Preparation, Conformational Structure, and Proteolytic Activity of Papain Covalently Conjugated to Poly(ethylene glycol)-Tethered Lipid Bilayer Membranes

IF 5.4 2区化学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Biomacromolecules Pub Date : 2025-04-14 Epub Date: 2025-04-02 DOI:10.1021/acs.biomac.4c01324

Yuya Takahashi, Kyohei Kubota, Makoto Yoshimoto, Noriko Yoshimoto

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Abstract

Conjugation of enzymes to lipid membranes is a key approach to reconstitute fascinating features of cell organelles and to deduce the nature of membrane-involved biological events. In this work, papain was covalently conjugated via a cross-linker to phospholipid vesicles (liposomes) tethered with poly(ethylene glycol) (PEG) at 25 °C and pH = 7.0, followed by chromatographic purification. The size of the PEG moiety and the type of cross-linker were optimized to obtain PEG-tethered liposome-conjugated papain (liposome-PEG-papain). Slight conformational changes of the membrane-conjugated papain in both its secondary and tertiary structures were revealed using circular dichroism and intrinsic fluorescence measurements. Notably, heat treatment of a liposome-PEG-papain dispersion at 77 or 84 °C caused permeabilization of the lipid membranes to 5(6)-carboxyfluorescein. Furthermore, liposome-PEG-papain exhibited the digestion activity of casein at 37 °C and pH = 7.6. The structural flexibility of liposomes as enzyme carriers may provide the opportunity to functionalize the membrane-conjugated biomacromolecules.

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聚乙二醇脂质双分子膜共价共轭木瓜蛋白酶的制备、构象结构和蛋白水解活性。

酶与脂质膜的结合是重建细胞器迷人特征和推断膜相关生物事件性质的关键途径。在这项工作中，木瓜蛋白酶通过交联剂在25°C和pH = 7.0的条件下与聚乙二醇（PEG）连接的磷脂囊泡（脂质体）共价偶联，然后进行色谱纯化。对PEG片段的大小和交联剂的类型进行优化，得到PEG系链脂质体共轭木瓜蛋白酶（liposome-PEG-papain）。利用圆二色性和固有荧光测量揭示了膜共轭木瓜蛋白酶在二级和三级结构上的轻微构象变化。值得注意的是，脂质体- peg -木瓜蛋白酶分散体在77°C或84°C下的热处理导致脂质膜对5(6)-羧基荧光素的渗透。此外，脂质体- peg -木瓜蛋白酶在37℃和pH = 7.6条件下具有酪蛋白的消化活性。脂质体作为酶载体的结构灵活性可能为膜共轭生物大分子的功能化提供了机会。

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来源期刊

Biomacromolecules 化学-高分子科学

CiteScore

10.60

自引率

4.80%

发文量

417

审稿时长

1.6 months

期刊介绍： Biomacromolecules is a leading forum for the dissemination of cutting-edge research at the interface of polymer science and biology. Submissions to Biomacromolecules should contain strong elements of innovation in terms of macromolecular design, synthesis and characterization, or in the application of polymer materials to biology and medicine. Topics covered by Biomacromolecules include, but are not exclusively limited to: sustainable polymers, polymers based on natural and renewable resources, degradable polymers, polymer conjugates, polymeric drugs, polymers in biocatalysis, biomacromolecular assembly, biomimetic polymers, polymer-biomineral hybrids, biomimetic-polymer processing, polymer recycling, bioactive polymer surfaces, original polymer design for biomedical applications such as immunotherapy, drug delivery, gene delivery, antimicrobial applications, diagnostic imaging and biosensing, polymers in tissue engineering and regenerative medicine, polymeric scaffolds and hydrogels for cell culture and delivery.