Duruksu Gökhan, Ozturk Bengu, Biely Peter, Bakir Ufuk, Ogel Zumrut B
Dept. of Biotechnology, Middle East Technical University, Ankara, Turkey.
Biotechnol Prog. 2009 Jan-Feb;25(1):271-6. doi: 10.1002/btpr.104.
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.
为了将其用于生物质转化，包括乙醇生产和半乳糖基化低聚糖（即益生元）的合成，烟曲霉IMI 385708（以前称为嗜热丝孢菌IMI 158749）的胞外内切β-1,4-甘露聚糖酶（EC 3.2.1.78）基因首先在大豆曲霉中表达，然后在甘油醛-3-磷酸脱氢酶（gpdA）和醇氧化酶（AOX1）启动子的控制下分别在巴斯德毕赤酵母中表达。在大豆曲霉中培养6天后观察到甘露聚糖酶的最高产量（352 U mL(-1)）。在巴斯德毕赤酵母中，用甲醇诱导10小时后观察到甘露聚糖酶的最高产量（61 U mL(-1)）。与烟曲霉相比，大豆曲霉和巴斯德毕赤酵母中甘露聚糖酶产量的增加倍数分别估计约为12倍和约2倍。两种重组酶的分子量均约为60 kDa，比活性相似（约350 U mg(-1)蛋白质）。大豆曲霉和巴斯德毕赤酵母的温度最适值分别为60℃和45℃，最大活性分别在pH 4.5和5.2时。巴斯德毕赤酵母的酶更稳定，在高达50℃时仍保留大部分活性，而大豆曲霉的酶在40℃以上迅速失去活性。
