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Biotechnology and Applied Biochemistry (2002) 35, (27–34) (Printed in Great Britain)

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Catalytic activities of intracellular dimeric neopullulanase on cyclodextrin, acarbose and maltose

Kyung-A Cheong*, Tae-Jip Kim*, Jong-Won Yoon*, Cheon-Seok Park†, Tae-Soo Lee‡, Young-Bae Kim§, Kwan-Hwa Park* and Jung-Wan Kim‡¹

*Research Center for New Bio-Materials in Agriculture and Department of Food Science and Technology, School of Agricultural Biotechnology, Seoul National University, Suwon, 441-744, South Korea, †Department of Food Science and Technology, KyungHee University, Suwon, 449-701, South Korea, ‡Department of Biology, University of Incheon, Incheon, 402-749, South Korea, and §The Graduate School of Biotechnology, Korea University, Seoul, 136-701, South Korea

Key words: amylase family 13, monomer–dimer equilibrium, multi-substrate specificity, transglycosylation.

Abbreviations used: CD, cyclodextrin; CDase, cyclomaltodextrinase; PTS, pseudotrisaccharide; nplA, neopullulanase gene; Ni-NTA, Ni²⁺-nitrilotriacetate; HPAEC, high-performance anion-exchange chromatography; BLMA, Bacillus licheniformis maltogenic amylase; ThMA, Thermus sp. IM6501 maltogenic amylase.

¹To whom correspondence should be addressed (e-mail kjw5864@incheon.ac.kr).

The nucleotide sequence of the nplA gene was deposited in the GenBank Nucleotide Sequence Database under the accession number AF233372.

Multi-substrate specificity of neopullulanase towards cyclodextrin, acarbose and maltose was investigated using a clone originating from Bacillus stearothermophilus IMA6503. The enzyme purified from Escherichia coli harbouring the corresponding nplA gene hydrolysed b-cyclodextrin (b-CD) to maltose and glucose. It exhibited substrate preference for b-CD, starch and pullulan in the proportions of 10.4:1.2:1. The enzyme not only hydrolysed acarbose, an a-amylase inhibitor, to a pseudotrisaccharide (PTS) and glucose, but also transferred PTS to glucose, forming isoacarbose. Moreover, it hydrolysed maltose to glucose and transferred the glucose to another maltose molecule to form panose when maltose was present at a low concentration (0.5%) in the reaction solution. The enzyme catalysed condensation between two maltose molecules and subsequent hydrolysis of the resulting 6²-O-a-maltosyl-maltose to glucose and panose, when maltose concentration was increased to 20%. Neopullulanase was likely to be present in monomer–dimer equilibrium with a molar ratio of 1:9 in 50 mM sodium acetate buffer (pH 6.0). The association–dissociation equilibrium of neopullulanase was shifted to monomerization by KCl. When the content of monomer increased in the reaction mixture, the specific activity towards soluble starch increased to 150%, while that towards b-CD decreased to 80%. Therefore, multi-substrate specificity of neopullulanase was likely to be modulated by the shift of monomer–dimer association equilibrium.

Received 6 July 2001/9 October 2001; accepted 23 October 2001

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