Cold-active proteases from Antarctic fungi as alternatives to heat-stabilisation with bentonite

The aim of this project was to seek an enzymatic replacement for bentonite suited to the low temperatures of winemaking. A cold-active protease would contribute to a new winemaking technology and, by precluding bentonite usage, could improve wine quality by reducing flavour losses and by facilitating membrane treatments for tailored wine styles. A successful cold-active protease would also reduce the environmental impact of winemaking and, as a product of biotechnology, would be far more sustainable than bentonite mining and hazardous waste removal.

Macquarie University has a collection of approximately 200 fungi from Antarctica, several of which were known to secrete cold-active proteases. In this project, the protease activities of all 200 isolates were screened using agar plate assays carried out at low temperature and low pH. The agar plates contained skim milk as a protein source, resulting in a cloudy appearance that was cleared as a result of proteases secreted by the fungal colonies. Twenty-four isolates were selected for their high protease activity ranked by the size of the clearing halo that appeared around the fungal colonies in relation to the colony size.

The 24 fungal isolates that displayed the highest protease activity in the plate assays were grown in liquid culture media. Supernatants were assessed for protease activity using liquid assays. Strains that exhibited the highest protease activity under acidic conditions at 10 ºC were Trichoderma, Glomerella, Embellisia, Chrysosporium, and Phoma species.

Protease activity against grape juice proteins was determined by incubating culture supernatants with grape juice at 10 ºC and 20 ºC and determining the extent of proteolysis by SDS-PAGE. Supernatant from a Phialophora stain (O1) caused some modification of chitinase, resulting in a reduction in molecular weight after two days incubation with grape juice at 10 ºC. Chrysosporium, Phoma, Glomerella, Embellisia, Aureobasidium, and Phialophora strains appeared to cause a very slight degradation of chitinase and thaumatin. The supernatant of a Chrysosporium strain (A6) caused significant degradation of both chitinase and thaumatin at 60 ºC. A serine protease was identified from the Chrysosporium A6 supernatant by mass spectrometry.