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来自Saccharomyces cerevisiae的一种histidine酸酸酶的功能表达,净化,生物化学和生物物理特征以及分子动力学模拟

Nima Ghahremani Nezhad^1,2, Siti Zahra Binti Jamaludin^1,2, Raja Noor Zaliha Raja Abd Rahman^1,3

¹Enzyme and Microbial Research Center, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, 43400 UPM, Selangor, Malaysia.

World journal of microbiology & biotechnology

|April 17, 2024

概括

这项研究成功地表达和表征了一种histidine酸酸酶 (HAP) 酶,PhySc,证明了其最佳活性和稳定性. 这些发现为HAP酶功能和潜在应用提供了宝贵的见解.

科学领域:

生物化学生化学
酶学是一种酶学.
蛋白质工程是指蛋白质工程.

背景情况:

酸酸酶 (HAPs) 是参与各种生物过程的关键酶.
了解HAP的结构功能关系对于其生物技术应用至关重要.

研究的目的:

在功能上表达一种新的HAP,PhySc,与Saccharomyces cerevisiae PHO5.5具有很高的序列相似性.
描述PhySc.的酶活性,运动参数和稳定性.
使用计算方法阐明PhySc的结构性质和催化残留物.

主要方法:

通过与分子伴侣共同表达的PhySc的功能表达.
在不同的pH值和温度下进行酶活性测定.
使用酸盐作为基质确定动力参数 (Km,Kcat).
使用半衰期测量进行热稳定性评估.
循环二重化 (CD) 光谱用于二次结构分析.
分子对接和分子动力学 (MD) 模拟用于结构洞察.

主要成果:

通过~110kDa的分子质量成功表达了PhySc.
在pH 5.5和40°C时观察到最佳活性,特异活性为28.75U/mg.
确定了动力参数 (Km = 0.608 mM,Kcat = 650.89 s-1) 的时间.
PhySc表现出显著的热稳定性,其半衰期为2.75分钟在55°C和55秒在60°C.
CD和MD模拟显示了一致的二次结构含量 (α螺旋,β片,转,线圈).
分子对接确定了His59和Asp322作为潜在的催化残留物.
MD模拟证实40°C的结构稳定性较高,而60°C和80°C的结构稳定性较高.

结论:

PhySc的成功表达和表征为HAP研究提供了一个强大的模型.
PhySc表现出有利的酶特性和热稳定性,表明工业应用的潜力.
综合实验和计算方法为HAP结构功能关系提供了全面的见解.

关键词:

生物物理化学特性表征功能表达式功能表达式希斯蒂丁酸酸酶是一种酸酶.

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