Enzymic interesterification of fats: The effect of non-lipase material on immobilized enzyme activity

An immobilized lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) suitable for fat interesterification has been prepared by precipitation onto diatomaceous earth (Celite) with acetone of a crude lipase preparation from an Aspergillus. Non-lipase material present in the preparation which precipitated at high acetone concentrations or ovalbumin added prior to the immobilization reduced the measured interesterification activity without affecting lipolytic activity. The non-lipase material reduced the interesterification activity by as much as 50%. The interesterification activity of immobilized preparations was enhanced by the use of higher concentrations of the crude lipase or, more substantially, by admixture of purified lipase.     

Surfactant-Modified lipase for the catalysis of the interesterification of triglycerides and fatty acids

The lipase-catalyzed intresterification of triglycerides and fatty acids in n-hexane was studied. Initially, lipase Saiken was modified with a surfactant of sorbitan esters so that its dispersibility in hydrophobic organic media was improved. The surfactant-modified lipase formed in the modification process carried out in a buffer solution has 1,3-positional specificity and predominantly catalyzed the interesterification reaction in a microaqueous n-hexane system. The modification technique converted inactive lipases to very active biocatalysts for the interesterification of triglycerides and fatty acids. The pH and the weight ratio of surfactant to enzyme used during the lipase modification process have shown significant effects in determining the recoveries of the protein and enzyme activity from the buffer solution, the protein content of the modified lipase complex after being freeze dried, and the interesterification activity of the complex. The water content in the reaction solution has strongly influenced the enzyme activity as well as the distribution of the products. (c) 1995 John Wiley & Sons, Inc.     

Kinetic modeling of interesterification reactions catalyzed by immobilized lipase

Kinetic data for lipase-catalyzed interesterification reactions between free fatty acids and triglycerides were collected and the dynamics of the interesterification reactions were successfully modeled using tow rate experssions requiring a total of five adjustable parameters. One rate expression describes the disappearance of the free fatty acid (octanoic or linolenic acid), and the second describes the rate of release of fatty acid residues from the triglycerides (olive oil or milkfat). This model is able to account for the effects of the concentration of all chemical species participating in interesterification throughout the entire reaction. When the data for both milkfat and olive oil were subjected to nonlinear regression analyses using the same mathematical model, the parameter estimates for both systems were comparable. In addition to reproducing the tendencies observed experimentally, simulations of the interesterification system under a variety of initial conditions provided insight into the effects of several reaction variables which could not be examined experimentally. Among the most significant findings of the simulation work are (1) there is a limit beyond which increasing the initial concentration of water produces no further increase in the initial rate of the interesterification reaction; (2) an increase in the initial concentration of lower glycerides produces a concomitant increase in the rate of the interesterification reaction; (3) the free fatty acids inhibit the rate of hydrolysis of the fatty acid residues of the triglycerides; (4) there is a limit beyond which increasing the initial concentration of triglycerides produces no significant increase in the rate of either the hydrolysis reaction or the interesterification reaction. (c) 1994 John Wiley & Sons, Inc.     

Enzymic interesterification of fats: Factors influencing the choice of support for immobilized lipase

Three grades of diatomaceous earth (Celite 560, Filtercel and Hyflo Supercel) and a controlled-pore silica have been examined for their suitability as support materials for lipase (triacyglycerol acylhydrolase, EC 3.1.1.3) catalysing the interesterification of fats. The controlled-pore silica gave a preparation with a low activity. Although all three Celites gave preparations with similar lipolytic activities, Hyflo Supercel gave the highest interesterification activity. The distribution of enzyme protein in Hyflo Supercel was examined by transmission electron microscopy.     

Lipase-catalyzed alcoholysis of triglycerides for the preparation of 2-monoglycerides

Rhizopus arrhizus lipase immobilized on celite was used to prepare isomerically pure 2-monoglycerides by alcoholysis of triglycerides in organic media. Reaction parameters such as choice of solvent, choice of alcohol, and alcohol concentration were studied. When 12.5 mM tripalmitin was used as substrate, methyl-tert-butyl ether was the best solvent for alcoholysis at water activity 0.11. Ethanol gave the highest yield (97%) at an optimal ethanol concentration of 200–300 mM. At higher alcohol concentrations, the enzyme activity was substantially lowered. The enzyme preparation showed high stability in repeated-batch reactions.     

An Efficient Method for Separating Ascospores from Sporulating Cultures in Saccharomyces cerevisiae by Hydrostatic Pressure

A new oxidative reaction of ethylene glycol was found with two alcohol oxidases from methanol yeast, Candida sp. and Pichia pastoris. Both alcohol oxidases oxidized ethylene glycol to glyoxal via glycolaldehyde. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Candida alcohol oxidase were around 8.5 and 5.5, respectively, and their apparent K_ms were 2.96 m and 28.6 mm, respectively. The optimum temperature was 40°C at pH 7.0. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Pichia alcohol oxidase were around 8.0 and 6.0, respectively, and their optimum temperatures were 50 and 45°C, respectively, at pH 7.0. The apparent K_m for glycolaldehyde was found to be 83.3 mm. For the accumulation of glyoxal, addition of catalase was effective, and a higher amount of glyoxal was obtained at a much lower temperature than the optimum for the alcohol oxidase. When 0.1 m ethylene glycol and glycolaldehyde were incubated with 80 units of the Pichia enzyme at 10°C, both substrates were almost completely converted to glyoxal after 10 and 3h of incubation, respectively.     

Stereoselectivity of lipases. II. Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward chemically alike, but sterically nonequivalent ester groups within one single triglyceride molecule was investigated. Lipolysis reactions were carried out on synthetic trioctanoin or triolein, which are homogenous, prochiral triglycerides, chosen as models for physiological lipase substrates. Diglyceride mixtures resulting from lipolysis were derivatized with optically active R-(+)-1-phenylethylisocyanate, to give diastereomeric carbamate mixtures, which were further separated by high performance liquid chromatography. Resolution of diastereomeric carbamates gave enantiomeric excess values, which reflect the lipases stereobias and clearly demonstrate the existence of a stereopreference by both gastric lipases for the sn-3 position. The stereoselectivity of human and rabbit gastric lipases, expressed as the enantiomeric excess percentage, was 54% and 70% for trioctanoin and 74% and 47% for triolein, respectively. The corresponding values with porcine pancreatic lipase were 3% in the case of trioctanoin and 8% in that of triolein. It is worth noting that rabbit gastric lipase, unlike human gastric lipase, became more stereoselective for the triglyceride with shorter acyl chains (trioctanoin). This is one of the most striking catalytic differences observed between these two gastric lipases.     

Phyllosilicate sol-gel immobilized lipases for the formation of partial acylglycerides**

Lipase PS-30 (pseudomonas cepacia) and Lipase F (Rhizopus oryzae), immobilized within a phyllosilicate sol-gel matrix, catalyzed the esterification of glycerol with short, medium and long-chain fatty acids to produce mono (MAG), di (DAG) and tri (TAG) acylglycerols. The results from the above esterification reactions were compared to reactions using a commercially available immobilized lipase, Lipozyme IM-60. Time course studies showed that free Lipase PS-30 or Lipase F enhanced esterification reactions with the use of silica-supported glycerol. In contrast, immobilized Lipase PS-30-catalyzed reactions occurred at the same conversion rate when using either free or silica-supported glycerol. For immobilized Lipase F and Lipozyme IM-60 reactions, the use of silica-supported glycerol favored the production of DAG and TAG over MAG. All three immobilized lipases could be reused for acylglycerol production.     

Continuous interesterification of butteroil and conjugated linoleic acid in a tubular reactor packed with an immobilized lipase

Modified milkfats were produced via interesterification (acidolysis) reactions of butteroil and conjugated linoleic acid (CLA) in a packed bed reactor containing an immobilized lipase preparation from Candida antarctica. The rate expression for the interesterification reaction is of the generalized Michaelis–Menten form. Significant enrichment of butteroil in CLA residues was accomplished at reactor space times (fluid residence times) of 2–4 h at 40–60°C, but the optimum operating temperature was ca. 50°C. Approximately 80–90% of the free CLA fed to the reactor can be converted to its esterified form.     

Hydrolysis of rice bran oil using an immobilized lipase from Candida rugosa in isooctane

The kinetics of enzymatic hydrolysis of rice bran oil in isooctane by immobilized Candida rugosa lipase in a batch reactor showed competitive inhibition by isooctane with a dissociation constant, K1, of 0.92 M. Continuous hydrolysis of rice bran oil was performed in recycling, packed bed reactor with 4352 U of immobilized lipase; the optimum recycle ratio was 9 and the operational half-life was 360 h without isooctane but 288 h with 25% (v/v) isooctane in rice bran oil.     

Enhancing operational stability and exhibition of enzyme activity by removing water in the immobilized lipase‐catalyzed production of erythorbyl laurate

Erythorbyl laurate was continuously synthesized by esterification in a packed‐bed enzyme reactor with immobilized lipase from Candida antarctica. Response surface methodology based on a five‐level three‐factor central composite design was adopted to optimize conditions for the enzymatic esterification. The reaction variables, such as reaction temperature (10–70°C), substrate molar ratio ([lauric acid]/[erythorbic acid], 5–15), and residence time (8–40 min) were evaluated and their optimum conditions were found to be 56.2°C, 14.3, and 24.2 min, respectively. Under the optimum conditions, the molar conversion yield was 83.4%, which was not significantly different (P < 0.05) from the value predicted (84.4%). Especially, continuous water removal by adsorption on an ion‐exchange resin in a packed‐bed enzyme reactor improved operational stability, resulting in prolongation of half‐life (2.02 times longer compared to the control without water‐removal system). Furthermore, in the case of batch‐type reactor, it exhibited significant increase in initial velocity of molar conversion from 1.58% to 2.04%/min. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:882–889, 2013     

Plant lipases: biocatalyst aqueous environment in relation to optimal catalytic activity in lipase-catalyzed synthesis reactions.

Adsorption and desorption isotherms of two commercial enzyme preparations of papain and bromelain were determined with a Dynamic Vapor System. The Guggenheim-Anderson-deBoer (GAB) modeling of the obtained sorption isotherms allowed the definition of different levels of hydration of those samples. Afterward, these enzyme preparations were used as biocatalysts in water and solvent-free esterification and alcoholysis reactions. The evolution of the obtained fatty acid ester level as a function of the initial hydration level of the biocatalyst, i.e., thermodynamic water activity (a(w)) and water content, was studied. The results show an important correlation between the initial hydration level of the biocatalyst and its catalytic activity during the lipase-catalyzed synthesis reactions. Thus, the Carica papaya lipase (crude papain preparation) catalytic activity is highly dependent on the biocatalyst hydration state. The optimized synthesis reaction yield is obtained when the a(w) value of the enzyme preparation is stabilized at 0.22, which corresponds to 2% water content. This optimal level of hydration occurs on the linear part of the biocatalyst's sorption isotherm, where the water molecules can form a mono- or multiple layer with the protein network. The synthesis reaction yield decreases when the a(w) of the preparation is higher than 0.22, because the excess water molecules modify the system equilibrium leading to the reverse and competitive reaction, i.e., hydrolysis. These results show also that an optimal storage condition for the highly hydrophilic crude papain preparation is a relative humidity strictly lower than 70% to avoid an irreversible structural transition leading to a useless biocatalyst. Concerning the bromelain preparation, no effect of the hydration level on the catalytic activity during esterification reactions was observed. This biocatalyst has too weak a catalytic activity which makes it difficult to observe any differences. Furthermore, the bromelain preparation is far more hydrophobic as it adsorbs only 18 g of water per 100 g of dry material at a(w) around 0.90. No deliquescence of this enzymatic preparation is observed at this a(w) value.     

Production of structured lipids by lipase-catalysed interesterification in an ultrafiltration membrane reactor

The application of membranes to the enzymatic production of structured lipids has been investigated using an ultrafiltration membrane reactor for a reaction between medium chain triacylglycerols (MCT) and n-3 polyunsaturated fatty acids from fish oil. Lipozyme IM was used as the biocatalyst. The incorporation of polyunsaturated fatty acids into MCT was increased by about 15% over 80 h by simultaneous separation of the released medium chain fatty acids compared to control.     

Substrate selectivity of lipases in the esterification of oleic acid, linoleic acid, linolenic acid and their all-trans-isomers and in the transesterification of cis/trans-isomers of linoleic acid methyl ester

The substrate selectivity of several microbial lipases has been examined in the esterification of oleic acid, linoleic acid, linolenic acid and their all-trans-isomers and in the alcoholysis of isomeric linoleic acid methyl esters with n-butanol. Lipases from Candida cylindracea and Mucor miehei preferred fatty acids and methyl esters with a (first) cis double bond in 9-position, while Chirazyme L-5, a Candida antarctica lipase A, had a preference for trans-9 unsaturated substrates.     

提高微生物脂肪酶产量、活性和稳定性的方法研究

微生物脂肪酶是一类广泛应用于诸多工业领域的生物催化剂。提高微生物脂肪酶的产量、活性和稳定性,增强产品的市场竞争力,一直是微生物脂肪酶研究的重点和热点。本文从产脂肪酶菌株的改造、脂肪酶基因的改良、脂肪酶发酵工程和脂肪酶后期处理等四个方面概述了提高微生物脂肪酶产量、活性和稳定性的方法,以期为微生物脂肪酶的规模化工业生产提供方法性指导。     

Convenient enzymatic resolution of (R,S)‐2‐methylbutyric acid catalyzed by immobilized lipases

The application of several immobilized lipases has been explored in the enantioselective esterification of (R,S)‐2‐methylbutyric acid, an insect pheromone precursor. With the use of Candida antarctica B, using hexane as solvent, (R)‐pentyl 2‐methylbutyrate was prepared in 2 h with c 40%, ee_p 90%, and E = 35, while Thermomyces lanuginosus leads to c 18%, ee_p 91%, and E = 26. The (S)‐enantiomer was obtained by the use of Candida rugosa or Rhizopus oryzae (2‐h reaction, c 34% and 35%, ee_p 75 and 49%, and E = 10 and 4, respectively). Under optimal conditions, the effect of the solvent, the molar ratio, and the nucleophile were evaluated.     

微水体系中荧光假单胞菌脂肪酶催化合成单甘酯

研究了无溶剂微水体系中荧光假单胞菌脂肪酶 (PFL)催化油脂甘油解合成单甘酯的反应因素以及多温程非均相固液反应对单甘酯产率的影响。以初始体系最低共熔点 (PFL)取代临界温度学说中的油脂初熔点 ,通过考察不同IEP体系的甘油解 ,发现PFL酶促油脂甘油解时存在碳链基质特异性的函数关系 ,即反应物油脂中饱和碳残基的质量百分含量 (C₁₆+C₁₈)与单甘酯产率间符合以下多项式:Y =- 0.0006X³ +0.0592X²-0.8909X+26.753(13%<X<76.5%),式中X为C₁₆+C₁₈,Y为40℃时等温反应条件下的单甘酯产率。IEP为40℃时,最适等温反应条件如下：加水量3%～4.5%,加酶量为500μ/g油酯摩尔比1：2.5-5.0(油酯：甘油)反应温度40℃.实验条件下多步等程序降温反应48h后单甘酯最高产率为81.4%.     

固定化脂肪酶合成二元酸酯

The syntheses of dicarboxylic esters by immobilized lipase from Candida sp. -1619 were investigated. The reaction system was composed of 1 mmol dicarboxylic acid, 2 mmol alcohol, 3 mL hexane and 15 mg celite-adsorbed im mobilized lipase(300 u), in a closed 100 mL Erlenmeyer flask, shaken at 40°C for 5h. Sebacic acid was the best substrate among nine dicarboxylic acids selected. Among the 18 saturated fatty n-alcohols, the alcohols with carbon chain length rangin from C4～C18 had good reactivity. The primary alcohols had much better reactivities than corresponding secondary alcohols and multihydroxy-alcohols. Tertiary alcohols showed no reactivity. Hydrocarbons, benzene, toluene, xylene and te trachloride were favorite reactants among 15 organic solvents selected, in none-solvent stationary system, (5 mmol sebacic acid, 10 mmol dndecanol, 150 mg immobilized lipase(3000 u))reacted without plug for 3.5h, the optimum temperature was 60°C. The conversion degree was over 92% when reaction carried out at 50～90°C for 17h. The suitable reaction pH ranged from 6～8. The reactant was developed on GF254 plate(hexane ethyl ether acetic acid = 30201 ( V V V).There were three spots with different Rf value at 0.96, 0.55 and 0 corresponding to product, oleyl alcohol and sebacic acids, respectively.     

Interesterification of butter fat by partially purified extracellular lipases from Pseudomonas putida,Aspergillus niger and Rhizopus oryzae

Three extracellular lipases were produced by batch fermentation of Pseudomonas putida ATCC 795, Aspergillus niger CBS 131.52 and Rhizopus oryzae ATCC 34612 during the late phase of growth, at 72, 96 and 96 h, respectively. The lipases were partially purified by (NH₄)₂SO₄ fractionation. The lipase of P. putida was optimal at pH 8.0 whereas those from A. niger and R. oryzae were optimal at pH 7.5. The A. niger lipase had the lowest V _max value (0.51×10^-3 U/min) and R. oryzae the highest (1.86×10^-3 U/min). The K _m values for P. putida, A. niger and R. oryzae lipases were 1.18, 0.97, and 0.98 mg/ml, respectively. Native PAGE of the partially-purified lipase extracts showed two to four major bands. The interesterification of butter fat by A. niger lipase decreased the water activity as well as the hydrolytic activity. The A. niger lipase had the highest interesterification yield value (26%) and the R. oryzae lipase the lowest (4%). In addition, A. niger lipase exhibited the highest decrease (17%) in long-chain hypercholesterolemic fatty acids (C12:0, C14:0 and C16:0) at the sn-2-position; the P. putida lipase demonstrated the least favourable changes in specificity at the same position.F. Pabai and S. Kermasha are with the Department of Food Science and Agricultural Chemistry, McGill University, 21111 Lakeshore, Ste Anne de Bellevue, Québec, H9X 3V9, Canada; A. Morin is with the Food Research and Development Centre, Agricultural and Agri-Food Canada, 3600 Casavant Blvd West, Saint-Hyacinthe, Québec, J2S 8E3, Canada.