A thermotolerant Endo-1,4-β-mannanase from Trichoderma virens UKM1: Cloning, recombinant expression and characterization

Sin Yee Chai, Farah Diba Abu Bakar, Nor Muhammad Mahadi, Abdul Munir Abd. Murad

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5 Citations (Scopus)

Abstract

A gene encoding a thermotolerant endo-1,4-β-mannanase belonging to glycosyl hydrolase family 5 (GH5) was isolated from the fungal strain Trichoderma virens UKM1 (manTV). The aim of this work was to heterologously express and characterize manTV for subsequent applications. The 1329 bp β-mannanase gene was cloned and expressed in Pichia pastoris X33 yeast cells, and the recombinant mannanase (rMANTV) was expressed as a His6-tagged glycoprotein of approximately 65-70 kDa. The purified rMANTV showed a specific activity of 415.49 U mg-1 for 0.5% locust bean gum (LBG). This enzyme had a high optimum temperature, 70°C, with stability from 20°C to 65°C. The rMANTV had its highest activity at pH 5, with a wide pH stability range of pH 3-9. It was relatively stable in the presence of several metal ions and chemical substances. In addition, rMANTV had Km values of 2.61 mg mL-1 and 1.49 mg mL-1 for LBG and Konjac glucomannan, respectively. Its catalytic efficiency (Kcat/Km) was 225.41 ± 20.14 mL mg-1 s-1 for LBG and 336.67 ± 27.39 mL mg-1 s-1 for Konjac glucomannan. The high temperature tolerance of this endo-1,4-β-mannanase makes it a good potential candidate for industrial applications.

Original languageEnglish
Pages (from-to)49-57
Number of pages9
JournalJournal of Molecular Catalysis B: Enzymatic
Volume125
DOIs
Publication statusPublished - 1 Mar 2016

Fingerprint

Trichoderma
Cloning
Organism Cloning
Hydrolases
Glycoproteins
Gene encoding
Yeast
Industrial applications
Metal ions
Temperature
Pichia
Enzymes
Genes
Cells
Yeasts
Metals
Ions
locust bean gum
konjac gluco-mannan

Keywords

  • Pichia pastoris
  • Recombinant protein
  • Thermotolerant
  • Trichoderma virens
  • β-mannanase

ASJC Scopus subject areas

  • Biochemistry
  • Bioengineering
  • Catalysis
  • Process Chemistry and Technology

Cite this

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title = "A thermotolerant Endo-1,4-β-mannanase from Trichoderma virens UKM1: Cloning, recombinant expression and characterization",
abstract = "A gene encoding a thermotolerant endo-1,4-β-mannanase belonging to glycosyl hydrolase family 5 (GH5) was isolated from the fungal strain Trichoderma virens UKM1 (manTV). The aim of this work was to heterologously express and characterize manTV for subsequent applications. The 1329 bp β-mannanase gene was cloned and expressed in Pichia pastoris X33 yeast cells, and the recombinant mannanase (rMANTV) was expressed as a His6-tagged glycoprotein of approximately 65-70 kDa. The purified rMANTV showed a specific activity of 415.49 U mg-1 for 0.5{\%} locust bean gum (LBG). This enzyme had a high optimum temperature, 70°C, with stability from 20°C to 65°C. The rMANTV had its highest activity at pH 5, with a wide pH stability range of pH 3-9. It was relatively stable in the presence of several metal ions and chemical substances. In addition, rMANTV had Km values of 2.61 mg mL-1 and 1.49 mg mL-1 for LBG and Konjac glucomannan, respectively. Its catalytic efficiency (Kcat/Km) was 225.41 ± 20.14 mL mg-1 s-1 for LBG and 336.67 ± 27.39 mL mg-1 s-1 for Konjac glucomannan. The high temperature tolerance of this endo-1,4-β-mannanase makes it a good potential candidate for industrial applications.",
keywords = "Pichia pastoris, Recombinant protein, Thermotolerant, Trichoderma virens, β-mannanase",
author = "Chai, {Sin Yee} and {Abu Bakar}, {Farah Diba} and Mahadi, {Nor Muhammad} and {Abd. Murad}, {Abdul Munir}",
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T1 - A thermotolerant Endo-1,4-β-mannanase from Trichoderma virens UKM1

T2 - Cloning, recombinant expression and characterization

AU - Chai, Sin Yee

AU - Abu Bakar, Farah Diba

AU - Mahadi, Nor Muhammad

AU - Abd. Murad, Abdul Munir

PY - 2016/3/1

Y1 - 2016/3/1

N2 - A gene encoding a thermotolerant endo-1,4-β-mannanase belonging to glycosyl hydrolase family 5 (GH5) was isolated from the fungal strain Trichoderma virens UKM1 (manTV). The aim of this work was to heterologously express and characterize manTV for subsequent applications. The 1329 bp β-mannanase gene was cloned and expressed in Pichia pastoris X33 yeast cells, and the recombinant mannanase (rMANTV) was expressed as a His6-tagged glycoprotein of approximately 65-70 kDa. The purified rMANTV showed a specific activity of 415.49 U mg-1 for 0.5% locust bean gum (LBG). This enzyme had a high optimum temperature, 70°C, with stability from 20°C to 65°C. The rMANTV had its highest activity at pH 5, with a wide pH stability range of pH 3-9. It was relatively stable in the presence of several metal ions and chemical substances. In addition, rMANTV had Km values of 2.61 mg mL-1 and 1.49 mg mL-1 for LBG and Konjac glucomannan, respectively. Its catalytic efficiency (Kcat/Km) was 225.41 ± 20.14 mL mg-1 s-1 for LBG and 336.67 ± 27.39 mL mg-1 s-1 for Konjac glucomannan. The high temperature tolerance of this endo-1,4-β-mannanase makes it a good potential candidate for industrial applications.

AB - A gene encoding a thermotolerant endo-1,4-β-mannanase belonging to glycosyl hydrolase family 5 (GH5) was isolated from the fungal strain Trichoderma virens UKM1 (manTV). The aim of this work was to heterologously express and characterize manTV for subsequent applications. The 1329 bp β-mannanase gene was cloned and expressed in Pichia pastoris X33 yeast cells, and the recombinant mannanase (rMANTV) was expressed as a His6-tagged glycoprotein of approximately 65-70 kDa. The purified rMANTV showed a specific activity of 415.49 U mg-1 for 0.5% locust bean gum (LBG). This enzyme had a high optimum temperature, 70°C, with stability from 20°C to 65°C. The rMANTV had its highest activity at pH 5, with a wide pH stability range of pH 3-9. It was relatively stable in the presence of several metal ions and chemical substances. In addition, rMANTV had Km values of 2.61 mg mL-1 and 1.49 mg mL-1 for LBG and Konjac glucomannan, respectively. Its catalytic efficiency (Kcat/Km) was 225.41 ± 20.14 mL mg-1 s-1 for LBG and 336.67 ± 27.39 mL mg-1 s-1 for Konjac glucomannan. The high temperature tolerance of this endo-1,4-β-mannanase makes it a good potential candidate for industrial applications.

KW - Pichia pastoris

KW - Recombinant protein

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KW - Trichoderma virens

KW - β-mannanase

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