A family 26 mannanase produced by Clostridium thermocellum as a component of the cellulosome contains a domain which is conserved in mannanases from anaerobic fungi

被引:40
作者
Halstead, JR
Vercoe, PE
Gilbert, HJ [1 ]
Davidson, K
Hazlewood, GP
机构
[1] Newcastle Univ, Dept Biol & Nutr Sci, Newcastle Upon Tyne NE1 7RU, Tyne & Wear, England
[2] Babraham Inst, Lab Mol Enzymol, Cambridge CB2 4AT, England
[3] Univ Western Australia, Dept Anim Sci, Nedlands, WA 6009, Australia
来源
MICROBIOLOGY-SGM | 1999年 / 145卷
关键词
mannanase; Clostridium thermocellum; cellulosome; family; 26;
D O I
10.1099/00221287-145-11-3101
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Cellulosomes prepared by the cellulose affinity digestion method from Clostridium thermocellum culture supernatant hydrolyses carob galactomannan during incubation at 60 degrees and pH 6.5. A recombinant phage expressing mannase activity was isolated from a library of C. thermocellum genomic DNA constructed in lambda ZAPII. The cloned fragment of DNA containing a putative mannanse gene (manA) was sequenced, revealing an ORF of 1767 nt, encoding in protein (mannanase A; Man26A) of 589 aa with a molecular mass of 66816 Da. The putative catalytic domain (CD) of Man26A, identified by gene sectioning and sequence comparisons, displayed up to 32% identity with other mannanases belonging to family 26. Immediately downstream of the CD and separated from it by a short proline/threonine linker was a duplicated 24-residue dockerin motif, which is conserved in all C. thermocellum cellulosomal enzymes described thus far and mediates their attachment to the cellulosome-integrating protein (CipA). Man26A consisting of the CD alone (Man26A') was hyperexpressed in Escherichia coli BL21(DSS) and purified. The truncated enzyme hydrolysed soluble and insoluble mannan, displaying a temperature optimum of 65 degrees C and a pH optimum of 6.5, but exhibited no activity against other plant cell wall polysaccharides. Antiserum raised against Man26A' crossreacted with a polypeptide with a molecular mass of 70 000 Da that is part of the C. thermocellum cellulosome. A second variant of Man26A containing the N-terminal segment of 130 residues and the CD (Man26A'') bound to ivory-nut mannan and weakly to soluble Carob galactomannan and insoluble cellulose. Man26A' consisting of the CD alone did not bind to these polysaccharides. These results indicate that the N-terminal 130 residues of mature Man26A may constitute a weak mannan-binding domain. Sequence comparisons revealed a lack of identity between this region of Man26A and other polysaccharide-binding domains, but significant identity with a region conserved in the three family 26 mannanases from the anaerobic fungus Piromyces equi.
引用
收藏
页码:3101 / 3108
页数:8
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