Department of Biotechnology, Cochin University of Science and Technology, Cochin, Kerala, 682022, India.
Department of Plant Biotechnology, College of Agriculture, Vellayani, Kerala, 695522, India.
Appl Microbiol Biotechnol. 2022 May;106(9-10):3583-3598. doi: 10.1007/s00253-022-11954-7. Epub 2022 May 17.
L-asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. It has application in the treatment of acute lymphoblastic leukemia in children, as well as in other malignancies, in addition to its role as a food processing aid for the mitigation of acrylamide formation in the baking industry. Its use in cancer chemotherapy is limited due to problems such as its intrinsic glutaminase activity and associated side effects, leading to an increased interest in the search for novel L-asparaginases without L-glutaminase activity. This study reports the cloning and expression of an L-asparaginase contig obtained from whole metagenome shotgun sequencing of Sardinella longiceps gut microbiota. Purified recombinant glutaminase-free L-asparaginase SlpA was a 74 kDa homodimer, with maximal activity at pH 8 and 30 °C. K and V of SlpA were determined to be 3.008 mM and 0.014 mM/min, respectively. SlpA displayed cytotoxic activity against K-562 (chronic myeloid leukemia) and MCF-7 (breast cancer) cell lines with IC values of 0.3443 and 2.692 U/mL, respectively. SlpA did not show any cytotoxic activity against normal lymphocytes and was proved to be hemocompatible. Pre-treatment of biscuit and bread dough with different concentrations of SlpA resulted in a clear, dose-dependent reduction of acrylamide formation during baking. KEY POINTS: • Cloned and expressed L-asparaginase (SlpA) from fish gut microbiota • Purified SlpA displayed good cytotoxicity against K-562 and MCF-7 cell lines • SlpA addition caused a significant reduction of acrylamide formation during baking.
天冬酰胺酶催化 L-天冬酰胺水解为 L-天冬氨酸和氨。它在治疗儿童急性淋巴细胞白血病以及其他恶性肿瘤方面具有应用价值，此外，它还作为一种食品加工助剂，用于减轻烘焙行业中天冬酰胺形成丙烯酰胺的问题。由于其内在的谷氨酰胺酶活性和相关的副作用等问题，其在癌症化疗中的应用受到限制，这导致人们对寻找新型无 L-谷氨酰胺酶活性的天冬酰胺酶产生了浓厚的兴趣。本研究报道了从沙尖鱼肠道微生物组宏基因组鸟枪法测序中获得的天冬酰胺酶基因簇的克隆和表达。纯化的重组无谷氨酰胺酶天冬酰胺酶 SlpA 是一个 74 kDa 的同源二聚体，最适 pH 为 8，最适温度为 30°C。SlpA 的 K 和 V 值分别为 3.008 mM 和 0.014 mM/min。SlpA 对 K-562（慢性髓性白血病）和 MCF-7（乳腺癌）细胞系表现出细胞毒性活性，IC 值分别为 0.3443 和 2.692 U/mL。SlpA 对正常淋巴细胞没有任何细胞毒性活性，并且被证明具有良好的血液相容性。用不同浓度的 SlpA 预处理饼干和面包面团，在烘焙过程中可明显、剂量依赖性地减少丙烯酰胺的形成。 要点： • 从鱼类肠道微生物组中克隆和表达天冬酰胺酶（SlpA） • 纯化的 SlpA 对 K-562 和 MCF-7 细胞系表现出良好的细胞毒性 • 添加 SlpA 可显著减少烘焙过程中天冬酰胺形成丙烯酰胺的量。
