麻疯树油脂肪酶产生菌的筛选鉴定及其催化特性

[目的]分离筛选具有脂解麻疯树油能力的脂肪酶产生菌株,为以麻疯树油为原料酶法生产生物柴油奠定基础.[方法]以麻疯树油为唯一碳源,从麻疯树种子粉末处理过的土壤中分离筛选出1株具有脂解疯树油能力的脂肪酶产生菌,考察该菌株及其脂肪酶对有机溶剂耐受性以及脂肪酶催化酯化和转酯反应的能力,并通过生理生化特征和16S rDNA序列分...     

Enzymatic synthesis of l-ascorbyl linoleate in organic media

A novel l-ascorbyl fatty acid ester, l-ascorbyl linoleate was successfully prepared by enzymatic esterification and transesterification in a non-aqueous medium using immobilized lipase as biocatalyst. Changes in enzymatic activity and product yield were studied for the following variable: the nature of the fatty acid, the fatty acid concentration and water content. The yield of synthesis for the C18 unsaturated fatty acids were higher than for the C18 saturated fatty acid. Initial enzyme concentration does not affect the equilibrium of the reaction. And the product yield (33.5%) in the transesterification was higher than that of the esterification (21.8%) at a high-substrate concentration 0.3 M. The medium water content was found to have a distinct influence on the l-ascorbyl linoleate synthesis.These authors contributed equally to the article.     

Preparation of short chain esters by the lipase from mucor miehei using heptane and silica gel

Butyl acetate, isoamyl acetate and isoamyl valerate were prepared by Mucor miehei lipase catalyzed esterification of free acids and alcohols carried out in non-aqueous systems using heptane and silica gel which removes water formed in the reaction. For butyl and isoamyl acetate 1:3 and for isoamyl acetate 1:2 molar proportions of acid to alcohol were found to be optimal. Heptane(5 ml) and 0.01g silica gel per 0.1M acid were found to improve the yields. Under optimum conditions using 60°C, within 48 hours 40% butyl acetate, 53% isoamyl acetate and 61% isoamyl valerate conversions were observed.     

Preparation of cross-linked enzyme aggregates by using bovine serum albumin as a proteic feeder

Addition of bovine serum albumin (BSA) as a proteic feeder facilitates obtaining cross-linked enzyme aggregates (CLEAs) in cases where the protein concentration in the enzyme preparation is low and/or the enzyme activity is vulnerable to the high concentration of glutaraldehyde required to obtain aggregates. CLEAs of Pseudomonas cepacia lipase and penicillin acylase were prepared. CLEA of lipase prepared in the presence of BSA retained 100% activity whereas CLEA prepared without BSA retained only 0.4% activity of the starting enzyme preparation. Lipase CLEA showed 12-fold increase in activity over free enzyme powder when the CLEA was used in transesterification of tributyrin. For the transesterification of Jatropha oil, while free enzyme powder required 8 h and 50 mg lipase to obtain 77% conversion, CLEA required only 6 h and 6.25 mg lipase to obtain 90% conversion. In the case of penicillin acylase, 86% activity could be retained in CLEA prepared with BSA whereas CLEA made without BSA retained only 50% activity. CLEA prepared without BSA lost 20% activity after 8 h at 45 degrees C whereas CLEA with BSA retained full activity. CLEA prepared with BSA showed Vmax/Km of 36.3 min-1 whereas CLEA prepared without BSA had Vmax/Km of 17.4 min-1 only. Scanning electron microscopy analysis showed that CLEAs prepared in the presence of BSA were less amorphous and closer in morphology to CLEAs of other enzymes described in the literature.     

A two steps enzymatic procedure to obtain sterol esters,tocopherols and fatty acid ethyl esters from soybean oil deodorizer distillate

Soybean oil deodorizer distillate (SODD) was enzymatically modified to obtain a product mixture comprised mainly of sterol esters, tocopherols, and fatty acid ethyl esters. Firstly, the original SODD was mixed with oleic acid to reduce its melting point from 65–70 to 30–35 °C and also to produce a reaction mixture with a ratio of free fatty acids (FFA) to sterols close to 2 to improve the progress of sterols esterification. Two enzymatic steps were used in order to separate sterols esterification and ethyl esterification in time and space. The first enzymatic step (in the presence of Candida rugosa lipase) allowed to efficiently transform more than 90% of the original sterols in a short period of time (5 h). The second enzymatic step (in the presence of Novozym 435) converted more than 95% of the FFA in less than 3 h. In addition, the stability of both biocatalysts has been evaluated and both bioprocesses have been scaled-up reutilizing the same batch of lipase up to 8 and 3 times for the first and the second enzymatic step, respectively. The final product obtained is intended to be used as starting material for the purification of sterol esters, tocopherols, and fatty acid ethyl esters via supercritical fluid extraction.     

Selection of lipase producing yeasts for methanol-tolerant biocatalyst as whole cell application for palm-oil transesterification

Methanol-tolerant lipase producing yeast was successfully isolated and selected thorough ecological screening using palm oil-rhodamine B agar as one step-approach. All 49 lipase-producing yeasts exhibited the ability to catalyze esterification reaction of oleic acid and methanol at 3 molar equivalents. However, only 16 isolates catalyzed transesterification reaction of refined palm oil and methanol. Rhodotorula mucilagenosa P11I89 isolated from oil contaminated soil showed the strongest hydrolytic lipase activity of 1.2U/ml against palm oil. The production of oleic methyl ester and fatty acid methyl ester (FAME) of 64.123 and 51.260% was obtained from esterification and transesterification reaction catalyzed by whole cell of R. mucilagenosa P11I89 in the presence of methanol at 3 molar equivalents against the substrates, respectively. FAME content increased dramatically to 83.29% when 6 molar equivalents of methanol were added. Application of the methanol-tolerant-lipase producing yeast as a whole cell biocatalyst was effectively resolved major technical obstacles in term of enzyme stability and high cost of lipase, leading to the feasibility of green biodiesel industrialization.     

Improvement of cell-bound lipase from Rhodotorula mucilaginosa P11I89 for use as a methanol-tolerant, whole-cell biocatalyst for production of palm-oil biodiesel

Rhodotorula mucilaginosa P11I89, isolated from oil-contaminated soil, was effectively used as the methanol-tolerant, whole-cell lipase for the synthesis of fatty acid methyl ester (FAME) via transesterification reaction in the presence of palm oil and methanol substrates at a 1:6 mole ratio. A combination of Taguchi experimental design and response surface methodology (RSM) were applied to systemically enhance transesterification activity of the whole-cell lipase or cell-bound lipase (CBL) from R. mucilaginosa P11I89 in a solvent-free system. The significant impacts of four factors including carbon sources, nitrogen sources, surfactants and pH on hydrolysis activity of extracellular and cell-bound lipases, and on the transesterification activity of CBL were evaluated using Taguchi design. Gum Arabic was the most significant component for high transesterification activity, whereas soybean oil was the most influential factor for the hydrolysis activity. Maximal CBL production of 272.72 U/L was obtained in the cultivation medium containing 2.1 % palm oil, 0.2 % NH₄NO₃ , and 0.45 % Gum Arabic, with initial pH 5.0 under shaking speed of 200 rpm at a temperature of 30?±?2 °C after 60 h incubation using Central Composite Design (CCD). Yeast cells grown under such conditions increased FAME yield from 84.0 to 92.98 % when the transesterification reaction was carried out, in comparison to those cultivated in the initial medium.     

Lysophosphatidylcholine synthesis with Candida antarctica lipase B (Novozym 435)

Immobilized lipase from Candida antarctica lipase B (Novozym 435) was effective in the synthesis of lysophosphatidylcholine (LPC). The transesterification of L-alpha-glycerophosphorylcholine (GPC) and vinyl laurate was carried out in a solvent free system or in the presence of 50% (v/v) t95%) were easily achieved. The lipase was selective for the sn10 times). High purity products could be produced by a decrease of the reaction temperature to induce precipitation of the product. The temperature needed depended on the fatty acid chain length. Thus, only lysophosphatidylcholine was produced with palmitic acid vinyl ester at 45 degrees C, whereas for the vinyl esters of lauric acid, capric acid, and caprylic acid, a lower reaction temperature (25 degrees C) was necessary to obtain solely the lysophospholipid products.     

Application of advanced materials as support for immobilisation of lipase from Candida rugosa

Ni/Al-layered double hydroxides (Ni-LDHs) and Ni/Al-sodium dodecyl sulfonate layered double hydroxide nanocomposites (Ni-SDS-LDHs) with a molar ratio of Ni:Al (4:1) have been prepared by a co-precipitation (or salt-base) method. Their structures were determined using Powder X-Ray Diffractometer (PXRD) and the spectra showed that basal spacings for Ni-LDHs and Ni-SDS-LDHs synthesised were around 8.1?Å and 34.8?Å, respectively. Lipase from Candida rugosa was immobilised onto these advanced materials, by physical adsorption. The activity of immobilised lipase was investigated through esterification of palmitic acid and isopropyl alcohol in hexane. The effects of reaction temperature, thermostability, stability in organic solvent, operational stability, leaching and storage studies of the immobilised lipase were investigated. These biocatalysts exhibited higher activities than the native lipase with an optimum temperature of 40°C. Immobilised lipases showed higher storage stability than native lipase (up to 60 days) and during operational studies at 30°C for 5?h, more than 50% of its activity was retained. Leaching studies showed that physical adsorption is suitable for the attachment of enzymes onto LDHs.     

非水相酶促合成癸酸偏甘油酯的研究

对无溶剂非水相中癸酸与甘油的酶促酯化反应进行了研究,发现Pseudomonas fluoresces脂肪酶（PFL）、Mucor miehei脂肪酶（MML）和Candida antarictica脂肪酶（CAL）均有较好的催化活性。CAL酶促转化癸酸的最适反应条件为：60℃,加酶量为20～100u/g,初始加水量为甘油质量的12%。CAL的1,3位置专一性在最终产物中未表达。CAL酶催化剂的失活主要与机械磨损有关,反应5批次后酶活残留量为96.4%。敞开物系、真空脱水或分子筛脱水均为有效脱水方式。敞开物系中反应物量比不影响平衡转化率而会影响单甘酯平衡产率。用碳酸氢钠水溶液萃取可有效脱除产品中的残余癸酸,终产品酸价为0.68mg KOH/g。提高甘油比例并使用非脱水原料,无外加水结合部分流加癸酸的工艺,可以减少减压脱水或敞开反应的时间,5h后癸酸最高转化率可达96.9%。      

Effects of capric acid on rumen methanogenesis and biohydrogenation of linoleic and α-linolenic acid

Capric acid (C10:0), a medium chain fatty acid, was evaluated for its anti-methanogenic activity and its potential to modify the rumen biohydrogenation of linoleic (C18:2n-6) and α-linolenic acids (C18:3n-3). A standard dairy concentrate (0.5 g), supplemented with sunflower oil (10 mg) and linseed oil (10 mg) and increasing doses of capric acid (0, 10, 20 and 30 mg), was incubated with mixed rumen contents and buffer (1 : 4 v/v) for 24 h. The methane inhibitory effect of capric acid was more pronounced at the highest (30 mg) dose compared to the medium (20 mg) (-85% v. -34%), whereas the lower dose (10 mg) did not reduce rumen methanogenesis. A 23% decrease in total short-chain fatty acid (SCFA) production was observed, accompanied by shifts towards increased butyrate at 20 mg and increased propionate at 30 mg of capric acid (P < 0.001). Capric acid linearly decreased the extent of biohydrogenation of C18:2n-6 and C18:3n-3, by up to 60% and 86%, respectively. This reduction was partially due to a lower extent of lipolysis when capric acid was supplemented. Capric acid at 20 and 30 mg completely inhibited the production of C18:0 (P < 0.001), resulting in an accumulation of biohydrogenation intermediates, mainly C18:1t10 + t11 and C18:2t11c15. In contrast to effects on rumen fermentation (methane production and proportions of SCFA), 30 mg of capric acid did not induce major changes in rumen biohydrogenation as compared to the medium (20 mg) dose. This study revealed the dual action of capric acid, being inhibitory to both methane production and biohydrogenation of C18:2n-6 and C18:3n-3.     

Mucor circinelloides whole-cells as a biocatalyst for the production of ethyl esters based on babassu oil

The intracellular lipase production by Mucor circinelloides URM 4182 was investigated through a step-by-step strategy to attain immobilized whole-cells with high lipase activity. Physicochemical parameters, such as carbon and nitrogen sources, inoculum size and aeration, were studied to determine the optimum conditions for both lipase production and immobilization in polyurethane support. Olive oil and soybean peptone were found to be the best carbon and nitrogen sources, respectively, to enhance the intracellular lipase activity. Low inoculum level and poor aeration rate also provided suitable conditions to attain high lipase activity (64.8 ± 0.8 U g^?1). The transesterification activity of the immobilized whole- cells was assayed and optimal reaction conditions for the ethanolysis of babassu oil were determined by experimental design. Statistical analysis showed that M. circinelloides whole-cells were able to produce ethyl esters at all tested conditions, with the highest yield attained (98.1 %) at 35 °C using an 1:6 oil-to-ethanol molar ratio. The biocatalyst operational stability was also assayed in a continuous packed bed reactor (PBR) charged with glutaraldehyde (GA) and Aliquat-treated cells revealing half-life of 43.0 ± 0.5 and 20.0 ± 0.8 days, respectively. These results indicate the potential of immobilized M. circinelloides URM 4182 whole-cells as a low-cost alternative to conventional biocatalysts in the production of ethyl esters from babassu oil.     

Biodiesel production from waste cooking oil by immobilized lipase on superparamagnetic Fe3O4 hollow sub-microspheres

Superparamagnetic Fe₃O₄ hollow sub-microspheres (FHSM) with strong response to an external magnet were prepared via a solvothermal method, followed by acid etching. Lipase from Candida sp. 99–125 was directly immobilized onto the amino-functional FHSM by simple adsorption, without glutaraldehyde linkage. The immobilized lipase was used to catalyze the esterification/transesterification of waste cooking oil with methanol to produce fatty acid methyl ester (FAME), a major source of biodiesel. FAME yield exceeded 93.4% over a wide range of temperatures from 10 to 40?°C. Notably, stability was clearly improved at the lower temperatures, in particular, giving a FAME yield of 89.6% after eight cycles of use at 10?°C.