Specific Ligation of Two Multimeric Enzymes with Native Peptides and Immobilization with Controlled Molar Ratio

化学 摩尔比 结扎 臼齿 固定化酶 色谱法 化学结扎 生物化学 组合化学 分子生物学 催化作用 医学 生物 牙科
作者
Kun Du,Jinjin Zhao,Jian Sun,Wei Feng
出处
期刊:Bioconjugate Chemistry [American Chemical Society]
卷期号:28 (4): 1166-1175 被引量:20
标识
DOI:10.1021/acs.bioconjchem.7b00043
摘要

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 H2O2 during 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 H2O2 from 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 the d-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.
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