J Integr Plant Biol. ›› 2010, Vol. 52 ›› Issue (10): 879-890.DOI: 10.1111/j.1744-7909.2010.00988.x

• Research Articles • Previous Articles     Next Articles

Characterization of a Novel β-thioglucosidase CpTGG1 in Carica papaya and its Substrate-dependent and Ascorbic Acid-independent O-β-glucosidase Activity

Han Nong1,3,†, Jia-Ming Zhang1,†, Ding-Qin Li2,4,†, Meng Wang2, Xue-Piao Sun2, Yun Judy Zhu2, Johan Meijer5 and Qin-Huang Wang1,*   

  1. 1National Center for Tropical Crops Engineering and Technology Research, Spice and Beverage Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences (CATAS), Wannin 571533, China
    2MOA Key Biotechnology Laboratory for Tropical Crops, Institute of Tropical Bioscience and Biotechnology, CATAS, Haikou 571101, China
    3College of Agriculture, Hainan University, Haikou 571101, China
    4Institute of Biotechnology, Yunnan Academy of Agricultural Sciences, Kunming 650205, China
    5Department of Plant Biology and Forest Genetics, BioCenter, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden
  • Received:2010-07-04 Accepted:2010-07-30 Published:2010-10-01
  • About author:*Corresponding author Tel: +86 898 6255 4427; Fax: +86 898 6256 1083; E-mail: lab.wangqh@hotmail.com
    These authors contributed equally.

Abstract:

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 variation 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 μmol 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 CpTGG1 belongs to the MYR II subfamily of myrosinases.

Nong H, Zhang JM, Li DQ, Wang M, Sun XP, Zhu YJ, Meijer J, Wang QH (2010) Characterization of a novel β-thioglucosidase CpTGG1 in Carica papaya and its substrate-dependent and ascorbic acid-independent O-β-glucosidase activity. J. Integr. Plant Biol. 52(10), 879–890.

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