| 摘要: |
| 【目的】 为了提高来源于宇佐美曲霉（Aspergillus usamii）的高比活木聚糖酶Xyn Ⅱ的热稳定性，对其热稳定性影响因素进行改造。【方法】 对木聚糖酶Xyn Ⅱ进行同源建模和序列比较，在Xyn Ⅱ的“Ser/Thr”平面引入精氨酸的四点诱变和五点诱变，对酶的热稳定性进行改造。【结果】 获得的2个突变酶在热稳定性上较野生型酶均有不同程度提高，突变酶ST4、ST5的最适温度分别由原酶的50 ℃提高为52和55 ℃。55 ℃保温15 min，ST4、ST5的残留酶活性由原酶的20%提高为65%和75%；保温1 h，ST4、ST5的残留酶活性由原酶的15%提高为50%和65%。【结论】 突变酶ST4、ST5在保持了Xyn Ⅱ优良性质的基础上，进一步提高了其热稳定性，具有更好的应用价值。 |
| 关键词: 宇佐美曲霉 木聚糖酶 热稳定性 定向诱变 “Ser/Thr”平面改造 |
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| 基金项目:国家“863”高新技术研究与发展计划项目（2006AA10Z305） |
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| Effect of engineering of multiple arginines into the Ser/Thr surface of xylanase XynⅡ on thermostability |
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ZHOU Chen-yan,WANG Wu and WU Min-chen,ZHOU Chen-yan,WANG Wu and WU Min-chen,ZHOU Chen-yan,WANG Wu and WU Min-chen
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| Abstract: |
| 【Objective】 The research was done in order to increase the thermotolerance of xylanase XynⅡ from Aspergillus usamii.【Method】 A homology modeling of XynⅡ was constructed by SWISS-MODEL and BLAST.Replacing several serine and threonine residues on the Ser/Thr surface of XynⅡ with arginines effectively increased the thermostability of the enzyme.【Result】 The substitution of Ser and Thr residues on the Ser/Thr surface of the enzyme with four (ST4) or five arginines (ST5) led to an increase in optimal temperature of the enzymes by 2 ℃ and 5 ℃ for the ST4 and ST5,respectively.The modified enzymes ST4 and ST5 showed 65% and 75% of maximal activities after incubated for 15 min at 55 ℃ compared to only 20% activity for wild-type enzyme.After incubated for 1 h at 55 ℃,ST4 and ST5 showed 50% and 65% of maximal activity compared to only 15% activity for wild-type enzyme.【Conclusion】 Having the good properties of XynⅡ,the mutants with higher thermostability are potentially useful in industrial applications. |
| Key words: Aspergillus usamii xylanase thermostability site-directed mutagenesis alteration of “Ser/Thr” surface |
