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Crude enzyme immobilization‑based cell‑free system for efficient N‑acetylneuraminic acid biosynthesis aided by N‑terminal coding sequence screening

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systems Microbiology and Biomanufacturing 2023年第3期3卷 479-488页

作者： Peng Wen Xueqin Lv Long Liu Jianghua Li Guocheng Du Yanfeng Liu Key Laboratory of Carbohydrate Chemistry and Biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi 214122China Science Center for Future Foods Jiangnan UniversityWuxi 214122China Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology Jiangnan UniversityWuxi 214122China Jiangsu Province Engineering Research Center of Food Synthetic Biotechnology Jiangnan UniversityWuxi 214122China

N-acetylneuraminic acid(NeuAc)is an important nutrient that plays a key role in brain development in infants NeuAc is mainly produced by extraction from natural resources such as edible birds’s nests,crucian eggs,caviar and human breast *** extraction process is complicated,resulting in the disadvantages of low NeuAc content and low recovery *** this study,a crude enzyme immobilization-based cell-free system(CEICFS)was developed for efficient NeuAc ***,N-terminal coding sequences that improved the expression levels of N-acetylglucosamine-2-epimerase(AGE)and N-acetylneuraminic acid aldolase(NanA)were obtained by high-throughput *** these sequences resulted in up to 1.5-fold(1.2-fold)increase in AGE(NanA)enzyme *** then,a CEICFS for NeuAc biosynthesis was proposed by directly immobilizing crude enzyme containing AGE and NanA on amino ***,NeuAc production from GlcNAc using CEICFS in one reactor was carried out,resulting 68 g/L of NeuAc and the highest productivity of 6.8 g/L/***,the enzyme activity was still higher than 75%after five repeated *** functional properties of CEICFS were studied and compared to those of the free enzyme,immobilization can extend the application of enzyme to some harsh environments,such as low temperature and acidic ***,CEICFS with excellent heat resistance,storage stability and reusability exhibit great potential for industrial application.

关键词： N-acetylneuraminic acid Immobilization cell-free system N-terminal coding sequences

In situ Development of cellulose-Based Nanocomposites Using a cell-free system for Potential Biomedical Applications

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In situ Development of Cellulose-Based Nanocomposites Using ...

Efficient production of human beta-defensin-2 in an E. coli cell-free system

第十一届全国高聚物分子与结构表征学术研讨会

作者： Muhammad Wajid Ullah Omar Mohammad Atta Manjila Adhikari Vincent Ikenna Iloh Stezhka Yulia Guang Yang Department of Biomedical Engineering College of Life Science and TechnologyHuazhong University of Science and Technology

＜正＞Bacterial cellulose has received immense considerations owing to its excellent structural, physico-chemical, mechanical, and biological properties. However, its broad spectrum applications have been limited by few limitations associated with its production and lack of antimicrobial, magnetic, antioxidant, and conducting properties and limited biocompatibility. The shortcomings associated with its production can be overcome by a cell-free system that offers several advantages compared to microbial system.

关键词： cellulose cell-free system Nanocomposites Characterization Antibacterial activity

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Efficient production of human beta-defensin-2 in an E. coli ...

体外重建用于研究参与自噬体形成的功能蛋白的cell-free system

第六届化学工程与生物技术展览暨会议

作者： Zhinan Xu,Haiqin Chen,Peilin Cen Institute of Bioengineering,Department of Chemical Engineering and Bioengineering,Zhejiang University, Hangzhou 310027,The People''s Republic of China

＜正＞ Human beta-defensin-2 （hBD-2）,a small cationic peptide,exhibits a broad range of antimicrobial activity without any observed acquired *** order to produce hBD-2 efficiently,an *** cell-free biosynthesis system was proposed as an alternative method to express the polypeptide.A specific plasmid pIVEX2.4c-trxA-hBD2 was first constructed for the cell-free expression in the form of fusion protein,in which hBD-2 was linked with His-Tag and Trx-Tag to enhance protein stability and facilitate downstream purification process.A significant amount of target fusion protein was synthesized in batch-mode bioreactor by optimizing the reaction conditions,and about five-fold improvement of productivity （ca.2.0 mg/ml soluble target protein） could be achieved by using a continuous exchange cell-free （CECF） *** biosynthesis of the hBD-2 fusion protein could be further confirmed by Western-Blotting,and a one-step purification process was developed to recover the target protein ***,a high purity （95.2%） of the fusion protein was obtained with the overall recovery rate of about 50%.Acknowledgement: This work was financially supported by National Natural Science Foundation of China （No.20276066）.

关键词： human beta-defensin-2 cell-free system soluble expression purification

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体外重建用于研究参与自噬体形成的功能蛋白的Cell-free System

cell-free systems in the new age of synthetic biology

低碳经济与高效养殖——第六次全国饲料营养学术研讨会暨动物营养学分会成立三十周年大会

作者：晏向华华中农业大学动物科技学院动物分子营养研究室

细胞自吞噬(Autophagy)是机体一种重要的防御和保护机制。细胞可以通过自噬体(Autophagosome)和溶酶体的融合形成自噬溶酶体(Autolysosome),进而消除、降解和消化受损、变性、衰老和失去功能的细胞、细胞器和变性蛋白质与核酸等生物大... 详细信息

细胞自吞噬(Autophagy)是机体一种重要的防御和保护机制。细胞可以通过自噬体(Autophagosome)和溶酶体的融合形成自噬溶酶体(Autolysosome),进而消除、降解和消化受损、变性、衰老和失去功能的细胞、细胞器和变性蛋白质与核酸等生物大分子。这一过程为细胞的重建、再生和修复提供必需原料,实细胞自吞噬(Autophagy)是机体一种重要的防御和保护机制。细胞可以通过自噬体(Autophagosome)和溶酶体的融合形成自噬溶酶体(Autolysosome),进而消除、降解和消化受损、变性、衰老和失去功能的细胞、细胞器和变性蛋白质与核酸等生物大分子。这一过程为细胞的重建、再生和修复提供必需原料,实

关键词：自吞噬自噬体 cell-free system

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Frontiers of Chemical Science and Engineering 2017年第1期11卷 58-65页

作者： Fernando Villarreal Cheemeng Tan Department of Biomedical Engineering University of California Davis Davis One Shields Avenue CA 95616 USA

The advent of synthetic biology has ushered in new applications of cell-free transcription-translation systems. These cell-free systems are reconstituted using cellular proteins, and are amenable to modular control of their composition. Here, we discuss the historical advance- ment of cell-free systems, as well as their new applications in the rapid design of synthetic genetic circuits and components, directed evolution ofbiomolecules, diagnosis of infectious diseases, and synthesis of vaccines. Finally, we present our vision on the future direction of cell-free synthetic biology.

关键词： cell-free system application

cell-free biocatalysis coupled with photo-catalysis and electro-catalysis: Efficient CO_(2)-to-chemical conversion

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Green Energy & Environment 2024年第9期9卷 1366-1383页

作者： Junzhu Yang Chi-Kit Sou Yuan Lu Key Laboratory of Industrial Biocatalysis Ministry of EducationTsinghua UniversityBeijing100084China Department of Chemical Engineering Tsinghua UniversityBeijing100084China

The increasing atmospheric carbon dioxide (CO_(2)) concentration has exposed a series of crises in the earth's ecological *** to effectively fix and convert carbon dioxide into products with added value has attracted the attention of many ***-free enzyme catalytic system coupled with electrical and light have been a promising attempt in the field of biological carbon fixation in recent *** this review,the research progresses of photoenzyme catalysis,electroenzyme catalysis and photo-electroenzyme catalysis for converting carbon dioxide into chemical products in cell-free systems are systematically *** focus on reviewing and comparing various coupling methods and principles of photoenzyme catalysis and electroenzyme catalysis in cell-free systems,especially the materials used in the construction of the coupling system,and analyze and point out the characteristics and possible problems of different coupling ***,we discuss the major challenges and prospects of coupling physical signals and cell-free enzymatic catalytic systems in the field of CO_(2) fixation,suggesting possible strategies to improve the carbon sequestration capacity of such systems.

关键词： CO_(2)fixation cell-free system Enzyme Photoenzyme catalysis Electroenzyme catalysis

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In vitro reassembly of nuclear envelopes and organelles in Xenopus egg extracts

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cell Research 2006年第7期16卷 632-640页

作者： Ping Lu~1 Hui Zheng~2 Zhonghe Zhai ~1College of Life Sciences ~2Institute of Molecular Medicine,Peking University Beijing 100871,China College of Life Sciences Peking University Beijing China Institute of Molecular Medicine Peking University Beijing China

We reconstituted bilayer nuclear membranes,multilayer membranes,and organelles from mixtures of Xenopus laevis eggextracts and demembranated Xenopus sperm *** proportions of the cytosolic and vesicular fractions from theeggs were used in the reconstitution mixtures.A cytosol:vesicle ratio of 10:1 promoted reassembly of the normal bilayernuclear membrane with inserted nuclear pore complexes around the decondensed Xenopus sperm chromatin.A cytosol:vesicle ratio of 5:1 caused decondensed and dispersed sperm chromatin to be either surrounded by or divided by unusualmultilayer membrane structures with inlaid pore complexes.A cytosol:vesicle ratio of 2.5:1 promoted reconstitution ofmitochondria,endoplasmic reticulum networks,and Golgi *** reassembly of the endoplasmic reticulumand Golgi apparatus,vesicular fragments of the corresponding organelles fused together and changed their shape to formflattened cisternae,which were then stacked one on top of another.

关键词： cell-free system endoplasmic reticulum Golgi apparatus mitochondria nuclear reconstitution Xenopus egg extracts

Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell-free extracts of Xenopus laevis eggs

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cell Research 2000年第2期10卷 127-137页

作者： LIU XIAO LING YAN SHEN ER JIE CHEN ZHONG HEZHAI(E-mail: zhaizh@*** Tel: 86-10-62754293 Fax: 86-10-62751526)(College of Life Sciences, Peking University, Beijing 100871,China) College of Life Sciences Peking University Beijing China

Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nuclear *** Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle. The assembled nuclei, being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii. However, incubation of dinoflagellate Crythecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinillm cohnii cells does not induce nuclear reconstitution.

关键词： Dinoflagellate Crythecodinium cohnii chromosome nuclear reconstitution cell-free system Xenopus laevis egg extracts

Comparative studies on in vitro sperm decondensation and pronucleus formation in egg extracts between gynogenetic and bisexual fish

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cell Research 2003年第3期13卷 159-169页

作者： CHANG JIAN LI,JIAN FANG GUIState. Key Laboratory of Freshwater Ecology and Biotechnology,Wuhan Center for Developmental Biology,Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan 430072,China State Key Laboratory of Freshwater Ecology and Biotechnology Wuhan Center for Developmental Biology Institute of Hydrobiology Chinese Academy of Sciences Wuhan China

A cell-free system based upon the egg extracts from gynogenetic gibel carp(Carassius auratus gibelio)or bisexual red common carp(Cyprinus carpio red variety)was developed to investigate developmentalbehaviors of the demembranated sperm *** red common carp and gibel carp sperm nuclei coulddecondense fully and form pronuclei in the red common carp egg *** carp sperm nuclei couldalso decondense fully and form pronuclei in the gibel carp egg extracts,but red common carp sperum nucleicould not decondense sufficiently in the same *** significant differences of morphological changeswere further confirmed by ultrastructural observation of transmission electron *** data furtheroffer cytological evidence for gonochoristic reproduction in the gynogenetically reproducing gibel ***,the sperm nuclei in vitro decondensation is dependent on the pH in the extracts,and the decon-densed efficiency is optimal at pH ***,no DNA replication was observed in two kinds of

关键词： decondensation pronucleus formation cell-free system gynogenesis gibel carp.