Study Of Electrochemical Method Detection Of Glucose Oxidase Activity During The Germination Of Aspergillus Spores

Spore is a special form of life of organisms with reproduction or dormant effects.Spores can sustain their own ability to spread in harsh environments and develops directly into new individuals under favorable conditions.Although spores are thought to be metabolically inert,they are excellent enzyme reservoirs and promising biocatalysts which is also the important stage of life reproduction.Studies of active substances in spore germination provide a theoretical basis which can be used to effectively utilize or control the living.The traditional methods include Spectrophotometry,chromatography which is time-consuming,labor-intensive,and costly and requires special facilities and can not carry out real-time,in situ research.The electrochemical method has the characteristics of high sensitivity,good selectivity and fast response.The electrochemical scanning microscope can realize the real-time,in-situ,non-destructive and on-line analysis of the biological system,combined with piezoelectric position meter and shows advantage in micro analysies in the study of morphology of the biological system.Therefore,we propose a new method and a new idea to study the active components in the process of spore germination by electrochemical method.In this paper,the glucose oxidase(GOx),a microbial source which is widely used in food,medicine,chemical industry and biotechnology industry,was used to study of the change of the GOx activity of Aspergillus sp.at different growth stages by electrochemical method to provide the theoretical basis and technical support for the further development of GOx biocatalyst activity or as target during the inhibition of harmful fungi.The results are as follows:(1)Electrochemical cyclic voltammetric method to the quantitative measurement of the activity of GOx by inducing Ferrocene methanol(Fc)as redox probe to get a bigger electron transfer in catalytic reaction.Since the orientation of the enzyme on the surface of the electrode is often detrimental to the electron transfer between its electroactive groups and the electrodes,the direct electron exchange between the enzyme and the electrode is hindered and the electrochemical detection of enzyme activity has always been a troubling problem.In this paper,The Fc was introducing as the redox probes on cyclic voltammetry,using glassy carbon electrode as working electrode,saturated calomel electrode as reference electrode,platinum wire electrode as the counter electrode and the peak current value of different enzyme activity was required.There was a linear relationship between the peak current in enzyme concentration of 0.128～2.377 U/mL and its regression equation was obtained as:I[10-5A]=4.427 E[U]+14.054,with the correlation coefficient as 0.998,the detection limitation as 1.282 U/mL.The recovery rate was between 97.208%and 104.538%though recovery test.At the same time of process of electrochemical experiment,the traditional titration method were compared and results indicated that cyclic voltammetry method for the detection of glucose oxidase activity is accurate and reliable and the operation is more simple and fast.(2)Determination of GOx in spore germination of Aspergillus niger by Electrochemical cyclic voltammetry.Based on the method of electrochemical cycling voltammetry established in the previous chapter to determine the activity of GOx,the principle and method of cyclic voltammetry for the determination of GOx activity in Aspergillus niger spores were proposed.The method was applied to the determination of GOx activity during the spore germination of Aspergillus niger,At the same time,the growth of Aspergillus niger spores was observed by biological microscope,and the changes of GOx activity during the spore germination of Aspergillus niger were obtained.The results showed that when cultured in 96-well plates,the activity of spores did not change obviously for 0～4 h.At the following 4～10 h,the rate of germination was gradually increased and the activity of GOx was gradually increased,reaching a maximum of 2.13 U/mL at 8 h.At 10～16 h,germination of spores was complete and the activity of GOx in spores was restored to the original level.The titration method was used to carry out the control experiment,and results showed that the two methods were similar.(3)Scanning electrochemical microscopy(SECM)monitor the the morphology and the changes of GOx activity of the single-spore germination process of Aspergillus flavus in real-time.Based on the principle of cyclic voltammetry for the detection of GOx,the morphological changes and the changes of GOx activity during the spore germination of Aspergillus flavus were studied in real-time in combination with SECM.spores of Aspergillus flavus was immobilized in polystyrene petri dish.Fc was used as the redox medium,using 10 um platimum electrode as working probe,silver/silver chloride as reference electrode,platinum wire electrode as auxiliary electrode,the SECM image of single Aspergillus flavus was used to observe the changes of the morphology and monitor the GOx activity of spores of Aspergillus flavus from dormancy to germination.From the SECM image,as the spores germinate,the spore shape is changed from the approximate sphere to having the germination tube that slowly grows on its side.At the same time,the permeability of the membrane of sporulated spore increased,Fc and glucose penetrated into the spore to react with GOx which recovered as the spores germinated,leading to higher current above the spores in germination stage than that in dormancy state.