Heterologous expression of a gene for thermostable xylanase from Chaetomium thermophilum in Pichia pastoris GS115.
We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml(-1) while that of recombinant without intron (xyn669) was 1.26 U ml(-1) after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5-8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.
Ghaffar
Abdul
A
Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad 38040, Pakistan. aghaffaruaf@yahoo.com
Khan
Sher Afzal
SA
Mukhtar
Zahid
Z
Rajoka
Muhammad Ibrahim
MI
Latif
Farooq
F
D000595:Amino Acid Sequence; D002597:Chaetomium / Q000201:enzymology*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D043325:Xylan Endo-1,3-beta-Xylosidase / Q000235:genetics* / Q000378:metabolism*
D005656:Fungal Proteins; D011994:Recombinant Proteins; D043325:Xylan Endo-1,3-beta-Xylosidase