Characterization of an Antifungal and Cryoprotective Class I Chitinase from Table Grape Berries (Vitis viniferaCv. Cardinal)

Abstract

Gene expression of a class I chitinase (Vcchit1b) in the skin of table grapes was analyzed as a molecular marker for changes induced at low temperature and also to study the effect of high CO(2) levels modulating transcript levels at 0 degrees C. An active recombinant VcCHIT1b was overexpressed in Escherichia coli, and as the protein was produced as insoluble inclusion bodies, it was solubilized and refolded. The purified recombinant chitinase showed an optimum pH of 6.0 and a temperature of 50 degrees C, retaining activity at 0 and -10 degrees C. Purified chitinase exerted in vitro antifungal activity against Botrytis cinerea. Furthermore, recombinant chitinase was able to cryoprotect lactate dehydrogenase against freeze/thaw inactivation. However, the recombinant VcCHIT1b did not show any antifreeze activity when the thermal hysteresis activity was measured using differential scanning calorimetry.

Potent cryoprotective activity of cold and CO2-regulated cherimoya (Annona cherimola) endochitinase

“…Although the molecular mass of chitinases from fruit is higher than that detected here (30-41 kDa: Sowka et al, 1998;Diaz-Perales et al, 1998;Sanchez-Monge et al, 1999;Fernandez-Caballero et al, 2009), there are currently a few examples of low molecular mass chitinases from plants (10-15 kDa: Lin et al, 1992;Lam and Ng, 2001). The purity of the final enzyme preparation was confirmed by the appearance of a single band in SDS-PAGE and the absence of any artifacts during MS protein analysis.…”

“…Comparing the thermodynamic parameters of BChi14 with other plant chitinases using the same substrate in identical solvents is particularly revealing. The activation energy for BChi14 (11.32 kJ mol −1 ) was intermediate between that of mesophilic plant chitinases (Chang et al, 1996;Kuo et al, 2008) and that grape chitinase significantly active at subzero temperatures purified previously by our group (Fernandez-Caballero et al, 2009). On the other hand, while the other thermodynamic activation parameters ( H ‡ , S ‡ and G ‡ ) have been extensively described for cold-adapted chitinases from a psychrophilic bacteria (Lonhienne et al, 2000), we did not find similar data for plant homologues.…”

“…Additionally, the purified chitinase showed relatively great pH stability, mainly at basic values. The diversity of chitinase isoenzymes in many tissues is associated with differences in their optimum pH or temperature and, accordingly, BChi14 had a significant lower optimum temperature than its counterparts (Li et al, 2003;Taira et al, 2005;Fernandez-Caballero et al, 2009). While the optimum temperature of almost all the fruit chitinases reported previously was in a broad range from 40 to 60 • C, this newly purified enzyme displayed moderate hydrolytic activity in a lower temperature range (30-40 • C).…”

“…To date, the most cryoactive members of the PR protein family are AHCSP33, a PR-5 protein from leaf groundnut (Dave and Mitra, 1998), and a class I recombinant chitinase PR-3 from the skin of table grapes (Fernandez-Caballero et al, 2009), which were 1.2-and 2.2-fold more active that BSA on a molar basis. A dose-response analysis showed that the purified BChi14 chitinase protein offers considerable enzymatic protection in freeze-thaw cycles at very low concentrations.…”

“…Although the defensive function of chitinases and 1,3-␤-glucanases has been the focus of most research, additional functions unrelated to their hydrolytic activity have also been described in plants (Van Loon et al, 2006). In this context, chitinase and 1,3-␤-glucanase enzymes that are induced in response to cold and low temperatures exhibit antifreeze (Griffith and Yaish, 2004) or cryoprotective activity in vitro (Hincha et al, 1997;Fernandez-Caballero et al, 2009). Generally, hydrolytic PR enzymes can exert a dual function, involving both direct degradation of their substrate and cold protection.…”