Cloning of genes encoding α-L-arabinofuranosidase and β-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae

Emilio Margolles-Clark, Maija Tenkanen, Tiina Nakari-Setälä, Merja Penttilä

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141 Citations (Scopus)

Abstract

A cDNA expression library of Trichoderma reesei RutC-30 was constructed in the yeast Saccharomyces cerevisiae. Two genes, abfI and bxII, were isolated by screening the yeast library for extracellular α-L- arabinofuranosidase activity with the substrate p-nitrophenyl-α-L- arabinofuranoside. The genes abfI and bxII encode 500 and 758 amino acids, respectively, including the signal sequences. The deduced amino acid sequence of ABFI displays high-level similarity to the α-L-arabinofuranosidase B of Aspergillus niger, and the two can form a new family of glycosyl hydrolases. The deduced amino acid sequence of BXLI shows similarities to the β- glucosidases grouped in family 3. The yeast-produced enzymes were tested for enzymatic activities against different substrates. ABFI released L-arabinose from p-nitrophenyl-α-L-arabinofuranoside and arahinoxylans and showed some β-xylosidase activity toward p-nitrophenyl-β-D-xylopyranoside. BXLI did not release L-arabinose from arabinoxylan. It showed α-L-arabinofuranosidase, α-L-arabinopyranosidase, and β-xylosidase activities against p- nitrophenyl-α-L-arahinofuranosidase, p-nitrophenyl-α-arabinopyranoside, and p-nitrophenyl-β-D-xylopyranoside, respectively, with the last activity being the highest. It was also able to hydrolyze xyhobiose and slowly release xylose from polymeric xylan. ABFI and BXLI correspond to a previously purified α-L-arabinofuranosidase and a β-xylosidase from T. reesei, respectively, as confirmed by partial amino acid sequencing of the Trichoderma-produced enzymes. Both enzymes produced in yeasts displayed hydrolytic properties similar to those of the corresponding enzymes purified from T. reesei.

Original languageEnglish
Pages (from-to)3840-3846
Number of pages7
JournalApplied and Environmental Microbiology
Volume62
Issue number10
Publication statusPublished - 1 Oct 1996
MoE publication typeA1 Journal article-refereed

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Keywords

  • 4 nitrophenol
  • alpha arabinofuranosidase
  • pyranoside
  • Xylan
  • xylan 1,4 beta xylosidase
  • Xylose
  • Amino Acid Sequence
  • Aspergillus niger
  • Hypocrea jecorina
  • Saccharomyces cerevisiae
  • Trichoderma

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