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Enhanced thermostability and catalytic efciency of glucose oxidase in Pichia Pastoris

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Systems Microbiology and Biomanufacturing 2022年第2期2卷 296-304页

作者： Hanlei Zhou Pu Zheng Pengcheng Chen Xiaowei Yu Dan Wu The Key Laboratory of Industrial Biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi 214122China

glucose oxidase(GOD)has many practical applications,but its poor thermostability limits its broader use.In this research,three primary mutants of wild-type GOD were designed using rational mutagenesis,and the GODm mutant was constructed by combinatorial design.The expression,purifcation,and enzymatic properties of the mutants were studied.The specifc enzyme activity of GODm was 2.10-fold higher than that of wild type,and the(k_(cat)/K_(m))value was increased by 1.45-fold.After treatment at 55℃for 3 h,GODm retained 37.5%of its enzymatic activity,and the half-life(t_(1/2))of GODm at 55℃and 65℃was 2.28-fold and 3.36-fold higher than that of wild type,respectively.By analyzing the three-dimensional structure of wild type and the GODm mutant,it was found that T30V formed a new hydrogen bond with FAD and strengthened the hydrophobic interaction,D315K optimized the surface electrostatic interaction,and A162T improved the efciency of the electron pathway.Thus,a novel mutant with improved thermostability and catalytic efciency was obtained in this research.

关键词： glucose oxidase Pichia pastoris Thermostability Catalytic efciency Rational mutagenesis

A direct electrochemical biosensor for rapid glucose detection based on nitrogen-doped carbon nanocages

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Rare Metals 2024年第5期43卷 2184-2192页

作者： Lan-Lan Li Yu Zhao Li-Jia Pan Jian-Bin Xu Yi Shi College of Mechanical and Electrical Engineering Henan Agricultural University School of Electronic Science and Engineering Collaborative Innovation Center of Advanced Microstructures Nanjing University Department of Electronic Engineering The Chinese University of Hong Kong

Given the increasing number of diabetic patients,rapid and accurate detection of glucose in body fluids is critical.This study developed a direct electrochemical biosensor for glucose based on nitrogen-doped carbon nanocages （NCNCs）.NCNCs possess a large specific surface area of 1395 m2·g-1,a high N atomic content of 9.37%and good biocompatibility,which is favorable for enzyme loading and electron transfer.The surface average concentration of electroactive glucose oxidase on NCNCs was 2.82×10–10mol·cm-2.The NCNC-based direct electrochemical biosensor exhibited a high sensitivity of 13.7μA·(mmol·L-1)-1·cm-2,rapid response time of 5 s and an impressive electron-transferrate constant （ks） of 1.87 s-1.Furthermore,we investigated an NCNC-based direct electron transfer （DET） biosensor for sweat glucose detection,which demonstrated tremendous promise for non-invasive wearable diabetes diagnosis

关键词： Direct electron transfer (DET) Nitrogen-doped carbon nanocages (NCNCs) glucose oxidase Biosensor

Regulation of glucose oxidase Activity through Interaction with Fullerene Derivatives

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Chinese Journal of Chemistry 2012年第2期30卷 418-426页

作者： Gao,Yunyan Wang,Zhongli Ou,Zhize Li,Yi Wang,Xuesong Yang,Guoqiang Ministry of Education Key Laboratory of Space Applied Physics and Chemistry&Department of Applied Chemistry School of ScienceNorthwestern Polytechnical UniversityXi'anShaanxi 710072China Technical Institute of Physics and Chemistry Chinese Academy of SciencesBeijing 100190China CAS Key Laboratory of Photochemistry Institute of ChemistryChinese Academy of SciencesBeijing 100190China

The 2-（hydroxymethyl）pyridine modified C60 （PY-C60） and methoxydiglycol modified C60 （MDG-C60） are synthesized using Bingel-Hirsch reaction and characterized by nuclear magnetic resonance （NMR） and mass spectra. PY-C60 and MDG-C60 can bind to glucose oxidase （GOx） and quench the fluorescence of tryptophan （Trp） residue in GOx through static mechanism. The conformation of GOx is disturbed after formation of complex with these fullerene derivatives. Kinetic analysis indicates that PY-C60 and MDG-C60 may affect the catalytic activity of GOx with a partial mixed-type inhibition mechanism. In the plasma glucose concentration range （3.6--5.2 mmol·L-1）, PY-C60 may significantly accelerate the catalytic velocity of GOx, however, MDG-C60 exerts almost no obvious change to the initial velocity of GOx, suggesting that elaborate design of molecular structure of fullerene derivative is very important for regulating the biological activity of fullerene-enzyme complex.

关键词： fullerene C60 glucose oxidase partial mixed-type inhibition mechanism redox mediator electron transfer

A Chemiluminescence Optical Fiber glucose Biosensor Based on Co-immobilizing glucose oxidase and Horseradish Peroxidase in a Sol-gel Film

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Chemical Research in Chinese Universities 2002年第1期18卷 12-15页

作者： LI Ying-xiu, ZHU Lian-de and ZHU Guo-yi State Key Lab of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P.R. ChinaZHAO Chun College of Chemistry, Jilin University, Changchun 130023, P Changchun Institute of Applied Chemistry Chinese Academy of Sciences State Key Lab of Electroanalytical Chemistry Changchun 130022 College of Chemistry Jilin University Changchun 130023

An optical fiber bienzyme sensor based on the luminol chemiluminescent reaction was developed and demonstrated to be sensitive to glucose. glucose oxidase(GOD) and horseradish peroxidase(HRP) were co-immobilized by microencapsulation in a sol-gel film derived from tetraethyl orthosilicate(TEOS). The calibration plots for glucose were established by the optical fiber glucose sensor fabricated by attaching the bienzyme silica gel onto the glass window of the fiber bundle. The linear range was 0 2-2 mmol/L and the detection limit was approximately 0 12 mmol/L. The relative standard deviation was 5.3% ( n =6). The proposed biosensor was applied to glucose assay in ofloxacin injection successfully.

关键词： Fiber optical biosensor Sol-gel Chemiluminescence glucose oxidase Horseradish peroxidase glucose assay

IMMOBILIZATION OF glucose oxidase AND CELLULASE BY CHITOSAN— POLYACRYLIC ACID COMPLEX

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Chinese Journal of Polymer Science 1990年第2期8卷 115-120页

作者：王玲治江英彦张长德黄德琇 Institute of Chemistry Academia Sinica Beijing Xi'an Jiaotong University Xi'an China

This study is concerned with chitosan-polyacrylic acid complex as a carrier to immobilize glucose oxidase (GOD)and cellulase. The optimum emperature of the immobilized GOD (IG) was determined to be 60℃ which is higher than that of the native GOD about 40℃. The optimum temperature of the immobilized cellulase (IC) was determined to be about 30℃ higher than that of native cellulase. Both of the optimum pH of IG and IC shifted one pH unit to acid. Immobilized enzyme may be used in more wide pH range. Their storage life are much longer compared with their native states. Both of them can be reused at least 12 times.

关键词： Immobilized enzyme Cellulase glucose oxidase Polymer complex

Direct Electron Communication Between glucose oxidase and Carbon Electrode Covered with Polypyrrole

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Chemical Research in Chinese Universities 1996年第1期12卷 37-43页

作者： CHI Qi-jin and DONG Shao-jun(Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry,Chinese Academp of Sciences, Changchun 130022) ACAD SINICA CHANGCHUN INST APPL CHEMELECTROANALYT CHEM LABCHANGCHUN 130022PEOPLES R CHINA

glucose oxidase can be effectively adsorbed onto the polypyrrole (PPy) thin film electrochemically formed on an anodized galssy carbon electrode (GCEa ). Direct electron communication between the redox of GOD and the modified electrode was successfully achieved, which was detected using cyclic voltammetry. GOD entrapped in PPy film still remained its biological activity and could catalyze the oxidation of glucose. As a third generation biosensor, GOD-PPy/GCEa responded linearly up to 20 mM glucose with a wider linear concentration range.

关键词： Biosensor Polypyrrole Anodization glucose oxidase

Immobilization of glucose oxidase on Ordered Macroporous Silicas Functionalized with Amino Group

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Chinese Journal of Chemistry 2012年第2期30卷 283-287页

作者： Wu,Quanzhou He,Jianfeng Zhang,Jingjia Ou,Jiming College of Chinese Materia Medica Guangzhou University of Chinese MedicineGuangzhouGuangdong 510006China

A novel glucose oxidase immobilized on three-dimensionally ordered macroporous （3DOM） material has been prepared by firstly preparation of hybrid 3DOM SiO2-NH2 materials using colloidal crystal method, and following covalent immobilization of glucose oxidase on the pore walls of the 3DOM materials. The materials were characterized by SEM, FTIR, DSC and BET techniques. SEM observation shows that the macropores are highly ordered and are interconnected by small windows. FTIR measurement shows that there are amino and organic groups in the pore walls. The surface area of the 3DOM SiO2-NH2 material is about 10.2 m2/g. The loaded amount of enzyme is increased with amino content in the materials. The immobilized enzyme has high activity, thermal stability and can be reused.

关键词： three-dimensionally ordered macropore SiO2 glucose oxidase supported catalyst materials science

Direct Electron Transfer between glucose oxidase and Multi-walled Carbon Nanotubes

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Chinese Journal of Chemistry 2003年第8期21卷 1088-1091页

作者：徐锦忠朱俊杰吴强胡征陈洪渊 Laboratory of Mesoscopic Materials Science Department of ChemistryNanjing University

The direct electrochemical behavior between the glucose oxidase (GOD) and the multi walled carbon nanotubes (MWNTs) has been studied. Two pairs of cyclic voltammetric peaks corresponding to the two different processes, i.e. mass transport and surface reaction of GOD are observed on this MWNTs. The formal potentials with E o′=-0.45 V and E o′=-0.55 V were obtained respectively. The GOD film was observed on the carbon nanotube by the TEM.

关键词： carbon nanotube glucose oxidase direct electron transfer

Immobilization and Characterization of glucose oxidase on Single-Walled Carbon Nanotubes and Its Application to Sensing glucose

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Chinese Journal of Chemistry 2007年第4期25卷 439-447页

作者：刘姝娜印亚静蔡称心 Jiangsu Key Laboratory for Molecular and Medical Biotechnology College of Chemistry and Environmental Science Nanjing Normal University Nanjing Jiangsu 210097 China

The negatively charged （at pH 8.2） glucose oxidase （GOx, pI ca. 4.2） was assembled onto the surface of single-walled carbon nanotubes （SWNT）, which was covered （or wrapped） by a layer of positively charged polyelectrolyte poly（dimethyldiallylammonium chloride）（PDDA）, via the electrostatic interaction forming GOx-PDDASWNT nanocomposites. Fourier transform infrared （FTIR）, UV-Vis and electrochemical impedance spectroscopy （EIS） were used to characterize the growth processes of the nanocomposites. The results indicated that GOx retained its native secondary conformational structure after it was immobilized on the surface of PDDA-SWNT. A biosensor （Nafion-GOx-PDDA-SWNT/GC） was developed by immobilization of GOx-PDDA-SWNT nanocomposites on the surface of glassy carbon （GC） electrode using Nafion （5%） as a binder. The biosensor showed the electrocatalytic activity toward the oxidation of glucose under the presence of ferrocene monocarboxylic acid （FcM） as an electroactive mediator with a good stability, reproducibility and higher biological affinity. Under an optimal condition, the biosensor could be used to detection of glucose, presenting a typical characteristic of Michaelis-Menten kinetics with the apparent Michaelis-Menten constant of KM^app ca. 4.5 mmol/L, with a linear range of the concentration of glucose from 0.5 to 5.5 mmol/L （with correlation coefficient of 0.999） and the detection limit of ca. 83 μmol/L （at a signal-to-noise ratio of 3）. Thus the biosensor was useful in sensing the glucose concentration in serum since the normal glucose concentration in blood serum was around 4.6 mmol/L. The facile procedure of immobilizing GOx used in present work would promote the developments of electrochemical research for enzymes （proteins）, biosensors, biofuel cells and other bioelectrochemical devices.

关键词： glucose oxidase carbon nanotube glucose biosenso nanocomposite