Unfolding and refolding of GH19 chitinase Chi19MK with antifungal activity from Lysobacter sp. MK9-1 at low pH and high temperature

Abstract

The GH19 chitinase Chi19MK from Lysobacter sp. MK9-1 inhibits fungal growth. In this study, the thermal stability of Chi19MK was investigated in buffers of different pH. Chi19MK retained approximately 100 % of its activity at 60 °C for 2 h in a sodium acetate buffer at pH 4.0. However, the enzyme retained approximately 15 % of its activity at 60 °C in potassium phosphate buffer (pH 6.0) and acetate (pH 5.0). Chi19MK was treated with acetate buffer (pH 4.0) for 1–5 h at a temperature range of 60 °C–100 °C, Chi19MK retained more than 70 % of its activity after heat treatment at 60 °C, 70 °C, and 80 °C for 5 h, and its activity was reduced to approximately 65 % after treatment at 90 °C for 2 h and at 100 °C for 1 h. The circular dichroism (CD) spectrum of Chi19MK in 10 mM acetate buffer (pH 4.0) was recorded at 80 °C and the CD spectrum showed the collapse of secondary structures. After decreasing the solution temperature from 80 °C to 25 °C, the spectrum was almost restored to the initial state. In contrast, the secondary structures of Chi19MK were not restored in acetate buffer (pH 5.0) or phosphate buffer (pH 6.0) by cooling after heat treatment at 80 °C. In an antifungal assay against Trichoderma reesei, Chi19MK retained its growth inhibition activity after heat treatment at 70 °C in acetate buffer at pH 4.0. These findings suggest that Chi19MK refolds in acetate buffer at pH 4.0, even when its conformation is disrupted by exposure to high-temperature conditions.