Enzymatic Properties Of Streptomyces Laccase LOOP Mutant And Its Applications On Catalytic Substrate Crosslinking Reactions

In this study,wild and mutant Streptomyces laccase were obtained by fermentation and purification in our laboratory.Based on the study of the enzymatic properties of wild-type and mutat-type Streptomyces laccase,the effect of laccase on the cross-linking of whey protein isolate and high methoxy citrus pectin was studied.The graft of carboxymethyl chitosan with gallic acid catalyzed by mutant-type laccase was studied.The graft product of carboxymethyl chitosan and gallic acid was applied to strawberry preservation.To provide a basis for the application of carboxymethyl chitosan and gallic acid graft products in the field of food.The main research results are summarized as follows:1.Comparison of the enzymatic properties of wild-type and mutant-type Streptomyces laccase.Fermentation and purification were used to prepare wild-type and mutant-type laccase.The recovery rate of wild-type laccase in fermentation and purification process was higher.In the study of the effect of metal ions on the activity of wild-type and mutant-type laccase,it was found that Fe2+had a significant activation effect on the enzyme activity of mutant-type laccase,while Fe3+had an obvious activation effect on the activity of wild-type laccase.In the study of the effect of protein inhibitors on the activity of laccase of wild-type and mutat-type,it was found that L-cysteine with a concentration of 100mM had a significant inhibitory effect on the enzyme activity of wild-type and mutat-type laccase.Substrates specific studies found that laccase had enhanced affinity for phenolic acids,such as gallic acid and guaiacol,after mutation.The results of optimal reaction temperature and pH showed that the two properties did not change significantly after mutation.Temperature and pH stability indicated that the stability of laccase decreased after mutation.The results of circular dichroism analysis of two laccases showed that the content of laccase’s flexible structure increased and the stability decreased after mutation.2.Study on the cross-linking of whey protein isolate with high methoxy citrus pectin by mutant-type Streptomyces laccase.Solution was prepared with whey protein isolated and pectin in a ratio of 2:1,and put different concentrations of laccase in it to motivate crosslinking reaction,after testing the physical properties of film,such as tensile strength and elongation at break,moisture content and barrier property.At the same time tested the Fourier infrared spectrum,differential thermal scanning,scanning electron microscopy(SEM)and other properties of film,to explore the mechanism of the change of the macroscopic properties.The results showed that with the increase of the concentration of laccase,the properties of the films changed from good to bad.When the laccase concentration was 6U/100mL,the tensile strength and elongation at break of the film reached the optimum,which were 3.23 MPa and 40.88%respectively,showing excellent strength and ductility.The transparency of the film is best when the laccase concentration is 4 U/100 mL,which is conducive to improving the acceptance of consumers.The addition of laccase with different concentrations had no significant effect on the color of the film.At the same time,the water content and barrier properties of laccase were studied.It was found that when the concentration of laccase was 6 U/100 mL,the film had low water content and excellent gas and water barrier properties,which was conducive to the storage and preservation of the film itself.The thermal stability,crystallinity and infrared spectroscopy of the film were studied.The results showed that when the laccase concentration in the film was 6 U/100 mL,the film had a good secondary structure.At the same time,the scanning electron microscope results further demonstrated that a good whey protein isolate-pectin composite film could be obtained at the concentration of laccase at 6 U/100mL from the microscopic structure.3.Mutant-type Laccase catalyzed graft of carboxymethyl chitosan with gallic acid and its application in strawberry preservation.With carboxymethyl chitosan and gallic acid as reaction substrate,adding laccase to grafting successfully.The radical scavenging ability of grafting product was increased obviously,and the diferent concentrations of the grafted product was applied to strawberry preservation.Analyzing the color,hardness,soluble solids content,the content of ascorbic acid,the respiration intensity,reducing GSH content,activity of catalase(CAT),ascorbic acid peroxidase(APX)and superoxide dismutase(SOD)and other properties of strawberry the during the storage time.The results showed that the strawberries treated with a concentration of 1.5%grafted product(GA-g-CMCS)had the lowest decay rate during storage,while the strawberries treated with a concentration of 1.0%GA-g-CMCS had the lowest weight loss and respiratory intensity and the greatest hardness during storage.There was no significant difference in strawberry color in each group during storage.Biochemical indexes showed that the soluble solids content,ascorbic acid content,reduced glutathione content,catalase,ascorbic acid peroxidase and superoxide dismutase activities of strawberries treated with GA-g-CMCS at a concentration of 1.5%were higher than those of other groups during storage time.Therefore,GA-g-CMCS with a concentration of 1.5%has a good preservation effect on strawberries.