Study On Light Absorption And Fluorescence Properties Of Laccase Immobilized On Sodium Alginate-Gelatin-Chitosan Composite

Laccase,a copper-containing polyphenol oxidase,is widely found in plants,fungi,bacteria and insects.This enzyme is known as a green catalyst because it can catalyze reactions with a wide range of compounds such as phenols,aromatic and aliphatic amines,hydroxyindoles,benzenethiols,polysaccharides and inorganic/organometallic complexes,with the product being only water.In nature,laccase exhibits a variety of biological functions,such as:plant laccase helps in the synthesis of lignin,flavonoids and secondary cell walls,and fungal laccase degrades lignin,etc.Laccase has a wide range of applications in industrial production,involving textiles,food,biofuels,organic synthesis,bioremediation,paper and pulp,pharmaceuticals,cosmetics,and other fields.Due to its high sensitivity and high-throughput detection,biosensors have been applied to various practical detections such as environment,food and water quality detection.This study investigates the light absorption and fluorescence properties of immobilized laccase by using a combination of bioinformatics and optics to provide a theoretical basis and experimental foundation for the construction of laccase optical biosensors,thereby extending their applications in medical,food,chemical and environmental detection fields.This thesis is organized around the following aspects:（1）Bioinformatics analysis of laccase.Genetic evolution and amino acid sequence analyses were performed on laccase from 18 common fungi,12 plants,5 bacteria and 12animals.The genetic evolution analysis revealed that the fungal laccase did not belong to the same major branch as the plant,bacterial and animal laccase,and it was hypothesized that there was diversity in the origin of laccase;the amino acid sequence analysis revealed that 27 out of 47 different species of laccase contained motif1,2,5 and 6.The comparative analysis（including amino acid sequence,secondary structure,subcellular localization and physicochemical properties）of Agaricus bisporus laccase（Accession No.Q12541）and Rhus vernicifera laccase（Accession No.Q8H979）revealed that the amino acid sequence and secondary structure of both were relatively similar and the specific catalytic sites were highly conserved.The isoelectric points of Agaricus bisporus laccase and Rhus vernicifera laccase were 5.60 and 7.34,respectively,indicating that they have different acidity and alkalinity in neutral solution.The subcellular localization analysis revealed that Agaricus bisporus laccase might be localized outside the cell,while Rhus vernicifera laccase was localized on the cell membrane,and it was speculated that their localizations were significantly different.（2）Study on the light absorption properties of immobilized laccase from sodium alginate chitosan gelatin composites.Immobilized Agaricus bisporus laccase and Rhus vernicifera laccase were reacted with gallic acid and scanned by light absorption spectra（wavelength range 250-800 nm）,and a significant absorption peak at 430 nm was found;the effect of sodium phosphate and sodium citrate buffer on the absorbance of the two immobilized laccases was compared,and the sodium phosphate buffer was found to be more suitable as the reaction solvent;the p H of the sodium phosphate buffer was adjusted to 3-8.The absorbance values of immobilized laccase from Agaricus bisporus were found to be highest at p H 7,while the absorbance values of immobilized laccase from Rhus vernicifera were highest at p H 6,indicating that the optimal p H of these two immobilized laccases were different;different substrates were selected for the reaction with the two immobilized laccases,and the highest absorbance values were found when they reacted with gallic acid,indicating that gallic acid was the optimal substrate for them in the reaction;different gallic acid concentrations were selected for the reaction with When different gallic acid concentrations were chosen to react with the two immobilized laccase,it was found that gallic acid concentrations as low as 0.2 m M could be detected;when common organic reagents（i.e.,ethanol and glycerol）were added to the reaction system,it was found that ethanol had less effect on the absorbance of immobilized Rhus vernicifera laccase when the volume of ethanol was 50%of the total system,while glycerol had less effect on the absorbance of immobilized laccase;different metal ions,on the absorbance of their two immobilized laccase reactions values had different degrees of interference,with Co²⁺having the strongest inhibitory effect on them their absorbance.（3）Study of the fluorescence properties of immobilized laccase from sodium alginate chitosan gelatin composites.Immobilized Agaricus bisporus laccase was reacted with gallic acid and scanned for fluorescence spectra,and it was found that the fluorescence intensity of its reaction was strongest when the excitation wavelength was 390 nm and the emission wavelength was 460 nm.Comparing the effect of sodium phosphate and sodium citrate buffer on the fluorescence intensity of its reaction,it was found that its fluorescence intensity was more stable in sodium phosphate buffer;its fluorescence intensity was highest when the p H of sodium phosphate buffer was 7;its fluorescence intensity was greater than that of ferulic acid and butyric acid when gallic acid was used as the reaction substrate;its fluorescence intensity was more inhibited in glycerol than that of ethanol;different metal ions,interfered with its fluorescence intensity to different degrees,among which Fe²⁺had the strongest inhibitory effect on its fluorescence intensity.