A gene library of the thermophilic eubacterium, Rkodotkermus marinus, strain 21, was prepared in pUC18 and used to transform Esckerickia 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 P-glucanase of Rkodothermus marinus with other glycosyl hydrolases showed 38.5 % identity to the C-terminal part of the P-1,3-glucanase of Bacillus circulans and limited identity to bacterial endo-~-1,3-1,4-glucanases. The amino acid sequence showed high similarity to regions surrounding the catalytic Glu residue of bacterial P-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, P-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°C and pH 7.0, respectively, and was shown to retain full activity after incubation for 16 h at 80°C and have a half life of 3 h at 85°C.Rhodotkermus marinus is a thermophilic heterotrophic eubacterium, isolated from a marine alkaline hot spring in Iceland. It is an obligate aerobe and has an optimum growth at 65°C pH7.0 and 2% NaCl [l]. Analysis of a 16s ribosomal RNA gene shows that R. marinus diverges from major bacterial phyla, being most closely allied to the FlexibacterCytophaga-Bacteoides group (Andresson, 0. A., unpublished results). It produces several polysaccharide-degrading enzymes such as cellulase, xylanase and glucanase but in very low amounts.The endosperm cell walls of oat grain and barley are rich in polymers of glucose units joined by both P-1,4 and P-1,3 linkages, called P-glucans. This mixed linkage is also found in lichenan, a similar polysaccharide from the lichen Cetraria islandica [2].Endo ~-1,3-1,4-glucanases, also called lichenases hydrolyse P-1,4-glycosyl linkages adjacent to /3-1,3 linkages, in barley glucan or lichenan [2]. Endo-P-l,3-1,4-~-glucanase genes have been cloned from several members of the Bacillaceae family, such as Bacillus subtilis Enzymes. Lichenase, endo p-1,3-1,4-glycanase (EC 3.2.1.73); laminarase, endo /~'-1,3-~-glucanohydrolase (EC 3.2.1 39).Note. The novel amino acid sequence data published here has been desposited with the GenBank sequence data bank and is available under accession number U04836. ~-1,3-d-glucanohydrolases) cleave 1,3-P-glucosyl linkages, such as those found in the algal polysaccharide laminarin. Genes coding for enzymes able to hydrolyse both P-1,3-1,4-glucans and P-1,3-glucans have been cloned from Cellvibrio...
A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6-7 and its highest measured initial activity at 100 degrees C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 degrees C.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.