Institute of Biomedicine, Pharmacology, Drug Development and Therapeutics/Laboratory of Synthetic Drug Chemistry and Department of Chemistry, University of Turku, Lemminkäisenkatu 5 C, FIN-20520, Turku, Finland.
Org Biomol Chem. 2010 Feb 21;8(4):886-95. doi: 10.1039/b920939p. Epub 2010 Jan 4.
Various commercial lyophilized and immobilized preparations of lipase A from Candida antarctica (CAL-A) were studied for their ability to catalyze the hydrolysis of amide bonds in N-acylated alpha-amino acids, 3-butanamidobutanoic acid (beta-amino acid) and its ethyl ester. The activity toward amide bonds is highly untypical of lipases, despite the close mechanistic analogy to amidases which normally catalyze the corresponding reactions. Most CAL-A preparations cleaved amide bonds of various substrates with high enantioselectivity, although high variations in substrate selectivity and catalytic rates were detected. The possible role of contaminant protein species on the hydrolytic activity toward these bonds was studied by fractionation and analysis of the commercial lyophilized preparation of CAL-A (Cat#ICR-112, Codexis). In addition to minor impurities, two equally abundant proteins were detected, migrating on SDS-PAGE a few kDa apart around the calculated size of CAL-A. Based on peptide fragment analysis and sequence comparison both bands shared substantial sequence coverage with CAL-A. However, peptides at the C-terminal end constituting a motile domain described as an active-site flap were not identified in the smaller fragment. Separated gel filtration fractions of the two forms of CAL-A both catalyzed the amide bond hydrolysis of ethyl 3-butanamidobutanoate as well as the N-acylation of methyl pipecolinate. Hydrolytic activity towards N-acetylmethionine was, however, solely confined to the fractions containing the truncated form of CAL-A. These fractions were also found to contain a trace enzyme impurity identified in sequence analysis as a serine carboxypeptidase. The possible role of catalytic impurities versus the function of CAL-A in amide bond hydrolysis is further discussed in the paper.
研究了来自南极假丝酵母（CAL-A）的各种商业冻干和固定化的脂肪酶 A 制剂，以研究其催化酰化α-氨基酸、3-丁酰胺丁酸（β-氨基酸）及其乙酯中酰胺键水解的能力。尽管与通常催化相应反应的酰胺酶具有密切的机制相似性，但对酰胺键的活性非常不典型。尽管检测到底物选择性和催化速率的高度变化，但大多数 CAL-A 制剂对各种底物的酰胺键具有高对映选择性地进行切割。通过对商业冻干的 CAL-A（Cat#ICR-112，Codexis）制剂进行分级和分析，研究了杂质蛋白对这些键的水解活性的可能作用。除了少量杂质外，还检测到两种同样丰富的蛋白质，它们在 SDS-PAGE 上的迁移位置与 CAL-A 的计算大小相差几个 kDa。基于肽片段分析和序列比较，两个条带都与 CAL-A 具有相当大的序列覆盖度。然而，在较小片段中没有鉴定出构成作为活性位点瓣的可移动结构域的 C 末端肽段。两种形式的 CAL-A 的分离凝胶过滤级分均催化乙基 3-丁酰胺丁酸的酰胺键水解以及甲基哌啶酸的 N-酰化。然而，对 N-乙酰蛋氨酸的水解活性仅局限于含有截断形式的 CAL-A 的级分中。在序列分析中，这些级分也被发现含有痕量的酶杂质，鉴定为丝氨酸羧肽酶。本文进一步讨论了催化杂质与 CAL-A 在酰胺键水解中的功能的可能作用。
