Brandt Sophie C, Ellinger Bernhard, van Nguyen Thuat, Harder Sönke, Schlüter Hartmut, Hahnke Richard L, Rühl Martin, Schäfer Wilhelm, Gand Martin
Department of Molecular Phytopathology, University of Hamburg, Hamburg, Germany.
Department ScreeningPort, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Hamburg, Germany.
Front Microbiol. 2020 Sep 18;11:2154. doi: 10.3389/fmicb.2020.573482. eCollection 2020.
A prerequisite for the transition toward a biobased economy is the identification and development of efficient enzymes for the usage of renewable resources as raw material. Therefore, different xylanolytic enzymes are important for efficient enzymatic hydrolysis of xylan-heteropolymers. A powerful tool to overcome the limited enzymatic toolbox lies in exhausting the potential of unexplored habitats. By screening a Vietnamese fungal culture collection of 295 undiscovered fungal isolates, 12 highly active xylan degraders were identified. One of the best xylanase producing strains proved to be an strain from shrimp shell (Fsh102), showing a specific activity of 0.6 U/mg. Illumina dye sequencing was used to identify our Fsh102 strain and determine differences to the CBS 593.65 reference strain. With activity based in-gel zymography and subsequent mass spectrometric identification, three potential proteins responsible for xylan degradation were identified. Two of these proteins were cloned from the cDNA and, furthermore, expressed heterologously in and characterized. Both xylanases, were entirely different from each other, including glycoside hydrolases (GH) families, folds, substrate specificity, and inhibition patterns. The specific enzyme activity applying 0.1% birch xylan of both purified enzymes were determined with 181.1 ± 37.8 or 121.5 ± 10.9 U/mg for xylanase I and xylanase II, respectively. Xylanase I belongs to the GH11 family, while xylanase II is member of the GH10 family. Both enzymes showed typical endo-xylanase activity, the main products of xylanase I are xylobiose, xylotriose, and xylohexose, while xylobiose, xylotriose, and xylopentose are formed by xylanase II. Additionally, xylanase II showed remarkable activity toward xylotriose. Xylanase I is stable when stored up to 30°C and pH value of 9, while xylanase II started to lose significant activity stored at pH 9 after exceeding 3 days of storage. Xylanase II displayed about 40% activity when stored at 50°C for 24 h. The enzymes are tolerant toward mesophilic temperatures, while acting in a broad pH range. With site directed mutagenesis, the active site residues in both enzymes were confirmed. The presented activity and stability justify the classification of both xylanases as highly interesting for further development.
向生物基经济转型的一个先决条件是识别和开发用于将可再生资源用作原材料的高效酶。因此，不同的木聚糖分解酶对于木聚糖杂聚物的高效酶促水解很重要。克服有限酶工具箱的一个有力工具在于挖掘未探索栖息地的潜力。通过筛选越南真菌培养物保藏中心的295株未发现的真菌分离株，鉴定出12株高活性木聚糖降解菌。其中产木聚糖酶最好的菌株之一是一株来自虾壳的菌株（Fsh102），其比活性为0.6 U/mg。使用Illumina染料测序来鉴定我们的Fsh102菌株，并确定其与CBS 593.65参考菌株的差异。通过基于活性的凝胶酶谱分析和随后的质谱鉴定，鉴定出三种负责木聚糖降解的潜在蛋白质。其中两种蛋白质从cDNA中克隆出来，此外，在大肠杆菌中进行了异源表达并进行了表征。两种木聚糖酶彼此完全不同，包括糖苷水解酶（GH）家族、折叠结构、底物特异性和抑制模式。两种纯化酶在应用0.1%桦木木聚糖时的比酶活性分别测定为木聚糖酶I为181.1±37.8 U/mg，木聚糖酶II为121.5±10.9 U/mg。木聚糖酶I属于GH11家族，而木聚糖酶II是GH10家族的成员。两种酶均表现出典型的内切木聚糖酶活性，木聚糖酶I的主要产物是木二糖、木三糖和木六糖，而木聚糖酶II形成木二糖、木三糖和木戊糖。此外，木聚糖酶II对木三糖表现出显著活性。木聚糖酶I在30°C和pH值为9的条件下储存时稳定，而木聚糖酶II在pH值为9的条件下储存超过3天后开始显著丧失活性。木聚糖酶II在50°C下储存24小时后仍显示约40%的活性。这些酶在中温温度下具有耐受性，同时在较宽的pH范围内起作用。通过定点诱变，确认了两种酶中的活性位点残基。所呈现的活性和稳定性证明将这两种木聚糖酶归类为具有进一步开发的高度吸引力是合理的。
