Purification and characterization of two forms of endo-beta-1,4-mannanase from a thermotolerant fungus, Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749)


Puchart V., Vrsanska M., Svoboda P., Pohl J., Ogel Z. , Biely P.

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, cilt.1674, sa.3, ss.239-250, 2004 (SCI İndekslerine Giren Dergi) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 1674 Konu: 3
  • Basım Tarihi: 2004
  • Doi Numarası: 10.1016/j.bbagen.2004.06.022
  • Dergi Adı: BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
  • Sayfa Sayıları: ss.239-250

Özet

Two extracellular endo-beta,4-mannanascs,MAN I (major form) and MAN II (minor form), were purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749). Molecular weights of MAN I and MAN II estimated by SDS-PAGE were 60 and 63 kDa, respectively. IEF afforded several glycoprotein bands with pI values in the range of 4.9-5.2 for MAN I and 4.75-4.9 for MAN 11, each exhibiting enzyme activity. MAN I as well as MAN 11 showed highest activity at pH 4.5 and 60 degreesC and were stable in the pH range 4.5-8.5 and up to 55 degreesC. In accordance with the ability of the enzymes to catalyze transglycosylation reactions, H-1 NMR spectroscopy of reaction products generated from mannopentaitol confirmed the retaining character of both enzymes. Both MAN I and MAN II exhibited essentially identical kinetic parameters for polysaccharides and a similar hydrolysis pattern of various oligomeric and polymeric substrates. Both beta-mannanases contained identical internal amino acid sequence corresponding to glycoside hydrolase family 5 and also a cellulose-binding module. These data suggested that both MAN I and MAN II are products of the same gene differing in posttranslational modification. Indeed, the corresponding gene was identified within the recently sequenced Aspergillusfumigatus genome (http://www.sanger.ac.uk/Projects/A-ftimigatus/). (C) 2004 Elsevier B.V. All rights reserved.