Bioinformatics Analysis And Optical Properties Of Microbial Laccase

Laccase(EC1.10.3.2)is a copper-containing polyphenol oxidase with low substrate specificity.It contains multiple substrates such as phenols,carboxylic acids,biological pigments,aromatic amines and metal organic compounds,and is widely distributed in higher plants,insects,fungi and bacteria.Its molecular structure contains four copper ions,divided into:type I copper ions,type II copper ions and two type III copper ions.Cu²⁺participates in the catalytic reaction of laccase:T1-Cu accepts electrons from the substrate,then transfers to T2/T3Cu,and finally transfers the electrons to oxygen.Among them,the structure of some non-blue laccases is different from the above.The yellow laccase contains four copper ions with varying oxidation states,while the white laccase contains one copper ion,one iron/manganese ion and two zinc ions.Because the laccase reaction is mild and stable,the by-products are non-toxic,copper ions participate in biochemical reactions and it has special optical properties,it can be used in the field of biosensors.The optical biosensor has the advantages of fast analysis speed,high accuracy and high sensitivity,and is widely used in the prevention and control of environmental pollutants,biomedical diagnosis and food detection.Therefore,more and more people are conducting research on laccase optical biosensors.However,the change mechanism of its optical properties is still unclear.In order to explore the change mechanism,the sequence of the microbial laccase is analyzed to study its structure,and the optical properties of Agaricus bisporus laccase are characterized to enhance the signal in the biological detection,it lays the foundation for the construction of optical biosensors based on laccase and gets more extensive application in food,environment and medicine.First of all,this project is based on bioinformatics to study the relationship between the structure and function of laccase,by analyzing the sequence of characteristic blue laccase and non-blue laccase,it is concluded that there is no significant difference between the glycosylation site,disulfide bond,phosphorylation site,signal peptide,transmembrane helix structure and amino acid content of the characteristic blue laccase and the non-blue laccase.But on the whole,it shows that laccase has not only monomers forms,but also multimeric forms,which are prone to phosphorylation,and most of them exist in the form of glycoproteins,showing diversity in cell position and sequence structure.Secondly,through single-factor experiments(such as pH,buffer,ions,etc.),the optical properties embodied in the biocatalytic reaction of Agaricus bisporus laccase were systematically characterized and analyzed,and the key parameters affecting its optical properties in biocatalysis were analyzed.In-depth study of related parameters(such as solvent ratio,substrate concentration,etc.)to enhance its optical signal in biological detection and make it be applied in the fields of food safety,environmental monitoring and biomedicine.Experimental results show:1.The laccase reaction of Agaricus bisporus has an obvious absorption peak at the absorbance wavelength of about 400 nm,and a strong fluorescence signal appears around the excitation wavelength of 280 nm and emission wavelength of 340 nm.Based on the above results,explore and the study.2.The absorbance of Agaricus bisporus laccase reaction in sodium phosphate buffer is stronger than that in citrate buffer;and the difference in fluorescence intensity is more obvious than that in sodium citrate buffer.3.The laccase reaction of Agaricus bisporus had the strongest absorbance at pH 7,and the strongest fluorescence intensity at pH 6.4.The absorbance of the laccase reaction of Agaricus bisporus gradually increases with the increase of ethanol concentration(0%,5%,10%,20%,50%).The absorbance in 50%ethanol is the strongest;The fluorescence intensity gradually decreases with time,and the fluorescence intensity is the strongest in 50%ethanol;its absorbance increases with the increase of glycerol concentration(0%,5%,10%,20%,50%),The absorbance first increases and then decreases with time,the highest absorbance in 5%glycerol;the fluorescence intensity gradually decreases with the increase of time,and with the glycerol concentration(0%,5%,10%,20%,50%)as it increases,its fluorescence intensity shows a trend of first decreasing and then increasing.The fluorescence intensity is the strongest in 50%glycerol.5.The absorbance produced by the laccase reaction of Agaricus bisporus catalyzed by the chemical reaction in different substrate concentrations gradually increased with the increase in concentration.When the substrate concentration was 0.5 m M,the difference in fluorescence intensity was more obvious than other concentrations.6.Different substrates have different effects on the optical properties of Agaricus bisporus laccase reaction.7.The effect of different metal ions on the optical properties of Agaricus bisporus laccase reaction:Cu²⁺and Fe²⁺inhibit the absorbance and fluorescence intensity in the catalyzed reaction of Agaricus bisporus laccase,K⁺promotes the absorbance and fluorescence intensity in the reaction.Ca²⁺?Co²⁺and Ni²⁺has a promoting effect on the absorbance of the reaction,but has an inhibitory effect on the fluorescence intensity.Finally,the laccase of Agaricus bisporus was immobilized by sol-gel technology to improve the stability of the laccase reaction and realize the reuse of laccase,laying a foundation for the manufacture of laccase optical biosensors.By comparing the immobilized Agaricus bisporus laccase with free Agaricus bisporus laccase,it was found that the absorbance and fluorescence intensity produced by the reaction become stronger,and the fluorescence intensity gradually increases as the number of times of using immobilized Agaricus bisporus laccase increases.This result indicates that the immobilized laccase is beneficial to the characterization of the optical signal generated by the reaction.