Purification of dual-functioning chitinases with hydrolytic and antifreeze activities from Hippophae rhamnoides seedlings

Abstract

Chitinases are glycosyl hydrolases which hydrolyse β-1,4-glycosidic bonds between N-acetylglucosamine residues of chitin. Seabuckthorn (Hippophae rhamnoides), a cold desert plant, is a storehouse of many cold-tolerant proteins including dual-functioning antifreeze proteins (AFPs) possessing both hydrolytic and antifreeze activities. Herein, we report the purification and characterization of antifreeze chitinases from seedlings grown in laboratory conditions. Chitin-affinity chromatography led to homogenous purification of two acidic chitinases HrS CHT1a (33 kDa) and HrS CHT1b (38 kDa) from seedlings. Antifreeze activity of purified AFPs was confirmed by the formation of hexagon-shaped ice crystals using nanolitre osmometer. Similarly, sucrose sandwich splat assay also confirmed their ice recrystallization inhibition activity (1.6-fold decrease in mean ice crystals). The chitinase activity of AFPs was confirmed by chitin hydrolytic assay where higher activity (1.8-fold) was observed in HrS CHT1b (500 U/mg) than HrS CHT1a (222 U/mg). MS identification showed homology of HrS CHT1b with provicilin while HrS CHT1a was identified as uncharacterized protein. In silico analysis showed that purified AFPs differ significantly in biochemical properties which suggests their different physiological roles. Protein association network analysis using string showed interaction of HrS CHT1b with enzymes involved majorly in pathogenic protection (pectinesterase, glycosyl hydrolase protein with chitinase domain). However, HrSCHT1a showed interaction with proteins associated with growth and energy regulation (glycine and purine synthesis, vitamin B metabolism) thereby indicating differential functional roles of both the chitinases. Conserved domain analysis also supported that these AFPs are multifunctional and exhibit differential regulatory roles in enabling the plant growth and defense responses. Further validation of these targets may open gates for commercial utilization of this plant growing abundantly in the Himalayan regions of India for protection of freeze-susceptible crops or biomedical applications.