Cloning and nucleotide sequence of b-mannanase and cellulase genes from Bacillus sp. 5H C. Khanongnuch, T. Ooi and S. Kinoshita* Department of Molecular Chemistry, Faculty of Engineering, Hokkaido Graduate University, N-13, W-8, Sapporo, Hokkaido 060-8628, Japan *Author for correspondence: Tel.: (+81-11)-706-6610; Fax: (+81-11)-706-6611; E-mail: shin-1@moby. hokudai.ac.jp Received in revised form 25 December 1998; accepted 6 January 1999 Keywords: Bacillus sp., cellulase gene, b-mannanase gene Summary The two genes for b-mannanase and cellulase of Bacillus sp. 5H have been cloned in Escherichia coli JM109 by a shotgun method, though the cellulase gene was not expressed in Bacillus sp. 5H. The nucleotide sequences of the b-mannanase gene and the cellulase gene revealed open reading frames of 1,086 and 1,503 base pairs, respectively, coding for a proteins of M r 40,803 Da (b-mannanase) and 55,420 Da (cellulase). The deduced primary structure of b-mannanase comprised 362 amino acids which had a mature protein of 336 amino acids and a signal peptide of 26 amino acids and that of cellulase comprised 501 amino acid residues. Introduction Hemicellulases have recently gained much atten- tion as possible biocatalysts in biobleaching of wood pulp (David et al. 1992) because of the re- placement of chlorinated materials used at present in the paper industry, where hemicellulase is ex- pected to decrease the environmental stress dras- tically (Viikari et al. 1993). In wood pulps, it is generally accepted that the lignin molecule is bound to cellulose through bonds with hemicellu- lose, mainly xylan. Therefore, a deligni®cation of wood pulp by hemicellulase treatment may reduce the amount of bleaching chemicals and increase the brightness of pulps. Beside xylanase, other hemicellulase such as b-mannanase have also been studied, because mannans are found in softwood pulp as a major hemicellulose (Viikari et al. 1987). Glucomannan is also found in hardwood as a minor component. Mannan consists of a b-1,4-mannose backbone commonly with branched side chains such as a-1,6-linked galactosyl residues. Therefore, the complete hydrolysis of mannan requires the co- operative action of endo-b-1,4-mannanase (EC 3.2.1.78), b-mannosidase (EC 3.2.1.25), and a series of enzymes to cleave side chains. So far, numerous b-mannanases have been puri®ed and characterized from dierent microorganisms including bacteria and fungi (Viikari et al. 1993). We have characterized the biobleaching of wood pulp by the xylanase and the b-mannanase from Bacillus sp. 5H (Khanongnuch et al. 1998). This bacterium did not produce cellulase. This seems to be a convenient characteristic for biobleaching of wood pulp. In this paper, we describe cloning and sequencing of the gene coding for the extracellular b-mannanase from this strain and compare the amino acid sequence with those of other b-man- nanases. During the cloning of the b-mannanase World Journal of Microbiology & Biotechnology 15: 249±258, 1999. 249 Ó 1999 Kluwer Academic Publishers. Printed in the Netherlands.