The cutinase CUTAB1 was cloned from a cutin induced culture of Alternaria brassicicola and heterologously expressed in Pichia pastoris under the control of the methanol-inducible AOX1 promoter. From a 400-ml culture, 36 mg of purified recombinant enzyme were obtained. Biochemical characterization revealed highest catalytic activity of the enzyme at 40 degrees C and pH 7-9 using p-nitrophenyl palmitate (p-NPP) as substrate. Among several fatty acid methyl and ethyl esters, glycerol esters and p-nitrophenyl esters tested, CUTAB1 showed highest activity towards tributyrin (3,302 +/- 160 U mg(-1)) and the activity decreased with increase in chain length of the investigated esters. Lowest activity was found for p-NPP. Replacing Leu80, Leu181 and Ile183, respectively, by the smaller alanine in the hydrophobic binding loop of CUTAB1, drastically reduced the overall activity of the enzyme. On the other hand, mutation A84F located in the small helical flap of CUTAB1 significantly increased the activity of the enzyme towards longer chain substrates like p-NPP.

Phenoxy radical coupling reactions are involved in the biosynthesis of lignans in planta. Interestingly, the reaction can be guided by dirigent proteins, which mediate the stereoselective formation of either (+) or (-)-pinoresinol from coniferyl alcohol. So far, the mechanism is poorly understood, and for detailed mechanistic studies, a heterologous expression platform which allows the cost-effective, fast, and robust expression in high yields is needed. We established a reliable, high-yield fed-batch fermentation process with Pichia pastoris resulting in 47 mg L⁻¹ of the dirigent protein AtDIR6, which represents a more than 250-fold increase compared to previous studies. Biochemical characterization of AtDIR6 produced with P. pastoris showed an overall agreement in protein structure, N-glycosylation sites, and dirigent activity compared to AtDIR6 produced by plant cell cultures of Solanum peruvianum. CD spectroscopy verified the β-barrel structure proposed by earlier studies and bioconversion experiments revealed similar activities to plant-derived protein, validating P. pastoris as a suitable expression system for dirigent proteins. Compared to the complex glycan structures of most plant cells, proteins produced with P. pastoris have the advantage that they can be enzymatically deglycosylated under non-denaturating conditions. With this study, we demonstrate that the glycan structures of AtDIR6 are essential for structure, solubility, and function of the protein as deglycosylation induced conformational changes leading to the complete loss in dirigent activity and subsequent protein aggregation.

No abstract

No abstract