López L M, Sequeiros C, Natalucci C L, Brullo A, Maras B, Barra D, Caffini N O
LIPROVE, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, 1900, Argentina.
Protein Expr Purif. 2000 Mar;18(2):133-40. doi: 10.1006/prep.1999.1165.
A new papain-like cysteine peptidase isolated from fruits of Pseudananas macrodontes (Morr.) Harms, a species closely related to pineapple (Ananas comosus L.), has been purified and characterized. The enzyme, named macrodontain I, is the main proteolytic component present in fruit extracts and was purified by acetone fractionation followed by anion-exchange chromatography. Separation was improved by selecting both an adequate pH value and a narrow saline gradient. Optimum pH range (more than 90% of maximum activity with casein) was achieved at pH 6.1-8.5. Homogeneity of the enzyme was confirmed by bidimensional electrophoresis and mass spectroscopy (MS). Molecular mass of the enzyme was 23,459 (MS) and its isoelectric point was 6.1. The alanine, glutamine, and tyrosine derivatives were strongly preferred when the enzyme was assayed on N-alpha-CBZ-l-amino acid p-nitrophenyl esters. The N-terminal sequence of macrodontain (by comparison with the N-terminus of 30 plant proteases with more than 50% homology) showed a great deal of sequence similarity to the other pineapple-stem-derived cysteine endopeptidases, being 85.7, 85. 2, and 77.8% identical to comosain, stem bromelain, and ananain, respectively. It seems clear that the Bromeliaceae endopeptidases are more closely related to each other than to other members of the papain family, suggesting relatively recent divergence.
从与菠萝（Ananas comosus L.）密切相关的物种大齿拟凤梨（Pseudananas macrodontes (Morr.) Harms）果实中分离出一种新的木瓜蛋白酶样半胱氨酸肽酶，并对其进行了纯化和表征。该酶命名为大齿拟凤梨蛋白酶I，是果实提取物中的主要蛋白水解成分，通过丙酮分级分离，然后进行阴离子交换色谱法进行纯化。通过选择合适的pH值和狭窄的盐梯度来改善分离效果。在pH 6.1 - 8.5时达到最佳pH范围（以酪蛋白计，活性超过最大值的90%）。通过双向电泳和质谱（MS）确认了该酶的同质性。该酶的分子量为23,459（MS），其等电点为6.1。当该酶作用于N-α-CBZ-L-氨基酸对硝基苯酯时，丙氨酸、谷氨酰胺和酪氨酸衍生物是其强烈偏好的底物。大齿拟凤梨蛋白酶的N端序列（与30种同源性超过50%的植物蛋白酶的N端进行比较）显示与其他菠萝茎来源的半胱氨酸内肽酶有很大的序列相似性，分别与菠萝蛋白酶、茎菠萝蛋白酶和菠萝蛋白酶的同一性为85.7%、85.2%和77.8%。很明显，凤梨科内肽酶彼此之间的关系比与木瓜蛋白酶家族的其他成员更为密切，这表明它们的分化相对较新。
