Study On Enzymatic Characterization Of Laccase Isoenzymes From Pleurotus Eryngii Var.Ferulae

Laccase（EC 1.10.3.2）is a copper-containing polyphenol oxidase,which oxidizes a variety of substrates into non-contaminating product,showing excellent potential in the fields of food manufacturing,textile printing and dyeing,and biosensor.LACC2 and LACC6 are the main components of laccases,whose gene transcription levels were significantly upregulated and downregulated respectively during the co-culture of Pleurotus eryngii var.ferulae and Rhodotorulla mucilaginosa.In the study,the enzymatic properties of LACC2 and LACC6were analyzed and compared,which provided the basis data for the mechanism of co-culture.The main results are as follows:（1）pPIC9K-lacc2 and pPIC9K-lacc6 were successfully constructed and transformed into Pichia pastoris GS115 to obtain P.pastoris GS115/pPIC9K-lacc2 and P.pastoris GS115/pPIC9K-lacc6.By excising the signal peptides of lacc2 and lacc6 and optimizing the induction conditions of the heterologous expression process of the recombinant strains,two recombinant strains were successfully constructed and their enzyme activities reached 351.75U·L^-1 and 8578.34 U·L^-1,respectively.（2）Electrophoretically pure LACC2 and LACC6 were purified and collected by Ni-NTA spin column.The results of enzymatic studies on LACC2 and LACC6 indicated:When ABTS was used as the substrate,the optimal pH and temperatures of both LACC2 and LACC6 were3 and 50°C,respectively.Moreover,low concentrations of K⁺,Cu²⁺,Co²⁺and Mn²⁺enhanced the activities of LACC2 and LACC6 in varying degrees,and the positive effect on LACC2was more obvious,however,Fe²⁺and Fe³⁺had very significant inhibitory effects on LACC2and LACC6.In addition,LACC2 showed better tolerance to organic reagents and surfactants than LACC6,while LACC2 was more vulnerable to the inhibitors than LACC6.The enzyme kinetics results showed that ABTS was the optimal substrate of LACC2 and LACC6,and the K_m of LACC2 and LACC6 were 0.13 mmol·L^-1 and 0.24 mmol·L^-1,respectively.Compared with other laccases from bacterial,fungal and insect,LACC2 and LACC6 from P.eryngii var.ferulae had higher catalytic efficiency on ABTS.（3）sspoxa3a and sspoxa3b were successfully expressed in P.pastoris and Escherichia coli.The addition of small subunits increased the relative enzyme activity of LACC2 by 24.44%and 29.94%,respectively.In addition,the stability and pH tolerance of LACC2 had also been improved.The combination of small subunits and LACC2 effectively promoted enzyme activity and stability of LACC2,which was consistent with the results of upregulated gene transcription levels of lacc2,sspoxa3a,and sspoxa3b after co-culture in the previous study.