Enhanced activity and stability of immobilized lipases by treatment with polar solvents prior to lyophilization

Lipases from Candida rugosa, Mucor javanicus and Rhizopus oryzae were respectively adsorbed on Amberlite XAD-7 followed by incubation in 2-propanol and then lyophilization. The activities of the immobilized enzymes were 1.6–3.4 times higher than those of the immobilized enzymes without incubation in the organic solvent before lyophilization for esterification of lauric acid (0.1 M) and 1-propanol (0.1 M) in isooctane at 37 °C. The immobilized C. rugosa lipase (Sigma) without the incubation did not show any activity but displayed considerable activity (19.8 μmol h⁻¹ mg⁻¹) after the incubation before lyophilization. Besides 2-propanol, acetone, 1-propanol and ethyl acetate were also found to be good solvents for treating M. javanicus lipase immobilized on Amberlite XAD-7 and acetone was the best among them. When incubated in isooctane at 25 °C for 120 h, the immobilized M. javanicus lipase prepared by incubation in acetone for 1 h before lyophilization retained 70% of its initial activity while the immobilized enzyme without the solvent treatment kept only 50% of its initial activity.     

On the activity loss of hydrolases in organic solvents: I. Rapid loss of activity of a variety of enzymes and formulations in a range of organic solvents

Dehydrated enzyme powders have been used extensively as suspensions in organic solvents to catalyze synthetic reactions. Prolonged enzyme activity is necessary to make such applications commercially successful. However, it has recently become evident that the stability and thus activity of many enzymes is compromised in organic solvents. Herein we explore the stability of various hydrolases (i.e., lipases from Mucor meihei and Candida rugosa, -chymotrypsin, subtilisin Carlsberg, and pig-liver esterase) and various formulations (lyophilized powder, cross-linked enzyme crystals, poly(ethylene glycol)-enzyme conjugates) in different organic solvents. The results show a roughly exponential activity decrease for all enzymes and formulations studied after exposure to organic solvents. Inactivation was observed independent of the enzyme, formulation details, and the solvent. In addition, no relationship was found between the magnitude of inactivation and the value of initial activity. Thus, quite active formulations lost their activity as quickly as less active formulations. The estimated half-times (t_1/2) for all enzymes and preparations ranged from 1.8 h for subtilisin C. co-lyophilized with methyl-β-cyclodextrin to 61.6 h for the most stable poly(ethylene glycol)--chymotrypsin preparation. The data here presented indicates that the inactivation is likely not related to changes in enzyme structure and dynamics.     

Cross-linked crystals of chloroperoxidase

Chloroperoxidase from Caldariomyces fumago was crystallized. The crystals were modified with several cross-linkers, but only glurataldehyde was able to produce catalytically active and insoluble crystals. Unlike other immobilized chloroperoxidase preparations, these catalytic crystals are more thermostable than the unmodified soluble enzyme. The enhanced stability is probably due to the structure conservation in the crystalline matrix. In addition, non-cross-linked chloroperoxidase crystals retained more activity than the soluble enzyme after incubation in an organic solvent with low water content. Although the cross-linked crystals were catalytically active, they showed lower specific activity than the soluble enzyme. This low activity may be due to non-specific reactions between the cross-linker and essential residues for catalysis. Alternative cross-linking strategies are discussed.     

Esterification of lauric acid with lauryl alcohol using cross-linked enzyme crystals: Solvent effect and kinetic study

In this paper, we report a comprehensive kinetic study on esterification of lauric acid with lauryl alcohol catalysed by commercial porcine pancreatic lipase (PPL) in the form of cross-linked enzyme crystals (CLEC) using glutaraldehyde as the cross linker. The stability of the CLEC was better than the immobilized enzyme for practical applications. Comparative studies using six different solvents having hydrophobicity (log p) values ranging from 0.70 to 3.50 revealed that the esterification reaction was favoured in hydrophobic solvents. The kinetics of the esterification reaction conformed with the so-called Ping-Pong-Bi-Bi mechanism with alcohol inhibition.     

Enantioselective affinity chromatography of a chiral drug by crystalline and carrier-bound antibody fab fragment

An antibody Fab fragment, ENA5His, capable of enantioselective affinity chromatographic separation of a chiral drug, finrozole, was stabilized against organic solvents by chemical cross-linking. High concentration of methanol is needed to release the bound drug from the antibody fragment. However, in native form the antibody fragment is unstable at these conditions. We used cross-linked protein crystal technology to stabilize the antibody fragment molecule. Glutaraldehyde cross-linked ENA5His crystals (CLAC) packed in a column separated pure enantiomers from the racemic mixture of the drug. CLAC was totally stable at the elution conditions, enabling reuse of the immunoaffinity column packed with CLAC. However, the specific drug enantiomer binding capacity of CLAC was only 50% of the corresponding capacity of immobilized ENA5His. We were also able to cross-link immobilized ENA5His by glutaraldehyde. This method produced a protein matrix with high activity and stability in the elution conditions.     

Esterification of lauric acid with lauryl alcohol using cross-linked enzyme crystals: Solvent effect and kinetic study

In this paper, we report a comprehensive kinetic study on esterification of lauric acid with lauryl alcohol catalysed by commercial porcine pancreatic lipase (PPL) in the form of cross-linked enzyme crystals (CLEC) using glutaraldehyde as the cross linker. The stability of the CLEC was better than the immobilized enzyme for practical applications. Comparative studies using six different solvents having hydrophobicity (log p) values ranging from 0.70 to 3.50 revealed that the esterification reaction was favoured in hydrophobic solvents. The kinetics of the esterification reaction conformed with the so-called Ping-Pong–Bi-Bi mechanism with alcohol inhibition.     

Influence of the immobilization chemistry on the properties of immobilized β-galactosidases

Neutral β-galactosidases (from E. coli and K. lactis) were bound to glutaraldehyde-agarose (Glut-agarose) through amino groups, and to thiolsulfinate-agarose (TSI-agarose) through thiol groups. In general, TSI-gels exhibited higher yields after immobilization (60–85%) than Glut-gels (36–40%). The kinetic parameters of the enzymes bound to TSI-gels (particularly those with lower concentration of active groups) were less affected than those of the Glut-gels. This might indicate that the binding to TSI-agarose is more conservative of the protein conformation. However, the Glut-derivatives exhibited in general better thermal and solvent stabilities than TSI-derivatives. The stability of the derivatives was studied in the presence of ethanol, dioxane and acetone (18% v/v). The stabilization of the immobilized enzymes, for some of the solvents assayed, was evidenced by the existence of final very stable enzyme states with high residual activities, thus allowing the utilization of the derivatives in the presence of organic cosolvents.     

Enhanced reactivity of Rhizopus oryzae lipase displayed on yeast cell surfaces in organic solvents: potential as a whole-cell biocatalyst in organic solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with alpha-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 x 10(4)-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 x 10(4)-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H2O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

枯草芽孢杆菌FM208849产果胶酶的固定化及酶学性质研究

为了提高游离果胶酶的稳定性,对罗布麻脱胶具有特异性的枯草芽孢杆菌(FM208849)进行产果胶酶发酵时,采用交联酶聚集体(CLEAs)技术制备固定化果胶酶,并对交联果胶酶聚集体的制备条件、酶学性质进行研究。结果表明,游离果胶酶经80%饱和硫酸铵沉淀后,在30℃,经4%的戊二醛溶液交联135 min,所形成的交联果胶酶聚集体的活回收率为61.5%,其最适反应温度45℃和最适pH10,在对交联果胶酶聚集体的热稳定性和有机溶剂稳定性分析中,均显示了比游离酶更高的稳定性。     

Amino silicones finished fabrics for lipase immobilization: Fabrics finishing and catalytic performance of immobilized lipase

Finishing of silk fabric was achieved by using amino-functional polydimethylsiloxane (PDMS) and lipase from Candida sp. 99-125 was immobilized on the treated silk fabrics. Hydrophobic fabrics were obtained by dipping the native fabric in 0.125–0.25% (w/v) PDMS solution and dried at 70 °C. The direct adsorption on PDMS-treated fabric was verified to be a better strategy for lipase immobilization than that by covalent binding. Compared to unfinished fabrics, the hydrolytic activity of immobilized enzyme on the finished fabric was improved by 1.6 times. Moreover, the activity of immobilized enzymes on hydrophobic fabrics was significantly improved in different concentrations of strong polar solvents such as methanol and ethanol, and in common organic solvents with different octanol–water partition coefficients (Log P). Enzymatic activity and stability in 15% water content system (added water accounted for the total reaction mixtures, v/v) showed more than 30% improvement in each batch. The amino–silicone finished fabric surface was investigated by scanning electron microscopy and X-ray photoelectron spectroscopy. The hydrophobic fabric immobilized enzyme could be recycled for more than 80 times with no significant decrease in esterification activity. PDMS-treated woven silk fabrics could be a potential support for lipase immobilization in catalytic esterification processes.     

Studies on crystallization and cross-linking of lipase for biocatalysis

The development of robust biocatalysts with increased stability and activity is a major challenge to industry. A major breakthrough in this field was the development of cross-linked enzyme crystals with high specificity and stability. A method is described to produce micro crystals of CLEC lipase, which is thermostable and solvent stable. Lipase from Burkholderia cepacia was crystallized using ammonium sulfate and cross-linked with glutaraldehyde to produce catalytically active enzyme. The maximum yield of CLEC was obtained with 70% ammonium sulfate and cross-linked with 5% (v/v) glutaraldehyde. SEM studies showed small hexagonal-shaped crystals of 2–5 μm size. CLEC lipase had improved thermal and reuse stability. It is versatile, having good activity in both polar and nonpolar organic solvents. CLEC lipase was coated using β cyclodextrin for improving the storage and reuse stability. CLEC was successfully used for esterification of Ibuprofen and synthesis of ethyl butyrate.     

Influence of compressed fluids treatment on the activity of Yarrowia lipolytica lipase

This work investigated the influence of temperature, pressure, exposure times and depressurization rate on the activity of a non-commercial immobilized lipase from Yarrowia lipolytica (YLL) submitted to compressed carbon dioxide, propane and n-butane. A high-pressure cell was employed in the experiments, in the pressure range of 10–280 bar, varying the temperature from 35 to 75 °C, exposure times from 1 to 6 h, and adopting distinct decompression rates. Results showed that significant activity losses were obtained when the treatment was conducted in carbon dioxide, while negligible losses were observed in both propane and n-butane. For the treatment with carbon dioxide, within the range studied, the decompression rate affected positively enzyme activity, while the exposure time and temperature presented an opposite effect on the non-commercial immobilized lipase from Y. lipolytica (YLL). Additionally, the performance of two commercial immobilized lipases (Lipozyme IM and Novozym 435) and the immobilized YLL in the three solvents was compared. Immobilized YLL has shown to be more suitable than Lipozyme IM for enzyme-catalyzed reactions using compressed propane and n-butane as solvents, but with inferior performance compared to Novozym 435 treated in these solvents.     

Immobilization of enzymes on magnetic particles

Trypsin (EC 3.4.4.4) was immobilized in low yield on aminoalkylsilylated magnetite (Fe₃O₄). Better results were obtained when trypsin was immobilized by crosslinking with glutaraldehyde on magnetite. The preparation contained 36 mg protein/g magnetite and the enzyme retained 46% and 11% of esterase and proteolytic activity. Immobilized trypsin was more heat stable than trypsin. Invertase (β-D -fructofuranoside fructohydrolase, EC 3.2.1.26) was cross-linked on magnetite with glutaraldehyde in low yield due to the inactivation of the enzyme. However in the presence of 1% sucrose, the total activity recovered was 79% of the initial activity and the preparation contained 4.4 mg/g of active invertase. Immobilized invertase was less active than invertase when acting on oligosaccharides of the raffinose family. The immobilized enzymes could be easily recovered, from solutions or suspensions, magnetically.     

Comparing the effect of immobilization methods on the activity of lipase biocatalysts in ester hydrolysis

The activity of various lipases was compared, in both free and immobilized forms, using the kinetics of the hydrolysis reaction of p-nitrophenyl butyrate, which was followed with in situ UV/Vis diode array spectrophotometry. Several enzymes were used to catalyze the reaction, namely Candida antarctica lipase B and Fusarium solani pisi cutinase wildtype and three single-mutation variants. The enzymes were tested in three different forms: free, immobilized as cross-linked aggregates and supported on zeolite NaY. A simple kinetic model was used to allow a quantitative comparison of the behavior of the different catalysts. It was concluded that although immobilization reduces the activity of the enzyme, the zeolite offers a much higher specific activity when compared to the cross-linked aggregates, thus supplying a heterogeneous catalyst with promising catalytic properties.     

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with α-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 × 10⁴-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 × 10⁴-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H₂O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

Pectolytic enzymes co-immobilization on γ-alumina spheres via organophosphate compounds

Endopectinlyase (EC 4.2.2.10), endopolygalacturonase (EC 3.2.1.15) and pectinesterase (EC 3.1.1.11) present in a commercial mixture were co-immobilized on γ-alumina spheres activated with organophosphate compounds. Staining of the alumina-enzyme complexes with a dye specific for protein showed that only the carrier external surface was available for protein binding. When confined in a packed bed reactor, the activity of the co-immobilized enzymes brought about a viscosity decrease of 70–90% in pectin and polygalacturonic acid solutions, respectively. Sixty per cent of the initial activity was retained from the immobilized enzymes after the sixth utilization cycle on both the substrates used. The immobilized enzymes were also active against fresh apple juice producing a 90% reduction in viscosity in the first five cycles of utilization.     

Polymethylglutamate as a new matrix for covalently immobilized enzymes: Preparation and properties of urease and uricase

Polymethylglutamate (PMG), a synthetic polypeptide, was used as a new carrier to immobilize urease (EC 3.5.1.5) and uricase (EC 1.7.3.3) by the azide method. The enzymes could be immobilized onto PMG in various forms, such as film, fiber, coating on various beads, and a silicon tube. The retained activities of the immobilized enzymes were excellent (more than 95%), therefore it was possible to immobilized almost all activities of the enzymes added in the coupling mixtures. Heat stabilities of the resulting immobilized enzymes were markedly improved, while the optimal pH and K_m values remained almost unchanged. The urease immobilized on the PMG-coated glass beads packed in a column, was found to retain its activity more than 80% of the initial value, even after the occasional use for a year. In view of the improved retained activities and stabilities of the immobilized enzymes, PMG may therefore be a very versatile matrix for the immobilized enzymes.     

Optimization of Pseudomonas cepacia lipase preparations for catalysis in organic solvents

The activity of different lipase (from Pseudomonas cepacia) forms, such as crude powder (crude PC), purified and lyophilized with PEG (PEG + PC), covalently linked to PEG (PEG-PC), cross-linked enzyme crystals (CLEC-PC), and immobilized in Sol-Gel-AK (Sol-Gel-AK-PC) was determined, at various water activities (aw), in carbon tetrachloride, benzene and 1,4-dioxane. The reaction of vinyl butyrate with 1-octanol was employed as a model and both transesterification (formation of 1-octyl butyrate) and hydrolysis (formation of butyric acid from vinyl butyrate) rates were determined. Both rates depended on the lipase form, solvent employed, and aw value. Hydrolysis rates always increased as a function of aw, while the optimum of aw for transesterification depended on the enzyme form and nature of the solvent. At proper aw, some lipase forms such as PEG + PC, PEG-PC, and Sol-Gel-AK-PC had a total activity in organic solvents (transesterification plus hydrolysis) which was close to (39 and 48%) or even higher than (130%) that displayed by the same amount of lipase protein in the hydrolysis of tributyrin-one of the substrates most commonly used as standard for the assay of lipase activity-in aqueous buffer. Instead, CLEC-PC and crude PC were much less active in organic solvents (2 and 12%) than in buffer. The results suggest that enzyme dispersion and/or proper enzyme conformation (favored by interaction with PEG or the hydrophobic Sol-Gel-AK matrix) are essential for the expression of high lipase activity in organic media.     

High stabilization of immobilized Rhizomucor miehei lipase by additional coating with hydrophilic crosslinked polymers: Poly-allylamine/Aldehyde–dextran

Immobilized enzymes have a very large surface region which is not in contact with the support surface and, thus, have potential as a target for novel stabilization strategies. In this paper, coating the surfaces of such enzymes with a highly hydrophilic and compact cross-linked poly-aminated polymer as a strategy to increase the thermal stability of the immobilized enzymes is proposed. In particular, Rhizomucor miehei lipase (RML) was immobilized by interfacial adsorption onto octyl-agarose and further coated with poly-allylamine (PAA), a polymer that is very rich in primary amino groups. Cross-linking of the PAA layer to coat the immobilized enzyme was carried out, in situ, by reaction with freshly oxidized dextran (aldehyde–dextran). The PAA layer only exerted moderate stabilizing effects (around 4-fold), but further cross-linking with aldehyde–dextran highly increased the stabilizing effects; the new derivative was 440-fold more stable than uncoated derivative at 55 °C and pH 7 and exhibited 6-fold more catalytic activity compared to the soluble enzyme used for immobilization. We hypothesize that the hydrophilicity of PAA reduces the exposure of internal hydrophobic pockets to the enzyme surface at high temperatures. Besides, the compactness of the polymer may reduce distortion of the enzyme surface during inactivation.     

Highly efficient solubilization of natural lignocellulosic materials by a commercial cellulase immobilized on various solid supports

Summary A commercial preparation of cellulase was immobilized on CNBr-sepharose, ConA-sepharose, and CNBr-glass beads. When filter paper was used as the substrate, the specific activity of the enzyme immobilized on ConA-sepharose was more than twice that of the soluble enzyme, while the activity of the enzymes immobilized on the other two substrates was either very slightly (CNBr-sepharose) or slightly (CNBr-glass beads) reduced. The immobilized enzymes showed alterations both in the Km and V max values: these were generally either slightly increased (Km) or reduced (V max). In addition, the immobilized enzymes were more resistant to inhibition both by glucose and cellobiose, they were all more stable than the soluble enzyme and solubilized three different natural lignocellulosic materials (alfa-alfa, wheat straw, and pine needles) to a much greater or significantly greater extext than the soluble enzyme: the ConA-sepharose cellulase was the most efficient. The possibility of reusing the immobilized enzyme was also tested. It was found that the ConA-sepharose cellulase could be reused five times with a final loss of activity that ranged between 30% and 50%.