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中文摘要：
智能酶是本项目小组在前期研究的基础上提出的概念，指的是通过蛋白的分子操作产生的具有快速监测和过程控制功能的酶，为实现酶的生产、分离纯化及催化过程的智能化控制和集成提供了重要思路。本项目以拥有的产气肠杆菌新型磷酸酶(PHO)为研究对象，在系统解析PHO的酶学特性的基础上，克隆分析PHO的全基因序列，通过分子改造，提高PHO特异转移磷酸的催化能力；分别构建PHO与麦芽糖结合蛋白(MBP)的两重融合蛋白、PHO与MBP和绿色荧光蛋白(GFP)三重融合蛋白的高效表达体系，利用GFP荧光的快速检测特点，建立融合蛋白的生产和催化过程的智能监控体系；建立定量表征MBP与不同状态麦芽糖和淀粉分子间相互作用的方法，利用MBP亲合吸附的基础数据，建立亲合吸附磁性纳米颗粒的规模化制备工艺；实现融合蛋白的分离和固定化一体化集成技术，构建固定化酶反应器，为智能磷酸酶催化生产呈味核苷酸技术的工业应用提供技术基础。
英文摘要：
fusion enzym；linker；GFP；magnetic nanoparticle；biocatalysis
结论摘要：
本项目创造性的提出刚柔度连续可调杂合连接肽库构建方法及其评价手段，并以磷酸转移酶、肝素酶、嗜热醇脱氢酶为模式体系，通过不同刚柔度的连接肽实现了酶的功能集成和活性显著提升，为利用连接肽调节融合酶的功能提供了重要的工具；成功制备了表面接枝麦芽糖分子的功能化磁性纳米粒子，能用于麦芽糖结合蛋白-肝素酶融合蛋白的特异性分离纯化和固定化，并发现其在融合酶的定向固定化中具有显著应用优势；实现了国内首套融合磷酸转移酶制备呈味核苷酸的中试工艺，该酶法技术与现有化学合成法工艺相比，具有明显的优势；实现了国际首套复合融合肝素酶法制备低分子量肝素的中试工艺，工艺的可控性及安全性优于现有化学法工艺，为低分子量肝素的清洁生产新技术发展提供了基础。项目执行期间，发表学术论文95篇，其中SCI收录47篇，参编1部专著（英文）；申请中国发明专利4项（其中授权专利4项）；国际学术会议邀请报告3次；主办国际会议2次，国际研究生学术会议2次，国内会议2次；培养博士后4名、博士生4名、硕士生4名。
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期刊论文
Cellulose-Based Dual Graft Molecular Brushes as Potential Drug Nanocarriers: Stimulus Responsive Mic
Fe3O4@poly (2-Hydroxyethyl Methacrylate)-graft-poly(ε-caprolactone) Magnetic Nanoparticles with Bran
Dual-sensing Porphyrin-containing Copolymer Nanosensor as Full-specturm Colorimeter and Ultra-sensit
Alteration of hydrogen metabolism of ldh-deleted Enterobacter aerogenes by overexpression of NAD(+)-
Rational Design of a Tripartite Fusion Protein of HeparinaseI Enables One-step Af?nity Puri?cation a
Biochemical Analysis and Kinetic Modeling of the Thermal Inactivation of MBP-Fused HeparinaseI: Impl
Reconstruction of the Violacein Biosynthetic Pathway from Duganella sp. B2 in Different Heterologous
Fluorescent Proteins as a Visible Molecular Signal for Rapid Quantification of Bioprocesses: Potenti
Dynamic Supramacromolecular Self-assembly: Deformable Polymer Fabricated Nanostructures through Host
Multifunctional Hybrid Magnetite Nanoparticles with pH-responsivity, Superparamagnetism, and Fluores
Synthesis and characterization of multi-functional hybrid magnetite nanoparticles with biodegradabil
Characteristics of low molecular weight heparin production by an ultrafiltration membrane bioreactor
Temperature influence on fluorescence intensity and enzyme activity of the fusion protein of GFP and
Systematic analysis of biochemical performance and the microbial community of an activated sludge pr
Synthesis, Characterization, and Controllable Drug Release of pH-sensitive Hybrid Magnetic Nanoparti
会议论文
Rational bioprocess design and manipulation of metabolic pathways for zero- and one-carbon sustainab
Deletion of Viod Gene in the Violacein Biosynthetic Pathway from Duganella sp. for the Efficient Pro
The characteristic of ARTP (Atmospheric and Room Temperature Plasma) Mutation System using Phenotypi
Construction of Efficient Expression System of Maltose Binding Protein (MBP) Fused Heparinase II/III
Construction of a Novel Cofactor Regeneration System by Using Thermostable NADH Oxidase from Hyperth
Rational bioprocess design and manipulation of metabolic pathways for zero- and one-carbon sustainab
Characterization of MBP-Fused HeparinaseⅠImmobilized on Magnetic Nanoparticles by Affinity Adsorptio
Rational bioprocess design and manipulation of metabolic pathways for zero- and one-carbon sustainab
Cloning, Overexpression, and Charcterization of Recombinant Chondroitinase AC from Pedobacter hepari
Characteristics of Low Molecular Weight Heparins (LMWHS) Produced by Different Combinations of Three
Fermentation performance of Pseudomonas putida with metabolic pathway reconstruction for efficient p
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