粪便微生物宏基因组来源L-天冬酰胺酶的性质表征及应用研究

Characterization and application of an L-asparaginase derived from the metagenome of fecal microbiota

摘要:

l-天冬酰胺酶是氨基酸代谢的关键酶，广泛应用于食品和医药领域，肠道菌群及其产生的l-天冬酰胺酶与宿主健康和疾病关系密切。【目的】获取肠道微生物来源的新型l-天冬酰胺酶，并对其进行性质表征和应用研究。【方法】以西黑冠长臂猿粪便微生物宏基因组为模板，克隆l-天冬酰胺酶基因，并在大肠杆菌中表达；对表达出的酶进行酶学性质研究，并用于处理薯条和癌细胞。【结果】克隆获得l-天冬酰胺酶基因NCasn5，全长996 bp，重组酶NCasn5分子量大小为37.296 kDa，最适pH为8.0，最适温度为60 ℃，K_m和V_max值分别为(3.33±0.21) mmol/L和(836.30±13.91)µmol/(min·mg)，37 ℃体外血清半衰期约69 h。NCasn5能降低薯条中69.35%的丙烯酰胺含量，抑制人肝癌细胞QGY-7703和人恶性黑色素瘤细胞A-375细胞的生长。【结论】本研究获得的新型l-天冬酰胺酶，具有良好的热稳定性和较长的血清半衰期，不仅无谷氨酰胺酶活性，还能减少油炸薯条中丙烯酰胺的含量，也能诱导癌细胞QGY-7703和A-375凋亡，在食品加工及医药领域具有潜在的应用价值。

Abstract:

l-asparaginase, a key enzyme in amino acid metabolism, is widely used in the food and pharmaceutical industries. The gut microbiota and its product l-asparaginase are closely associated with host health and diseases. [Objective] This study aims to acquire a novel l-asparaginase gene from gut microbiota and explore its enzymatic characteristics and potential applications. [Methods] An l-asparaginase gene was cloned from the metagenome of the fecal microbiota of Nomascus concolor and heterologously expressed in Escherichia coli BL21(DE3). The enzymatic properties of the expressed protein were determined. Furthermore, the potential applications of this protein were explored, including processing potato chips and treating cancer cells. [Results] The cloned l-asparaginase gene, NCasn5, was 996 bp. It encoded the recombinant enzyme NCasn5 with a molecular weight of 37.296 kDa, optimal activity at pH 8.0 and 60 °C, K_m of (3.33±0.21) mmol/L, V_max of (836.30±13.91) µmol/(min·mg), and a serum half-life of about 69 h in vitro at 37 °C. NCasn5 reduced the acrylamide content in potato chips by 69.35% and inhibited the growth of human liver cancer cells (QGY-7703) and human melanoma cells (A-375). [Conclusion] We obtained a novel l-asparaginase demonstrating good thermal stability and a prolonged serum half-life. This enzyme lacks the glutaminase activity and reduces acrylamide levels in potato chips. Moreover, it can induce apoptosis in the cancer cell lines QGY-7703 and A-375. These findings suggest the potential applicability of l-asparaginase in both food processing and pharmaceutical industries.