β-氨基肽酶具有独特的水解N-末端β-氨基酸的能力,对β-氨基酸侧链的性质具有不同的偏好。这种独特的能力使它们可用作合成β-肽的生物催化剂,并可在动力学上拆分β-肽和酰胺以生产对映体纯的β-氨基酸。迄今为止,已经发现了六种β-氨基肽酶并对其进行了功能表征,五个来自革兰氏阴性细菌,一个来自真菌,曲霉在这里,我们报告了另外四种β-氨基肽酶的纯化和表征,一个来自革兰氏阳性细菌,耻垢分枝杆菌(BapAMs),一个来自酵母,Yarrowialipolytica(BapAYlip),从活性污泥中分离出的两种革兰氏阴性菌被鉴定为伯克霍尔德菌。(BapABcA5和BapABcC1)。克隆了编码β-氨基肽酶的基因,在大肠杆菌中表达,和纯化。β-氨肽酶作为无活性的前蛋白产生,其经历自切割以形成由两个不同亚基组成的活性酶。亚单位,指定为α和β,似乎紧密联系在一起,由于固定化金属亲和色谱(IMAC)纯化后回收了活性酶,即使只有α亚基是6-组氨酸标记的。这些酶显示出具有高活性的N端l-构型β-homo-Gly(βhGly)和β3-homo-Leu(β3hLeu)的显色底物。与先前表征的来自其他微生物的酶相比,这些酶对H-βhGly-对硝基苯胺(H-βhGly-pNA)的活性更高。这些数据表明,新的β-氨基肽酶是全功能的,添加到可用于生产β肽的酶的工具箱中。铜绿假单胞菌的过表达研究还表明,β-氨基肽酶可能在某些细胞功能中起作用。重要性β-氨基肽酶是在不同范围的微生物中发现的独特酶,其可以利用合成的β-肽作为唯一碳源。先前已经表征了六种β-氨肽酶对不同β-氨基酸底物的偏好,并且已经证明了不仅催化合成β-肽的降解而且催化短β-肽的合成的能力。其他β-氨肽酶的鉴定增加了具有不同β-氨基酸底物偏好和动力学的酶的工具箱。这些酶具有用于可持续制造用于生物医学和生物材料应用的β-氨基酸衍生物和β-肽的潜力。这很重要,因为β-氨基酸和β-肽赋予生物活性化合物增加的蛋白水解抗性并形成新的结构以及类似于α-肽的结构。新酶的发现还将提供对这些酶在自然界中的生物学重要性的了解。
β-Aminopeptidases have the unique capability to hydrolyze N-terminal β-amino acids, with varied preferences for the nature of β-amino acid side chains. This unique capability makes them useful as biocatalysts for synthesis of β-peptides and to kinetically resolve β-peptides and amides for the production of enantiopure β-amino acids. To date, six β-aminopeptidases have been discovered and functionally characterized, five from Gram-negative bacteria and one from a fungus, Aspergillus Here we report on the purification and characterization of an additional four β-aminopeptidases, one from a Gram-positive bacterium, Mycolicibacterium smegmatis (BapAMs), one from a yeast, Yarrowia lipolytica (BapAYlip), and two from Gram-negative bacteria isolated from activated sludge identified as Burkholderia spp. (BapABcA5 and BapABcC1). The genes encoding β-aminopeptidases were cloned, expressed in Escherichia coli, and purified. The β-aminopeptidases were produced as inactive preproteins that underwent self-cleavage to form active enzymes comprised of two different subunits. The subunits, designated α and β, appeared to be tightly associated, as the active enzyme was recovered after immobilized-metal affinity chromatography (IMAC) purification, even though only the α-subunit was 6-histidine tagged. The enzymes were shown to hydrolyze chromogenic substrates with the N-terminal l-configurations β-homo-Gly (βhGly) and β3-homo-Leu (β3hLeu) with high activities. These enzymes displayed higher activity with H-βhGly-p-nitroanilide (H-βhGly-pNA) than previously characterized enzymes from other microorganisms. These data indicate that the new β-aminopeptidases are fully functional, adding to the toolbox of enzymes that could be used to produce β-peptides. Overexpression studies in Pseudomonas aeruginosa also showed that the β-aminopeptidases may play a role in some cellular functions.IMPORTANCE β-Aminopeptidases are unique enzymes found in a diverse range of microorganisms that can utilize synthetic β-peptides as a sole carbon source. Six β-aminopeptidases have been previously characterized with preferences for different β-amino acid substrates and have demonstrated the capability to catalyze not only the degradation of synthetic β-peptides but also the synthesis of short β-peptides. Identification of other β-aminopeptidases adds to this toolbox of enzymes with differing β-amino acid substrate preferences and kinetics. These enzymes have the potential to be utilized in the sustainable manufacture of β-amino acid derivatives and β-peptides for use in biomedical and biomaterial applications. This is important, because β-amino acids and β-peptides confer increased proteolytic resistance to bioactive compounds and form novel structures as well as structures similar to α-peptides. The discovery of new enzymes will also provide insight into the biological importance of these enzymes in nature.