Cloning and characterization of a novel acidic cutinase from Sirococcus conigenus

Research output: Contribution to journalArticleScientificpeer-review

13 Citations (Scopus)

Abstract

A cutinase gene (ScCut1) was amplified by PCR from the genomic DNA of the ascomycetous plant pathogen Sirococcous conigenus VTT D-04989 using degenerate primers designed on the basis of conserved segments of known cutinases and cutinase-like enzymes. No introns or N- or O-glycosylation sites could be detected by analysis of the ScCut1 gene sequence. The alignment of ScCut1 with other fungal cutinases indicated that ScCut1 contained the conserved motif G-Y-S-Q-G surrounding the active site serine as well as the aspartic acid and histidine residues of the cutinase active site. The gene was expressed in Pichia pastoris, and the recombinantly produced ScCut1 enzyme was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His-tag translationally fused to the protein. The purified ScCut1 exhibited activity at acidic pH. The K m and V max values determined for pNP-butyrate esterase activity at pH 4.5 were 1.7 mM and 740 nkat mg -1, respectively. Maximal activities were determined at between pH 4.7 and 5.2 and at between pH 4.1 and 4.6 with pNP-butyrate and tritiated cutin as the substrates, respectively. With both substrates, the enzyme was active over a broad pH range (between pH 3.0 and 7.5). Activity could still be detected at pH 3.0 both with tritiated cutin and with p-nitrophenyl butyrate (relative activity of 25 %) as the substrates. ScCut1 showed activity towards shorter (C2 to C3) fatty acid esters of p-nitrophenol than towards longer ones. Circular dichroism analysis suggested that the denaturation of ScCut1 by heating the protein sample to 80°C was to a great extent reversible.

Original languageEnglish
Pages (from-to)3639-3650
Number of pages12
JournalApplied Microbiology and Biotechnology
Volume98
Issue number8
DOIs
Publication statusPublished - 1 Jan 2014
MoE publication typeA1 Journal article-refereed

Fingerprint

Organism Cloning
Catalytic Domain
Enzymes
Plant DNA
Genes
Pichia
Butyrates
Circular Dichroism
cutinase
Glycosylation
Affinity Chromatography
Histidine
Aspartic Acid
Heating
Introns
Serine
Esters
Proteins
Fatty Acids
Metals

Keywords

  • Cutin
  • Cutinase
  • Esterase
  • Polyester
  • Suberin

Cite this

@article{2500de60783f413ca1c8c832daa94f59,
title = "Cloning and characterization of a novel acidic cutinase from Sirococcus conigenus",
abstract = "A cutinase gene (ScCut1) was amplified by PCR from the genomic DNA of the ascomycetous plant pathogen Sirococcous conigenus VTT D-04989 using degenerate primers designed on the basis of conserved segments of known cutinases and cutinase-like enzymes. No introns or N- or O-glycosylation sites could be detected by analysis of the ScCut1 gene sequence. The alignment of ScCut1 with other fungal cutinases indicated that ScCut1 contained the conserved motif G-Y-S-Q-G surrounding the active site serine as well as the aspartic acid and histidine residues of the cutinase active site. The gene was expressed in Pichia pastoris, and the recombinantly produced ScCut1 enzyme was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His-tag translationally fused to the protein. The purified ScCut1 exhibited activity at acidic pH. The K m and V max values determined for pNP-butyrate esterase activity at pH 4.5 were 1.7 mM and 740 nkat mg -1, respectively. Maximal activities were determined at between pH 4.7 and 5.2 and at between pH 4.1 and 4.6 with pNP-butyrate and tritiated cutin as the substrates, respectively. With both substrates, the enzyme was active over a broad pH range (between pH 3.0 and 7.5). Activity could still be detected at pH 3.0 both with tritiated cutin and with p-nitrophenyl butyrate (relative activity of 25 {\%}) as the substrates. ScCut1 showed activity towards shorter (C2 to C3) fatty acid esters of p-nitrophenol than towards longer ones. Circular dichroism analysis suggested that the denaturation of ScCut1 by heating the protein sample to 80°C was to a great extent reversible.",
keywords = "Cutin, Cutinase, Esterase, Polyester, Suberin",
author = "Antti Nyyss{\"o}l{\"a} and Ville Pihlajaniemi and Mari H{\"a}kkinen and Hanna Kontkanen and Markku Saloheimo and Tiina Nakari-Set{\"a}l{\"a}",
year = "2014",
month = "1",
day = "1",
doi = "10.1007/s00253-013-5293-z",
language = "English",
volume = "98",
pages = "3639--3650",
journal = "Applied Microbiology and Biotechnology",
issn = "0175-7598",
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Cloning and characterization of a novel acidic cutinase from Sirococcus conigenus. / Nyyssölä, Antti; Pihlajaniemi, Ville; Häkkinen, Mari; Kontkanen, Hanna; Saloheimo, Markku; Nakari-Setälä, Tiina.

In: Applied Microbiology and Biotechnology, Vol. 98, No. 8, 01.01.2014, p. 3639-3650.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Cloning and characterization of a novel acidic cutinase from Sirococcus conigenus

AU - Nyyssölä, Antti

AU - Pihlajaniemi, Ville

AU - Häkkinen, Mari

AU - Kontkanen, Hanna

AU - Saloheimo, Markku

AU - Nakari-Setälä, Tiina

PY - 2014/1/1

Y1 - 2014/1/1

N2 - A cutinase gene (ScCut1) was amplified by PCR from the genomic DNA of the ascomycetous plant pathogen Sirococcous conigenus VTT D-04989 using degenerate primers designed on the basis of conserved segments of known cutinases and cutinase-like enzymes. No introns or N- or O-glycosylation sites could be detected by analysis of the ScCut1 gene sequence. The alignment of ScCut1 with other fungal cutinases indicated that ScCut1 contained the conserved motif G-Y-S-Q-G surrounding the active site serine as well as the aspartic acid and histidine residues of the cutinase active site. The gene was expressed in Pichia pastoris, and the recombinantly produced ScCut1 enzyme was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His-tag translationally fused to the protein. The purified ScCut1 exhibited activity at acidic pH. The K m and V max values determined for pNP-butyrate esterase activity at pH 4.5 were 1.7 mM and 740 nkat mg -1, respectively. Maximal activities were determined at between pH 4.7 and 5.2 and at between pH 4.1 and 4.6 with pNP-butyrate and tritiated cutin as the substrates, respectively. With both substrates, the enzyme was active over a broad pH range (between pH 3.0 and 7.5). Activity could still be detected at pH 3.0 both with tritiated cutin and with p-nitrophenyl butyrate (relative activity of 25 %) as the substrates. ScCut1 showed activity towards shorter (C2 to C3) fatty acid esters of p-nitrophenol than towards longer ones. Circular dichroism analysis suggested that the denaturation of ScCut1 by heating the protein sample to 80°C was to a great extent reversible.

AB - A cutinase gene (ScCut1) was amplified by PCR from the genomic DNA of the ascomycetous plant pathogen Sirococcous conigenus VTT D-04989 using degenerate primers designed on the basis of conserved segments of known cutinases and cutinase-like enzymes. No introns or N- or O-glycosylation sites could be detected by analysis of the ScCut1 gene sequence. The alignment of ScCut1 with other fungal cutinases indicated that ScCut1 contained the conserved motif G-Y-S-Q-G surrounding the active site serine as well as the aspartic acid and histidine residues of the cutinase active site. The gene was expressed in Pichia pastoris, and the recombinantly produced ScCut1 enzyme was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His-tag translationally fused to the protein. The purified ScCut1 exhibited activity at acidic pH. The K m and V max values determined for pNP-butyrate esterase activity at pH 4.5 were 1.7 mM and 740 nkat mg -1, respectively. Maximal activities were determined at between pH 4.7 and 5.2 and at between pH 4.1 and 4.6 with pNP-butyrate and tritiated cutin as the substrates, respectively. With both substrates, the enzyme was active over a broad pH range (between pH 3.0 and 7.5). Activity could still be detected at pH 3.0 both with tritiated cutin and with p-nitrophenyl butyrate (relative activity of 25 %) as the substrates. ScCut1 showed activity towards shorter (C2 to C3) fatty acid esters of p-nitrophenol than towards longer ones. Circular dichroism analysis suggested that the denaturation of ScCut1 by heating the protein sample to 80°C was to a great extent reversible.

KW - Cutin

KW - Cutinase

KW - Esterase

KW - Polyester

KW - Suberin

U2 - 10.1007/s00253-013-5293-z

DO - 10.1007/s00253-013-5293-z

M3 - Article

VL - 98

SP - 3639

EP - 3650

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

IS - 8

ER -