Cloning, expression and characterization of endo-beta-1,4-mannanase from Aspergillus fumigatus in Aspergillus sojae and Pichia pastoris.
To be utilized in biomass conversion, including ethanol production and galactosylated oligosaccharide synthesis, namely prebiotics, the gene of extracellular endo-beta-1,4-mannanase (EC 3.2.1.78) of Aspergillus fumigatus IMI 385708 (formerly known as Thermomyces lanuginosus IMI 158749) was expressed first in Aspergillus sojae and then in Pichia pastoris under the control of the glyceraldehyde triphosphate dehydrogenase (gpdA) and the alcohol oxidase (AOX1) promoters, respectively. The highest production of mannanase (352 U mL(-1)) in A. sojae was observed after 6 days of cultivation. In P. pastoris, the highest mannanase production was observed 10 h after induction with methanol (61 U mL(-1)). The fold increase in mannanase production was estimated as approximately 12-fold and approximately 2-fold in A. sojae and P. pastoris, respectively, when compared with A. fumigatus. Both recombinant enzymes showed molecular mass of about 60 kDa and similar specific activities ( approximately 350 U mg(-1) protein). Temperature optima were at 60 degrees C and 45 degrees C, and maximum activity was at pH 4.5 and 5.2 for A. sojae and P. pastoris, respectively. The enzyme from P. pastoris was more stable retaining most of the activity up to 50 degrees C, whereas the enzyme from A. sojae rapidly lost activity above 40 degrees C.
Duruksu
Gökhan
G
Dept. of Biotechnology, Middle East Technical University, Ankara, Turkey.
Ozturk
Bengu
B
Biely
Peter
P
Bakir
Ufuk
U
Ogel
Zumrut B
ZB
D001230:Aspergillus / Q000235:genetics / Q000378:metabolism*; D001232:Aspergillus fumigatus / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008361:Mannosidases / Q000235:genetics* / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase