CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China.
J Appl Microbiol. 2014 Jul;117(1):151-9. doi: 10.1111/jam.12519. Epub 2014 May 2.
The study was to examine whether glycosylation could improve the thermostability of recombinant Thermomyces lanuginosus lipase (Tll) expressed in Pichia pastoris.
The Tll gene was synthesized and transformed into Pichia pastoris GS115.The recombinant Tll protein was expressed and purified, and its glycosylation site was identified by LCMS/MS as Asn-33. Two nonglycosylated mutants were constructed and the variant proteins were also expressed and purified. Effects of temperature on activities of the wild-type Tll and variants were analysed. The glycosylated Tll exhibited better thermostability than nonglycosylated variants.
Our experiments have demonstrated the improvement of Tll thermostability by Asn-33 glycosylation.
This work has deepened our understanding in the mechanism of Tll thermostability and will guide us to directional improvement of lipases and even other industrial enzymes.
本研究旨在探讨糖基化是否能提高毕赤酵母表达的重组嗜热真菌脂肪酶（Tll）的热稳定性。
Tll 基因被合成并转化到毕赤酵母 GS115 中。重组 Tll 蛋白被表达和纯化，并通过 LCMS/MS 鉴定其糖基化位点为 Asn-33。构建了两个非糖基化突变体，并表达和纯化了变体蛋白。分析了温度对野生型 Tll 和变体蛋白活性的影响。糖基化 Tll 表现出比非糖基化变体更好的热稳定性。
我们的实验证明了 Asn-33 糖基化可以提高 Tll 的热稳定性。
这项工作加深了我们对 Tll 热稳定性机制的理解，并将指导我们对脂肪酶甚至其他工业酶的定向改进。
