Journal Article

FZJ-2015-04682

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Purification and simultaneous immobilization of Arabidopsis thaliana hydroxynitrile Lyase using a Family 2 carbohydrate-binding module

Kopka, B.FZJ* ; Diener, M.FZJ* ; Wirtz, A.FZJ* ; Pohl, M.FZJ* ; Jaeger, K.-E.FZJ* ; Krauss, U. (Corresponding Author)FZJ*

2015
Wiley-VCH Weinheim

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Please use a persistent id in citations: doi:10.1002/biot.201400786

Abstract: Tedious, time- and labor-intensive protein purification and immobilization procedures still represent a major bottleneck limiting the widespread application of enzymes in synthetic chemistry and industry. We here exemplify a simple strategy for the direct site-specific immobilization of proteins from crude cell extracts by fusion of a family 2 carbohydrate-binding module (CBM) derived from the exoglucanase/xylanase Cex from Cellulomonas fimi to a target enzyme. By employing a tripartite fusion protein consisting of the CBM, a flavin-based fluorescent protein (FbFP), and the Arabidopsis thaliana hydroxynitrile lyase (AtHNL), binding to cellulosic carrier materials can easily be monitored via FbFP fluorescence. Adsorption properties (kinetics and quantities) were studied for commercially available Avicel PH-101 and regenerated amorphous cellulose (RAC) derived from Avicel. The resulting immobilizates showed similar activities as the wild-type enzyme but displayed increased stability in the weakly acidic pH range. Finally, Avicel, RAC and cellulose acetate (CA) preparations were used for the synthesis of (R)-mandelonitrile in micro-aqueous methyl tert-butyl ether (MTBE) demonstrating the applicability and stability of the immobilizates for biotransformations in both aqueous and organic reaction systems.

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Contributing Institute(s):

1. Biotechnologie (IBG-1)
2. Institut für Molekulare Enzymtechnologie (HHUD) (IMET)

Research Program(s):

1. 581 - Biotechnology (POF3-581) (POF3-581)

Appears in the scientific report 2015

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Database coverage:
; BIOSIS Previews ; IF < 5 ; JCR ; NCBI Molecular Biology Database ; SCOPUS ; Science Citation Index Expanded ; Thomson Reuters Master Journal List ; Web of Science Core Collection

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