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

Research output: Contribution to journalArticleScientificpeer-review

135 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

Fingerprint

Xylosidases
alpha-N-arabinofuranosidase
Trichoderma reesei
Trichoderma
yeast
Saccharomyces cerevisiae
Organism Cloning
molecular cloning
amino acid
Yeasts
enzyme
yeasts
Arabinose
gene
Enzymes
arabinose
enzymes
Genes
Amino Acid Sequence
amino acid sequences

Keywords

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

Cite this

@article{cd4c81678ad04763a48f62d5169ae84d,
title = "Cloning of genes encoding α-L-arabinofuranosidase and β-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae",
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.",
keywords = "4 nitrophenol, alpha arabinofuranosidase, pyranoside, Xylan, xylan 1,4 beta xylosidase, Xylose, Amino Acid Sequence, Aspergillus niger, Hypocrea jecorina, Saccharomyces cerevisiae, Trichoderma",
author = "Emilio Margolles-Clark and Maija Tenkanen and Tiina Nakari-Set{\"a}l{\"a} and Merja Penttil{\"a}",
year = "1996",
month = "10",
day = "1",
language = "English",
volume = "62",
pages = "3840--3846",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
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}

Cloning of genes encoding α-L-arabinofuranosidase and β-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae. / Margolles-Clark, Emilio; Tenkanen, Maija; Nakari-Setälä, Tiina; Penttilä, Merja.

In: Applied and Environmental Microbiology, Vol. 62, No. 10, 01.10.1996, p. 3840-3846.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Cloning of genes encoding α-L-arabinofuranosidase and β-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae

AU - Margolles-Clark, Emilio

AU - Tenkanen, Maija

AU - Nakari-Setälä, Tiina

AU - Penttilä, Merja

PY - 1996/10/1

Y1 - 1996/10/1

N2 - 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.

AB - 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.

KW - 4 nitrophenol

KW - alpha arabinofuranosidase

KW - pyranoside

KW - Xylan

KW - xylan 1,4 beta xylosidase

KW - Xylose

KW - Amino Acid Sequence

KW - Aspergillus niger

KW - Hypocrea jecorina

KW - Saccharomyces cerevisiae

KW - Trichoderma

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M3 - Article

VL - 62

SP - 3840

EP - 3846

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 10

ER -