CN108342373B - 一种自组装纳米多肽纤维的制备方法及其应用 - Google Patents

一种自组装纳米多肽纤维的制备方法及其应用 Download PDF

Info

Publication number
CN108342373B
CN108342373B CN201710054635.3A CN201710054635A CN108342373B CN 108342373 B CN108342373 B CN 108342373B CN 201710054635 A CN201710054635 A CN 201710054635A CN 108342373 B CN108342373 B CN 108342373B
Authority
CN
China
Prior art keywords
polypeptide
self
cellobiose
conh
enzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201710054635.3A
Other languages
English (en)
Other versions
CN108342373A (zh
Inventor
王硕
何兴兴
刘继峰
王俊平
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tianjin University of Science and Technology
Original Assignee
Tianjin University of Science and Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin University of Science and Technology filed Critical Tianjin University of Science and Technology
Priority to CN201710054635.3A priority Critical patent/CN108342373B/zh
Publication of CN108342373A publication Critical patent/CN108342373A/zh
Application granted granted Critical
Publication of CN108342373B publication Critical patent/CN108342373B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明公开了一种自组装纳米多肽纤维的制备方法及其作为模拟酶催化水解纤维二糖的应用。所制备的多肽模拟酶溶液均可自组装形成纳米纤维网状结构，纤维直径大约为几十纳米。该纳米纤维模拟酶具有催化纤维二糖水解的特性，且肽④Ac‑FEFEAEA‑CONH2序列对纤维二糖的水解活性最高。与现有技术相比，该多肽纤维模拟酶具有合成简单，不易失活等特点。

Description

一种自组装纳米多肽纤维的制备方法及其应用
技术领域
本发明涉及的是能自组装形成纳米纤维多肽的设计、表征及其作为纤维二糖水解酶的应用，属于纳米材料及仿生催化技术领域。
背景技术
分子自组装是指在一定条件下，通过分子间或者分子中某一片段与另一片段间的相互识别，在氢键、静电力、疏水作用力、范德华力以及π-π相互作用等弱的非共价键力的作用下，自发形成稳定的、具有某种结构和功能的分子聚集体的过程。而多肽自组装是生命体中广泛存在的一种现象，是众多生命活动和生物学功能得以实现的基础。具有特定氨基酸序列的多肽可以组装形成结构和形态不同的功能体系，可以产生单分子层、胶束、纳米纤维、纳米管、纳米棒等各种形态。此外，由于多肽本身就是生物活性物质，具有良好的生物相容性和生物降解性，深受国内外研宄者广泛的重视。
酶是一类具有高效催化功能的生物催化剂，自然界的一切生命现象都与酶的参与有关。酶催化反应因其具有高的催化效率、高的反应专一性和温和的反应条件等特点，使其在医药、化工、食品和农业等领域发挥着重要作用。然而天然酶在实际应用上存在明显缺陷，比如来源有限、提纯困难、易失活、价格昂贵、使用条件苛刻等等。这些都使得天然酶的应用受到了极大的限制。因而，开发具有酶功能的模拟酶的体系便应运而生。
诺贝尔奖获得者Cram、Pederson与Lehn根据酶催化反应机理，提出了主-客体化学和超分子化学，奠定了模拟酶的重要理论基础。到目前为止，学者们开发并研究了一系列主体试剂模拟酶以及纳米粒子模拟酶等，取得了一定的发展。但近年来，多肽自组装模拟酶由于其来源组成接近天然酶，具有较高生物相容性，合成方便，具有二级结构等特点逐渐成为目前模拟酶的研究热点，相继开展了以组氨酸催化酯水解、脯氨酸催化醛醇缩合为核心的多肽自组装模拟酶。
纤维素的有效降解已成为制约木质纤维素物质有效利用的瓶颈之一。将纤维素水解为葡萄糖是纤维素利用的重要途径，已成为当今科技界的研究热点，这对解决当前的资源、能源和环境问题都具有重要意义。而纤维二糖作为纤维素的基本结构单元也是纤维素水解为葡萄糖的重要组成部分，其模拟酶的设计及活性研究将会为纤维素的水解提供一条新的思路。
设计合成纤维二糖水解酶模型，需要考虑较多因素，关键在于活性位点谷氨酸(E)或天冬氨酸(D)的排列分布以及自组装骨架的选择。过去的研究中，人们用羧基耦合在不同纳米材料等方法合成了两种多糖水解模型物。
发明内容
本发明的目的在于设计一系列可自组装形成纳米纤维的多肽模拟纤维二糖水解酶的特性，提供了一种纤维二糖水解的新方法。
为了实现上述目的，本发明采用以下技术方案：
一种自组装纳米多肽纤维的制备方法，包括如下步骤：
A)将2mg多肽冻干粉溶于少量的二甲基亚砜中，充分震荡后加入1mM pH3.0柠檬酸-磷酸缓冲液，混匀超声90s，得到终浓度为5mM的多肽溶液；
B)将步骤B溶解的肽液于室温、避光条件下静置24h，使其缓慢自组装纤维化并形成凝胶。
反应原理是：以苯丙氨酸(Phe,F)间的π-π堆积作用以及异亮氨酸(Ile,I)间的疏水相互作用为驱动力自组装形成纤维骨架，将水解纤维二糖的活性位点谷氨酸或天冬氨酸均一的嵌入到多肽纤维结构中，设计合成一系列能自组装形成纳米纤维且具有水解酶活性的多肽。
进一步，所述能自组装形成纳米纤维且具有水解酶活性的多肽共有五种氨基酸序列排布：①Ac-FEFEIEI-CONH2；②Ac-EFEFEIE-CONH2；③Ac-FDFDIDI-CONH2；④Ac-FEFEAEA-CONH2；⑤Ac-FEFEVEV-CONH2；
其中Ac为乙酰基；F为苯丙氨酸；E为谷氨酸；I为异亮氨酸；CONH2为甲酰胺基；D为天冬氨酸；A为丙氨酸；V为缬氨酸。
自组装纳米多肽纤维作为催化纤维二糖水解反应的水解模拟酶的应用，其特征在于，包括如下步骤：
A)将上述得到的纤维化凝胶液先用漩涡震荡仪震碎，再用1mM pH3.0柠檬酸-磷酸缓冲液稀释，最后与纤维二糖混合；
B)室温孵育48小时，使纤维二糖充分水解生成产物葡萄糖。
本发明的有益效果是所制备的纤维二糖模拟酶是以生物体中的多肽作为基础材料，因此该模拟酶具有较高的生物相容性和安全性。且本发明设计的五种多肽均能自组装形成交联清晰的纤维结构，其直径大约在几十纳米左右，该模拟酶具有催化水解纤维二糖的功能，且催化效率远远高于其他非肽材料的模拟酶，是一种新型的β-1,4糖苷键水解模拟酶。此外，本发明得到的模拟酶与天然β-糖苷酶相比，具有合成纯化简单，成本低，不易失活等特点
附图说明
图1为本发明的5mM多肽纤维凝胶液。图中从左到右依次是肽①到肽⑤。
图2为本发明的多肽纤维模拟酶的透射电镜表征图(标尺是200nm)。
图3为本发明的多肽纤维模拟酶对纤维二糖水解活性最适pH值测定。
图4为本发明的多肽纤维模拟酶对纤维二糖水解活性最适温度测定。
图5为本发明的五条多肽模拟酶在最适条件下对纤维二糖水解活性的测定。
具体实施方式
实施例1：自组装多肽纤维模拟酶的制备步骤如下：
各取2mg多肽干粉溶于20μL DMSO中，充分震荡后加入一定体积的1mM pH3.0柠檬酸-磷酸缓冲液，混匀并超声90s，得到终浓度为5mM的肽液。将上述肽液于室温、避光条件下静置48h，使其缓慢纤维化并形成凝胶液，测定活性前将该凝胶剧烈漩涡震荡打碎，得到多肽纤维模拟酶。图1为所制备模拟酶的凝胶液。
实施例2：自组装多肽纤维特性表征步骤如下：
上述制备的5mM肽液用1mM pH3.0柠檬酸-磷酸缓冲液稀释到200μM，取7μL200μM肽液于碳膜铜网1h，用滤纸吸去多余肽液，然后立即滴加7μL新鲜过膜的2％磷钨酸溶液染色1.5min，再用超纯水清洗三次，将自然晾干后的透射电镜样品通过样品架置于JEM-1011透射电子显微镜的镜筒中，在合适的放大倍数下观察多肽的形貌，将图像调节清晰后通过CCD采集图像。图2为所制备多肽纤维模拟酶的TEM表征图。
从表征结果图2可以看出，多肽自组装能形成清晰的纤维网状结构。
实施例3：多肽纤维模拟酶对纤维二糖的水解活性：
取30μl上述制备的模拟酶溶液与40μl 15mM底物纤维二糖混合，分别加入30μl不同pH值的1mM柠檬酸-磷酸缓冲液，室温震荡孵育48h，以待反应充分进行，然后在同一离心管中加入300μl葡萄糖氧化酶试剂盒工作液，混匀后37℃恒温反应20min，用酶标仪测定OD505nm处的吸光度。图3为所制备多肽模拟酶催化水解纤维二糖的最适pH条件。
取30μl上述制备的模拟酶溶液与40μl 15mM底物纤维二糖混合，再加入30μl 1mMpH3.0d的柠檬酸-磷酸缓冲液，分别在20℃、25℃、37℃、45℃、55℃和65℃条件下震荡孵育48h，以待反应充分进行，然后在同一离心管中加入300μl葡萄糖氧化酶试剂盒工作液，混匀后37℃恒温反应20min，用酶标仪测定OD505nm处的吸光度。图4为所制备多肽模拟酶催化水解纤维二糖的最适温度条件。
从图3数据可以看出，本发明所设计的五条多肽对纤维二糖有较高的水解活性且该模拟酶体系均在pH3.0的条件下对纤维二糖的水解活性最高。从图4数据可以看出，肽①、肽③、肽④和肽⑤模拟酶在25℃的条件下对纤维二糖的水解活性最高，而肽②模拟酶在55℃的条件下对纤维二糖的水解活性最高。从图5数据可以看出，本发明所设计的肽④模拟酶对纤维二糖的水解活性最高。
可以理解的是，对本领域普通技术人员来说，可以根据本发明的技术方案及其发明构思加以等同替换或改变，而所有这些改变或替换(例如多肽序列Fmoc-IEIEIEI-CONH2)都应属于本发明所附的权利要求的保护范围。

Claims (3)

1.一种自组装纳米多肽纤维的制备方法，包括如下步骤：
A)将2mg多肽冻干粉溶于少量的二甲基亚砜中，充分震荡后加入1mM pH3 .0柠檬酸-磷酸缓冲液，混匀超声90s，得到终浓度为5mM的多肽溶液；多肽选自如下五种氨基酸序列之一：①Ac-FEFEIEI-CONH2；②Ac-EFEFEIE-CONH2；③Ac-FDFDIDI-CONH2；④Ac-FEFEAEA-CONH2；⑤Ac-FEFEVEV-CONH2；
其中Ac为乙酰基；F为苯丙氨酸；E为谷氨酸；I为异亮氨酸；CONH2为甲酰胺基；D为天冬氨酸；A为丙氨酸；V为缬氨酸；
B)将步骤A溶解的多肽溶液于室温、避光条件下静置24h，使其缓慢自组装纤维化并形成凝胶。
2.权利要求1所述的制备方法得到的自组装纳米多肽纤维作为催化纤维二糖水解反应的水解模拟酶的应用。
3.权利要求2所述的自组装纳米多肽纤维作为催化纤维二糖水解反应的水解模拟酶的应用，其特征在于，包括如下步骤：
A)将上述得到的纤维化凝胶液先用漩涡震荡仪震碎，再用1mM pH3 .0柠檬酸-磷酸缓冲液稀释，最后与纤维二糖混合；
B)室温孵育48小时，使纤维二糖充分水解生成产物葡萄糖。
CN201710054635.3A 2017-01-23 2017-01-23 一种自组装纳米多肽纤维的制备方法及其应用 Active CN108342373B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710054635.3A CN108342373B (zh) 2017-01-23 2017-01-23 一种自组装纳米多肽纤维的制备方法及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710054635.3A CN108342373B (zh) 2017-01-23 2017-01-23 一种自组装纳米多肽纤维的制备方法及其应用

Publications (2)

Publication Number Publication Date
CN108342373A CN108342373A (zh) 2018-07-31
CN108342373B true CN108342373B (zh) 2022-11-01

Family

ID=62962906

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710054635.3A Active CN108342373B (zh) 2017-01-23 2017-01-23 一种自组装纳米多肽纤维的制备方法及其应用

Country Status (1)

Country Link
CN (1) CN108342373B (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110028551A (zh) * 2019-04-18 2019-07-19 福州大学 一种多肽水凝胶及其制备方法
CN110028552A (zh) * 2019-04-18 2019-07-19 福州大学 一种自组装多肽及其水凝胶的制备方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105073987A (zh) * 2013-03-27 2015-11-18 本田技研工业株式会社 耐热性纤维二糖水解酶

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6429377B2 (ja) * 2014-12-12 2018-11-28 本田技研工業株式会社 耐熱性セロビオハイドロラーゼ

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105073987A (zh) * 2013-03-27 2015-11-18 本田技研工业株式会社 耐热性纤维二糖水解酶

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Probing the structure-activity relationship of a novel artificial cellobiose hydrolase;Xingxing He等;《J Mater Chem B》;20170608;第5卷;第5225-5233页 *

Also Published As

Publication number Publication date
CN108342373A (zh) 2018-07-31

Similar Documents

Publication Publication Date Title
Poorakbar et al. Synthesis of magnetic gold mesoporous silica nanoparticles core shell for cellulase enzyme immobilization: improvement of enzymatic activity and thermal stability
Satyamurthy et al. A novel process for synthesis of spherical nanocellulose by controlled hydrolysis of microcrystalline cellulose using anaerobic microbial consortium
Huang et al. Preparation and characterization of stable chitosan nanofibrous membrane for lipase immobilization
Paz-Cedeno et al. Magnetic graphene oxide as a platform for the immobilization of cellulases and xylanases: Ultrastructural characterization and assessment of lignocellulosic biomass hydrolysis
Ariaeenejad et al. Immobilization of enzyme cocktails on dopamine functionalized magnetic cellulose nanocrystals to enhance sugar bioconversion: A biomass reusing loop
Khoshnevisan et al. Immobilization of cellulase enzyme on superparamagnetic nanoparticles and determination of its activity and stability
Huang et al. Covalent immobilization of lipase from Candida rugosa onto poly (acrylonitrile-co-2-hydroxyethyl methacrylate) electrospun fibrous membranes for potential bioreactor application
Huang et al. Immobilization of Candida rugosa lipase on electrospun cellulose nanofiber membrane
Ye et al. Nanofibrous poly (acrylonitrile-co-maleic acid) membranes functionalized with gelatin and chitosan for lipase immobilization
Liu et al. Cellobiohydrolase hydrolyzes crystalline cellulose on hydrophobic faces
Kaur et al. Characterization of magnetic nanoparticle–immobilized cellulases for enzymatic saccharification of rice straw
Liu et al. Electrospun nanofibrous membranes containing epoxy groups and hydrophilic polyethylene oxide chain for highly active and stable covalent immobilization of lipase
Wang et al. Covalent immobilization of redox enzyme on electrospun nonwoven poly (acrylonitrile‐co‐acrylic acid) nanofiber mesh filled with carbon nanotubes: A comprehensive study
Blanchette et al. Enhanced cellulose degradation using cellulase-nanosphere complexes
Je et al. Cellulose nanofibers for magnetically-separable and highly loaded enzyme immobilization
El-Aassar Functionalized electrospun nanofibers from poly (AN-co-MMA) for enzyme immobilization
Wang et al. Cellulase immobilized by sodium alginate-polyethylene glycol-chitosan for hydrolysis enhancement of microcrystalline cellulose
Husain Nanomaterials immobilized cellulolytic enzymes and their industrial applications
Wan et al. Immobilization of Candida antarctic lipase B on MWNTs modified by ionic liquids with different functional groups
Gokhale et al. Cellulase immobilized nanostructured supports for efficient saccharification of cellulosic substrates
CN108342373B (zh) 一种自组装纳米多肽纤维的制备方法及其应用
Ulu et al. The carboxylated multi-walled carbon nanotubes/l-asparaginase doped calcium-alginate beads: Structural and biocatalytic characterization
Abraham et al. Nano-immobilized cellulases for biomass processing with application in biofuel production
Dwivedee et al. Tailoring a robust and recyclable nanobiocatalyst by immobilization of Pseudomonas fluorescens lipase on carbon nanofiber and its application in synthesis of enantiopure carboetomidate analogue
Quan et al. Fabrication of glycopolymer/MWCNTs composite nanofibers and its enzyme immobilization applications

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant