Engineering increased thermostability in the thermostable GH-11 xylanase from Thermobacillus xylanilyticus
Engineering increased thermostability in the thermostable GH-11 xylanase from Thermobacillus xylanilyticus
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DOI：
10.1016/j.jbiotec.2006.03.025
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发表时间：
2006-09-18
影响因子：
4.1
通讯作者：
O'Donohue, Michael J.
中科院分区：
文献类型：
--
作者：
Paes, Gabriel;O'Donohue, Michael J.
酶水解是对丰富的低成本农业副产品（如麦麸和秸秆）进行生物精炼的一种有吸引力的策略。然而，要采用这种方法，需要高效的酶，特别是木聚糖酶。为了促进麦麸中复杂细胞壁网络的热诱导解聚，从而提高酶水解效率，我们试图提高一种已经具有中等热稳定性的GH - 11木聚糖酶的耐热性。采用先前描述的涉及引入二硫键的工程策略，获得了一种显示出增强的热稳定性和热活性的突变体（Tx - xyl - SS3）。Tx - xyl - SS3在70℃下的半衰期（180分钟）是野生型酶的10倍，其比活性几乎翻倍（3500 IU/mg）。尽管有这些改进，Tx - xyl - SS3并不适合在明显更高的反应温度（即85 - 95℃）下使用，因此本研究的最初目标仍未实现。然而，出乎意料的是，即使在正常水解温度（60℃）下，Tx - xyl - SS3也能够溶解50%的麦麸阿拉伯木聚糖，比平行反应中的野生型酶高出10个百分点。这里呈现的数据表明，这种改进与热稳定性和/或热活性的提高没有直接联系，而是与可能使Tx - xyl - SS3更好地穿透麦麸细胞壁网络的其他未确定的理化性质变化有关。（c）2006 Elsevier B.V.保留所有权利。
Enzymatic hydrolysis constitutes an attractive strategy for biorefining of abundant, low-cost agricultural by-products such as wheat bran and straw. However, to adopt such an approach, efficient enzymes are required, in particular xylanases. To promote heat-induced disorganization of the complex cell wall network in wheat bran and thus increase enzymatic hydrolysis, we have attempted to improve the thermoresistance of a GH-11 xylanase that is already moderately thermostable. Using a previously described engineering strategy that involves the introduction of disulphide bridges, a mutant (Tx-xyl-SS3) displaying enhanced thermostability and thermoactivity was obtained. The half life at 70 degrees C (180 min) of Tx-xyl-SS3 is 10-fold greater than that of the wild type enzyme and its specific activity is almost doubled (3500 IU mg(-1)). Despite these improvements, Tx-xyl-SS3 was unsuitable for use at significantly higher reaction temperatures (i.e. 85-95 degrees C and thus the initial objective of this study remained unaccomplished. However, unexpectedly even at the normal hydrolytic temperature (60 degrees C), Tx-xyl-SS3 was able to solubilize 50% of the wheat bran arabinoxylans, 10 points more than the wild type enzyme in parallel reactions. The data presented here show that this improvement is not directly linked to the increase in thermostability and/or thermoactivity, but rather to other unidentified changes to physico-chemical properties that may allow Tx-xyl-SS3 to better penetrate the cell wall network in wheat bran. (c) 2006 Elsevier B.V. All rights reserved.