• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.289-296
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• 2017

Lysine decarboxylase (CadA) converts ${\small{L}}-lysine$ into cadaverine (1,5-pentanediamine), which is an important platform chemical with many industrial applications. Although there have been many efforts to produce cadaverine through the soluble CadA enzyme or Escherichia coli whole cells overexpressing the CadA enzyme, there have been few reports concerning the immobilization of the CadA enzyme. Here, we have prepared a cross-linked enzyme aggregate (CLEA) of E. coli CadA and performed bioconversion using $CadA^{CLEA}$. $CadA^{free}$ and $CadA^{CLEA}$ were characterized for their enzymatic properties. The optimum temperatures of $CadA^{free}$ and $CadA^{CLEA}$ were $60^{\circ}C$ and $55^{\circ}C$, respectively. The thermostability of $CadA^{CLEA}$ was significantly higher than that of $CadA^{free}$. The optimum pH of both enzymes was 6.0. $CadA^{free}$ could not be recovered after use, whereas $CadA^{CLEA}$ was rapidly recovered and the residual activity was 53% after the $10^{th}$ recycle. These results demonstrate that $CadA^{CLEA}$ can be used as a potential catalyst for efficient production of cadaverine.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.325-331
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• 2010

Rhodococcus erythropolis amidase was expressed in Escherichia coli cells. The crude amidase in the cell-free extract was immobilized using the cross-linked enzyme aggregate (CLEA) method. The crude amidase was mixed with bovine serum albumin and then precipitated with ammonium sulfate. The resultant precipitant was subsequently cross-linked with glutaraldehyde. Scanning electron microscopy revealed that this co-CLEA had a ball-like shape with a diameter of approximately $1\;{\mu}m$. This co-CLEA evidenced hydrolytic activity toward a variety of amide substrates. The amidase co-CLEA evidenced an optimum temperature of $60^{\circ}C$ and an optimum pH of 8.0, results that were similar to those of the soluble amidase. The reaction stability of the co-CLEA was increased. That is, it was stable up to $50^{\circ}C$ and in a pH range of 5.0-12.0. Additionally, the co-CLEA could be recovered by centrifugation, and retained 96% activity after 3 repeated cycles. This amidase co-CLEA may prove useful as a substitute for soluble amidase as a biocatalyst in the pharmaceutical and chemical 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.

• Microbiology and Biotechnology Letters
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• v.46 no.3
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• pp.234-243
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• 2018

The Antarctic Ocean contains numerous microorganisms that produce novel biocatalysts that can have applications in various industries. We screened various psychrophilic bacterial strains isolated from the Ross Sea and found that a Croceibacter atlanticus strain (Stock No. 40-F12) showed high lipolytic activity on a tributyrin plate. We isolated the corresponding lipase gene (lipCA) by shotgun cloning and expressed the LipCA enzyme in Escherichia coli cells. Homology modeling of LipCA was carried out using the Spain Arreo lake metagenome alpha/beta hydrolase as a template. According to the model, LipCA has an ${\alpha}/{\beta}$ hydrolase fold, Gly-X-Ser-X-Glymotif, and lid sequence, indicating that LipCA is a typical lipase enzyme. Active LipCA enzyme was purified fromthe cell-free extract by ammonium sulfate precipitation and gel filtration chromatography. We determined its enzymatic properties including optimum temperature and pH, stability, substrate specificity, and organic solvent stability. LipCA was immobilized by the cross-linked enzyme aggregate (CLEA) method and its enzymatic properties were compared to those of free LipCA. After cross-linking, temperature, pH, and organic solvent stability increased considerably, whereas substrate specificities did not changed. The LipCA CLEA was recovered by centrifugation and showed approximately 40% activity after 4th recovery. This is the first report of the expression, characterization, and immobilization of a C. atlanticus lipase, and this lipase could have potential industrial application.

• 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.