Template enhanced activity of lipase accommodated in siliceous mesocellular foams

Lipases were adsorbed in siliceous mesocellular foams containing different amounts of residual template in the nanopores. It is found that the hydrolytic activities of the adsorbed lipases are increased with increasing the contents of template in the mesopores. The triacetin hydrolytic activity of the lipase adsorbed in the foam containing 46% of template can be 13 times higher than that of the lipase adsorbed in the foam without template in the nanopores, and its specific activity is about three times higher than that of the free lipase, showing the hyperactivation effect on lipase resulting from the interaction between the lipase and the surfactant in the nanopores. The immobilized lipase cross-linked with glutaraldehyde can retain up to 88% of its original activity after six hydrolysis reaction test. This work provides a new strategy to enhance the activity of immobilized lipase in mesoporous materials.     

Immobilization of Lipase from Rhizopus Niveus: A Way to Enhance its Synthetic Activity in Organic Solvent

Lipase (EC 3.1.1.3) from Rhizopus niveus was immobilized by physical adsorption on various carriers, including different types of Celite, Spherosil and Duolite. After the enzyme immobilization, the recovered hydrolytic and synthetic activities on the different carriers were then determined. The results showed that the highest synthetic activity was obtained when Duolite XAD 761 was used as the carrier. However the recovered hydrolytic activity after the immobilization on this resin was relatively low although this carrier showed the best protein loading capacity. The highest recovered hydrolytic activity was observed when the lipase was immobilized on Celite Hyflo-Supercel using an immobilization buffer adjusted to pH 4. The comparison of the free and immobilized lipase specific activities suggest that the immobilization on Celite Hyflo-Supercel, Spherosil XOA 200 and silica has enhanced the lipase hydrolytic activity. On the other hand, the use of the lipase immobilized on Duolite XAD 761 as biocatalyst of synthetic reaction, compared to that of the free enzyme, allows the reaction initial velocity to be increased 12.2-fold. In addition, the synthetic activity of the lipase immobilized on Duolite XAD 761 was shown to be maximum at a water activity in the range of 0.32-0.52.     

华根霉脂肪酶有机相合成酶活的研究

通过比较7种微生物脂肪酶的有机相合成酶活、水相水解酶活及在正庚烷中催化己酸乙酯合成的能力,证明了合成酶活与水解酶活相关性不高,合成酶活比水解酶活更能反映脂肪酶的合成能力。通过比较两株华根霉(Rhizopus chinensis)脂肪酶酶活,发现合成酶活相差较大,表明相同种属微生物的脂肪酶合成酶活存在不同。对．Rhizopus chinensis-2液态发酵产脂肪酶进程研究发现,水解酶活高峰先于合成酶活高峰大约12h。将不同培养时间的Rhizopus chinensis-2全细胞脂肪酶用于催化己酸乙酯合成,具有高合成酶活的全细胞脂肪酶催化己酸乙酯合成反应较快。因此,全细胞脂肪酶用于催化有机相酯合成反应时,具有高脂肪酶合成酶活的菌体具有较好的催化酯合成能力。     

Enzymic interesterification of fats: Laboratory and pilot-scale studies with immobilized lipase from Rhizopus arrhizus

An immobilized lipase suitable for fat interesterification has been prepared by precipitation with acetone of a commercial lipase from Rhizopus arrhizus onto diatomaceous earth. As observed previously with a less active enzyme from Aspergillus sp., the interesterification activity was enhanced by addition of purified lipase or by high loadings of commercial enzyme. The interesterification activities reached maximum values in both cases. For immobilized preparations with purified enzyme, interesterification activity was also enhanced by the presence of a precoat of glutaraldehyde cross-linked commercial lipase. A 2.9-L column of immobilized lipase was used to interesterify batches of shea oleine (67 kg) and shea oil (40 kg). Little activity was lost processing shea oleine, but slow poisoning of the bed occurred when shea oil was fed to the column.     

Interesterification of fats and oils by immobilized fungus at constant water concentration

The kinetics of enzymatic interesterification of oils and fats, using acetone-dried cells of Rhizopus chinensis immobilized on biomass support particles as a lipase catalyst, were investigated in batch operations at several constant water concentrations.Even under microaqueous (i.e., low-water-content) conditions, not only interesterification but also hydrolysis occured, and the water content in the reaction system decreased. The reaction rates of interesterification and hydrolysis at constant water concentrations were determined.For the reactions between olive oil and methyl stearate at several water concentrations, the parameters involved in the reaction model were determined by a trial-and-error method so as to make the calculated results correlate with the experimental data. The relationship between the parameters obtained and water concentration were examined.The rate constants involved in the reaction model of both interesterification and hydrolysis increased or decreased monotonically with the increasing water content, while the apparent activity of the lipase catalyst for interesterification had a maximum value at a water concentration of about 50 ppm. This suggests that when the water content is excessive the hydrolysis activity of lipase is accelerated more than its interesterification activity, and that when the water content is too little lipase activity can not be activated for either hydrolysis or interesterification.     

Simple screening method for lipase for transesterification in organic solvent

We investigated lipase-catalyzed hydrolysis in water and dioxane—water with a simple colorimetric method. We screened 24 lipases for the ability to hydrolyze p-nitrophenyl esters as chromogenic substrates. Their hydrolytic activities were varied by adding dioxane. Most of the lipases showed high activity in hydrolysis in water, but some showed activity in 50% dioxane—water several tens times higher than those in water. Moreover, several lipases with hydrolytic abilities in 50% dioxane—water also catalyzed the transesterification of p-nitrophenol using fatty acid vinyl esters. We found it possible that a useful lipase for transesterification can be selected by measuring the hydrolysis activity of p-nitrophenyl ester in 50% dioxane—water.     

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.     

Production of diacylglycerols from glycerol monooleate and ethyl oleate through free and immobilized lipase-catalyzed consecutive reactions

The ability of free and immobilized lipase on the production of diacylglycerols (DAG) by transesterification of glycerol monooleate (GMO) and ethyl oleate was investigated. Among three free lipases such as lipase G (Penicillium cyclopium), lipase AK (Pseudomonas fluorescens) and lipase PS (Pseudomonas cepacia), lipase PS exhibited the highest DAG productivity, and the DAG content gradually increased up to 24 hours reaction and then remained steady. The comparative result for DAG productivity between free lipase PS and immobilized lipases (lipase PS-D and Lipozyme RM IM) during nine times of 24 hours reaction indicated that total DAG production was higher in immobilized lipase PS-D (183.5mM) and Lipozyme RM IM (309.5mM) than free lipase PS (122.0mM) at the first reaction, and that the DAG production rate was reduced by consecutive reactions, in which more sn-1,3-DAG was synthesized than sn-1,2-DAG. During the consecutive reactions, the activity of lipase PS was relatively steady by showing similar DAG content, whereas DAG production of lipase PS-D and Lipozyme RM IM was gradually decreased to 69.9 and 167.1mM at 9th reaction, respectively, resulting in 62% and 46% reduced production when compared with 1st reaction. Interestingly, from 7th reaction lipase PS produced more DAG than immobilized lipase PS-D, and exhibited a stable activity for DAG production. Therefore, the present study suggested that DAG productivity between GMO and ethyl oleate was higher in immobilized lipases than free lipases, but the activity was reduced with repeated uses.     

Lipase-mediated hydrolysis of blackcurrant oil

Four commercially available lipases, both free and immobilized, were tested for their ability to catalyze hydrolysis of blackcurrant (Ribes nigrum) oil using two different approaches. The lipase from Mucor miehei was studied free and immobilized in two different ways. The former series of enzymic reactions were performed in tap water at 40 degrees C, but the latter series of enzymic processes were carried out in mixtures of isooctane and phosphate buffer (in a typical 2/1 ratio of the components) at 30 degrees C. These conditions were optimized to increase and/or to maximize the yields of the products, which were priority targets in this study. A rate of hydrolysis and a selective preference of the hydrolytic enzymes towards fatty acids, with a special focus on enrichment of alpha-linolenic acid and/or gamma-linolenic acid, were studied. Higher rates of hydrolysis of the blackcurrant oil in the former series of reactions were observed with the immobilized lipase from Pseudomonas cepacia used as biocatalyst. In the latter approach, the most favorable results of the rate of hydrolysis of the target blackcurrant oil were achieved with the immobilized lipase from Mucor miehei employed as biocatalyst. Only three lipases, selected from a series of lipases tested during this investigation, displayed specificity towards alpha-linolenic acid and gamma-linolenic acid, i.e. the immobilized lipase from P. cepacia, lipase from M. miehei and lipase from P. fluorescens.     

Immobilization of lipases for non-aqueous synthesis

Immobilization of lipases involves many levels of complications relating to the structure of the active site and its interactions with the immobilization support. Interaction of the so called hydrophobic ‘lid’ with the support has been reported to affect synthetic activity of an immobilized lipase. In this work we evaluate and compare the synthetic activity of lipases from different sources immobilized on different kinds of supports with varying hydrophobicity. Humicola lanuginosa lipase, Candida antarctica lipase B and Rhizomucor miehei lipase were physically adsorbed onto two types of hydrophobic carriers, namely hydrophilic carriers with conjugated hydrophobic ligands, and supports with base matrix hydrophobicity. The prepared immobilized enzymes were used for acylation of n-butanol with oleic acid as acyl donor in iso-octane with variable water content (0–2.8%, v/v) as reaction medium. Enzyme activity and effect of water on the activity of the immobilized derivatives were compared with those of respective soluble lipases and a commercial immobilized lipase Novozyme 435. Both R. miehei and H. lanuginosa immobilized lipases showed maximum activity at 1.39% (v/v) added water concentration. Sepabeads, a methacrylate based hydrophilic support with conjugated octadecyl chain showed highest immobilized esterification (synthetic) activity for all three enzymes, and of the three R. miehei lipase displayed maximum esterification activity comparable to the commercial enzyme.     

Immobilization of lipases on different carriers and their use in synthesis of pentyl isovalerates

Porcine pancreatic lipase (PPL) and Candida cylindracea lipase (CCL) were immobilized on Celite and Amberlite IRA 938 by deposition from the aqueous solution by the addition of hexane. The influence of the immobilization on the activities of the immobilized lipase derivatives has been studied. The immobilized lipases were used in synthesis of pentyl isovalerates. Various reaction parameters affecting the synthesis of pentyl isovalerates were investigated. The reaction rates were compared with the rates of esterification with free lipases. The immobilized lipases were found to be very effective in the esterification reaction. The lipases immobilized on Celite 545 exhibited better operational stabilities than that of immobilized on Amberlite IRA-938.     

Amino acid modified chitosan beads: Improved polymer supports for immobilization of lipase from Candida rugosa

Amino acid modified chitosan beads (CBs) for immobilization of lipases from Candida rugosa were prepared by activation of a chitosan backbone with epichlorohydrin followed by amino acid coupling. The beads were analyzed by elemental analysis and solid state NMR with coupling yields of the amino acids ranging from 15 to 60%. The immobilized lipase on unmodified chitosan beads showed the highest immobilization yield (92.7%), but its activity was relatively low (10.4%). However, in spite of low immobilization yields (15–50%), the immobilized lipases on the amino acid modified chitosan beads showed activities higher than that of the unmodified chitosan beads, especially on Ala or Leu modified chitosan beads (Ala-CB or Leu-CB) with 49% activity for Ala-CB and 51% for Leu-CB. The immobilized lipases on Ala-CB improved thermal stability at 55 °C, compared to free and immobilized lipases on unmodified chitosan beads and the immobilized lipase on Ala-CB retained 93% of the initial activity when stored at 4 °C for 4 weeks. In addition, the activity of the immobilized lipase on Ala-CB retained 77% of its high initial activity after 10 times of reuse. The kinetic data (k_cat/K_m) supports that the immobilized lipase on Ala-CB can give better substrate specificity than the unmodified chitosan beads.     

Immobilization of lipase in organic solvent in the presence of fatty acid additives

In this study porcine pancreatic lipase (PPL) was covalently immobilized on cross-linked polyvinyl alcohol (PVA) in organic media in the presence of fatty acid additives in order to improve its immobilized activity. The effects of fatty acid additions to the immobilization media were investigated choosing tributyrin hydrolysis in water and ester synthesis by immobilized PPL in n-hexane. Various fatty acids which are also the substrates of lipases in esterification reactions were used as active site protecting agents during the immobilization process in an organic solvent. The obtained results showed that covalent immobilization carried out in the presence of fatty acids as protective ligands improved the hydrolytic and esterification activity of immobilized enzyme. A remarkable increase in activity of the immobilized PPL was obtained when octanoic acid was used as an additive and the hydrolytic activity was increased from 5.2 to 19.2 μmol min⁻¹ mg⁻¹ as compared to the non-additive immobilization method. With the increase of hydrolytic activity of immobilized lipase in the presence of octanoic acid, in an analogous manner, the rate of esterification for the synthesis of butyl octanoate was also increased from 7.3 to 26.3 μmol min⁻¹ g⁻¹ immobilized protein using controlled thermodynamic water activities with saturated salt solutions. In addition, the immobilized PPL activity was maintained at levels representing 63% of its original activity value after 5 repeated uses. The proposed method could be adopted for a wide variety of other enzymes which have highly soluble substrates in organic solvent such as other lipases and esterases.     

Enzymatic interesterification of triglyceride with surfactant-coated lipase in organic media

Several surfactant-coated enzymes have been prepared by coating lipases of various origins with a nonionic surfactant, glutamic acid dioleylester ribitol (2C(18)Delta(9)GE). Enzymatic interesterification of tripalmitin with oleic acid using the surfactant-coated lipase was carried out in organic media. The surfactant-coated lipases could effectively catalyze the interesterification of glycerides better than did the powder lipases. A suitable organic solvent was an aliphatic hydrocarbon such as isooctane. The enzymatic activity for the interesterification strongly depended on the origin of the lipase. The surfactant-coated lipase prepared by Mucor javanicus showed the highest enzymatic activity for the interesterification of glycerides, although its powder lipase did not show enzymatic activity. Selective interesterification of glycerides could be performed by adjusting the concentration ratio of oleic acid to tripalmitin in isooctane. Di-substituted glyceride could be selectively produced when the concentration ratio of carboxylic acid to glycerides was 7. (c) 1995 John Wiley & Sons, Inc.     

Synthesis and characterization of cyclodextrin-based polymers as a support for immobilization of Candida rugosa lipase

The objective of this study was to prepare cross-linked β-cyclodextrin polymers for immobilization of Candida rugosa lipase. The structures of synthesized macrocyclic compounds were characterized by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. Properties of the immobilized systems were assessed and their performance on hydrolytic reaction were evaluated and compared with the free enzyme. The influence of activation agents (glutaraldehyde (GA) and hexamethylene diisocyanate (HMDI)) and thermal and pH stabilities of the biocatalyst was evaluated. After the optimization of immobilization process, the physical and chemical characterization of immobilized lipase was performed. Obtained data showed that the immobilized enzyme seemed better and offered some advantages in comparison with free enzyme. It can be observed that the free lipase loses its initial activity within around 80 min at 60 °C, while the immobilized lipases retain their initial activities of about 56% by HMDI and 82% by GA after 120 min of heat treatment at 60 °C.Results showed that the specific activity of the immobilized lipase with glutaraldehyde was 62.75 U/mg protein, which is 28.13 times higher than that of the immobilized lipase with HMDI.     

Interesterification of phosphatidylcholine with lipases in organic media

Summary Lipases were investigated with respect to their ability to catalyse the incorporation of fatty acids into phosphatidylcholine (PC) by interesterification reactions. The enzymes were dried onto solid support materials and the conversions were carried out in water-saturated toluene. Three lipases (two fungal and one plant enzyme) had the desired activity; immobilized lipase from Mucor miehei (Lipozyme) was the most active enzyme. The Lipozyme-catalysed interesterification was selective for the sn-1 position of PC and during 48 h of reaction around 50% of the fatty acids in this position were replaced with heptadecanoic acid, a fatty acid which was practically absent in the original phospholipid. Due to adsorption on the support material and the competing hydrolysis reaction the total amount of PC in the reaction solution decreased to about 40% of the original amount. Higher interesterification rates were obtained with free fatty acids as acyl donors than with fatty acid esters. Offprint requests to: I. Svensson     

Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil

Enzymatic transesterification of soybean oil with methanol and ethanol was studied. Of the nine lipases that were tested in the initial screening, lipase PS from Pseudomonas cepacia resulted in the highest yield of alkyl esters. Lipase from Pseudomonas cepacia was further investigated in immobilized form within a chemically inert, hydrophobic sol-gel support. The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and iso-butyltrimethoxysilane. Using the immobilized lipase PS, the effects of water and alcohol concentration, enzyme loading, enzyme thermal stability, and temperature in the transesterification reaction were investigated. The optimal conditions for processing 10 g of soybean oil were: 35 degrees C, 1:7.5 oil/methanol molar ratio, 0.5 g water and 475 mg lipase for the reactions with methanol, and 35 degrees C, 1:15.2 oil/ethanol molar ratio, 0.3 g water, 475 mg lipase for the reactions with ethanol. Subject to the optimal conditions, methyl and ethyl esters formation of 67 and 65 mol% in 1h of reaction were obtained for the immobilized enzyme reactions. Upon the reaction with the immobilized lipase, the triglycerides reached negligible levels after the first 30 min of the reaction and the immobilized lipase was consistently more active than the free enzyme. The immobilized lipase also proved to be stable and lost little activity when was subjected to repeated uses.     

Immobilization of Mucor javanicus lipase on effectively functionalized silica nanoparticles

Mucor javanicus lipase was effectively immobilized on silica nanoparticles which were prepared by Stöber method. Glycidyl methacrylate (GMA), which bears a reactive epoxide group, was incorporated onto the surface of the nanoparticles and the epoxide groups were directly used for multipoint coupling of the enzyme. We also introduced amine residues by coupling ethylene diamine (EDA) to the epoxide group of GMA. M. javanicus lipase was covalently immobilized onto the amine-activated silica nanoparticles by using glutaraldehyde (GA) or 1,4 phenylene diisothiocyanate (NCS) as a coupling agent. The lipase loading capacities of the EDA-GA and EDA-NCS nanoparticles (81.3 and 60.9 mg g⁻¹, respectively) were much higher than that of the unmodified GMA nanoparticles (18.9 mg g⁻¹). The relative hydrolytic activities in an aqueous medium of the lipases immobilized on EDA-GA and EDA-NCS attached silica nanoparticles (115% and 107%, respectively) were significantly high and almost in the same range with the free enzyme. This may be due to the improvement of the enzyme–substrate interaction by avoiding the potential aggregation of free lipase molecules. The immobilized lipases were also more resistant to temperature inactivation than the free form. This work demonstrates that the size-controlled silica nanoparticles can be efficiently employed as host materials for enzyme immobilization leading to high activity and stability of the immobilized enzymes.     

Meetings & Symposia

Abstract

Porcine pancreatic lipase (PPL) and Candida cylindracea lipase (CCL) were immobilized on Celite and Amberlite IRA 938 by deposition from the aqueous solution by the addition of hexane. The influence of the immobilization on the activities of the immobilized lipase derivatives has been studied. The immobilized lipases were used in synthesis of pentyl isovalerates. Various reaction parameters affecting the synthesis of pentyl isovalerates were investigated. The reaction rates were compared with the rates of esterification with free lipases. The immobilized lipases were found to be very effective in the esterification reaction. The lipases immobilized on Celite 545 exhibited better operational stabilities than that of immobilized on Amberlite IRA‐938.     

Culture conditions for intracellular lipase production by Rhizopus chinensis and its immobilization within biomass support particles

Acetone-dried cells of Rhizopus chinensis (with a 1,3-positional specificity lipase) were investigated for the interestierification reaction of olive oil and methyl stearate. First, the culture conditions for intracellular lipase production were examined, and then the activities of dried cells obtained from immobilization in Biomass Support Particles (BSPs) were compared with those of freely suspended cells.It was clear from cultivation of freely suspended cells that intracellular lipase activity for the interesterification reaction was enhanced sifnificantly by the presence of oleic acid, oil, and tea oil, but that the presence of glucose reduced the activity.The specific activity of dried cells within BSPs increased 7-fold compared with that obrained from freely suspended cells.The process presented here, using immobilization within BSPs, can provide cells directly as a catalyst with high activity, where cells become immobilized simply during batch operation, and no special preparation of cells is necessary. Therefore, the reaction system using dried cells immobilized within BSPs is a promising interesterifcation process for industrial applications.