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. 1995 Jun;61(6):2262–2269. doi: 10.1128/aem.61.6.2262-2269.1995

Correction of the beta-mannanase domain of the celC pseudogene from Caldocellulosiruptor saccharolyticus and activity of the gene product on kraft pulp.

D D Morris 1, R A Reeves 1, M D Gibbs 1, D J Saul 1, P L Bergquist 1
PMCID: PMC167498  PMID: 7793947

Abstract

The celA, manA, and celB genes from Caldocellulosiruptor saccharolyticus compose a cellulase-hemicellulase gene cluster and are arranged on a 12-kb C. saccharolyticus genomic fragment of the recombinant lambda bacteriophage NZP lambda 2. The beginning of a fourth open reading frame (celC) which was homologous to the C. saccharolyticus manA and celA genes was located at the 3' end of the 12-kb NZP lambda 2 genomic fragment. Genome-walking PCR was used to isolate DNA fragments downstream of the C. saccharolyticus celB gene, and the entire nucleotide sequence of celC was obtained. From the preliminary nucleotide sequence, celC appeared to encode yet another multidomain bifunctional enzyme (CelC) consisting of an N-terminal endo-1,4-beta-D-glucanase domain (75% similar to CelA domain 1), two central cellulose-binding domains, and a C-terminal endo-1,4-beta-D-mannanase domain (98% similar to ManA domain 1). However, upon completion of the celC sequencing, two -1 frameshifts were identified in the region encoding the putative CelC mannanase domain. The isolated CelC mannanase domain exhibited no beta-mannanase activity, which supported this observation. Recombinant PCR was used to correct the celC frameshifts by inserting the appropriate nucleotides into the gene. The repaired celC fragment containing the base insertions (manB) expressed strong beta-mannanase activity on soluble mannan substrates and showed significant activity on kraft pulp as judged by the release of reducing sugars.

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Selected References

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  1. Akino T., Kato C., Horikoshi K. Two Bacillus beta-mannanases having different COOH termini are produced in Escherichia coli carrying pMAH5. Appl Environ Microbiol. 1989 Dec;55(12):3178–3183. doi: 10.1128/aem.55.12.3178-3183.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Araujo A., Ward O. P. Hemicellulases of Bacillus species: preliminary comparative studies on production and properties of mannanases and galactanases. J Appl Bacteriol. 1990 Mar;68(3):253–261. doi: 10.1111/j.1365-2672.1990.tb02572.x. [DOI] [PubMed] [Google Scholar]
  3. Arcand N., Kluepfel D., Paradis F. W., Morosoli R., Shareck F. Beta-mannanase of Streptomyces lividans 66: cloning and DNA sequence of the manA gene and characterization of the enzyme. Biochem J. 1993 Mar 15;290(Pt 3):857–863. doi: 10.1042/bj2900857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Copley C. G., Boot C., Bundell K., McPheat W. L. Unknown sequence amplification: application to in vitro genome walking in Chlamydia trachomatis L2. Biotechnology (N Y) 1991 Jan;9(1):74–79. doi: 10.1038/nbt0191-74. [DOI] [PubMed] [Google Scholar]
  5. Gibbs M. D., Saul D. J., Lüthi E., Bergquist P. L. The beta-mannanase from "Caldocellum saccharolyticum" is part of a multidomain enzyme. Appl Environ Microbiol. 1992 Dec;58(12):3864–3867. doi: 10.1128/aem.58.12.3864-3867.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gilkes N. R., Henrissat B., Kilburn D. G., Miller R. C., Jr, Warren R. A. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991 Jun;55(2):303–315. doi: 10.1128/mr.55.2.303-315.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1991 Dec 1;280(Pt 2):309–316. doi: 10.1042/bj2800309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lever M. Colorimetric and fluorometric carbohydrate determination with p-hydroxybenzoic acid hydrazide. Biochem Med. 1973 Apr;7(2):274–281. doi: 10.1016/0006-2944(73)90083-5. [DOI] [PubMed] [Google Scholar]
  9. Lüthi E., Jasmat N. B., Bergquist P. L. Xylanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum": overexpression of the gene in Escherichia coli and characterization of the gene product. Appl Environ Microbiol. 1990 Sep;56(9):2677–2683. doi: 10.1128/aem.56.9.2677-2683.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lüthi E., Jasmat N. B., Grayling R. A., Love D. R., Bergquist P. L. Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for a beta-mannanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum". Appl Environ Microbiol. 1991 Mar;57(3):694–700. doi: 10.1128/aem.57.3.694-700.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lüthi E., Love D. R., McAnulty J., Wallace C., Caughey P. A., Saul D., Bergquist P. L. Cloning, sequence analysis, and expression of genes encoding xylan-degrading enzymes from the thermophile "Caldocellum saccharolyticum". Appl Environ Microbiol. 1990 Apr;56(4):1017–1024. doi: 10.1128/aem.56.4.1017-1024.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Matsushita O., Russell J. B., Wilson D. B. A Bacteroides ruminicola 1,4-beta-D-endoglucanase is encoded in two reading frames. J Bacteriol. 1991 Nov;173(21):6919–6926. doi: 10.1128/jb.173.21.6919-6926.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Parker J. D., Rabinovitch P. S., Burmer G. C. Targeted gene walking polymerase chain reaction. Nucleic Acids Res. 1991 Jun 11;19(11):3055–3060. doi: 10.1093/nar/19.11.3055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rainey F. A., Donnison A. M., Janssen P. H., Saul D., Rodrigo A., Bergquist P. L., Daniel R. M., Stackebrandt E., Morgan H. W. Description of Caldicellulosiruptor saccharolyticus gen. nov., sp. nov: an obligately anaerobic, extremely thermophilic, cellulolytic bacterium. FEMS Microbiol Lett. 1994 Jul 15;120(3):263–266. doi: 10.1111/j.1574-6968.1994.tb07043.x. [DOI] [PubMed] [Google Scholar]
  15. Reynolds P. H., Sissons C. H., Daniel R. M., Morgan H. W. Comparison of Cellulolytic Activities in Clostridium thermocellum and Three Thermophilic, Cellulolytic Anaerobes. Appl Environ Microbiol. 1986 Jan;51(1):12–17. doi: 10.1128/aem.51.1.12-17.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Saul D. J., Williams L. C., Grayling R. A., Chamley L. W., Love D. R., Bergquist P. L. celB, a gene coding for a bifunctional cellulase from the extreme thermophile "Caldocellum saccharolyticum". Appl Environ Microbiol. 1990 Oct;56(10):3117–3124. doi: 10.1128/aem.56.10.3117-3124.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schauder B., Blöcker H., Frank R., McCarthy J. E. Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region. Gene. 1987;52(2-3):279–283. doi: 10.1016/0378-1119(87)90054-0. [DOI] [PubMed] [Google Scholar]
  18. Sissons C. H., Sharrock K. R., Daniel R. M., Morgan H. W. Isolation of cellulolytic anaerobic extreme thermophiles from new zealand thermal sites. Appl Environ Microbiol. 1987 Apr;53(4):832–838. doi: 10.1128/aem.53.4.832-838.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Teather R. M., Wood P. J. Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol. 1982 Apr;43(4):777–780. doi: 10.1128/aem.43.4.777-780.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]

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