State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
J Microbiol Biotechnol. 2009 Dec;19(12):1506-13. doi: 10.4014/jmb.0902.0096.
Alpha-dextranase, which can hydrolyze dextran, is largely used in the sugar industry. However, a thermostable alpha- dextranase is needed to alleviate the viscosity of syrups and clean blocked machines. Thus, to improve the optimal temperature of Lipomyces starkeyi alpha-dextranase expressed by Pichia pastoris, the rational introduction of a de novo designed disulfide bond was investigated. Based on the known structure of Penicillium minioluteum dextranase, L. starkeyi alpha-dextranase was constructed using homology modeling. Four amino acids residues were then selected for site-directed mutagenesis to cysteine. When compared with the wildtype dextranase, the mutant DexM2 (D279C/S289C) showed a more than 13oC improvement on its optimal temperature. DexM2 and DexM12 (T245C/N248C, D279C/S289C) also showed a better thermal stability than the wild-type dextranase. After the introduction of two disulfide bonds, the specific activity of DexM12 was evaluated and found to be two times higher than that of the wild-type. Moreover, DexM12 also showed the highest Vmax.
α-葡聚糖酶能够水解葡聚糖，在制糖工业中得到广泛应用。然而，需要一种热稳定的α-葡聚糖酶来减轻糖浆的黏度并清洁堵塞的机器。因此，为了提高毕赤酵母表达的里氏木霉α-葡聚糖酶的最适温度，研究了合理引入从头设计的二硫键。基于已知的小毛壳菌葡聚糖酶结构，使用同源建模构建了 L. starkeyiα-葡聚糖酶。然后选择四个氨基酸残基进行定点突变生成半胱氨酸。与野生型葡聚糖酶相比，突变体 DexM2（D279C/S289C）的最适温度提高了 13°C 以上。DexM2 和 DexM12（T245C/N248C、D279C/S289C）的热稳定性也优于野生型葡聚糖酶。引入两个二硫键后，评估了 DexM12 的比活性，发现比野生型高两倍。此外，DexM12 还表现出最高的 Vmax。
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