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ISSN 0582-9879 Acta Biochimica et Biophysica Sinica 2004, 36(7): 477–484 CN 31-1300/Q
Cloning, Expression and Identification of a New Trehalose Synthase
Gene from Thermobifida fusca Genome
Yu-Tuo WEI, Qi-Xia ZHU1, Zhao-Fei LUO1,
Fu-Shen LU1, Fa-Zhong CHEN1, Qing-Yan WANG1,
Kun HUANG, Jian-Zhong MENG, Rong WANG, and Ri-Bo HUANG*
Guangxi Key Laboratory of Subtropical Bioresource Conservation and
Utilization, Guangxi University, Nanning 530004, China; 1Sinozyme
Biotech Co. Ltd., Nanning 530004, China
Abstract A new open reading frame
in Thermobifida fusca sequenced genome was identified to encode a new
trehalose synthase, annotated as “glycosidase” in the GenBank database, by
bioinformatics searching and experimental validation. The gene had a length of
1830 bp with about 65% GC content and encoded for a new trehalose synthase with
610 amino acids and deduced molecular weight of 66 kD. The high GC content
seemed not to affect its good expression in E. coli BL21 in which the
target protein could account for as high as 15% of the total cell proteins. The
recombinant enzyme showed its optimal activities at 25 °C and pH 6.5 when it
converted substrate maltose into trehalose. However it would divert a high
proportion of its substrate into glucose when the temperature was increased to
37 °C, or when the enzyme concentration was high Its activity was not inhibited
by 5 mM heavy metals such as Cu2+, Mn2+, and Zn2+ but affected by high concentration of glucose. Blasting against the
database indicated that amino acid sequence of this protein had maximal 69%
homology with the known trehalose synthases, and two highly conserved segments
of the protein sequence were identified and their possible linkage with
functions was discussed.
Key words trehalose synthase; Thermobifida
fusca; open reading frame (ORF); gene expression
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Received: April 8, 2004 Accepted: June 2, 2004
This work was supported by a grant of the National High Technology Research
and Development Program of China (2001AA214171)
*Corresponding author: Tel, 86-771-3235706; Fax, 86-771-3238107;
E-mail, [email protected]