N-terminal truncation contributed to increasing thermal stability of mannanase Man1312 without activity loss.
N-terminal truncation contributed to increasing thermal stability of mannanase Man1312 without activity loss.
复制标题
DOI：
10.1002/jsfa.7240
复制
发表时间：
2016-03
影响因子：
4.1
通讯作者：
Haiyan Zhou;Wenjiao Yang;Yun Tian;H. Peng;Yongyao Wu
中科院分区：
文献类型：
--
作者：
Haiyan Zhou;Wenjiao Yang;Yun Tian;H. Peng;Yongyao Wu
研究背景末端环上的无序残基影响分子结构的稳定性，在某些情况下对催化反应具有调节作用。为了进一步了解远端残基突变的影响，本研究探讨了甘露聚糖酶Man 1312缺失突变体的热稳定性和酶活性。重点是位于N-末端区域的残基，因为它们更无序和可变。采用分光光度法、圆二色谱法和差示扫描量热法研究了N-末端截短对酶活性和热动力学的影响。结果V3、N7和Q11缺失突变体的稳定性明显提高，而三联体缺失后酶活性明显提高。三联体缺失MandVNQ的稳定性约为其相应的单位点和双位点缺失突变体的两倍。MandVNP的Tm值比Man 1312高约8 °C。与Man 1312相比，ManVNP的Topt特性提高了10 °C，t1/2提高了10 min，催化活性提高了11%。光谱分析和建模表明，MandVNQ增加了螺旋和链含量。结论N端截短对甘露聚糖酶的热稳定性和酶活有积极的影响。
BACKGROUND The disordered residues on distal loops affect the molecular structural stability and on some occasions have regulatory roles in catalytic reaction. To increase understanding of the influence of distal residue mutation, this study explored the thermostability and enzymatic activity of mannanase Man1312 deletion mutants. The focus was on residues located on the N-terminal region because they are more disordered and changeable. The effects of N-terminal truncation on enzymatic activity and thermal dynamics were investigated by spectrophotometry, circular dichroism and differential scanning calorimetry assays. RESULTS The deletion mutants on V3, N7 and Q11 showed a marked increase in stability, while the enzymatic activity was significantly improved when triplet deletion was carried out. Triplet deletion MandVNQ showed around double the stability of its corresponding single-site and double-site deletion mutants. The Tm value of MandVNP was about 8 °C higher than that of Man1312. MandVNP had improved characteristics of Topt by 10 °C, t1/2 by 10 min and catalytic activity by 11% in comparison with Man1312. Analysis of spectra and modeling showed that MandVNQ had increased helix and strand contents. CONCLUSION N-terminal truncation had positive effects on the thermostability and activity of mannanase.