Molecular cloning and nucleotide sequence of the glycogen branching enzyme gene (glgB) from Bacillus stearothermophilus and expression in Escherichia coli and Bacillus subtilis. |
| |
Authors: | J. A. K. W. Kiel J. M. Boels G. Beldman G. Venema |
| |
Affiliation: | (1) Department of Genetics, Center of Biological Sciences, Kerklaan 30, NL-9751 NN Haren, The Netherlands;(2) Netherlands Institute for Carbohydrate Research (NIKO-TNO), Rouaanstraat 27, NL-9723 CC Groningen, The Netherlands;(3) Present address: Department of Food Science, Agricultural University Wageningen, P.O. box 8129, NL-6700 EV Wageningen, The Netherlands |
| |
Abstract: | Summary The structural gene for the Bacillus stearothermophilus glycogen branching enzyme (glgB) was cloned in Escherichia coli. Nucleotide sequence analysis revealed a 1917 nucleotide open reading frame (ORF) encoding a protein with an Mr of 74787 showing extensive similarity to other bacterial branching enzymes, but with a shorter N-terminal region. A second ORF of 951 nucleotides encoding a 36971 Da protein started upstream of the glgB gene. The N-terminus of the ORF2 gene product had similarity to the Alcaligenes eutrophus czcD gene, which is involved in cobalt-zinc-cadmium resistance. The B. stearothermophilus glgB gene was preceded by a sequence with extensive similarity to promoters recognized by Bacillus subtilis RNA polymerase containing sigma factor H (E - H). The glgB promoter was utilized in B. subtilis exclusively in the stationary phase, and only transcribed at low levels in B. subtilis spoOH, indicating that sigma factor H was essential for the expression of the glgB gene in B. subtilis. In an expression vector, the B. stearothermophilus glgB gene directed the synthesis of a thermostable branching enzyme in E. coli as well as in B. subtilis, with optimal branching activity at 53° C. |
| |
Keywords: | Branching enzyme Gram-positive Glycogen biosynthesis Sigma factor H Thermostability |
本文献已被 SpringerLink 等数据库收录! |
|