Study of novel alkalikeratinase enzyme

我们分离到一株能降解羽毛的嗜碱芽孢杆菌。FA30-01从家禽养殖场的堆肥中提取。该菌在液体培养(pH 10.5)条件下，30℃培养3d，羽毛完全降解。FA30-01菌株为革兰氏阳性、芽胞形成和杆状细菌，经鉴定为假固件芽孢杆菌。通过16SrDNA角质酶分析，用硫酸铵沉淀、Deab Toyota opearl阴离子交换层析和羟基磷灰石层析对FA30-01产生的角蛋白酶进行了精制。精细化水平为14.5倍。该酶的相对分子质量为27.5 kDa，等电点为5.9。该酶对偶氮角蛋白在pH5.1-11.5，30-80℃范围内具有较强的催化活性。角蛋白酶最适pH为8.8~10.3，最适温度为60℃。该酶与枯草杆菌碱性磷酸酶在N末端氨基酸序列上有90%的同源性。由于该酶在相对分子质量、耐热性或等电点上与ALP I不同，因此推测这两种酶是不同的酶。

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.