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Title 

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

Authors 

Y H HongDong-Woo LeeY R PyunS H Lee

Publisher 

American Chemical Society

Issue Date 

2011

Citation 

Journal of Agricultural and Food Chemistry, vol. 59, no. 24, pp. 12939-12947

Keywords 

activitychimeraL-arabinose isomerasemetalthermostability

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.

ISSN 

0021-8561

Link 

http://dx.doi.org/10.1021/jf203897a

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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