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Cloning and heterologous expression of the exo-β-d-glucosaminidase-encoding gene (gls93) from a filamentous fungus, Trichoderma reesei PC-3-7

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Abstract

We have previously reported on purification and characterization of an exo-β-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-β-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-β-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-β-d-glucosaminidase but not those of β-galactosidase, β-glucuronidase, and β-mannosidase belonging to GHF2.

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Author notes

  1. Masahiro Nogawa

    Present address: Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda, Nagano, 386-8567, Japan

Authors and Affiliations

  1. Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188, Japan

    Masakazu Ike, Koji Isami, Yoshio Tanabe, Masahiro Nogawa, Wataru Ogasawara, Hirofumi Okada & Yasushi Morikawa

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  1. Masakazu Ike
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  2. Koji Isami
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  3. Yoshio Tanabe
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  4. Masahiro Nogawa
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  5. Wataru Ogasawara
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  6. Hirofumi Okada
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  7. Yasushi Morikawa
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Correspondence to Yasushi Morikawa.

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Ike, M., Isami, K., Tanabe, Y. et al. Cloning and heterologous expression of the exo-β-d-glucosaminidase-encoding gene (gls93) from a filamentous fungus, Trichoderma reesei PC-3-7. Appl Microbiol Biotechnol 72, 687–695 (2006). https://doi.org/10.1007/s00253-006-0320-y

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  • DOI: https://doi.org/10.1007/s00253-006-0320-y

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