Functional Expression And Characterization Of Acid α-galactosidases In Cucumber (Cucumis Sativus L.)

Stachyose is the major carbohydrate translocated by cucumber(Cucumis sativus L.)in the phloem. The initial step in stachyose catabolism is thought to be hydrolysis byα-galactosidase. To study the detailed mechanism of stachyose unloading in cucumber, we have cloned the full length sequences of two putative aicdα-galactosidase cDNA. In this study, the two putative acidα-galactosidase genes were functional expressed using the baculovirus expression vector system(BEVS) in order to verify two cloned genes and study their enzyme characteristics.Two pairs of gene specific primers were designed and the entire coding region of the genes were amplified by polymerase chain reactions (PCR), then engineered into the baculovirus recombinant vector pVL 1392 and transformed into E.coli DH5α. Wild baculovirus and the expression vectors were used to co-transfect the healthy insect cell lines Sf9 in order to produce recombinant virus and heterologous express two putative aicdα-galactosidase genes. The results showed that two recombinant proteins produced in the insect cells are functional acidα-galactosidases.To investigate the enzyme characteristics of two recombinant acidα-galactosidases, the substrate specificity with artificial substrate p-nitrophenyl-α-D-galactoside (pNPG) and natural substrates, raffinose and stachyose, were investigated. The results showed that when use pNPG as substrate, as for acidα-galactosidaseⅠ, the best response pH is 4.8, the optimum temperature is 30℃, the Km is 8.99×10-4mol/L and Vm is 3.34×10-2 mol/L·min; as for acidα-galactosidaseⅡ, the best response pH is 6.1, the optimum temperature is 35-40℃, the Km is 1.35×10-3mol/L and Vm is 3.36 mol/L·min. the activity of two acidα-galactosidases were significantly inhibited by D-galactosidase with low concentrations. Furthermore, Two enzymes have high affinity with raffinose, but relatively low affinity with stachyose.