The Breeding Of Keratinase High-yielding Strain And Optimization Of Ferment Condition

Keratinase is a kind of inducible enzymes which can specificly degrade keratin into amino acids. It also can inhibit microorganism, tenderize meat, improve feed digestibility, improve the skin permeability and remove aging skin horny and hair, etc. Therefore, it has a broad application prospects in the food, feed, agriculture, environmental protection, pharmaceutical, leather, cosmetics, detergent and other industries. However, keratinase industrial production is not achieved in the domestic still now and the low enzyme activity has been one of the major bottlenecks. This study around the core content of improving the keratinase production, the fermentation characteristics of feather keratin, methods for the enzyme activity assay, mutation breeding of high-yielding strains, optimization of fermentation conditions were system investigated by modern breeding technology and modern fermentation technology. The main results achieved from this research are as follows:1. A keratinase producing bacteria was isolated and screened from soil of long-time deposit of waste feather, based on the detailed analysis of morpho.logica, physiological-biochemical properties and16S rDNA sequencing, the bacteria was identified as Bacillus pumilus, named Bacillus pumilus strain JYL. It could utilize feather as the sole carbon source to grow. Feathers have been completely degraded after shake flask fermentation2d.2. The fermentation characteristics of feathers and feather meal by strain JYL was studied. The results show that the keratinase production using complete feather as substrate is significantly higher than fermentation feather meal. The enzyme production period of integrated feather fermentation is longer than feather powder and the enzyme activity remained at a higher level within24h-36h. But the degradation rate and soluble protein content of feather fermentation are less than feather powder.3. Based on the method of Gradisar, the keratinase activity determination condition was studied systematically using feather meal as substrate. The optimum condition of keratinase activity determination is as following:fermentation broth centrifuged directly under the low temperature to extract crude enzyme, the regents adding order TSE, feather powder granularity20mesh, substrate45mg, reaction pH8.5, reaction temperature45℃, reaction time1h, oscillation frequency30min per time, detemination wavelength280nm. The keratinase activity was2.87times as many as the traditional method.4. The initial medium composition was optimized by single-factor experiment, the Plackett-Burman design and the Box-Benhnken response surface method. The optimized medium was composed of feather0.5%, NaCl0.5%, FeSO40.036%, MgSO40.025%, K2HPO40.073%, KH2PO40.035%, corn flour0.5%, initial pH6.5. The keratinase activity was increased by49.3%compared to before optimization.5. The high-yield keratinase strain Bacillus pumilus UW-27was obtained by UV mutagenesis and microwave mutagenesis on the original strain Bacillus pumilus JYL. The mutant strain UW-27has a higher keratinase production and better genetic stability, the production of keratinase was112.36U/mL which was increased by96.64%compared to before mutagenesis.6. The optimum fermentation conditions were gained by orthogonal experiment design that were temperature37℃, rotation speed160r/min, inocula2%, liquid medium volume80mL/250mL. Under the above conditions, the production of keratinase was124.12U/mL which was10.47%higher than that before optimization.