COMPARATIVE PRODUCTION AND RAPID PURIFICATION OF CANDIDA ACID PROTEINASE FROM PROTEIN-SUPPLEMENTED CULTURES
COMPARATIVE PRODUCTION AND RAPID PURIFICATION OF CANDIDA ACID PROTEINASE FROM PROTEIN-SUPPLEMENTED CULTURES
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DOI：
10.1128/iai.58.2.508-514.1990
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发表时间：
1990-02-01
影响因子：
3.1
通讯作者：
PAYNE, CD
中科院分区：
文献类型：
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作者：
RAY, TL;PAYNE, CD
6株念珠菌以前被确定为皮肤致病性的人在添加白蛋白、氮素限制的培养基中对念珠菌酸性蛋白酶(CAP)的产生进行了评估。比较了添加白蛋白、酪蛋白、胶原、血红蛋白或角蛋白的培养液和TC培养液中白念珠菌CAP的产量。白色念珠菌、星形念珠菌和热带念珠菌是小鼠感染中的皮肤病原体，它们产生的CAP比非病原体近平假丝酵母菌和革兰氏杆菌多3.3-4.7倍。克鲁斯梭菌是一种非病原菌，其产酶量可以忽略不计。白念珠菌CAP的产量在每种添加蛋白质的培养基中都是相似的，并且每种都只能回收一种酸性蛋白酶。通过中空纤维和搅拌细胞超滤、Affi-Gel蓝和Sephacryl柱层析，实现了对培养上清液中CAP的快速纯化。从添加角蛋白的培养上清液中获得了最高的CAP产量、纯度和比活性，在7L的培养中生产了2.86 mg的CAP。CAP是一种41,500-道尔顿糖蛋白，等电点为4.5；pH范围为2.5-5.5；底物专一性广，包括角蛋白、变性胶原、血红蛋白、酪蛋白和白蛋白。CAP的分离也分离了念珠菌的角蛋白水解酶。胃抑素A对CaP有抑制作用，EDTA和苯甲基磺酰氟对其无抑制作用。免疫印迹分析表明，在小鼠体内产生了针对CAP的单特异性抗体，并仅与41,500-Dalton蛋白发生反应。皮肤致病念珠菌产CAP量高。支持CAP是一个潜在的毒力因子，可能促进念珠菌的定植和对皮肤的侵袭。在添加角蛋白的培养液中，CAP的产量高于其他供试的培养基产酶，所产的酶足以用于CAP抗原和抗体的免疫检测。
Six Candida spp. that were previously characterized for cutaneous pathogenicity were assessed for Candida acid proteinase (CAP) production in albumin-supplemented, nitrogen-restricted media. C. albicans CAP production was compared in media supplemented with albumin, casein, collagen, hemoglobin, or keratin and in TC medium 199. C. albicans, C. stellatoidea, and C. tropicalis, which are cutaneous pathogens in murine infections, produced 3.3 to 4.7 times more CAP than did the nonpathogens C. parapsilosis and C. guilliermondii. C. krusei, a nonpathogen, produced negligible amounts of enzyme. C. albicans CAP production was similar in each protein-supplemented medium, and only a single acid proteinase was recovered from each one. Rapid CAP purification from culture supernatants was achieved by hollow fiber and stirred cell ultrafiltration followed by Affi-Gel blue and Sephacryl column chromatographies. The highest yield, purity, and specific activity of CAP were obtained from keratin-supplemented medium supernatants, producing 2.86 mg of purified CAP from a 7-liter culture. CAP was characterized as a 41,500-dalton glycoprotein, with a pI of 4.5; a pH range of 2.5 to 5.5; and broad substrate specificity, including that for keratin, denatured collagen, hemoglobin, casein, and albumin. Isolation of CAP also isolated the keratinolytic proteinase of Candida spp. CAP was inhibited by pepstatin A, but not by EDTA or phenylmethylsulfonyl fluoride. Monospecific antibody to CAP was produced in mice and reacted only to the 41,500-dalton protein, as determined by immunoblot analysis. High CAP production by cutaneous pathogenic Candida spp. supports the fact that CAP is a potential virulence factor that may facilitate Candida colonization and invasion of skin. CAP production from keratin-supplemented medium was superior to that from the other media that were tested and yielded sufficient and suitable enzyme for use in immunoassays of CAP antigen and antibody.