Abstract:

Myrosinase widely exists in the Cruciferae plant, which can catalyse the hydrolysis of
thioglucosides. Myrosinase and glucosinolate are mixed upon infestation by pests, and thioglucosides are
hydrolyzed into toxic compounds to serve as defense weapons. A new myrosinase gene was found, denoted as
TGG5 . The research of myrosinase geneTGG5 could explore the function of myrosinase defense system. In this
research, the cDNA sequence of TGG5 was cloned, and the pichia expression vector pPIC3.5K was
constructed, then transformation of pichia GS115 was made. The expression of recombinantTGG5 protein was
induced by Methanol, and purified by Ni-chelating colum. SDS-PAGE analysis showed that the recombinant
protein concentrated in the 58 kD molecular weight around and showed typical features of protein glycosylation
with a more visible tail. The myrosinase activity of recombinantTGG5 protein was future researched after
separation and purification. The results indicated that theK m andV max of the enzyme were 1.96 mmol/L and
0.084 mmol/(min·mg)respectively. The optimal pH of tested myrosinase was 5.5. The myrosinase showed the
wider adaptability in the temperature range of 37~80℃, and took on better stability on 80℃ high temperature.
NaCl had an obvious inhibitory effect on activity of tested myrosinase, but low concentration of ascorbic acidcould activate the myrosinase, which reached to a climax with an ascorbic acid final concentration of 1.5 mmol/L.
RT-RCR detection confirmed the TGG5 gene only specificlly expressed in roots, which showed that TGG5
represent a novel myrosinase gene subfamily.