Production, partial purification and efficacy of keratinase from <i>Bacillus halotolerans</i> L2EN1 isolated from the poultry farm of Himachal Pradesh as a potential laundry additive

To augment the keratinolytic ability of <i>Bacillus halotolerans</i> L2EN1 isolated from the poultry farm (Nahan, District Sirmour) of Himachal Pradesh, different cultural conditions were optimised using One Variable at a Time (OVAT) approach accompanied by Response Surface Methodology (RSM). Optimisation (OVAT) results revealed that after 3rd day of incubation, maximum enzyme activity was attained at 45 °C, pH 11.0 with 12.5% of inoculum size in the presence of Mn2<b> </b>+ and EDTA in the production medium. Sucrose (1.5%) and yeast extract (2.0%) were observed to be best carbon and nitrogen sources, respectively. A significant increase of 73.19 per cent in the keratinase activity was observed using Central Composite Design (CCD) of RSM. The SDS-PAGE results revealed that crude keratinase is a heterotetramer made up of four polypeptide chains with molecular weights of 17, 37, 40 and 60 kDa. Partial purification by 90 − 100 percent ammonium sulphate gave maximum keratinase production of 22.66 U/mL with purification of 1.68 and yield of 11.47 per cent. The enzyme showed compatibility with different commercial detergents and retained its activity in the order: Reshma (97.77%) &gt; Speed (93.44%) &gt; Tide (79.93%) &gt;Ariel (70.18%) &gt; Surf excel (67.98%) at 50 °C after 1 h of incubation. Wash performance analysis demonstrated that washing with tap water at 18, 28, 35 and 45 °C for 30, 45 and 60 min removed some amount of blood stains from the cotton cloth pieces. However, replacement of detergent’s enzyme (Reshma) with crude keratinase achieved complete blood stain removal under same conditions, suggesting its suitability as a potential cleaning additive in detergents for the removal of blood (proteinaceous) stains for long washing cycles (1 h).为提升从印度喜马偕尔邦锡莫尔区纳汉家禽养殖场分离得到的耐盐芽孢杆菌（*Bacillus halotolerans*）L2EN1的角蛋白降解能力，本研究采用单因子变量法（One Variable at a Time, OVAT）结合响应面法（Response Surface Methodology, RSM）对其发酵培养条件进行优化。单因子变量法优化结果显示，培养至第3天时，在温度45℃、初始pH 11.0、接种量12.5%，且发酵培养基中添加Mn²+与乙二胺四乙酸（EDTA）的条件下，酶活性达到峰值。研究发现，蔗糖（1.5%）与酵母提取物（2.0%）分别为最优碳源与氮源。通过响应面法中的中心复合设计（Central Composite Design, CCD），角蛋白酶活性较优化前显著提升73.19%。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）结果表明，粗提角蛋白酶为异四聚体，由分子量分别为17、37、40与60 kDa的四条多肽链构成。采用90%~100%饱和度硫酸铵进行部分纯化，获得的角蛋白酶最高酶活为22.66 U/mL，纯化倍数达1.68，回收率为11.47%。该酶与多款商用洗涤剂具有良好相容性，在50℃下孵育1小时后，酶活性保留率依次为：Reshma（97.77%）> Speed（93.44%）> Tide（79.93%）> Ariel（70.18%）> Surf excel（67.98%）。洗涤性能测试表明，在18、28、35与45℃下分别以30、45及60分钟时长使用自来水洗涤棉布块，可去除其上部分血渍。但若将商用洗涤剂Reshma中的酶替换为粗提角蛋白酶，在相同条件下可完全去除血渍，表明该酶可作为潜在的洗涤剂清洁添加剂，适用于长洗涤周期（1小时）下血渍（蛋白质类污渍）的去除。