State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu, PR China; School of Food Science, Henan University of Technology, 100 Lianhua Street, Zhengzhou 450001, Henan, PR China.
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu, PR China.
Int J Biol Macromol. 2020 Jul 1;154:1314-1323. doi: 10.1016/j.ijbiomac.2019.11.009. Epub 2019 Nov 13.
An in situ and real-time investigation was performed using an optical cell system and in-silico analysis to reveal the impacts of pressure and temperature on the conformational state and behaviours of Rhizopus chinensis lipase (RCL). The fluorescence intensity (FI) of RCL increased remarkably under high pressure, and part of this increase was recovered after depressurization. This result displayed the partially reversible conformational change of RCL, which may be associated with the local change of Trp224 near the catalytic centre. High temperature caused a significant loss of secondary structure, whereas the α-helical segments including the lid were preserved by high pressure even at temperatures over 60 °C. The parameters of enzymatic reaction monitored by UV showed that the hydrolysis rate was remarkably enhanced by the pressure of 200 MPa. In the pressure range of 0.1-200 MPa, the active volume measured by the in situ system decreased from -2.85 to -6.73 mL/mol with the temperature increasing from 20 °C to 40 °C. The high catalytic capacity of the lipase under high pressure and high temperature was primarily attributed to pressure protection on RCL.
采用光学池系统和计算机模拟分析对常压和高温下对根霉脂肪酶（RCL）构象状态和行为的影响进行了原位实时研究。RCL 的荧光强度（FI）在高压下显著增加，部分增加在减压后恢复。该结果显示 RCL 的部分可逆构象变化，可能与靠近催化中心的色氨酸 224 附近的局部变化有关。高温导致二级结构显著损失，而包括盖子在内的α-螺旋片段在 60°C 以上的高压下得以保留。通过 UV 监测的酶反应参数表明，在 200 MPa 的压力下，水解速率显著提高。在 0.1-200 MPa 的压力范围内，随着温度从 20°C 升高到 40°C，原位系统测量的活性体积从-2.85 减少到-6.73 mL/mol。在高压和高温下脂肪酶的高催化能力主要归因于对 RCL 的压力保护。
