Cloning and sequencing of a Rhodothermus marinus gene, bglA, coding for a thermostable beta-glucanase and its expression in Escherichia coli.
A gene library of the thermophilic eubacterium, Rhodothermus marinus, strain 21, was prepared in pUC18 and used to transform Escherichia coli. Of 5400 transformants, two produced halos on lichenan plates after Congo-red staining. Restriction mapping showed that the two clones shared an overlapping 1200-bp DNA fragment, which was used for DNA sequencing. Five potential methionine (Met) translational-initiation codons were identified. A putative signal peptide of 30 amino acids was identified with a hydrophobic core of nine hydrophobic amino acids. The molecular mass of the mature enzyme was estimated to be 29.7 kDa. A comparison of the primary protein sequence of beta-glucanase of Rhodothermus marinus with other glycosyl hydrolases showed 38.5% identity to the C-terminal part of the beta-1,3-glucanase of Bacillus circulans and limited identity to bacterial endo-beta-1,3-1,4-glucanases. The amino acid sequence showed high similarity to regions surrounding the catalytic Glu residue of bacterial beta-glucanases. A gene fragment of 889 bp containing the catalytic domain was overexpressed in E. coli using the pET23, T7-phage RNA polymerase system. The enzyme showed activity on lichenan, beta-glucan and laminarin but not on CMC cellulose or xylan. The expressed enzyme was purified by heat treatment of the host. The enzyme had a temperature and pH optima of 85 degrees C and pH 7.0, respectively, and was shown to retain full activity after incubation for 16 h at 80 degrees C and have a half life of 3 h at 85 degrees C.
Spilliaert
R
R
Institute of Biology, University of Iceland, Reykjavík.
Hreggvidsson
G O
GO
Kristjansson
J K
JK
Eggertsson
G
G
Palsdottir
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli; D005798:Genes, Bacterial; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000235:genetics* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D006026:Glycoside Hydrolases; D004693:Glucan Endo-1,3-beta-D-Glucosidase; C028020:licheninase