Laboratory for Genetics and Biochemistry of Microorganisms, National Center for Biotechnology, Astana, Kazakhstan.
Faculty of Natural Sciences, L.N. Gumilyev Eurasian National University, Astana, Kazakhstan.
PLoS One. 2024 Oct 25;19(10):e0312679. doi: 10.1371/journal.pone.0312679. eCollection 2024.
Keratinases, a subclass of proteases, are used to degrade keratin thereby forming peptones and free amino acids. Bacillus paralicheniformis strain T7 was isolated from soil and exhibited high keratinase, protease, collagenase, amylase, xylanase, lipase, and phosphatase activities. Keratinases of the strain showed maximum activity at 70°C and pH 9.0 as well as high thermal stability. A mass-spectrometric analysis identified seven peptidases with molecular masses of 26.8-154.8 kDa in the secretory proteome. These peptidases are members of S8 and S41 serine peptidase families and of M14, M42, and M55 metallopeptidase families. Additionally, α-amylase (55.2 kDa), alkaline phosphatase (59.8 kDa), and esterase (26.8 kDa) were detected. The strong keratinolytic properties of the strain were confirmed by degradation of chicken and goose feathers, which got completely hydrolyzed within 4 days. Submerged fermentation by strain B. paralicheniformis T7 was carried out in a pilot bioreactor, where the highest keratinase production was noted after 19 h of cultivation. After the fermentation, in the culture fluid, the keratinase activity toward keratin azure was 63.6 ± 5.8 U/mL. The protease activity against azocasein was 715.7 ± 40.2 U/mL. The possibility of obtaining enzyme preparations in liquid and powder form was demonstrated, and their comparative characteristics are given. In the concentrate, the keratinase, protease, α-amylase, phosphatase, and esterase/lipase activities were 2,656.7 ± 170.4, 29,886.7 ± 642.9, 176.1 ± 16.3, 23.9 ± 1.8, and 510.9 ± 12.2 U/mL, respectively. In the lyophilizate, these activities were 57,733.3 ± 8,911.4, 567,066.7 ± 4,822.2, 2,823.0 ± 266.8, 364.2 ± 74.8, and 17,618.0 ± 610.3 U/g, respectively. In the preparation obtained by air flow drying at 55°C, these activities were 53,466.7 ± 757.2, 585,333.3 ± 4,277.1, 2,395.8 ± 893.7, 416.7 ± 52.4, and 15,328.1 ± 528.6 U/g, respectively. The results show high potential of B. paralicheniformis strain T7 as a producer of keratinases and other enzymes for applications in agricultural raw materials and technologies for processing of keratin-containing animal waste.
角蛋白酶是蛋白酶的一个子类，用于降解角蛋白，从而形成蛋白胨和游离氨基酸。从土壤中分离到一株芽孢杆菌 paralicheniformis 菌株 T7，该菌株表现出很高的角蛋白酶、蛋白酶、胶原酶、淀粉酶、木聚糖酶、脂肪酶和磷酸酶活性。该菌株的角蛋白酶在 70°C 和 pH9.0 时表现出最大活性，并且具有较高的热稳定性。质谱分析鉴定出分泌蛋白质组中 7 种分子量为 26.8-154.8 kDa 的肽酶。这些肽酶属于 S8 和 S41 丝氨酸肽酶家族以及 M14、M42 和 M55 金属肽酶家族。此外，还检测到α-淀粉酶（55.2 kDa）、碱性磷酸酶（59.8 kDa）和酯酶（26.8 kDa）。该菌株具有很强的角蛋白降解特性，通过对鸡和鹅羽毛的降解得到了证实，4 天内羽毛完全水解。在小型生物反应器中进行了芽孢杆菌 paralicheniformis T7 的发酵，培养 19 小时后角蛋白酶的产量最高。发酵后，在培养液中，角蛋白酶对角蛋白天蓝的活性为 63.6±5.8 U/mL。对偶氮酪蛋白的蛋白酶活性为 715.7±40.2 U/mL。证明了可以在液体和粉末形式获得酶制剂，并给出了它们的比较特性。在浓缩物中，角蛋白酶、蛋白酶、α-淀粉酶、磷酸酶和脂肪酶/脂肪酶的活性分别为 2656.7±170.4、29886.7±642.9、176.1±16.3、23.9±1.8 和 510.9±12.2 U/mL。在冻干物中，这些活性分别为 57733.3±8911.4、567066.7±4822.2、2823.0±266.8、364.2±74.8 和 17618.0±610.3 U/g。在 55°C 下通过气流干燥获得的制剂中，这些活性分别为 53466.7±757.2、585333.3±4277.1、2395.8±893.7、416.7±52.4 和 15328.1±528.6 U/g。结果表明，芽孢杆菌 paralicheniformis 菌株 T7 具有作为角蛋白酶和其他酶的生产者的巨大潜力，可用于农业原料和加工含角蛋白动物废物的技术。
