Goedegebuur Frits, Dankmeyer Lydia, Gualfetti Peter, Karkehabadi Saeid, Hansson Henrik, Jana Suvamay, Huynh Vicky, Kelemen Bradley R, Kruithof Paulien, Larenas Edmund A, Teunissen Pauline J M, Ståhlberg Jerry, Payne Christina M, Mitchinson Colin, Sandgren Mats
From DuPont Industrial Biosciences, Archimedesweg 30, Leiden 2333CN, The Netherlands,
From DuPont Industrial Biosciences, Archimedesweg 30, Leiden 2333CN, The Netherlands.
J Biol Chem. 2017 Oct 20;292(42):17418-17430. doi: 10.1074/jbc.M117.803270. Epub 2017 Aug 31.
Secreted mixtures of cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 °C increase in and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 °C. The X-ray crystal structure of the catalytic domain was determined at 2.1 Å resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites.
纤维素酶的分泌混合物能够在大规模、具有商业相关性的规模下有效地将纤维素生物质降解为可发酵糖。来自糖苷水解酶家族7的纤维二糖水解酶I（Cel7A）是该过程的主力酶。然而，Cel7A的热稳定性限制了其在温度不高于50°C的过程中的使用。提高热稳定性对于能够使用更高的加工温度和提高工业生物质转化的经济可行性是可取的。在这里，我们通过定向进化提高了Cel7A的热稳定性。将具有增强热稳定性特性的位点进行组合，获得了一个Cel7A变体（FCA398），与野生型酶相比，其熔点提高了10.4°C，半衰期延长了44倍。这个Cel7A变体包含18个突变位点，在至少75°C的应用条件下仍具有活性。催化结构域的X射线晶体结构在2.1 Å分辨率下确定，结果表明突变的影响是局部的，不会引起主要的主链构象变化。分子动力学模拟显示，野生型Cel7A和FCA398变体的催化结构域在300 K时表现出相似的行为，而在升高的温度（475和525 K）下，FCA398变体波动较小，并且随着时间的推移保持更多的天然接触。结合结构和动力学研究，为许多突变位点的稳定作用提出了理论依据。
