Mutation Breeding Of A High-yielding Hoof Keratinase Strain And Optimization Of Ferment Condition

There are plenty of resources on hoof keratin in our country. Livestock and poultry hoof accumulate an abundance of potential protein and amino acids, which could be used for the source of excellent feedstuff protein. It is the key to find and appropriate way to degrade keratin, as the keratin is very difficult to be decomposed due to the steady configuration. Different from conventional methods such as physical or chemical degradation, biodegradation holds outstanding effect and no defects like low efficiency, high expend and excessive contamination, which not only makes use of the protein source but also helps to protect environment. As the applications of keratin and keratinase to all kinds of industry emerged more, those are considered to be provided with more values of economic and social, and with brilliant prospect.This study is mainly focused on the utilization of keratin, starting from mutagenesis-screening optimal strain; the characterization of crude enzyme and the fermentation process have been researched. The results completed are reported as follows:1. The strain N5 was identified as Chryseobacterium sp. based on morphology observation,physicochemical characteristics, 16SrDNA sequence analysis .2. The high-yield hoof keratinase strain U3-22 was obtained by UV mutagenesis on the original strain N5. The hoof keratinase production reached 69.85U/mL using method of coomassie brilliant blue colorimetric, which was 2.75 times as higher as that of the original strain with 25.43U/mL.3. Effects of medium components on keratinase production were carried out by orthogonal experimental design of the four factors, the optimal fermentation medium was determined as follows: starch soluble 1.5%, Yeast extract powder 1.5%, K2HPO4, 0.05%, hoof 0.4%; The best fermentation conditions were: the optimum cultural temperature 37℃, inoculating amount 3%, medium volume 50mL in 300mL flask, optimal pH 8.5. Under these conditions, the keratinase activity was 160.34U/mL, which was 2.3 times of the previous.4. The crude keratinase had been extracted from the fermentation liquid of U3-22 strain, and the enzymatic characteristics were analyzed. The optimum reaction temperature and pH were 70℃and 7.5 respectively. The enzyme activity could be clearly activated by K⁺, Mg²⁺and Na₂SO₃, and inhibited by Mn²⁺ and Zn²⁺. The keratinase could degrade the hoof effectively. These results suggest that the keratinase from the mutant strain U3-22 is novel, thermal stable and pH-tolerant and it can be applied in waste disposal of hoof and feather.