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The Keratinase Enzyme: Development of Suitable Application ParametersEPA Grant Number: U914946
Title: The Keratinase Enzyme: Development of Suitable Application Parameters
Investigators: Carter, Scott D.
Institution: North Carolina State University
EPA Project Officer: Just, Theodore J.
Project Period: January 1, 1996 through January 1, 1999
Project Amount: $102,000
RFA: STAR Graduate Fellowships (1996) RFA Text | Recipients Lists
Research Category: Fellowship - Molecular Biology/Genetics , Academic Fellowships , Biology/Life Sciences
The objective of this research project is to develop the keratinase enzyme for large-scale field application with the intent of reducing the large amount of keratin waste produced by the poultry industry each year. In 1992, the U.S. poultry industry produced 54 million tons of manure and 15 million tons of scrape manure and litter, with a substantial portion of this being feather-keratin waste. Keratinase, an extracellular protease produced by Bacillus licheniformis PWD-1, can degrade this keratin waste. Following the application of the enzyme, this waste can be fed to chickens as a high-protein supplement, resulting in the recycling of this product. If practical methods of enzyme production and application can be developed and implemented, the poultry industry could utilize this technology to reduce the overall production of feather-keratin waste.
A double protease assay with trypsin and keratinase is used to determine the in vitro hydrolysis of feather keratin under a variety of application parameters. The effectiveness of the assay conditions is quantitated by the DA280 of the reaction supernatant. Using this assay, the influence of the sample's pH, fat content, temperature, and other controllable parameters can be determined. Wild-type PWD-1 has largely been optimized for fermentation, and the methods used for determining optimal parameters also are being used for optimization of keratinase-overproducing mutants. The factors influencing optimal enzyme production include the age of innoculum at each scaleup step, the need for pH control during fermentation and the pH level, the level of dissolved oxygen, the level of feather-keratin substrate, and the temperature of fermentation. Fermentations are conducted in-house using a New Brunswick Bio-Flow III 10 L fermentation unit for small-scale fermentation optimization, and a New Brunswick IF150 ML4100 microprocessor-controlled 150 L fermentation unit for large-scale enzyme production. Parameters determined to be important using the small-scale assay are used to predict scale-up parameters, with the enzyme produced by 100 L, in-house fermentation used for large-scale application. Large-scale application consists of applying enzyme under predicted parameters to commercial feather meal as a protein supplement in chicken feed. Chickens are fed this diet from 1 to 3 weeks of age, with body-weight gain and feed/gain ratios taken as quantitative data. Application parameters are evaluated as weight gain and feed/gain ratios versus a soybean meal positive-control diet. The in vitro assay is routinely used to test different parameters on enzyme application. Twelve keratinase-overproducing mutants have been isolated, and the best four mutants are being optimized for fermentation. Optimization is complete for the best mutant. Keratinase is routinely being produced by large-scale fermentation, and the application of this enzyme is underway, with two chicken-feeding trials already completed. An additional two to three feeding trials are planned, followed by a large-scale, 7-week feeding trial once optimal application parameters are determined.