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Issue 2, 2011
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Oriented irreversible immobilization of a glycosylated Candida antarctica B lipase on heterofunctional organoborane-aldehyde support

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Abstract

The immobilization of Candida antarctica (fraction B) lipase expressed in Pichia pastoris, a selective glycosylated protein at Asn 74, on a new heterofunctional support consisted of phenylboronic acid and aldehyde groups (Borald) has been performed. This method occurs via a two step mechanism: first orientation by organoborane interaction at neutral pH and a consecutive multi-point covalent attachment by aldehyde reaction at alkaline pH. The enzyme was specifically immobilized on this support in 70% yield at pH 7, oriented by the reaction of the hydroxyl groups on the sugar moiety with boronic acid on the support, whereas commercial CAL-B from Novozymes (non-glycosylated) was hardly immobilized at this pH (<10%). The consecutive incubation at pH 10 permitted the reaction of amine groups of the protein with aldehyde groups on the support. After a reductive amination, an irreversible immobilization methodology on organoborane-aldehyde support was possible with >99% final immobilization yield. The Borald-CAL-B preparation was a very stable biocatalyst in the presence of high amount of solvent or high temperature (e.g. more than 10 fold in the presence of 60% (v/v) acetonitrile). An improvement of the specific activity up to 5 fold for example in the hydrolysis of methyl phenyl acetate was obtained compared with a one-point covalent preparation. An ee of 89% towards R isomer was achieved with this new immobilized biocatalyst in the enantioselective hydrolysis of methyl mandelate.

Graphical abstract: Oriented irreversible immobilization of a glycosylated Candida antarctica B lipase on heterofunctional organoborane-aldehyde support

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Article information


Submitted
15 Dec 2010
Accepted
19 Jan 2011
First published
11 Feb 2011

Catal. Sci. Technol., 2011,1, 260-266
Article type
Paper

Oriented irreversible immobilization of a glycosylated Candida antarctica B lipase on heterofunctional organoborane-aldehyde support

M. L. E. Gutarra, C. Mateo, D. M. G. Freire, F. A. G. Torres, A. M. Castro, J. M. Guisan and J. M. Palomo, Catal. Sci. Technol., 2011, 1, 260
DOI: 10.1039/C0CY00087F

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