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

Abstract

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.

Molecular strategies to enhance the keratinase gene expression and its potential implications in poultry feed industry

“…The main factors that may affect the yield and production rate depend upon the choice of appropriate host for preparing recombinant enzyme protein that is expressed in bacteria strains like lactic acid bacteria, Bacillus and Escherichia coli, it would be used in Pichia pastoria (yeast) and Aspergillus (fungi) ( Liu et al, 2013 ; Hou et al, 2019 ).In a bacterial host, it's more convenient to express recombinant enzyme protein as it cannot over express large proteins that may need to post translational modifications ( Gong et al, 2020 ; Ruiz-Villafán et al, 2023 ).Through xylose inducible expression arrangements in B. megaterium and E.coli ; keratinase of Bacillus licheniformis was cloned successfully with the use of 17 promotors ( Radha and Gunasekaran 2007 ; Gong et al, 2020 ). Response surface methodology ( RSM ) had been used to maximize the process that inference in 3-time high keratinase production through B. megaterium (pWHK3 recombinant than that of strain B. licheniformis MKU3 ( Radha and Gunasekaran 2007 ; Tian et al, 2019 ; Su et al, 2023 ). Many studies have reported the different factors affecting the keratinase enzyme activity of different strains of bacteria on various waste products of poultry industry.…”

“…The integrated vectors containing kerA, either with or without the P43-promoter, were created and subsequently subcloned into Bacillus subtilis DB104 and B. licheniformis T399D. Southern blot analysis was used to identify and determine the presence of multiple copies of kerA integration into the chromosome in T399D ( Rahimnahal et al, 2023 ; Su et al, 2023 ). Three to 5 copies were found to be the ideal range for kerA integration.…”

Perspective on Agricultural Industrialization: Modification Strategies for Enhancing the Catalytic Capacity of Keratinase

“…20 In the process of feed pelleting, there is a short step involving a high-temperature steam process, which requires a certain degree of heat resistance for the keratinases; moreover, the dehairing step in the leather industry and the keratinases in the washing industry both require resistance alkali. 21 Along with the modification of the extremophilicity of keratinases, acid and alkali resistance, high temperature resistance, and protease resistance will be the main directions of keratinase modification. Many agricultural and industrial applications have used microbial-produced keratinases in recent decades.…”