Molecular cloning of a gene encoding endo-beta-D-1,4-glucanase PCE1 from Phycomyces nitens

• Published in: Bioscience, biotechnology, and biochemistry Vol. 68; no. 11; pp. 2299 - 2305
• Main Authors: Shimonaka, A. (Meiji Seika Kaisha Ltd., Sakado, Saitama (Japan)), Baba, Y, Koga, J, Nakane, A, Kubota, H, Kono, T
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01.01.2004; Japan Society for Bioscience Biotechnology and Agrochemistry
• Subjects: Amino Acid Sequence; AMINO ACIDS; Base Sequence; Biological and medical sciences; CELLULASE; Cellulase - genetics; Cellulase - metabolism; CELLULOLYTIC MICROORGANISMS; Cloning, Molecular; DNA, Complementary - biosynthesis; DNA, Complementary - genetics; Electrophoresis, Polyacrylamide Gel; endoglucanase; ENZYMES; family 45 glycoside hydrolase; Fundamental and applied biological sciences. Psychology; Genes, Fungal - genetics; Hydrogen-Ion Concentration; MOLECULAR CLONING; Molecular Sequence Data; PHYCOMYCES; Phycomyces - enzymology; Phycomyces - genetics; Phycomyces nitens; Plasmids - genetics; Reverse Transcriptase Polymerase Chain Reaction; Rhizopus oryzae; Saccharomyces cerevisiae - metabolism; Temperature; Zygomycota; Phycomyces; Zygomycota; Enzyme; Phycomycetes; family 45 glycoside hydrolase; Fungi; endoglucanase; Cellulase; Glycosidases; Hydrolases; Glycoside; Molecular cloning; Phycomyces nitens; O-Glycosidases; Thallophyta; Glucanase
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.68.2299

We previously cloned three endoglucanase genes, rcel, rce2, and rce3, from Rhizopus oryzae as the first cellulose genes from the subdivision Zygomycota. In this study, an endoglucanase gene, designated a peel gene, was cloned by plaque hybridization with the codon usage-optimized reel gene as a probe from Phycomyces nitens, a member of the subdivision Zygomycota. The peel gene had an open reading frame of 1,038 nucleotides encoding an endoglucanasee (PCE1) of 346 amino acid residues. The amino acid sequence deduced from the pcel gene consisted of a cellulose-binding domain (CBD) at the N terminus and of a catalytic domain belonging to family 45 glycoside hydrolase at the C terminus. PCE1 was purified to apparent homogeneity from the culture supernatant of P. niters and the molecular mass was found to be 45 kDa. The optimum pH for the CMCase activity of PCE1 was 6.0, and the optimum temperature was 50°C, the lowest among the family 45 endoglucanases.