Molecular and enzymic properties of recombinant 1,2-α-mannosidase from Aspergillus saitoi overexpressed in Aspergillus oryzae cells

Eiji Ichishima, Noriyuki Taya, Masamichi Ikeguchi, Yasunori Chiba, Motoyoshi Nakamura, Choko Kawabata, Takashi Inoue, Koji Takahashi, Toshiki Minetoki, Kenji Ozeki, Chieko Kumagai, Katsuya Gomi, Takashi Yoshida, Tasuku Nakajima

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41 Citations (Scopus)

Abstract

For the construction of an overexpression system of the intracellular 1,2-α-mannosidase (EC 3.2.1.113) gene (msdS) from Aspergillus saitoi (now designated Aspergillus phoenicis), the N-terminal signal sequence of the gene was replaced with that of the aspergillopepsin I (EC 3.4.23.18) gene (apnS) signal, one of the same strains as described previously. Then the fused 1,2-α-mannosidase gene (f-msdS) was inserted into the NotI site between P-No8142 and T-agdA in the plasmid pNAN 8142 (9.5 kbp) and thus the Aspergillus oryzae expression plasmid pNAN-AM1 (11.2 kbp) was constructed. The fused f-msdS gene has been overexpressed in a transformant A. oryzae niaD AM1 cell. The recombinant enzyme expressed in A. oryzae cells was purified to homogeneity in two steps. The system is capable of making as much as about 320 mg of the enzyme/litre of culture. The recombinant enzyme has activity with methyl-2-O-α-D-mannopyranosyl α-D-mannopyranoside at pH 5.0, while no activity was determined with methyl-3-O-α-D-mannopyranosyl α-D-mannopyranoside or methyl-6-O-α-D-mannopyranosyl α-D-mannopyranoside. The substrate specificity of the enzyme was analysed by using pyridylaminated (PA)-oligomannose-type sugar chains, Man9-6(GlcNAc)2-PA (Man is mannose; GlcNAc is N-acetylglucosamine). The enzyme hydrolysed Man8GlcNAc2-PA (type 'M8A') fastest, and 'M6C' {Manα1-3[Manα1-2Manα1-3(Manα1-6)Manα1-6]Manβ1-4GlcNAcβ1-4GlcNAc-PA} slowest, among the PA-sugar chains. Molecular-mass values of the enzyme were determined to be 63 kDa by SDS/PAGE and 65 kDa by gel filtration on Superose 12 respectively. The pI value of the enzyme was 4.6. The N-terminal amino acid sequence of the enzyme was GSTQSRADAIKAAFSHAWDGYLQY, and sequence analysis indicated that the signal peptide from apnS gene was removed. The molar absorption coefficient, ε, at 280 nm was determined as 91,539 M-1 cm-1. Contents of the secondary structure (α-helix, β-structure and the remainder of the enzyme) by far-UV CD determination were about 55, 38 and 7% respectively. The melting temperature, T(m), of the enzyme was 71°C by differential scanning calorimetry. The calorimetric enthalpy, ΔH(cal), of the enzyme was calculated as 13.3 kJ.kg of protein-1. Determination of 1 g-atom of Ca2+/mol of enzyme was performed by atomic-absorption spectrophotometry.

Original languageEnglish
Pages (from-to)589-597
Number of pages9
JournalBiochemical Journal
Volume339
Issue number3
DOIs
Publication statusPublished - 1999 May 1

Keywords

  • 1,2-α-mannosidase
  • Aspergillus oryzae
  • Aspergillus saitoi
  • Overexpression
  • Recombinant α-mannosidase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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  • Cite this

    Ichishima, E., Taya, N., Ikeguchi, M., Chiba, Y., Nakamura, M., Kawabata, C., Inoue, T., Takahashi, K., Minetoki, T., Ozeki, K., Kumagai, C., Gomi, K., Yoshida, T., & Nakajima, T. (1999). Molecular and enzymic properties of recombinant 1,2-α-mannosidase from Aspergillus saitoi overexpressed in Aspergillus oryzae cells. Biochemical Journal, 339(3), 589-597. https://doi.org/10.1042/0264-6021:3390589