Studies of expression and analysis of recombinant Arabidopsis myrosinases in Pichia pastoris

Abstract: Myrosinases (thioglucoside glucohydrolase, EC 3.2.3.147) are present in Brassicales plants. This enzyme can hydrolyze glucosinolates into various products of some which are toxic against pathogens and insects, thus serving as an important defense mechanism. Many vegetables and oil crops contain the myrosinase-glucosinolate system making studies on myrosinase very significant to understand how this type of plant protection has evolved. The performed experiments concerned expression of myrosinases in Pichia pastoris aiming to test their functionality based on recombinant enzyme activity and to compare different expression and cell breakage systems. Two myrosinase cDNAs from Arabidopsis thaliana (TGG1 and TGG4) had been cloned earlier into the pPIC3.5 vector behind an alcohol inducible promoter and with a histidine-tag added to the 3´-end (C-terminus of the protein). Pichia cells had earlier been transformed with the constructs and the transgene integrated into Pichia genome by homologous recombination. The purity and functionality of the TGG1 wild-type enzyme and TGG4 wild-type and mutated enzymes were analyzed by a coupled colorimetric activity assay, protein determination and SDS-PAGE. In addition, the expression of TGG1 and TGG4 were determined in Arabidopsis transformants containing GUS fusions with myrosinase gene promoters. GUS staining in these plant lines was investigated after treatment with beneficial Bacillus bacteria and challenge with pathogens. The basis of the study was to analyze the myrosinase activity and function necessary for plant protection and from the analysis we found that under stress conditions plants showed a more tissue specific myrosinase expression compared with control.