Development of a Growth-Dependent System to Regulate Cell Growth and Keratinase Production in B. subtilis

Keratinases specifically degrade insoluble keratin waste, thus contributing to environmental protection and sustainable biomass development. However, their industrial application is hindered by inefficient enzyme production and poor biomass generation. In this study, the heterologous expression of keratinase was found to have cytotoxicity and might block host cell growth due to its proteolytic property. To address this problem, an autoregulatory expression system based on quorum sensing was developed to synergistically regulate cell growth and keratinase production in Bacillus subtilis. The growth-dependent promoter P_aprE was chosen and shown to be effective in delaying keratinase production while promoting host cell proliferation. Copy number screening and core region mutations further balanced the two states. Carbon supplement optimization indicated that addition of 2% glucose facilitated biomass accumulation during the early stage of fermentation. Cell density increased to 15.6 (OD_{600 nm}) from 8 with keratinase activity raised to 4200 U·mL^-1 from 1162 U·mL^-1. Keratinase was then utilized in the bioconversion of feather waste to prepare soluble keratins, polypeptides, and amino acids. This study provides a powerful system for efficient production of keratinase and paves the way for keratin waste recycling.