| Morchella importuna SCYDJ1-A1 is a black morel strain that was domesticated and successfully cultivated under artificial condition.Due to the oxidative decomposition of enzymes,such as,laccase,manganese peroxidase and lignin peroxidase,etc,are important enzymes in plant-litter decay of soil-inhabiting ascomycetes,this study determined the key laccase gene in the life-cycle of the morel and further characterized the enzyme.Two laccase-like multicopper oxidase（LMCO）genes were identified in the genome of M.importuna SCYDJ1-A1 as putative laccase-encoding genes,thus named as Mi Lac A and Mi Lac B.They belonged to Auxiliary Activity family 1 subfamily 3.Phylogenetic analysis of deduced amino acid sequences showed that Mi Lac A was closest to a LMCO of M.importuna 22J1,while Mi Lac B had low similarity with known Morchella LMCOs.Real-time quantitative PCR results showed that Mi Lac A was expressed at much higher levels than Mi Lac B throughout the whole artificial cultivation course.Mi Lac A was overexpressed in Pichia pastoris as a recombinant protein.Biochemical characterization of purified enzyme showed that Mi Lac A simultaneously possessed activities of laccase as well as polyphenol oxidase.The enzyme received strong inhibition from Fe2+,which is unusual.The optimum p H was 4,optimum temperature was 60°C and molecular weight was 66 KDa.The enzyme could retain over 74%of the laccase activity after 16 h incubation at 60°C,which means that the thermostability is in a front position among the currently known laccases.The findings might help understanding how the laccase of M.importuna is involved in decaying lignin in soil plant-litter,and could also provide a candidate thermostable laccase for potential industrial application.There is a ferroxidase of M.importuna SCYJD1-A1 which is most active in the formation and mushrooming stages of the life history of the morel earlier before this experiment.Ferroxidase may be involved in the degradation of key nutrients for mushroom production.In this experiment,we used the E.coli expression system Aretic Express（DE3）to express the foreign protein of the ferroxidase gene Mi FOX,and further studied its biochemical characteristics,which provided a reference for us to better understand the nutrient utilization of the morel.The relative molecular weight of Mi FOX is about 64 KDa.Substrate specificity studies show that Mi FOX enzyme both has laccase activity and freeoxidase activity.Mi FOX maintains laccase activity and stability under acidic to neutral conditions,after 16h,the Mi FOX enzyme still maintained at least 70%activity.Although the Mi FOX enzyme has high activity at 0 to 80°C,it is obviously not resistant to high temperature environment.And only known from the data shows that the influence of most metal ions and SDS and urea on Mi FOX enzymes is positive or extremely negative. |
