Development of simple, scalable protease production from Botrytis cinerea

Heat haze-forming proteins are stable during winemaking and are typically removed via adsorption to bentonite. Proteolytic degradation is an alternative method to prevent wine-haze and offers the opportunity to reduce the environmental impacts and labor cost of the process. Herein, we describe the development of a production system for Botrytis cinerea proteases for the enzymatic degradation of heat haze-forming proteins. The effect of culture medium on the secretion of glucan by B. cinerea was investigated and methods to inactivate B. cinerea laccase in liquid culture medium were assessed. Protease production by B. cinerea was scaled up from 50 mL in shake flasks to 1 L in bioreactors, resulting in an increase in protease yield from 0.30 to 3.04 g L^-1. Glucan secretion by B. cinerea was minimal in culture medium containing lactose as a carbon source and either lactic or sulfuric acid for pH control. B. cinerea laccases were inactivated by reducing the pH of culture supernatant to 1.5 for 1 h. B. cinerea proteases were concentrated and partially purified using ammonium sulfate precipitation. SWATH-MS identified aspartic acid protease BcAP8 amongst the precipitated proteins. These results demonstrate a simple, affordable, and scalable process to produce proteases from B. cinerea as a replacement for bentonite in winemaking. KEY POINTS: • Isolates of B. cinerea that produce proteases with potential for reducing wine heat-haze forming proteins were identified. • Media and fermentation optimization increased protease yield tenfold and reduced glucan secretion. • Low pH treatment inactivated laccases but not proteases.