Expression And Enzymatic Properties Of Linamarase In Cassava

Cyanogenic glucosidase catalyzes the degradation of cyanogenic glucoside in the Euphorbiaceae, Leguminosae, Rosaceae, and other crops, yeilding hydrogen cyanide. As a result, the reaction is called cyanogenesis. It is utilized by plants to form a unique cyanogenic defense system. Cyanogenic glucoside glucohydrolase is separately stored from cyanogenic glucoside, when the plant gets hurt from outside, the cyanogenic glucohydrolase reacts with substrates to produce toxic hydrogen cyanide, forming an essential chemical defense system. Moreover, the defense system has gradually been used for the treatment of cancer through the continuous efforts of the researchers, which indicates that it is quite necessary and urgent to carry out study on cyanogenic glycosidase.Linamarase, a kind of cyanogenic glucosidase, plays an important role in the degradation of cyanogenic glucoside in crops such as cassava, croton linamarin (linseed glycosides). It shares a lot of similarities with myrosinase in the structure. Based on bioinformatics and biochemical studies, it was hypothesized that cyanogenic glucosidase may be the precursor of myrosinase. The origin of myrosinase and the relationship between the cyanogenic glucosidase and myrosinase could be uncovered if we focus on linamarase.In this study, further research about linamarase in cassava was carried out, including tissue-specific expression in cassava SC8, high-cyanide cassava and low-cyanide cassava, the enzymatic properties. The results of PT-PCR confirmed that linamarase was synthesized on aboveground of plant, and then transported to the underground part; no significant difference in expression was found on the terminal bud of high cyanide and low-cyanide cassava, although higher expression appeared in leaves and stems of low cyanide cassava and no expression in the tuber. Linamarase gene was cloned into the yeast expression vector then transformed into Pichia pastoris GS115. Purified recombinant linamarase was obtained after inducible expression and affinity purification. SDS-PAGE analysis showed that the molecular weight of recombinant protein was about71kDa. The optimum temperature and pH of the enzyme was about37℃and5respectively. The Km was1.70mmol/L and the maximum reaction rate was8.36μmol/(min·mg) when pNPG was its substrate.