摘要
Plant thioglucosidases are the only known S-glycosidases in the large superfamily of glycosidases. These enzymes evolved more recently and are distributed mainly in Brassicales. Thioglucosidase research has focused mainly on the cruciferous crops due to their economic importance and cancer preventive benefits. In this study, we cloned a novel myrosinase gene, CpTGG1, from Carica papaya Linnaeus. and showed that it was expressed in the aboveground tissues in planta. 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, suggesting substrate priority varia- tion of the non-cruciferous myrosinases. Using sinigrin as substrate, the apparent Km and Vmax values of recombinant CpTGG1 were 2.82 mM and 59.9 μmoI min-1 mg protein-1, respectively. The KcatlKm 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 CpTGG1 belongs to the MYR II subfamily of myrosinases.
Plant thioglucosidases are the only known S-glycosidases in the large superfamily of glycosidases. These enzymes evolved more recently and are distributed mainly in Brassicales. Thioglucosidase research has focused mainly on the cruciferous crops due to their economic importance and cancer preventive benefits. In this study, we cloned a novel myrosinase gene, CpTGG1, from Carica papaya Linnaeus. and showed that it was expressed in the aboveground tissues in planta. 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, suggesting substrate priority varia- tion of the non-cruciferous myrosinases. Using sinigrin as substrate, the apparent Km and Vmax values of recombinant CpTGG1 were 2.82 mM and 59.9 μmoI min-1 mg protein-1, respectively. The KcatlKm 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 CpTGG1 belongs to the MYR II subfamily of myrosinases.
基金
supported by grants from the National Natural Science Foundation of China (30810103007)
the Natural Science Foundation of Hainan Province (80669)
the Key Scienceand Technology Program of Hainan Province (KJXM20100003)
the National Nonprofit Institute Research Grant of CATASITBB (KF1005)
