Table_1_First Evidence of Acyl-Hydrolase/Lipase Activity From Human Probiotic Bacteria: Lactobacillus rhamnosus GG and Bifidobacterium longum NCC 2705.docx

Lactobacillus rhamnosus GG (ATCC 53103) and Bifidobacterium longum NCC 2705 are among the most studied probiotics. However, the first evidence of acyl hydrolase/lipase of two annotated proteins, one in each genome of these strains, is reported in this work. Signal peptide analysis has predicted that these proteins are exported to the extracellular medium. Both proteins were produced in Escherichia coli, purified and characterized. Molecular masses (without signal peptides) were 27 and 52.3 kDa for the proteins of L. rhamnosus and B. longum, respectively. Asymmetrical flow field-flow fractionation analysis has shown that both proteins are present as monomers in their native forms at pH 7. Both have shown enzymatic activity on pNP-laurate at pH 7 and 37°C. The enzyme from L. rhamnosus was characterized deeper, showing preference on pNP-esters with short chain fatty acids. In addition, a computational model of the 3D structure has allowed the prediction of the catalytic amino acids. The enzymatic activities using synthetic substrates were very low for both enzymes. The investigation of natural substrates and biological functions of these enzymes is still open.

鼠李糖乳杆菌GG（Lactobacillus rhamnosus GG，ATCC 53103）与长双歧杆菌NCC 2705（Bifidobacterium longum NCC 2705）均为当前研究最为广泛的益生菌菌株。本研究首次报道了这两株菌株各自基因组中各一个注释蛋白所具备的酰基水解酶/脂肪酶（acyl hydrolase/lipase）活性相关证据。信号肽分析（signal peptide analysis）预测，这两类蛋白均可分泌至胞外培养基中。研究人员已在大肠杆菌（Escherichia coli）中成功表达这两类蛋白，并完成了纯化与功能表征。去除信号肽后，鼠李糖乳杆菌GG与长双歧杆菌NCC 2705对应的蛋白分子质量分别为27 kDa与52.3 kDa。不对称流场流分级分离（asymmetrical flow field-flow fractionation）分析结果显示，在pH 7条件下，两类蛋白的天然构象均以单体形式存在。二者在pH 7、37℃条件下均表现出对硝基苯酚月桂酸酯（pNP-laurate）的酶促活性。针对鼠李糖乳杆菌GG所产酶的表征更为深入，结果显示其偏好作用于带有短链脂肪酸的对硝基苯酚酯（pNP-esters）类底物。此外，通过三维结构计算模型，研究人员成功预测了该酶的催化氨基酸残基。两类酶在合成底物上的酶促活性均极低。针对这两类酶的天然底物与生物学功能的研究仍有待开展。