Mwanza Elebert Pauline, van der Westhuizen Wouter André, Boucher Charlotte Enastacia, Charimba George, Hugo Celia
Department of Microbiology and Biochemistry, PO Box 339, University of the Free State, Bloemfontein, Free State, South Africa.
Department of Food Science & Technology, Cape Peninsula University of Technology, Bellville, Western Cape, South Africa.
Protein Expr Purif. 2021 Oct;186:105926. doi: 10.1016/j.pep.2021.105926. Epub 2021 Jun 4.
Chryseobacterium carnipullorum 9_R23581, isolated from raw chicken meat, was evaluated for its potential to degrade keratin found in feathers. The focus of this study was to heterologously express and characterise a keratinolytic enzyme produced by C. carnipullorum. Chryseobacterium carnipullorum secretes proteolytic enzymes that have feather degrading capabilities during its exponential growth phase. This study concluded that the most likely main component of the keratinolytic enzymes of C. carnipullorum was peptidase M64, a serine-endopeptidase with a molecular weight in crude form of 49.46 kDa. Primers were designed on the selected gene of interest, which was amplified from the genome of C. carnipullorum (accession number NZ-FRCD01000002.1). The gene coding for peptidase M64 was further cloned, propagated and expressed in E. coli BL21 [DE3] cells. Purification was by Immobilised Metal Affinity Chromatography (IMAC). The molecular weight of the keratinase was about 50 kDa after purification while its optimum temperature and pH were 50 °C and 8.5, respectively. The activity of this keratinase was inhibited by phenylmethylsulfonyl fluoride (PMSF) and it was enhanced by the presence of divalent metal ions such as Mg and Ca. Enzyme activity was further assayed by application to chicken feathers and observed degradation was an indication of keratinolytic potential.
从生鸡肉中分离出的肉拉氏金黄杆菌9_R23581，对其降解羽毛中角蛋白的潜力进行了评估。本研究的重点是异源表达和表征肉拉氏金黄杆菌产生的一种角蛋白酶。肉拉氏金黄杆菌在其指数生长期分泌具有羽毛降解能力的蛋白水解酶。本研究得出结论，肉拉氏金黄杆菌角蛋白酶最可能的主要成分是肽酶M64，一种粗形式分子量为49.46 kDa的丝氨酸内肽酶。在从肉拉氏金黄杆菌基因组（登录号NZ-FRCD01000002.1）中扩增出的选定目的基因上设计引物。编码肽酶M64的基因进一步在大肠杆菌BL21 [DE3]细胞中克隆、繁殖和表达。通过固定化金属亲和色谱法（IMAC）进行纯化。纯化后角蛋白酶的分子量约为50 kDa，其最适温度和pH分别为50℃和8.5。该角蛋白酶的活性受到苯甲基磺酰氟（PMSF）的抑制，而二价金属离子如Mg和Ca的存在则增强了其活性。通过将其应用于鸡毛进一步测定酶活性，观察到的降解表明具有角蛋白分解潜力。
