• Analytical Science and Technology
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• v.29 no.3
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• pp.105-113
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• 2016

The present study investigated the immobilization of lipases on silica nanoparticles and silica-coated magnetite nanoparticles as supports with a functional group to enhance the stability of lipase. The influence of functional groups, such as the epoxy group and the amine group, on the activity and stability of immobilized lipase was also studied. The epoxy group and the amino group were introduced onto the surface of nanoparticles by glycidyl methacrylate and aminopropyl triethoxysilane, respectively. Immobilized Candida rugosa lipase on silica nanoparticles and silica-coated magnetite nanoparticles with a functional group showed slightly lower initial enzyme activities than free enzyme; however, the immobilized Candida rugosa lipase retained over 92 % of the initial activity, even after 3 times reuse. Lipase was also immobilized on the silica-coated magnetite nanoparticles by cross-linked enzyme aggregate (CLEA) using glutaraldehyde and covalent binding, respectively, were also studied. Immobilized Candida rugosa lipase on silica nanoparticles and silica-coated magnetite nanoparticles by CLEA and covalent binding showed higher enzyme activities than free enzyme, while immobilized Candida rugosa lipase retained over 73 % of the initial activity after 5 times reuse.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.6
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• pp.367-370
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• 2002

Candida cylindracea lipase was immobilized by adsorption on acid washed glass beads. It was observed that protein loading of the support depends on the size of the particle, with smaller particle containing higher amount of protein per unit weight. Initial reaction rate linearly varied up to enzyme concentration of 17.25 U/mL. Amount of free fatty acids produced was linearly proportional up to the enzyme loading of 1650 $\mu$g/g of bead. Achievement of chemical equilibrium took longer time in the case of less protein loading. Degree of hydrolysis was found to decrease in second and third consecutive batch operations on repeated use of immobilized lipase.

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.836-839
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• 2000

Beef tallow, which is an industrial lipid substrate, was hydrolyzed by lipase immobilized on a high-density polyethylene (HDPE) powder. Ethanol pre-washing process affected the immobilization efficiency. Half-life of storage of the HDPE at $4^{\circ}C$ was 150 days. And after 10 times of repeated use, more than 50% of initial activity remained. An apparent Michaelis constant ($K_m$) and maximum velocity ($V_{max}$) were 2.7M, and 1.4 mmol/min/l for immobilized lipase, and 0.5 M, and 1.9 mmol/min/l for soluble lipase, respectively.

• Journal of Microbiology and Biotechnology
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• v.24 no.10
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• pp.1382-1388
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• 2014

Proteus vulgaris K80 lipase was expressed in Escherichia coli BL21 (DE3) cells and immobilized on amine-terminated magnetic microparticles (Mag-MPs). The immobilization yield and activity retention were 84.15% and 7.87%, respectively. A homology model of lipase K80 was constructed using P. mirabilis lipase as the template. Many lysine residues were located on the protein surface, remote from active sites. The biochemical characteristics of immobilized lipase K80 were compared with the soluble free form of lipase K80. The optimum temperature of K80-Mag-MPs was $60^{\circ}C$, which was $20^{\circ}C$ higher than that of the soluble form. K80-Mag-MPs also tended to be more stable than the soluble form at elevated temperatures and a broad range of pH. K80-Mag-MP maintained its stable form at up to $40^{\circ}C$ and in a pH range of 5.0-10.0, whereas soluble K80 maintained its activity up to $35^{\circ}C$ and pH 6.0-10.0. K80-Mag-MPs had broader substrate specificity compared with that of soluble K80. K80-Mag-MPs showed about 80% residual relative activity after five recovery trials. These results indicate the potential benefit of K80-Mag-MPs as a biocatalyst in various industries.

• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1159-1165
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• 2011

Biodiesel is methyl and ethyl esters of long-chain fatty acids produced from vegetable oils or animal fats. Lipase enzymes have occasionally been used for the production of this biofuel. Recently, biodiesel production using immobilized lipase has received increased attention. Through enhanced stability and reusability, immobilized lipase can contribute to the reduction of the costs inherent to biodiesel production. In this study, methanol-tolerant lipase M37 from Photobacterium lipolyticum was immobilized using the cross-linked enzyme aggregate (CLEA) method. Lipase M37 has a high lysine content (9.7%) in its protein sequence. Most lysine residues are located evenly over the surface of the protein, except for the lid structure region, which makes the CLEA preparation yield quite high (~93%). CLEA M37 evidences an optimal temperature of $30^{\circ}C$, and an optimal pH of 9-10. It was stable up to $50^{\circ}C$ and in a pH range of 4.0-11.0. Both soluble M37 and CLEA M37 were stable in the presence of high concentrations of methanol, ethanol, 1-propanol, and n-butanol. That is, their activities were maintained at solvent concentrations above 10% (v/v). CLEA M37 could produce biodiesel from olive oil and alcohols such as methanol and ethanol. Additionally, CLEA M37 generated biodiesel via both 2-step methanol feeding procedures. Considering its physical stability and reusability, CLEA M37 may potentially be used as a catalyst in organic synthesis, including the biodiesel production reaction.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.2
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• pp.57-66
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• 2002

This review focuses on the use of immobilized lipase technology for the hydrolysis of oils. The importance of lipase catalyzed fat splitting process, the various immobilization procedures, kinetics, deactivation kinetics, New immobilized lipases for chiral resolution, reactor configurations, and process considerations are all reviewed and discussed.

• Journal of the Korean Society of Food Science and Nutrition
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• v.43 no.7
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• pp.1025-1035
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• 2014

This study assessed the effect of immobilized lipase on weak base styrene resin using polyethyleneimine (PEI) with cross-linking. Two procedures were used in this study. The first one, "mono-layer" lipase immobilization, involves washing PEI after adsorption. The second procedure, "multi-layer" lipase immobilization, has no washing before the cross-linking step. Treverlite XS-100200 (weak base styrene resin) was immersed with PEI solution (2.2 mg/mL). Lipase AH (from Burkholderia cepacia) was adsorbed onto the support coated with PEI before cross-linking with glutaraldehyde. Structured lipid was synthesized by immobilized lipase-catalyzed interesterification using canola oil, palmitic ethyl ester (PEE), and stearic ethyl ester (StEE). Total fatty acid contents of triacylglycerol (TAG) in structured lipids were analyzed to investigate activity, properties, and reusability of immobilized lipases. Activities of immobilized lipases on the multi-layer and mono-layer increased at a high concentration (8 mg/mL) of lipase solution used for immobilization. The results show that immobilized lipase with the mono-layer method at pH 8.0 on resin had the highest total saturated fatty acid content (26.17 area%). Activity of immobilized lipase with the multi-layer method at pH 7.5 on support was lower than that of the mono-layer, but total saturated fatty acid content was 16.79 area% higher than that of lipase AH (15.01 area%).

• Korean Journal of Chemical Engineering
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• v.35 no.11
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• pp.2220-2231
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• 2018

Lipase from Candida rugosa was immobilized on $MgO{\cdot}SiO_2$ hybrid grafted with amine, thiol, cyano, phenyl, epoxy and carbonyl groups. The products were analyzed using Fourier transform infrared spectroscopy, nuclear magnetic resonance, low-temperature $N_2$ sorption and elemental analysis. Additionally, the degree of coverage of the oxide material surface with different functional groups and the number of surface functional groups were estimated. The Bradford method was used to determine the quantity of immobilized enzyme. The largest quantity of enzyme (25-28 mg/g) was immobilized on the hybrid functionalized with amine and carbonyl groups. On the basis of hydrolysis reaction of p-nitrophenyl palmitate to p-nitrophenol, it was determined how the catalytic activity of the obtained biocatalysts is affected by pH, temperature, storage time, and repeated reaction cycles. The best results for catalytic activity were obtained for the lipase immobilized on $MgO{\cdot}SiO_2$ hybrids with amine and carbonyl groups. The biocatalytic system demonstrated activity above 40% in the pH range 4-10 and in the temperature range $30-70^{\circ}C$. Lipase immobilized on the $MgO{\cdot}SiO_2$ systems with amine and epoxy groups retains, respectively, around 80% and 60% of its initial activity after 30 days of storage, and approximately 60-70% after 10 reaction cycles.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.735-741
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• 2008

In our previous study, a surfactant-coated Candida rugosa lipase immobilized in microemulsion-based organogels was exploited for the synthesis of ethyl isovalerate. In the present study, we are focusing on the effective reuse of lipase immobilized in microemulsion-based organogels (MBGs) in terms of retainment of the catalytic activity. As water is one of the co-products in esterification reactions, the removal of water becomes a priority to allow the reaction to work in the forward direction and to prevent back hydrolysis. Taking this fact into consideration, the lipase-containing microemulsion-based organogels were given pretreatment and/or several intermittent treatments with dry reverse micellar solution of AOT in organic solvent during repeated cycles of ester synthesis. The pretreated MBGs with dry reverse micellar solution exhibited lower water content and higher initial rates of esterification in comparison with untreated freshly prepared MBGs. The esterification efficiency of untreated MBGs started decreasing after 5 cycles of reuse and was almost completely lost by the end of the $8^{th}$ cycle. In contrast, pretreated MBGs exhibited a gradual decrease in esterification efficiency after 5 cycles and retained about 80% of the initial activity at the end of the $8^{th}$ cycle. The intermittent treatment of MBGs after every 3 cycles resulted in enhanced reusability of immobilized lipase for up to 9 cycles without significant loss in esterification activity, after which it resulted in a slow decrease in activity with about 27% lower activity at the end of the $12^{th}$ cycle. Furthermore, the treatment conditions such as concentration of AOT in liquid dessicant and time of treatment were optimized with respect to our system. The granulated MBGs proved to be better in terms of initial esterification rates (1.2-fold) as compared with the pelleted MBGs.

• Microbiology and Biotechnology Letters
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• v.15 no.2
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• pp.122-128
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• 1987

Lipases from Candida rogosa and Rhizopus arrhizus were immobilized by entrapment with photo-crosslinkable resin prepolymer for the study of fat splitting and interesterification in isooctane-two phase system. Dioctylsulfosuccinate was selected as the most suitable surfactant during the immobilization. Lipase entrapped with hydrophobic photo-crosslinkable resin prepolymer(ENTP-3000) exhibited the highest activity, whereas lipase entrapped with hydrophilic gel(ENT-4000) was more stable in organic solvent. As the degree of hydrophobicity of the immobilization matrix was increased, Vm(app) of the lipase entrapped was increased, but Km(app) was approximately constant. While the optimum pH of the lipases entrapped on hydrophilic gel (ENT-4000) were around pH 7.0 for Candida lipase and Rhizopus lipase, the reaction rate of the lipases entrapped on hydrophobic gel were less dependent on pH variations for short reaction time. However, for longer reaction time, the lipnses from C. rugosa and R. arrhizus entrapped on hydrophobic gel yielded maximum rate at pH 6.0 and 6.5, respectively, Entrapment method endowed the lipase with thermal stability.