Immobilization of lipase on a new inorganic ceramics support, toyonite, and the reactivity and enantioselectivity of the immobilized lipase

A novel porous ceramics support, named "Toyonite," for the immobilization of enzymes was prepared from the minerals of kaolinite under acidic conditions. Modification of the porous surface of Toyonite with two different organic coating agents gave Toyonite 200-M (TN-M), and Toyonite 200-A (TN-A), possessing methacryloyloxy and amino groups on the respective surfaces. Compared with other solid supports, TN-M and TN-A supports exhibited high selectivity for lipase PS (Pseudomonas cepacia, Amano) and glucoamylase (Gluczyme AF 6, Amano) proteins, respectively. The activities of both the transesterification of rac-1 with TN-M PS lipase and the hydrolysis of starch with TN-A glucoamylase were greater than those of similar reactions with these two enzymes immobilized on other solid supports. Further, TN-M PS lipase showed higher reactivity toward synthetic substrates, including aromatic and aliphatic secondary alcohols, than the free enzyme powder.

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“…8 The IR and 1 H-NMR spectral data and the optical rotation values of all chiral products are available.…”

Section: Lipase Ps-catalyzed Transesterificationmentioning

confidence: 99%

Enzyme immobilization utilizing a porous ceramics support for chiral synthesis

2002

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“…However, in many cases, because the emulsion was destroyed during the separation of the product, enzymes has to be handled again to make a new emulsion, which increases the possibility to denature enzymes. It is well known that, compared to free enzyme in emulsion, the immobilization of enzyme on a membrane is an advisable method to keep enzyme activity stable and simplify the downstream processing (Kamori et al, 2000;Arica et al, 2001;Giorno et al, 1995Giorno et al, , 1997Li et al, 2002). For example, Arica et al (2001) showed that the free lipase lost all its initial activity within 21 days, while immobilized lipase lost 24% of its activity during the same period.…”

Section: Introductionmentioning

confidence: 99%

Use of stable emulsion to improve stability, activity, and enantioselectivity of lipase immobilized in a membrane reactor

Giorno

Drioli

2003

Biotech & Bioengineering

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The enantiocatalytic performance of immobilized lipase in an emulsion membrane reactor using stable emulsion prepared by membrane emulsification technology was studied. The production of optical pure (S)-naproxen from racemic naproxen methyl ester was used as a model reaction system. The O/W emulsion, containing the substrate in the organic phase, was fed to the enzyme membrane reactor from shell-to-lumen. The enzyme was immobilized in the sponge layer (shell side) of capillary polyamide membrane with 50 kDa cut-off. The aqueous phase was able to permeate through the membrane while the microemulsion was retained by the thin selective layer. Therefore, the substrate was kept in the enzyme-loaded membrane while the water-soluble product was continuously removed from the reaction site. The results show that lipase maintained stable activity during the entire operation time (more than 250 h), showing an enantiomeric excess (96 +/- 2%) comparable to the free enzyme (98 +/- 1%) and much higher compared to similar lipase-loaded membrane reactors used in two-separate phase systems (90%). The results demonstrate that immobilized enzymes can achieve high stability as well as high catalytic activity and enantioselectivity.

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“…However, the practical use of enzymes in organic solvents has been limited due to the lower catalytic activity and lower stability compared with the activity and stability of enzymes in an aqueous phase. Numerous immobilization techniques, including adsorption, entrapment, emulsion, and covalent attachment, have been proposed as alternatives to enhance activity and stability (10,20,22). However, many of these processes are expensive and have various limitations for actual application to industrial production.…”

Section: Discussionmentioning

confidence: 99%

Cell Surface Display of Lipase in Pseudomonas putida KT2442 Using OprF as an Anchoring Motif and Its Biocatalytic Applications

Lee¹,

Lee

Park

2005

Appl Environ Microbiol

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We developed a new cell surface display system in Pseudomonas putida KT2442 using OprF, an outer membrane protein of Pseudomonas aeruginosa, as an anchoring motif in a C-terminal deletion-fusion strategy. The Pseudomonas fluorescens SIK W1 lipase gene was fused to two different C-terminal truncated OprF genes, and the fusion genes were cloned into the broad-host-range plasmid pBBR1MCS2 to make pMO164PL and pMO188PL. Plasmid pMO188PL allowed better display of lipase and thus was chosen for further study. The display of lipase on the surface of P. putida KT2442 was confirmed by Western blot analysis, immunofluorescence microscopy, and measurement of whole-cell lipase activity. The whole-cell lipase activity of recombinant P. putida KT2442 harboring pMO188PL was more than fivefold higher than that of recombinant Escherichia coli displaying lipase in the same manner. Cell surface-displayed lipase exhibited the highest activity at 47°C and pH 9.0, and the whole-cell lipase activity was greater than 90% of the initial activity in organic solvents at 47°C for 1 week. In a biocatalytic application, enantioselective resolution of 1-phenyl ethanol was carried out in an organic solvent. (R)-Phenyl ethyl acetate was successfully produced with 41.9% conversion and an enantiomeric excess of more than 99% in a 36-h reaction. These results suggest that the OprF anchor can be used for efficient display of proteins in P. putida KT2442 and consequently for various biocatalytic applications.

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Immobilization of lipase on a new inorganic ceramics support, toyonite, and the reactivity and enantioselectivity of the immobilized lipase

Cited by 65 publications

References 13 publications

Enzyme immobilization utilizing a porous ceramics support for chiral synthesis

Enzyme immobilization utilizing a porous ceramics support for chiral synthesis

Use of stable emulsion to improve stability, activity, and enantioselectivity of lipase immobilized in a membrane reactor

Cell Surface Display of Lipase in Pseudomonas putida KT2442 Using OprF as an Anchoring Motif and Its Biocatalytic Applications

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