Specific Ligation of Two Multimeric Enzymes with Native Peptides and Immobilization with Controlled Molar Ratio
Specific Ligation of Two Multimeric Enzymes with Native Peptides and Immobilization with Controlled Molar Ratio
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两种多聚酶与天然肽的特异性连接和受控摩尔比的固定化
DOI：
10.1021/acs.bloconjchem.7b00043
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发表时间：
2017
影响因子：
4.7
通讯作者：
Feng Wei
中科院分区：
文献类型：
--
作者：
Du Kun;Zhao Jinjin;Sun Jian;Feng Wei
D-氨基酸氧化酶(DAAOs)是一种风味酶，已被用于拆分外消旋氨基酸和医药生产。然而，催化过程中产生的过氧化氢具有有害和抑制作用。过氧化氢酶(CAT)分解过氧化氢可以消除这些负面影响。DAAo和CAT分别是二聚体和四聚体蛋白。在此，通过内含子介导的体内蛋白质剪接，DAAO亚基的N-端与CAT亚基的C-端与天然多肽特异性地连接。圆二色谱(CD)证实体内剪接对酶的二级结构影响不大，荧光光谱表明剪接产物DAAO&CAT具有比DAAO更高的稳定性。在剪接产物DAao&CAT中，DAao亚基与CAT亚基紧密相连，有助于过氧化氢从一个催化部位立即转移到另一个催化部位，从而能够有效地分解生成的过氧化氢。DAAO亚基的还原辅因子被周围释放的分子氧重新氧化。动力学分析表明，丙氨酸底物符合Michaelis-Menten动力学。DAAO和CAT的催化效率是DAAO的22.4倍。此外，还将剪接产物DAAO&CAT包埋在配位聚合物中，包封率为91.3±2.7%。包埋的DAAO&CAT在30℃和40℃时分别保持了98.1±3.1%和94.9±2.9%的游离DAAO和CAT活性。
d-Amino acid oxidases (DAAOs) are flavor enzymes and have been used in resolution of racemic amino acids and manufacturing of pharmaceuticals. However, the evolved H2O2during the catalysis has deleterious and inhibitory effects. Decomposition of the hydrogen peroxide by catalase (CAT) can eliminate the negative effects. DAAO and CAT are dimeric and tetrameric proteins, respectively. Here, the N-terminus of the DAAO subunits has been specifically ligated to the C-terminus of the CAT subunits with native peptides through intein-mediated in vivo protein splicing. The in vivo splicing has little effect on the secondary structures of the enzymes as confirmed by circular dichroism (CD) spectra, and fluorescence spectra showed that the spliced product DAAO&CAT has a higher stability than DAAO. In the spliced product DAAO&CAT, the DAAO subunits are in close proximity to the CAT subunits, facilitating immediate transfer of H2O2from one catalytic site to the other, enabling efficient decomposition of the generated H2O2. The reduced cofactors of the DAAO subunits were reoxidized by the evolved molecular oxygen around. Kinetics analysis showed that thed-alanine substrate follows Michaelis–Menten kinetics. The catalytic efficiency of DAAO&CAT is 22.4-fold that of DAAO. Furthermore, the spliced product DAAO&CAT has been encapsulated within a coordination polymer with an encapsulation efficiency of 91.3 ± 2.7%. The encapsulated DAAO&CAT has retained 98.1 ± 3.1% and 94.9 ± 2.9% of the activity of free DAAO&CAT at 30 and 40 °C, respectively.