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来自Aspergillus oryzae的帕拉本化酶和化酶的基质特异性修改

Michiko Hakoda¹, Tomoe Kato¹, Chihiro Takahashi¹

¹Department of Biosciences, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka 997-8555, Japan.

Enzyme and microbial technology

|August 9, 2024

概括

酶工程研究修改了Aspergillus oryzae的基酶 (AoPrbA) 和酶 (AoTanB),以改变它们的基质特异性. 变种表现出改变的活动,一些AoPrbA变种获得了酶活性,扩大了它们的生物化学效用.

科学领域:

生物化学和分子生物学
酶工程和定向进化研究
微生物生物技术微生物生物技术

背景情况:

来自Aspergillus oryzae的帕拉本化酶 (AoPrbA) 和化酶 (AoTanB) 具有狭窄的基质特异性.
这些酶属于ESTHER数据库中的酶家族.
对于酶应用来说,理解和工程基质歧视至关重要.

研究的目的:

通过局部定向突变发生的AoPrbA和AoTanB之间的基质歧视进行调查.
为了设计AoPrbA和AoTanB变体,改变基质特异性.
探索在工程变体中获得新的酶活性.

主要方法:

基于结构信息,构建了五种AoPrbA和四种AoTanB的单个变体.
在Pichia pastoris中对工程变异的表达和净化.
酶分析测定野生类型和变种酶的基酶和酶活性.

主要成果:

所有工程AoPrbA和AoTanB变种都表现出减少的野生类型活动.
三种AoPrbA变体 (Thr200Glu, Leu232Gln, Leu428Ser) 获得了酸甲基的水解能力,证明了新的化活性.
AoTanB Glu203Thr 变体对测试基质失去活性,而 Ser440Leu 变体显示了基酶与酶活性的比率显著增加.

结论:

位点定向突变可以有效地改变AoPrbA和AoTanB的基质特异性.
工程AoPrbA变种可以获得新的酶功能,例如酶活性.
特定突变可以调节这些酶的基质偏好和活性比率,为定制生物催化剂开发提供潜力.

关键词:

亚斯伯吉illus oryzae 是一个植物.帕拉本氨酸酸酶是什么基质特异性修改的基质特异性修改化剂 (tannase) 是一种化剂.

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