Kytococcus sedentarius, the organism associated with pitted keratolysis, produces two keratin-degrading enzymes
Kytococcus sedentarius, the organism associated with pitted keratolysis, produces two keratin-degrading enzymes
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DOI：
10.1046/j.1365-2672.2002.01742.x
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发表时间：
2002-01-01
影响因子：
4
通讯作者：
Holland, KT
中科院分区：
文献类型：
--
作者：
Longshaw, CM;Wright, JD;Holland, KT
方法与结果：对具有愈伤组织降解活性的克氏杆菌连续培养上清液浓缩后，进行一系列层析纯化。该酶的活性可归因于两种蛋白酶。它们均能降解天然愈伤组织和提取的角蛋白多肽，并经纯化后均一，经SDS-PAGE银染显示。酶P1和P2的大小分别为30 kDa和50 kDa，等电点分别为4.6和2.7。降解愈伤组织的最适条件为：温度40℃，pH 7.1，P2温度50℃，pH 7.5。P2在800 mmoL L(-1)氯化钠存在下表现出较强的活性，但这两种酶均被PMSF(1 mmoL-1苯甲基硫酰氟)和1 mmoL L(-1)EDTA抑制。P1和P2与胰岛素β链孵育后，确定主要的酶切位点为Lys-Trp、Val-Lys、Gly-Asp和Asp-Arg。结论：K.sedentarius产生两种胞外酶，它们独立地降解天然的、不溶性的人愈伤组织。这两种酶都是丝氨酸蛋白酶，并具有存在于一系列人类角蛋白中的切割偏好部位。这项研究的意义和影响：在K.sedentarius培养物中发现了两种可以降解人类愈伤组织的酶，这强化了这样的假设，即这种生物对在凹陷性角化溶解中观察到的人类表皮的凹陷性负责。这些酶可能用于一系列角蛋白聚合物的生物降解、生物洗衣粉的生物降解，以及治疗人体皮肤上不想要的老茧。
Aims: To determine characteristics of the extracellular enzyme activity of Kytococcus sedentarius on human callus.Methods and Results: A concentrate of a continuous culture supernatant fluid of K. sedentarius, which had callus-degrading activity, was subjected to a series of chromatographic purification procedures. The enzyme activity was found to be attributable to two proteases. These were capable of degrading both native callus and extracted keratin polypeptides and were purified to homogeneity, as shown by SDS-PAGE with silver staining. The enzymes P1 and P2 were 30 kDa and 50 kDa in size with isoelectric points of 4.6 and 2.7, respectively. The optimum conditions for callus-degrading activity were 40degreesC, pH 7.1 for P1 and 50degreesC, pH 7 5 for P2. P2 displayed increased activity in the presence of 800 mmol l(-1) NaCl and both enzymes were inhibited by PMSF (1 mmol(-1) Phenylmethylsulphoryl fluoride) and 1 mmol l(-1) EDTA. The main enzyme cleavage sites were Lys-Trp, Val-Lys, Gly-Asp and Asp-Arg, as determined after incubation of P1 and P2 with the beta-chain of insulin.Conclusions: K. sedentarius produces two extracellular enzymes that independently degrade natural, insoluble human callus. Both enzymes are serine proteases and have cleavage preference sites that are present in a range of human keratins.Significance and Impact of the Study: The identification, in K. sedentarius cultures, of two enzymes which can degrade human callus strengthens the hypothesis that this organism is responsible for the pitting in human epidermis observed in pitted keratolysis. These enzymes may be of commercial use in the biodegradation of a range of keratin polymers, biological washing powders and in the treatment of unwanted callus on human skin.