Batool Hira, Maqsood Beenish, Muzzamal Hira, Islam Butt Hamama, Gul Roquyya, Latif Farooq, Saleem Mahjabeen
School of Biochemistry and Biotechnology, University of the Punjab, Lahore-54590, Pakistan.
School of Medical Lab Technology, Minhaj University Lahore, Lahore-54770, Pakistan.
Protein Pept Lett. 2025 Jul 21. doi: 10.2174/0109298665378063250628211031.
Keratinases have an established role in degrading highly stable and insoluble fibers of keratin proteins, which are otherwise difficult to be hydrolyzed by conventional proteases. Keratinases find promising application in degrading poultry waste to valuable products. Moreover, their role in cosmetics, detergents, agriculture and the leather industry is well recognized.
In this study, the keratinase gene from locally isolated Brevibacillus agri bacteria was cloned and expressed in Escherichia coli, and some of its potential applications were explored.
1300 bp amplified gene from Brevibacillus agri was cloned into E. coli DH5α competent cells using pTZ57R/T vector. After blue-white screening, the positive clone was confirmed by colony PCR and restriction analysis. Purified keratinase gene KerH from recombinant pTZR/KerH plasmid was ligated into pET-28a (+) and transferred into competent cells of E. coli DH5α. Following conformation through colony PCR, and restriction analysis, recombinant plasmid (pET-28a/Ker) from the positive clone was transferred into competent E. coli BL21 cells. The transformed cells were then cultured for up to 8 hours after induction with 0.8 mM IPTG and lysed by sonication. The resulting recombinant keratinase (KerH) was purified by heat treatment and Ni-affinity column and characterized.
The blast analysis and homologous sequences in the NCBI database established a close link to Brevibacillus agri. The highest expression from transformed E. coli BL21 was achieved with 0.8 mM IPTG following 6 hours of induction. The resulting recombinant keratinase (KerH), purified by Ni-affinity chromatography, possessed 283 U/mg specific activity and displayed ~45 kDa band on SDS-PAGE and zymogram. Secondary structure analysis and active site prediction was performed computationally. Considering the extensive applications of keratinase, KerH was found to be useful in dehairing animal skin surfaces without any damage. The encapsulated KerH possessed improved stability and better compatibility with commercial detergents. It efficiently removed blood, turmeric, strawberry, and egg yolk stains from the fabric. Furthermore, KerH significantly degraded the poultry feathers and provided a protein hydrolysate that helped in converting damaged, dull and curly hair into healthier, shiny and straightened hair.
The recombinant KerH from Brevibacillus agri can be considered as a valuable microbial keratinase that can be used as an alternative to the eco hazardous chemicals used in commercial applications of feather degradation, hair protein treatment, feather keratin hydrolysate production and hide dehairing.
角蛋白酶在降解角蛋白高度稳定且不溶性纤维方面具有既定作用，而传统蛋白酶难以水解这些纤维。角蛋白酶在将家禽废弃物降解为有价值产品方面有广阔应用前景。此外，其在化妆品、洗涤剂、农业和皮革工业中的作用也得到了广泛认可。
本研究克隆了从本地分离的农业短芽孢杆菌中提取的角蛋白酶基因，并在大肠杆菌中进行表达，同时探索了其一些潜在应用。
使用pTZ57R/T载体将从农业短芽孢杆菌扩增得到的1300 bp基因克隆到大肠杆菌DH5α感受态细胞中。经过蓝白斑筛选后，通过菌落PCR和限制性分析确认阳性克隆。从重组pTZR/KerH质粒中纯化出角蛋白酶基因KerH，将其连接到pET - 28a(+)中，并转入大肠杆菌DH5α感受态细胞。通过菌落PCR和限制性分析确认后，将阳性克隆的重组质粒(pET - 28a/Ker)转入大肠杆菌BL21感受态细胞。用0.8 mM IPTG诱导后，将转化后的细胞培养长达8小时，然后通过超声破碎裂解。所得重组角蛋白酶(KerH)通过热处理和镍亲和柱进行纯化并进行表征。
Blast分析和NCBI数据库中的同源序列显示与农业短芽孢杆菌有密切联系。用0.8 mM IPTG诱导6小时后，转化的大肠杆菌BL21实现了最高表达。通过镍亲和层析纯化得到的重组角蛋白酶(KerH)，比活性为283 U/mg，在SDS - PAGE和酶谱上显示约45 kDa的条带。通过计算进行了二级结构分析和活性位点预测。考虑到角蛋白酶的广泛应用，发现KerH可用于动物皮肤表面脱毛且无任何损伤。包封后的KerH具有更高的稳定性以及与商业洗涤剂更好的兼容性。它能有效去除织物上的血渍、姜黄渍、草莓渍和蛋黄渍。此外，KerH能显著降解家禽羽毛，并提供一种蛋白质水解产物，有助于将受损、暗淡和卷曲的头发转变为更健康、有光泽且顺直的头发。
来自农业短芽孢杆菌的重组KerH可被视为一种有价值的微生物角蛋白酶，可替代用于羽毛降解、毛发蛋白处理、羽毛角蛋白水解产物生产和皮革脱毛等商业应用中的生态有害化学物质。
