Expression And Enzymatic Properties Of Myrosinase Gene CpTGG1 In Carica Papaya

Glucosinolate-myrosinase system which is peculiar to cruciferous crops, endow them with the superiority of ecological expansion and the notable insect defense function. Glucosinolates and myrosinases are located spatially from each other in plant, and they come into contact upon tissue disruption and glucosinolates are degraded into toxic products, such as isothiocyanates, thiocyanates, nitriles or epithionitriles, oxazolidine-thione and so on. The breakdown products also have important value in the preventive work of tumor and heart disease.Carica papaya is one of the three main tropical herbaceons plants, which is the only known plant that has non-cruciferous myrosinases. Myrosinase in papaya has both S-β-glucosidases and cyanogenic O-β-glucosidases activity. In papaya there are Three myrosinase genes CpTGG1, CpTGG2 and CpTGG3 have been cloned. The recombinant CpTGG1 and CpTGG2 were successfully expressed in Pichia pastoris. To adopt biochemistry and bioinformatics, we belived that myrosinases are evolved from cyanogenic O-β-glucosidases only recently. And the glucosinolate-myrosinase system in papaya is good for our study in molecular evolution of myrosinase which is believed to belong to the early stage of the evolution of the glucosinolate-myrosinase system in plant.Here, we study the recombinant CpTGGl intensively, included kinetic parameters analysis, functional allocation, substrate-dependent activity and phylogenetic relationships among the family of myrosinase, which verify CpTGG1 with the nature of myrosinase and it has important value in the evolution of myrosinase. In this study we found that CpTGGl was expressed in the aboveground tissues in plant, CpTGG2 was specifically expressed in the root, and CpTGG3 was expressed in flower and pulp. The recombinant CpTGG1 expressed in Pichia pastoris catalyzed the hydrolysis of both sinigrin and glucotropaeolin (the only thioglucoside present in papaya), showing that CpTGG1 was indeed a functional myrosinase gene. Sequence alignment analysis indicated that CpTGG1 contained all the motifs conserved in functional myrosinases from crucifers, except for two aglycon-binding motifs. Using sinigrin as substrate, the apparent Km and Vmax values of recombinant CpTGGl were 2.82 mM and 59.9μmol min-1 mg protein-1, respectively. The Kcat/Km value was 23 s-1.mM-1. O-β-glucosidase activity towards a variety of substrates were tested, CpTGG1 displayed substrate-dependent and ascorbic acid-independent O-β-glucosidase activity towards 2-nitrophenyl-β-D-glucopyranoside and 4-nitrophenyl-β-D-glucopyranoside, but was inactive towards glucovanillin and n-octyl-β-D-glucopyranoside. Phylogenetic analysis indicated CpTGGs belong to the MYRⅡsubfamily of myrosinases.