Eibinger Manuel, Sigl Karin, Sattelkow Jürgen, Ganner Thomas, Ramoni Jonas, Seiboth Bernhard, Plank Harald, Nidetzky Bernd
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, 8010 Graz, Austria.
Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology, Steyrergasse 17, 8010 Graz, Austria.
Biotechnol Biofuels. 2016 Aug 26;9(1):178. doi: 10.1186/s13068-016-0590-2. eCollection 2016.
Through binding to cellulose, expansin-like proteins are thought to loosen the structural order of crystalline surface material, thus making it more accessible for degradation by hydrolytic enzymes. Swollenin SWO1 is the major expansin-like protein from the fungus Trichoderma reesei. Here, we have performed a detailed characterization of a recombinant native form of SWO1 with respect to its possible auxiliary role in the enzymatic saccharification of lignocellulosic substrates.
The swo1 gene was overexpressed in T. reesei QM9414 Δxyr1 mutant, featuring downregulated cellulase production, and the protein was purified from culture supernatant. SWO1 was N-glycosylated and its circular dichroism spectrum suggested a folded protein. Adsorption isotherms (25 °C, pH 5.0, 1.0 mg substrate/mL) revealed SWO1 to be 120- and 20-fold more specific for binding to birchwood xylan and kraft lignin, respectively, than for binding to Avicel PH-101. The SWO1 binding capacity on lignin (25 µmol/g) exceeded 12-fold that on Avicel PH-101 (2.1 µmol/g). On xylan, not only the binding capacity (22 µmol/g) but also the affinity of SWO1 (K d = 0.08 µM) was enhanced compared to Avicel PH-101 (K d = 0.89 µM). SWO1 caused rapid release of a tiny amount of reducing sugars (<1 % of total) from different substrates (Avicel PH-101, nanocrystalline cellulose, steam-pretreated wheat straw, barley β-glucan, cellotetraose) but did not promote continued saccharification. Atomic force microscopy revealed that amorphous cellulose films were not affected by SWO1. Also with AFM, binding of SWO1 to cellulose nanocrystallites was demonstrated at the single-molecule level, but adsorption did not affect this cellulose. SWO1 exhibited no synergy with T. reesei cellulases in the hydrolysis of the different celluloses. However, SWO1 boosted slightly (1.5-fold) the reducing sugar release from a native grass substrate.
SWO1 is a strongly glycosylated protein, which has implications for producing it in heterologous hosts. Although SWO1 binds to crystalline cellulose, its adsorption to xylan is much stronger. SWO1 is not an auxiliary factor of the enzymatic degradation of a variety of cellulosic substrates. Effect of SWO1 on sugar release from intact plant cell walls might be exploitable with certain (e.g., mildly pretreated) lignocellulosic feedstocks.
通过与纤维素结合，类伸展蛋白被认为可以松解晶体表面物质的结构秩序，从而使其更易于被水解酶降解。肿胀素SWO1是里氏木霉中主要的类伸展蛋白。在此，我们对重组天然形式的SWO1在木质纤维素底物酶促糖化过程中可能的辅助作用进行了详细表征。
swo1基因在纤维素酶产量下调的里氏木霉QM9414 Δxyr1突变体中过表达，并从培养上清液中纯化出该蛋白。SWO1进行了N-糖基化修饰，其圆二色光谱表明该蛋白具有折叠结构。吸附等温线（25°C，pH 5.0，1.0 mg底物/mL）显示，SWO1与桦木木聚糖和硫酸盐木质素的结合特异性分别比与微晶纤维素PH-101高120倍和20倍。SWO1对木质素的结合能力（25 μmol/g）超过对微晶纤维素PH-101的12倍（2.1 μmol/g）。与微晶纤维素PH-101（Kd = 0.89 μM）相比，SWO1在木聚糖上不仅结合能力（22 μmol/g）增强，亲和力（Kd = 0.08 μM）也有所提高。SWO1能使不同底物（微晶纤维素PH-101、纳米晶纤维素、蒸汽预处理麦秸、大麦β-葡聚糖、纤维四糖）快速释放少量还原糖（<总量的1%），但不能促进持续糖化。原子力显微镜显示，无定形纤维素膜不受SWO1影响。同样通过原子力显微镜，在单分子水平上证明了SWO1与纤维素纳米晶体的结合，但吸附并未影响这种纤维素。在不同纤维素的水解过程中，SWO1与里氏木霉纤维素酶没有协同作用。然而，SWO1能使天然草类底物的还原糖释放量略有增加（1.5倍）。
SWO1是一种高度糖基化的蛋白，这对在异源宿主中生产它具有重要意义。尽管SWO1能与结晶纤维素结合，但其与木聚糖的吸附作用更强。SWO1不是多种纤维素底物酶促降解的辅助因子。SWO1对完整植物细胞壁糖释放的影响可能在某些（如轻度预处理的）木质纤维素原料中得到利用。
