基于体外分子进化技术提高弯曲芽孢杆菌CCTCC 2015368 β-淀粉酶的热稳定性

Enhancing thermostability of β-amylase from Bacillus flexus CCTCC 2015368 based on in vitro molecular evolution

摘要:

运用体外分子进化技术易错PCR方法，高通量筛选热稳定性提高的弯曲芽孢杆菌Bacillus flexus CCTCC 2015368 β-淀粉酶突变体。利用LB琼脂淀粉板显色、96-孔板DNS法测酶活和酶标仪检测等，最终筛选到了一株热稳定性显著提高的突变体D476N。野生型和突变体D476N 分别纯化后，酶学性质测定表明：突变体D476N的最适pH为6.5，与野生型相比降低了0.5。突变体D476N和野生型的最适温度均为55 ℃，突变体D476N在55 ℃下的半衰期为35 min，比野生型提高了95%。突变体D476N的T50值比野生型提高4 ℃。突变体D476N的Km值为97.98 μmol/L，是野生型 (85.86 μmol/L) 1.14倍；突变体稳定性提高的同时，催化活力相对于野生型有略微下降。通过SWISS-MODEL同源模拟野生型和突变体D476N的三维结构，并通过PyMol软件分析，发现突变后的氨基酸残基Asn476位于蛋白质表面的loop环上，通过MOE软件计算，D476N的分子自由能 (ΔG) 为106.01 kcal/mol，比野生酶降低10.3%，这一结果与蛋白质分子自由能和热稳定性呈负相关的理论相符。

Abstract:

We used in vitro molecular evolution technology by error-prone PCR and high-throughput screening to improve thermostability of Bacillus flexus CCTCC 2015368 β-amylase. Mutant D476N with significant thermostability increase was selected by LB agar starch plate colorimetric assay and 96-well plate enzyme activity assay. The optimum pH was 6.5 for the mutant D476N, compared to 7.0 of the wild type. The optimal temperature was 55 ℃ for both mutant D476N and the wild type. The T50 value of the mutant D476N was 4 ℃ higher than that of the wild type. The half-life of mutant D476N at 55 ℃ was 35 min, 95% higher than that of the wild type. The Km of the mutant D476N was 97.98 μmol/L, 1.14 times of that of the wild type (85.86 μmol/L). The thermostability of the mutant D476N was slightly lower than that of the wild type. The three-dimensional structure of wild type and mutant D476N was simulated by SWISS-MODEL and analyzed by PyMol software. The mutated amino acid residue Asn476 was located on the loop of protein surface. The molecular free energy(ΔG) of D476N was calculated by MOE software was 106.0 kcal/mol, reduced by 10.3% compared to the wild enzyme. These results were consistent with the theory that the protein molecular free energy and thermostability were negatively correlated.