Study of novel alkalikeratinase enzyme

从某家禽饲养场的堆肥中分离出一株羽毛降解性嗜碱芽孢杆菌FA30-01。分离物在30℃的液体培养条件下（pH 10.5），培养3天，完全降解羽片。菌株FA30-01为革兰氏阳性、芽孢形成、棒状细菌，与假firmus芽孢杆菌鉴定。通过分析FA30-01产生的16SrDNAKeratinase酶，采用硫酸铵沉淀法、DEAB - Toyopearl交换负离子层析法和羟基磷灰石层析法进行提纯。细化程度为14.5倍。该酶分子量为27.5kDa，等电点为5.9。该酶在pH 5.1 ~ 11.5和30 ~ 80℃条件下对偶氮角蛋白具有活性。角化酶活性的最佳pH值为8.8 ~ 10.3，温度为60℃。该酶属于丝氨酸型蛋白酶。枯草菌素ALP I与该酶n端氨基酸序列同源性达90%。由于该酶与ALP I在分子量、耐热性、等电点等方面存在差异，推测两者为不同酶。

We isolated a feather-degrading alkalophilic Bacillus sp. FA30-01 from the compost of a poultry farm. The isolate degraded completely the pieces of feather in liquid culture (pH 10.5) under the culture condition 30℃ for 3 days. FA30-01 strain was Gram-positive, spore-forming and rod-shaped bacterium and was identified with Bacillus pseudofirmus. by the analysis of 16SrDNAKeratinase enzyme produced by FA30-01 was refined by using the ammonium sulfate precipitation, the negative ion of DEAB Toyopearl exchange chromatography, and the hydroxyapatite chromatography. The refinement level was 14.5 times. The molecular weight of this enzyme was 27.5kDa and an isoelectric point of 5.9.The enzyme had the activity at pH 5.1-11.5 and 30-80℃ for azokeratin. The optimum pH and temperature for keratinase activity were pH 8.8-10.3 and 60℃. And this enzyme belonged to the serine-type protease.Subtilisin ALP I and this enzyme had 90% of homology in N-terminal amino acid sequence. Since this enzyme differed from ALP I in a molecular weight, heat resistance or isoelectric point, these two enzymes were suggested to be different enzyme.