Tyurin A А, Sadovskaya N S, Nikiforova Kh R, Mustafaev O N, Komakhin R A, Fadeev V S, Goldenkova-Pavlova I V
Institute of Plant Physiology, Russian Academy of Sciences, ul. Botanicheskaya 35, Moscow 127276, Russia; Department of Genetics and Biotechnology, Russian State Agrarian University-Moscow Timiryazev Agricultural Academy, ul. Timiryazevskaya 49, Moscow 127550, Russia.
Institute of Plant Physiology, Russian Academy of Sciences, ul. Botanicheskaya 35, Moscow 127276, Russia.
Biochim Biophys Acta. 2015 Jan;1854(1):10-9. doi: 10.1016/j.bbapap.2014.10.012. Epub 2014 Oct 23.
The Clostridium thermocellum lichenase (endo-β-1,3;1,4-glucan-D-glycosyl hydrolase) displays a high thermostability and specific activity and has a compact protein molecule, which makes it attractive, in particular, for protein engineering. We have utilized in silico analysis to construct circularly permuted (CP) variants and estimated the retained activity and thermostability. New open termini in the region of residues 53 or 99 in two lichenase CP variants (CN-53 and CN-99) had no effect on their activity and thermal tolerance versus another variant CP variant, CN-140 (cut in the region of residue 140), which displayed a dramatic decrease in the activity and thermostability. Construction and further activity and thermostability testing of the modified lichenase variants (M variants) and CP variants with peptides integrated via insertion fusion have demonstrated that the N-terminal regions in the lichenase catalytic domain (53 and 99 amino acid residues) that permit circular permutations with retention of activity and thermostability of the enzyme as well as the region between the C and N termini of the native lichenase in thermostable and active lichenase variants (CN-53 and CN-99) may be used for integrating small peptides without the loss of activity and thermostability. These findings not only suggest that CP predictions can be used in search for internal integration sites within protein molecule, but also form the background for further enzymatic engineering of the C. thermocellum thermostable lichenase aiming to create new fusion proteins.
嗜热栖热放线菌地衣酶（内切-β-1,3；1,4-葡聚糖-D-糖苷水解酶）具有很高的热稳定性和比活性，并且蛋白质分子结构紧凑，这使其在蛋白质工程领域具有很大吸引力。我们利用计算机分析构建了环形排列（CP）变体，并评估了其保留活性和热稳定性。两种地衣酶CP变体（CN-53和CN-99）中残基53或99区域的新开放末端对其活性和耐热性没有影响，而另一种CP变体CN-140（在残基140区域切割）的活性和热稳定性则显著下降。对修饰的地衣酶变体（M变体）和通过插入融合整合了肽的CP变体进行构建并进一步测试其活性和热稳定性，结果表明，地衣酶催化结构域中的N端区域（53和99个氨基酸残基）允许进行环形排列，同时保留酶的活性和热稳定性，并且在热稳定且有活性的地衣酶变体（CN-53和CN-99）中，天然地衣酶C端和N端之间的区域可用于整合小肽而不会丧失活性和热稳定性。这些发现不仅表明CP预测可用于寻找蛋白质分子内的内部整合位点，还为嗜热栖热放线菌热稳定地衣酶的进一步酶工程改造奠定了基础，旨在创造新的融合蛋白。
