title: Cloning and heterologous expression of the exo-beta-D-glucosaminidase-encoding gene (gls93) from a filamentous fungus, Trichoderma reesei PC-3-7.
abstract: We have previously reported on purification and characterization of an exo-beta-D-glucosaminidase (Gls93) from culture filtrate of Trichoderma reesei PC-3-7 grown on N-acetyl-D-glucosamine (GlcNAc). The corresponding gene of Gls93 was cloned and characterized in this work. To our knowledge, this is the first report on cloning of the gene encoding fungal exo-beta-D-glucosaminidase. This gene has no introns and encodes a polypeptide of 892 amino acids (aa) containing a secretion signal of 28 amino acids. Comparison of the amino acid sequence to known proteins and phylogenetic analysis indicated that gls93 belongs to the glycoside hydrolase family (GHF) 2 and should be further classified into a new subgroup, exo-beta-D-glucosaminidase subgroup. The gls93 transcription was biphasic when T. reesei was grown on GlcNAc, suggesting that the expression of this gene may be regulated by a complex mechanism, in which multiple regulatory proteins are involved. Furthermore, gls93 could be expressed in Pichia pastoris (ca. 0.49-mg/ml culture). The recombinant Gls93 had the two molecular forms, ca. 105 and 100 kDa, whose difference is caused by N-glycosylation. Both of them had the same properties such as specific activity and substrate specificity and showed only the activity of exo-beta-D-glucosaminidase but not those of beta-galactosidase, beta-glucuronidase, and beta-mannosidase belonging to GHF2.
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Ike
authors.forename: Koji
authors.initials: K
authors.lastname: Isami
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Tanabe
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nogawa
authors.forename: Wataru
authors.initials: W
authors.lastname: Ogasawara
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Okada
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Morikawa
mesh_terms: D000595:Amino Acid Sequence; D002686:Chitin / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D006596:Hexosaminidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010843:Pichia / Q000235:genetics; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
chemical_list: D002686:Chitin; D006596:Hexosaminidases; C064499:exo-beta-D-glucosaminidase