Department of Chemical & Biological Engineering, Princeton University, Princeton, NJ 08544, USA.
Biotechnol Lett. 2012 Jan;34(1):91-6. doi: 10.1007/s10529-011-0743-0. Epub 2011 Sep 29.
Cellulose degradation is essential for the future production of many advanced biofuels. Cellulases from the filamentous fungus Trichoderma reesei are among the most efficient enzymes for the hydrolysis of cellulosic materials. One of the cellulases from T. reesei, cellobiohydrolase II (CBH2), was studied because of its industrial relevance and proven enzymatic activity. Using both crude and rigorous membrane fractionation methods we show that full length T. reesei CBH2 is exclusively localized to the outer membrane when expressed recombinantly in Escherichia coli. Even fusing signal sequence-free maltose-binding protein to the N-terminus of CBH2, which has been shown to increase solubility of other proteins, did not prevent the outer membrane localization of CBH2. These results highlight the difficulties in producing fungal cellulases in bacterial hosts and provide a stepping stone for future cellulase engineering efforts.
纤维素降解对于未来许多先进生物燃料的生产至关重要。丝状真菌里氏木霉的纤维素酶是水解纤维素材料最有效的酶之一。里氏木霉的一种纤维素酶，即纤维二糖水解酶 II（CBH2），因其工业相关性和已被证实的酶活性而受到研究。我们使用粗分级和严格的膜分级方法表明，当在大肠杆菌中重组表达全长里氏木霉 CBH2 时，它完全定位于外膜。即使将无信号序列的麦芽糖结合蛋白融合到 CBH2 的 N 端，这已被证明可以增加其他蛋白质的溶解度，也不能阻止 CBH2 的外膜定位。这些结果突出了在细菌宿主中生产真菌纤维素酶的困难，并为未来的纤维素酶工程努力提供了一个垫脚石。
