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ISSN 0582-9879 Acta Biochimica et Biophysica Sinica 2004, 36(7): 477–484 CN 31-1300/Q

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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 ºC 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 ºC, 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]