Zhou Haiyan, Yong Jie, Gao Han, Li Tong, Xiao Hongshi, Wu Yongyao
College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, China.
J Sci Food Agric. 2017 May;97(7):2199-2204. doi: 10.1002/jsfa.8029. Epub 2016 Oct 13.
Mannanases are important enzymes which are widely used as a tool in agriculture and food industries. To improve the performance of mannanase Man23, a mutant library was created with rational design, and mutations were introduced on loops around the catalytic region. The Brevibacillus brevis B16 cell-free system which was created in this experiment provided the ability to express the mutant library efficiently. The activities of mutants were measured with a multi-volume spectrophotometer.
The mutant Man1606 gained from this system is a sextet which has mutations of N146G, S147H, S156P, T157Y, Q206S and T249H simultaneously on loops 6, 8 and 10. Man1606 showed higher activity and stability than Man23. The optimal temperature of Man1606 rose by 5 °C (from 55 to 60 °C) and the optimal pH increased slightly but its range became broader.
This experiment demonstrated the B. brevis cell-free system shortens the expression time and is an efficient tool for mannanase engineering. © 2016 Society of Chemical Industry.
甘露聚糖酶是重要的酶类，在农业和食品工业中被广泛用作工具。为了提高甘露聚糖酶Man23的性能，通过合理设计构建了一个突变文库，并在催化区域周围的环上引入了突变。本实验构建的短短芽孢杆菌B16无细胞系统具备高效表达突变文库的能力。使用多体积分光光度计测量突变体的活性。
从该系统获得的突变体Man1606是一个六重突变体，在环6、8和10上同时具有N146G、S147H、S156P、T157Y、Q206S和T249H突变。Man1606比Man23表现出更高的活性和稳定性。Man1606的最适温度提高了5℃（从55℃升至60℃），最适pH略有升高但其范围变宽。
本实验证明短短芽孢杆菌无细胞系统缩短了表达时间，是甘露聚糖酶工程的有效工具。©2016化学工业协会。
