Characterisation and stabilisation of recombinant humicola insolens endoglucanase produced in pichia pastoris

S. S. Abdul Fattah, R. Mohamed, W. M K Wan Seman, Jamaliah Md Jahim, R. M. Illias, Farah Diba Abu Bakar, Abdul Munir Abd. Murad

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Cellulases are industrially important hydrolytic enzymes that are applicable in the bioconversion of cellulosic biomass to simple sugars. In this work, Pichia pastoris carrying an endoglucanase cDNA (CMC3) from a thermophilic fungus, Humicola insolens, was grown in a 30 L bioreactor to produce recombinant CMC3 in a fed-batch cultivation mode. After optimisation of the cultivation conditions, a total of 5.3 gL -1 proteins were obtained in a 20 L working volume after a 40 h induction with methanol. CMC3 expresses a β-1,4-endoglucanase with a specific activity of 62.83 U mg -1 , demonstrating its specificity for hydrolysing carboxymethyl cellulose as a substrate. No detectable hydrolysis on Sigmacell ® cellulose, Avicel or beechwood xylan was observed. The recombinant CMC3 displayed moderate thermostability, being stable at up to 50°C for more than 72 h. Metal ions such as Mn² + and Co² + enhanced the CMC3 activity, while Ni + , Zn² + and Cu² + inhibited the enzyme activity. The CMC3 produced in P. pastoris was stable under long-term storage, retaining 84% and 75% of its initial activity after 4 months of storage at 4°C and 25°C, respectively. The addition of stabilisers further improved the enzyme stability by 7% and 5% at 4°C and 25°C, respectively.

Original languageEnglish
Pages (from-to)155-159
Number of pages5
JournalMalaysian Applied Biology
Volume44
Issue number1
Publication statusPublished - 2015

Fingerprint

Humicola insolens
Pichia pastoris
Pichia
Cellulase
endo-1,4-beta-glucanase
Cellulose
thermophilic fungi
enzyme stability
Enzyme Stability
Cellulases
Xylans
Carboxymethylcellulose Sodium
cellulases
carboxymethylcellulose
xylan
Bioreactors
Enzymes
bioreactors
biotransformation
thermal stability

Keywords

  • Characterisation
  • Endoglucanase
  • Humicola insolens
  • Production
  • Stabilisation

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)

Cite this

Characterisation and stabilisation of recombinant humicola insolens endoglucanase produced in pichia pastoris. / Abdul Fattah, S. S.; Mohamed, R.; Wan Seman, W. M K; Md Jahim, Jamaliah; Illias, R. M.; Abu Bakar, Farah Diba; Abd. Murad, Abdul Munir.

In: Malaysian Applied Biology, Vol. 44, No. 1, 2015, p. 155-159.

Research output: Contribution to journalArticle

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AB - Cellulases are industrially important hydrolytic enzymes that are applicable in the bioconversion of cellulosic biomass to simple sugars. In this work, Pichia pastoris carrying an endoglucanase cDNA (CMC3) from a thermophilic fungus, Humicola insolens, was grown in a 30 L bioreactor to produce recombinant CMC3 in a fed-batch cultivation mode. After optimisation of the cultivation conditions, a total of 5.3 gL -1 proteins were obtained in a 20 L working volume after a 40 h induction with methanol. CMC3 expresses a β-1,4-endoglucanase with a specific activity of 62.83 U mg -1 , demonstrating its specificity for hydrolysing carboxymethyl cellulose as a substrate. No detectable hydrolysis on Sigmacell ® cellulose, Avicel or beechwood xylan was observed. The recombinant CMC3 displayed moderate thermostability, being stable at up to 50°C for more than 72 h. Metal ions such as Mn² + and Co² + enhanced the CMC3 activity, while Ni + , Zn² + and Cu² + inhibited the enzyme activity. The CMC3 produced in P. pastoris was stable under long-term storage, retaining 84% and 75% of its initial activity after 4 months of storage at 4°C and 25°C, respectively. The addition of stabilisers further improved the enzyme stability by 7% and 5% at 4°C and 25°C, respectively.

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