Université de Toulouse, INSA, UPS, INP, France.
Bioresour Technol. 2010 Nov;101(21):8237-43. doi: 10.1016/j.biortech.2010.05.097. Epub 2010 Jun 25.
To implement a protein engineering strategy for the improvement of enzyme performance on biomass, a straightforward, robust high-throughput method was devised and tested with recombinant GH11 xylanase as acting on wheat straw. The method requires automated liquid handling equipment, but avoids the need for specialized milling and powder weighing devices and the use of labour intensive steps such as manual cutting of pipette tips. After expression in Escherichia coli cells grown in microtiter plates, recombinant xylanase was released into the culture medium and used directly for biomass hydrolysis. Reactions were monitored using a micro-3,5-dinitrosalicylic acid assay. The cumulative error of the method was less than 15%. To validate the method, randomly generated xylanase mutants were analyzed. This allowed the detection of one mutant, which produced a 74% increase in hydrolysis compared to the parental enzyme. Closer analysis revealed that this increase in activity was correlated with a twofold increase in xylanase expression.
为了在生物质上实施改进酶性能的蛋白质工程策略，设计并测试了一种直接、稳健的高通量方法，该方法以作用于小麦秸秆的重组 GH11 木聚糖酶为研究对象。该方法需要自动化液体处理设备，但避免了专用粉碎和粉末称重设备的需求，以及使用劳动力密集的步骤，如手动切割移液管吸头。在大肠杆菌细胞中表达后，重组木聚糖酶被释放到培养基中，并直接用于生物质水解。使用微 3,5-二硝基水杨酸法监测反应。该方法的累积误差小于 15%。为了验证该方法，分析了随机生成的木聚糖酶突变体。这使得能够检测到一种突变体，与亲本酶相比，其水解活性提高了 74%。进一步分析表明，这种活性的提高与木聚糖酶表达量增加两倍相关。
