Cloning, expression and identification of a new trehalose synthase gene from Thermobifida fusca genome.
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 degrees 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 degrees, 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.
Wei
Yu-Tuo
YT
Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization, Guangxi University, Nanning 530004, China.
Zhu
Qi-Xia
QX
Luo
Zhao-Fei
ZF
Lu
Fu-Shen
FS
Chen
Fa-Zhong
FZ
Wang
Qing-Yan
QY
Huang
Kun
K
Meng
Jian-Zhong
JZ
Wang
Rong
R
Huang
Ri-Bo
RB
D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D016680:Genome, Bacterial; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D017931:DNA Primers; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C099692:trehalose synthase