Taipa M A, Liebeton K, Costa J V, Cabral J M, Jaeger K E
Laboratório de Engenharia Bioquímica, Instituto Superior Técnico, Lisboa, Portugal.
Biochim Biophys Acta. 1995 Jun 6;1256(3):396-402. doi: 10.1016/0005-2760(95)00052-e.
Previous purification of a commercial lipolytic preparation from Chromobacterium viscosum using gel filtration chromatography yielded two enzymatically active fractions, named lipases A and B. Characterization of these fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that lipase A consisted of a high molecular weight aggregate of lipase protein with lipopolysaccharides. This complex could be dissociated by treatment with EDTA-Tris buffer containing the non-ionic detergent n-octyl-beta-D-glucopyranoside and subsequent isoelectric focusing in an agarose gel containing the same detergent. Both lipases A and B revealed a major peak corresponding to an isoelectric point of 7.1. SDS-PAGE analysis of lipases A and B after purification by gel filtration or by IEF revealed one major protein band of M(r) of 33 K. Determination of N-terminal amino acid sequences confirmed that both fractions A and B contained the same lipase protein. Furthermore, the N-terminal amino acid sequence of the C. viscosum lipase was identical to the one of Pseudomonas glumae lipase.
此前，使用凝胶过滤色谱法从粘稠色杆菌中纯化一种商业脂解制剂，得到了两个具有酶活性的组分，分别命名为脂肪酶A和B。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳对这些组分进行表征，结果显示脂肪酶A由脂肪酶蛋白与脂多糖形成的高分子量聚集体组成。用含有非离子去污剂正辛基-β-D-吡喃葡萄糖苷的EDTA- Tris缓冲液处理，随后在含有相同去污剂的琼脂糖凝胶中进行等电聚焦，可使该复合物解离。脂肪酶A和B均显示出一个对应等电点为7.1的主峰。对通过凝胶过滤或等电聚焦纯化后的脂肪酶A和B进行SDS-PAGE分析，结果显示一条主要蛋白带，其相对分子质量（M(r)）为33 K。N端氨基酸序列测定证实，组分A和B均含有相同的脂肪酶蛋白。此外，粘稠色杆菌脂肪酶的N端氨基酸序列与稻谷假单胞菌脂肪酶的N端氨基酸序列相同。
