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 language | English |
|---|---|
| Pages (from-to) | 49-57 |
| Number of pages | 9 |
| Journal | Journal of Molecular Catalysis B: Enzymatic |
| Volume | 125 |
| DOIs | |
| Publication status | Published - 1 Mar 2016 |
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Keywords
- Pichia pastoris
- Recombinant protein
- Thermotolerant
- Trichoderma virens
- β-mannanase
ASJC Scopus subject areas
- Biochemistry
- Bioengineering
- Catalysis
- Process Chemistry and Technology
Cite this
A thermotolerant Endo-1,4-β-mannanase from Trichoderma virens UKM1 : Cloning, recombinant expression and characterization. / Chai, Sin Yee; Abu Bakar, Farah Diba; Mahadi, Nor Muhammad; Abd. Murad, Abdul Munir.
In: Journal of Molecular Catalysis B: Enzymatic, Vol. 125, 01.03.2016, p. 49-57.Research output: Contribution to journal › Article
}
TY - JOUR
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
KW - Thermotolerant
KW - Trichoderma virens
KW - β-mannanase
UR - http://www.scopus.com/inward/record.url?scp=84955584157&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84955584157&partnerID=8YFLogxK
U2 - 10.1016/j.molcatb.2015.12.011
DO - 10.1016/j.molcatb.2015.12.011
M3 - Article
AN - SCOPUS:84955584157
VL - 125
SP - 49
EP - 57
JO - Journal of Molecular Catalysis - B Enzymatic
JF - Journal of Molecular Catalysis - B Enzymatic
SN - 1381-1177
ER -