Optimisation of Recombinant Myrosinase Production in Pichia pastoris

Abstract

Myrosinase is a plant defence enzyme catalysing the hydrolysis of glucosinolates, a group of plant secondary metabolites, to a range of volatile compounds. One of the products, isothiocyanates, proved to have neuroprotective and chemo-preventive properties, making myrosinase a pharmaceutically interesting enzyme. In this work, extracellular expression of TGG1 myrosinase from Arabidopsis thaliana in the Pichia pastoris KM71H (Mut^S) strain was upscaled to a 3 L laboratory fermenter for the first time. Fermentation conditions (temperature and pH) were optimised, which resulted in a threefold increase in myrosinase productivity compared to unoptimised fermentation conditions. Dry cell weight increased 1.5-fold, reaching 100.5 g/L without additional glycerol feeding. Overall, a specific productivity of 4.1 U/L_medium/h was achieved, which was 102.5-fold higher compared to flask cultivations.

Keywords: Arabidopsis thaliana; Pichia pastoris; fermentation; myrosinase; upscale.

Figure 1

Scheme of glucosinolate hydrolysis. Bioactive products formation after myrosinase-catalysed hydrolysis of glucosinolates, which is affected by pH, the epithiospecifier protein (ESP), and the structure of the amino acid-derived side chain (R).

Figure 2

The effect of ascorbic acid on glucose determination assays. Inhibition of the colour development of glucose oxidase (GOD) kit by ascorbic acid (a). Ascorbic acid-independent glucose dehydrogenase (GDH) activity (b) and the dependence of maximal velocity in GDH catalysed reactions with various glucose concentrations (c).

Figure 3

The effect of decreased induction temperature on myrosinase production in mini fermenters. Increase in myrosinase activity (a) and specific productivity (b) at decreased induction temperature in high cell density cultivation of Pichia pastoris KM71H (Mut^S) in mini fermenters.

Figure 4

Temperature and pH optimisation of upscaled high-cell density cultivation of Pichia pastoris expressing myrosinase. Fermentation of P. pastoris KM71H (Mut^S) expressing AtTGG1 myrosinase at 30 (a) and 20 °C (b) at pH 5; and at pH 4 (c), and 6 (d) at 20 °C.

Figure 5

Catalytic properties of recombinant myrosinase. Temperature profile (a), operational temperature stability (b), pH profile (c), and ascorbic acid-dependent activation (d) of recombinant AtTGG1 myrosinase in the form of culture supernatant.

Figure 6

Long-term storage of recombinant myrosinase. Residual activity of recombinant AtTGG1 myrosinase stored in the form of culture supernatant with and without glycerol at 4, −20 and −80 °C.