DNA shuffling是蛋白质定向进化的一种常用策略,其优点是可以快速积累多突变效果,但同时由于突变数较多,其中真正发挥作用的突变及其结构基础往往不清楚。β-葡萄糖苷酶是纤维素高效降解的限速酶,良好的热稳定性是影响其实际催化效率的关键因素。该研究以DNA shuffling策略产生的热稳定性β-葡萄糖苷酶突变体Bgl3-6511(含60个突变,T50值比野生型提高4.6 ℃)为研究对象,通过序列比对、定点突变和热稳定性测定,对其中的有益突变进行鉴定。结果显示,6个单点突变Y50F、R52H、R56K、V65I、T67A和P143A分别将该酶的T50值提高2.9、4.2、1.5、2.8、3.2、1.2 ℃。同时,鉴定到5个有害突变将该酶的T50值降低1.0~3.4 ℃。将获得单点有益突变进行组合,获得T50值提高13.4 ℃的M6(Y50F/R52H/R56K/V65I/T67A/P143A),说明DNA shuffling策略积累的有害突变确实损害了性能优化。结构分析和分子动力学模拟显示,有益突变主要是通过增强分子内氢键、π-π键和稳定二级结构发挥作用。该研究对Bgl3-6511中的单点有益突变进行鉴定,对其结构基础进行了分析,并获得了热稳定性更加优良的突变酶,相关信息可为其他酶的分子改造提供有益借鉴。
DNA shuffling is a wildly used strategy of directed evolution.It can quickly test the effects of multiple mutations, however, the actual beneficial mutations among them and their structural basis are often unclear.β-Glucosidase is the rate-limiting enzyme for efficient degradation of cellulose, and good thermostability is a key factor affecting its practical catalytic efficiency.In this work, the beneficial mutations of a thermostable β-glucosidase mutant Bgl3-6511 (containing 60 single mutations and has a T50 value 4.6 ℃ higher than wild-type) were identified through sequence alignment, site-directed mutagenesis, and thermostability assay.Results showed that six single point mutations, Y50F, R56K, V65I, T67A, and P143A, increased the T50 of the enzyme by 2.9, 4.2, 1.5, 2.8, 3.2, and 1.2 ℃, respectively.At the same time, 5 single mutations decreased T50 by 1.0-3.4 ℃ were identified.A combination of beneficial mutations gave rise to mutant M6 (Y50F/R52H/R56K/V65I/T67A/P143A) with a T50 increase of 13.4 ℃, indicating that the harmful mutations in 6511 indeed hampered the thermostability optimization.Structural analysis and molecular dynamic simulations showed that the beneficial mutations mainly increased/enhanced hydrogen bonds and Π-Π interactions, and made the local loops more rigid.In summary, this work identified the beneficial mutations in Bgl3-6511, analyzed their structural basis, and obtained a more thermostable mutant M6.These results may be useful for the protein engineering of other enzymes.
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