β-半乳糖苷酶(EC3.2.1.23)是外糖苷酶,可催化β1,3-末端β-D-半乳糖残基的糖缀合物的裂解,β1,4-或β1,6-连接。虽然这个家族的外切糖苷酶已经在脊椎动物中进行了广泛的研究,植物,酵母,和细菌,关于软体动物的信息很少。软体动物是一类多样化且非常成功的动物,在其生态系统中扮演着许多不同的角色,包括滤嘴和有害金属。这里,来自太平洋牡蛎的第一个β-半乳糖苷酶,Crassostreagigas被发现了,生化特征,并与我们先前表征的来自Arionvulgaris(UniProtRef。Nr.:A0A0B7AQJ9)。总的来说,贻贝酶表现出与蜗牛酶相似的生化参数。来自C.gigas的酶在酸性环境(pH3.5)和50°C的反应温度下最活跃。最佳储存条件高达37°C。与来自A.vulgaris的酶相反,补充阳离子(Ni2+,Co2+,Mn2+,Mg2+,Ca2+,Cu2+,Ba2)增加了来自C.gigas的酶的活性。来自贻贝的β-半乳糖苷酶的底物特异性研究,C.gigas,和鼻塞,A.vulgaris,揭示了两种酶对末端β1,3-和β1,4-连接的半乳糖残基的活性。使用标记和未标记形式的相同底物,我们能够使用MALDI-TOFMS检测标记对β-半乳糖苷酶活性的影响,HPTLC,和HPLC。当乳糖在未标记或标记状态下被酶裂解时,一旦添加2-氨基苯甲酸标记,半乳糖-N-二糖就不会裂解。在这项研究中,我们介绍了太平洋牡蛎中第一个重组表达的β-半乳糖苷酶的生化特征,C.gigas,我们比较了使用C.gigas和A.vulgaris酶测定β-半乳糖苷酶活性的不同分析方法。
β-
Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal β-D-galactose residues in β1,3-, β1,4- or β1,6-linkage. Although this family of exoglycosidases has been extensively studied in vertebrates, plants, yeast, and bacteria, little information is available for mollusks. Mollusks are a diverse and highly successful group of animals that play many different roles in their ecosystems, including filter feeders and detritivores. Here, the first β-galactosidase from the Pacific oyster, Crassostrea gigas was discovered, biochemically characterized, and compared to our previously characterized slug enzyme from Arion vulgaris (UniProt Ref. Nr.: A0A0B7AQJ9). Overall, the mussel enzyme showed similar biochemical parameters to the snail enzyme. The enzyme from C. gigas was most active in an acidic environment (pH 3.5) and at a reaction temperature of 50 °C. Optimal storage conditions were up to 37 °C. In contrast to the enzyme from A. vulgaris, the supplementation of cations (Ni2+, Co2+, Mn2+, Mg2+, Ca2+, Cu2+, Ba2+) increased the activity of the enzyme from C. gigas. Substrate specificity studies of the β-
galactosidases from the mussel, C. gigas, and the slug, A. vulgaris, revealed activity towards terminal β1,3- and β1,4-linked galactose residues for both enzymes. Using the same substrates in labeled and unlabeled form, we were able to detect the effect of labeling on the β-galactosidase activity using MALDI-TOF MS, HPTLC, and HPLC. While lactose was cleaved by the enzymes in an unlabeled or labeled state, galacto-N-biose was not cleaved as soon as a 2-amino benzoic acid label was added. In this study we present the biochemical characterization of the first recombinantly expressed β-galactosidase from the Pacific oyster, C. gigas, and we compare different analytical methods for the determination of β-galactosidase activity using the enzyme from C. gigas and A. vulgaris.