Thermostable carbohydrate binding module increases the thermostability and substrate-binding capacity of Trichoderma reesei xylanase 2
Thermostable carbohydrate binding module increases the thermostability and substrate-binding capacity of Trichoderma reesei xylanase 2
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DOI：
10.1016/j.nbt.2009.04.002
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发表时间：
2009-10-01
影响因子：
5.4
通讯作者：
Chen Daiwen
中科院分区：
文献类型：
--
作者：
He Jun;Yu Bing;Chen Daiwen
为了提高里氏木霉木聚糖酶2（Trichoderma reesei木聚糖酶2，Xyn 2）的热稳定性，将海栖热袍菌（Thermotoga maritima）XynA的热稳定结构域（A2）改造到Xyn 2蛋白的N端。利用甲醇诱导型醇氧化酶1（AOX 1）启动子和酵母分泌信号序列，在毕赤酵母中成功表达了xyn 2和hybrid基因。酿酒酵母（α-因子）。与表达天然Xyn 2基因的相应菌株相比，表达所产生的杂合基因的转化体明显增加了热稳定性和底物结合能力。杂合酶的活性在65 ℃时最高，比天然Xyn 2高10 ℃。杂交酶在60 ℃下是稳定的，在此温度下孵育30分钟后保留了85%以上的活性。该酶对木聚糖具有高度的专一性，对桦木木聚糖降解产物的分析表明，该酶是一种内切木聚糖酶，主要降解产物为木二糖和木三糖。这些属性应使其成为各种应用的有吸引力的申请人。我们的研究结果还表明，N端结构域A2负责T的热稳定性和底物结合能力。海百合
To improve the thermostability of Trichoderma reesei xylanase 2 (Xyn2), the thermostabilizing domain (A2) from Thermotoga maritima XynA were engineered into the N-terminal region of the Xyn2 protein. The xyn2 and hybrid genes were successfully expressed in Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from S. cerevisiae (alpha-factor). The transformants expressed the hybrid gene produced clearly increased both the thermostability and substrate-binding capacity compared to the corresponding strains expressed the native Xyn2 gene. The activity of the hybrid enzyme was highest at 65 degrees C that was 10 degrees C higher than the native Xyn2. The hybrid enzyme was stable at 60 degrees C and retained more than 85% of its activity after 30-min incubation at this temperature. The hybrid enzyme was highly specific toward xylan and analysis of the products from birchwood xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylotriose as the main degradation products. These attributes should make it an attractive applicant for various applications. Our results also suggested that the N-terminal domain A2 is responsible for both the thermostability and substrate-binding capacity of T. maritima XynA.