[Expression of endo-beta-mannanase gene from Trichoderma reesei in Pichia pastoris].
Complete mannanase gene with two introns was cloned from Trichoderrna reesei by PCR. The two introns were then removed by overlap extension PCR. The gene encoding the mature mannanase protein was inserted into the expression vector pPIC9K, downstream of a alpha-factor signal peptide sequence. The resultant recombinant vector was named pM242. After linearized with Sac I , pM242 was transformed to Pichia pastoris GS115 by electroporation. After screening, the recombinant strain Gpmf25 that expresses the secretory protein at high level was obtained. The activity of the recombinant mannanase reached 12.5 IU/mL. Optimum pH and temperature for the recombinant enzyme were 5.0 and 80 degrees C, respectively. The enzyme was stable at pH 5.0-6.0 and maintained over 50% of original activity after incubation at 70 degrees C for 30 min.
State Key Laboratory of Microbial Resources of Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
Yue-Hua
YH
Wei
Ai-Jun
AJ
Mao
Yong-Zhi
YZ
He
Yu
Y
Qiao
Zhi-Yang
ZY
Dong
D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics*; D044902:beta-Mannosidase / Q000096:biosynthesis* / Q000235:genetics
D005656:Fungal Proteins; D011994:Recombinant Proteins; D044902:beta-Mannosidase