适用于蛋白质生物材料合成的祖先分选酶E的设计,ACS Catalysis

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适用于蛋白质生物材料合成的祖先分选酶E的设计
ACS Catalysis ( IF 11.3 ) Pub Date : 2024-02-20 , DOI: 10.1021/acscatal.4c00487
Azusa Miyata ₁ , Taichi Chisuga ₁ , Akira Kambe ₁ , Ryo Miyata ₁ , Yui Kawamura ₁ , Hiroyuki Takeda ₂ , Sohei Ito ₁ , Shogo Nakano _{1,

3}

Affiliation

蛋白质生物材料将有潜力解决全球健康和环境挑战。存在多种生物材料的生产方法，其中分选酶介导的连接（SML）是代表性技术之一。SML 促进两种化合物的位点特异性缀合：C 末端具有细胞壁分选信号的供体肽或蛋白质，以及 N 末端具有寡甘氨酸的亲核体。在我们的研究中，我们通过序列数据挖掘和祖先序列重建相结合，重建了祖先分选酶E，命名为AcSE5。AcSE5 是一种独立于 Ca ^{2+ 的}分选酶，可识别 C 末端带有 LAETG 的供体，并且可以使用 N 末端带有 GGG 或 GAA 的肽以及直链烷基胺作为亲核试剂。该酶可以实现高效的肽缀合，在最佳条件下超过70%。利用 AcSE5，我们合成了两种蛋白缀合物：金星缀合的鲨鱼可变新抗原受体 (VNAR) 和通过聚乙二醇二胺双缀合的 VNAR。使用 AcSE5 还实现了将酶直接固定到氨基封端的聚苯乙烯珠上。当与超热稳定的祖先l-氨基酸氧化酶(HTAncLAAO2)缀合时，所得珠子可用于将各种外消旋氨基酸去外消旋化成d-形式。对于三种苯丙氨酸衍生物，可以实现制备规模（100mg规模）的去消旋化。该工艺通过化学酶反应提供高对映纯度 (>99% ee ) 和分离产率 (>64%)。固定化的 HTAncLAAO2 对 10 次重复反应（总共 240 小时）表现出完全耐受性。AcSE5 是一种适用于 SML 应用的出色酶。

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Design of Ancestral Sortase E that is Applicable in Protein Biomaterial Synthesis

Protein biomaterials would have the potential to address global challenges in health and environment. Numerous production methods of the biomaterials exist, with sortase-mediated ligation (SML) being one of the representative technique. SML facilitates the site-specific conjugation of two compounds: donor peptides or proteins with a cell wall sorting signal at their C-terminus and nucleophiles that have oligoglycine at their N-terminal. In our research, we reconstructed an ancestral sortase E, named AcSE5, through a combination of sequence data mining and ancestral sequence reconstruction. AcSE5, a Ca²⁺ independent sortase, recognizes donors with LAETG at their C-termini and can employ both peptides bearing GGG- or GAA-at N-terminus and straight-chained alkylamines as nucleophiles. The enzyme can achieve efficient peptide conjugation, exceeding 70% under optimal conditions. With AcSE5, we synthesized two protein conjugates: Venus-conjugated shark variable new antigen receptor (VNAR) and dual-conjugated VNAR via poly(ethylene) glycol diamine. Direct enzyme immobilization to amino-terminated polystyrene beads was also achieved using AcSE5. The resultant beads, when conjugated with hyper-thermostable ancestral l-amino acid oxidases (HTAncLAAO2), can be employed for deracemization of various racemic amino acids into d-form. For three of phenylalanine derivatives, preparative-scale (100 mg scale) deracemization can be achieved. This process provides high enantiopurity (>99% ee) and isolation yields (>64%) through chemoenzymatic reactions. The immobilized HTAncLAAO2 showed complete resistance to 10 repeated reactions for a total of 240 h. AcSE5 is an excellent enzyme for SML applications.

更新日期：2024-02-20

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