Thermostable xylanases, Xyn10A and Xyn11A, from the actinomycete Nonomuraea flexuosa: solation of the genes and characterization of recombinant Xyn11A polypeptides produced in Trichoderma reesei

S. Leskinen, Arja Mäntylä (Corresponding Author), Richard Fagerström, Jari Vehmaanperä, Raija Lantto, Marja Paloheimo, Pirkko Suominen

Research output: Contribution to journalArticleScientificpeer-review

44 Citations (Scopus)

Abstract

Two endoxylanases, Nf Xyn11A and Nf Xyn10A, were cloned from a Nonomuraea flexuosa (previously Actinomadura flexuosa) DSM43186 genomic expression library in Escherichia coli. The coding sequences of xyn11A and xyn10A consist of 344 and 492 amino acids, respectively. The catalytic domains belong to family 11 and family 10 of glycoside hydrolases. The C-termini share strong amino acid sequence similarity to carbohydrate-binding module (CBM) families CBM2 and CBM13, respectively. Native Nf Xyn11A, and recombinant Xyn11A expressed in the filamentous fungus Trichoderma reesei, were purified from cultivation media and characterized. The molecular masses of the full-length enzymes determined by mass spectrometry were 32.9 kDa and 33.4 kDa, the recombinant enzyme having higher molecular mass due to glycosylation. In addition, shorter polypeptides with molecular masses of 23.8 kDa and 22.0 kDa were characterized from the T. reesei culture medium, both lacking the C-terminal CBM and the 22.0 kDa polypeptide also lacking most of the linker region. The recombinant polypeptides were similar to each other in terms of specific activity, pH and temperature dependence. However, the 23.8 kDa and 22.0 kDa polypeptides were more thermostable at 80°C than the full-length enzyme. All polypeptide forms were effective in pretreatment of softwood kraft pulp at 80°C.
Original languageEnglish
Pages (from-to)495 - 505
Number of pages11
JournalApplied Microbiology and Biotechnology
Volume67
Issue number4
DOIs
Publication statusPublished - 2005
MoE publication typeA1 Journal article-refereed

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Trichoderma
Actinobacteria
Peptides
Genes
Enzymes
Carbohydrates
Endo-1,4-beta Xylanases
Genomic Library
Glycoside Hydrolases
Glycosylation
Culture Media
Amino Acid Sequence
Mass Spectrometry
Catalytic Domain
Fungi
Escherichia coli
Amino Acids
Temperature

Cite this

@article{f860b8b764c04b65baa13615271e74a5,
title = "Thermostable xylanases, Xyn10A and Xyn11A, from the actinomycete Nonomuraea flexuosa: solation of the genes and characterization of recombinant Xyn11A polypeptides produced in Trichoderma reesei",
abstract = "Two endoxylanases, Nf Xyn11A and Nf Xyn10A, were cloned from a Nonomuraea flexuosa (previously Actinomadura flexuosa) DSM43186 genomic expression library in Escherichia coli. The coding sequences of xyn11A and xyn10A consist of 344 and 492 amino acids, respectively. The catalytic domains belong to family 11 and family 10 of glycoside hydrolases. The C-termini share strong amino acid sequence similarity to carbohydrate-binding module (CBM) families CBM2 and CBM13, respectively. Native Nf Xyn11A, and recombinant Xyn11A expressed in the filamentous fungus Trichoderma reesei, were purified from cultivation media and characterized. The molecular masses of the full-length enzymes determined by mass spectrometry were 32.9 kDa and 33.4 kDa, the recombinant enzyme having higher molecular mass due to glycosylation. In addition, shorter polypeptides with molecular masses of 23.8 kDa and 22.0 kDa were characterized from the T. reesei culture medium, both lacking the C-terminal CBM and the 22.0 kDa polypeptide also lacking most of the linker region. The recombinant polypeptides were similar to each other in terms of specific activity, pH and temperature dependence. However, the 23.8 kDa and 22.0 kDa polypeptides were more thermostable at 80°C than the full-length enzyme. All polypeptide forms were effective in pretreatment of softwood kraft pulp at 80°C.",
author = "S. Leskinen and Arja M{\"a}ntyl{\"a} and Richard Fagerstr{\"o}m and Jari Vehmaanper{\"a} and Raija Lantto and Marja Paloheimo and Pirkko Suominen",
year = "2005",
doi = "10.1007/s00253-004-1797-x",
language = "English",
volume = "67",
pages = "495 -- 505",
journal = "Applied Microbiology and Biotechnology",
issn = "0175-7598",
publisher = "Springer",
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}

Thermostable xylanases, Xyn10A and Xyn11A, from the actinomycete Nonomuraea flexuosa : solation of the genes and characterization of recombinant Xyn11A polypeptides produced in Trichoderma reesei. / Leskinen, S.; Mäntylä, Arja (Corresponding Author); Fagerström, Richard; Vehmaanperä, Jari; Lantto, Raija; Paloheimo, Marja; Suominen, Pirkko.

In: Applied Microbiology and Biotechnology, Vol. 67, No. 4, 2005, p. 495 - 505.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Thermostable xylanases, Xyn10A and Xyn11A, from the actinomycete Nonomuraea flexuosa

T2 - solation of the genes and characterization of recombinant Xyn11A polypeptides produced in Trichoderma reesei

AU - Leskinen, S.

AU - Mäntylä, Arja

AU - Fagerström, Richard

AU - Vehmaanperä, Jari

AU - Lantto, Raija

AU - Paloheimo, Marja

AU - Suominen, Pirkko

PY - 2005

Y1 - 2005

N2 - Two endoxylanases, Nf Xyn11A and Nf Xyn10A, were cloned from a Nonomuraea flexuosa (previously Actinomadura flexuosa) DSM43186 genomic expression library in Escherichia coli. The coding sequences of xyn11A and xyn10A consist of 344 and 492 amino acids, respectively. The catalytic domains belong to family 11 and family 10 of glycoside hydrolases. The C-termini share strong amino acid sequence similarity to carbohydrate-binding module (CBM) families CBM2 and CBM13, respectively. Native Nf Xyn11A, and recombinant Xyn11A expressed in the filamentous fungus Trichoderma reesei, were purified from cultivation media and characterized. The molecular masses of the full-length enzymes determined by mass spectrometry were 32.9 kDa and 33.4 kDa, the recombinant enzyme having higher molecular mass due to glycosylation. In addition, shorter polypeptides with molecular masses of 23.8 kDa and 22.0 kDa were characterized from the T. reesei culture medium, both lacking the C-terminal CBM and the 22.0 kDa polypeptide also lacking most of the linker region. The recombinant polypeptides were similar to each other in terms of specific activity, pH and temperature dependence. However, the 23.8 kDa and 22.0 kDa polypeptides were more thermostable at 80°C than the full-length enzyme. All polypeptide forms were effective in pretreatment of softwood kraft pulp at 80°C.

AB - Two endoxylanases, Nf Xyn11A and Nf Xyn10A, were cloned from a Nonomuraea flexuosa (previously Actinomadura flexuosa) DSM43186 genomic expression library in Escherichia coli. The coding sequences of xyn11A and xyn10A consist of 344 and 492 amino acids, respectively. The catalytic domains belong to family 11 and family 10 of glycoside hydrolases. The C-termini share strong amino acid sequence similarity to carbohydrate-binding module (CBM) families CBM2 and CBM13, respectively. Native Nf Xyn11A, and recombinant Xyn11A expressed in the filamentous fungus Trichoderma reesei, were purified from cultivation media and characterized. The molecular masses of the full-length enzymes determined by mass spectrometry were 32.9 kDa and 33.4 kDa, the recombinant enzyme having higher molecular mass due to glycosylation. In addition, shorter polypeptides with molecular masses of 23.8 kDa and 22.0 kDa were characterized from the T. reesei culture medium, both lacking the C-terminal CBM and the 22.0 kDa polypeptide also lacking most of the linker region. The recombinant polypeptides were similar to each other in terms of specific activity, pH and temperature dependence. However, the 23.8 kDa and 22.0 kDa polypeptides were more thermostable at 80°C than the full-length enzyme. All polypeptide forms were effective in pretreatment of softwood kraft pulp at 80°C.

U2 - 10.1007/s00253-004-1797-x

DO - 10.1007/s00253-004-1797-x

M3 - Article

VL - 67

SP - 495

EP - 505

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

IS - 4

ER -