Department of Pharmaceutical Sciences, Saurashtra Univeristy, Rajkot-360005, India.
Department of Pharmaceutical Sciences, Saurashtra Univeristy, Rajkot-360005, India.
Int J Biol Macromol. 2016 Jun;87:256-62. doi: 10.1016/j.ijbiomac.2016.02.067. Epub 2016 Mar 3.
The Bacillus subtilis DP1 was isolated from poultry farm soil at Anand district, India. The highest enzyme production (379.65U/ml) was obtained at pH 10.0, a temperature of 37°C and a growth period of 72h. The extracellular keratinase was purified by gel filtration chromatography with 27.98 purification fold. Purity was also confirmed by High-Performance Liquid Chromatography (HPLC) analysis, where a major peak having retention time of 2.5min was obtained on C18 column using photo diode array detector. Purified keratinase was stable in a broad range of pH (8-12) and temperature (20-50°C) with optimum at pH 10.0 and 37°C. The metallic ions, Ca(2+) and Mg(2+) enhance keratinase activity. Secondary structure from Circular Dichroism (CD) spectra implies that purified keratinase is largely β-pleated sheet rich protein. For preparation of dehairing cream formulation, compatibility studies of excipients were carried out. Fourier transform infrared spectroscopy (FTIR) spectra of sodium stearate, calcium carbonate and sodium lauryl sulphate shows no reactivity of functional groups and hence mixture was compatible for formulation of keratinase dehairing cream. Prepared biological depilatory was able to remove hair more efficiently compared to marketed formulations.
从印度安得地区的家禽养殖场土壤中分离到枯草芽孢杆菌 DP1。在 pH 值为 10.0、温度为 37°C 和生长周期为 72h 的条件下，酶产量最高（379.65U/ml）。采用凝胶过滤色谱法对细胞外角蛋白酶进行了纯化，得到了 27.98 倍的纯化倍数。高效液相色谱（HPLC）分析也证实了纯度，在 C18 柱上使用光电二极管阵列检测器，得到一个主要保留时间为 2.5min 的主峰。纯化的角蛋白酶在较宽的 pH（8-12）和温度（20-50°C）范围内稳定，最适 pH 值为 10.0，最适温度为 37°C。金属离子 Ca(2+)和 Mg(2+)增强角蛋白酶的活性。圆二色性（CD）光谱的二级结构表明，纯化的角蛋白酶主要富含β-折叠片层的蛋白质。为了制备脱毛膏制剂，对赋形剂进行了相容性研究。硬脂酸钠、碳酸钙和十二烷基硫酸钠的傅里叶变换红外光谱（FTIR）显示没有官能团的反应性，因此混合物适合用于角蛋白酶脱毛膏的制剂。制备的生物脱毛剂与市售制剂相比，能更有效地去除毛发。
