Creation of metal-independent hyperthermophilic L-arabinose isomerase by homologous recombination

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Title
Creation of metal-independent hyperthermophilic L-arabinose isomerase by homologous recombination
Author(s)
Y H Hong; Dong-Woo Lee; Y R Pyun; S H Lee
Bibliographic Citation
Journal of Agricultural and Food Chemistry, vol. 59, no. 24, pp. 12939-12947
Publication Year
2011
Abstract
Hyperthermophilic l-arabinose isomerases (AIs) are useful in the commercial production of d-tagatose as a low-calorie bulk sweetener. Their catalysis and thermostability are highly dependent on metals, which is a major drawback in food applications. To study the role of metal ions in the thermostability and catalysis of hyperthermophilic AI, four enzyme chimeras were generated by PCR-based hybridization to replace the variable N- and C-terminal regions of hyperthermophilic Thermotoga maritima AI (TMAI) and thermophilic Geobacillus stearothermophilus AI (GSAI) with those of the homologous mesophilic Bacillus halodurans AI (BHAI). Unlike Mn 2+-dependent TMAI, the GSAI- and TMAI-based hybrids with the 72 C-terminal residues of BHAI were not metal-dependent for catalytic activity. By contrast, the catalytic activities of the TMAI- and GSAI-based hybrids containing the N-terminus (residues 1-89) of BHAI were significantly enhanced by metals, but their thermostabilities were poor even in the presence of Mn 2+, indicating that the effects of metals on catalysis and thermostability involve different structural regions. Moreover, in contrast to the C-terminal truncate (Δ20 residues) of GSAI, the N-terminal truncate (Δ7 residues) exhibited no activity due to loss of its native structure. The data thus strongly suggest that the metal dependence of the catalysis and thermostability of hyperthermophilic AIs evolved separately to optimize their activity and thermostability at elevated temperatures. This may provide effective target regions for engineering, thereby meeting industrial demands for the production of d-tagatose.
Keyword
activitychimeraL-arabinose isomerasemetalthermostability
ISSN
0021-8561
Publisher
Amer Chem Soc
DOI
http://dx.doi.org/10.1021/jf203897a
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
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