Adedibu Peter Adeolu, Noskova Yulia Aleksandrovna, Yugay Yulia Anatolievna, Ovsiannikova Daria Mikhailovna, Vasyutkina Elena Anatolievna, Kudinova Olesya Dmitrievna, Grigorchuk Valeria Petrovna, Shkryl Yury Nikolaevich, Tekutyeva Liudmila Aleksandrovna, Balabanova Larissa Anatolievna
School of Advanced Engineering Studies, Institute of Biotechnology, Bioengineering and Food Systems, FEFU, 10 Ajax Bay, 690922 Vladivostok, Russia.
Laboratory of Marine Biochemistry, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Prospect 100-letya Vladivostoka 152, 690022 Vladivostok, Russia.
Plants (Basel). 2024 Dec 21;13(24):3570. doi: 10.3390/plants13243570.
Alkaline phosphatase (ALP) of the PhoA family is an important enzyme in mammals, microalgae, and certain marine bacteria. It plays a crucial role in the dephosphorylation of lipopolysaccharides (LPS) and nucleotides, which overstimulate cell signaling pathways and cause tissue inflammation in animals and humans. Insufficient ALP activity and expression levels have been linked to various disorders. This study aims to produce recombinant ALP from the marine bacterium KMM 296 (CmAP) in transformed leaves and calli of and to elucidate the influence of the plant host on its physical and chemical properties. has proven to be versatile and is extensively used as a heterologous host in molecular farming. The gene encoding for CmAP was cloned into the binary vectors pEff and pHREAC and transformed into leaves through agroinfiltration and the leaf disc method for callus induction using strain EHA105. Transformed plants were screened for recombinant CmAP (rCmAP) production by its enzymatic activity and protein electrophoresis, corresponding to 55 kDa of mature CmAP. A higher rCmAP activity (14.6 U/mg) was detected in a homogenate of leaves bearing the pEFF-CmAP construct, which was further purified 150-fold using metal affinity, followed by anion exchange chromatography. Enzymatic activity and stability were assessed at different temperatures (15-75 °C) and exposure times (≤1 h), with different buffers, pHs, divalent metal ions, and salt concentrations. The results show that rCmAP is relatively thermostable, retaining its activity at 15-45 °C for up to 1 h. Its activity is highest in Tris HCl (pH 9.0-11.0) at 35 °C for 40 min. rCmAP shows higher salt-tolerance and divalent metal-dependence than obtained in . This can be further explored for cost-effective and massively scalable production of LPS-free CmAP for possible biomedical and agricultural applications.
PhoA家族的碱性磷酸酶（ALP）是哺乳动物、微藻和某些海洋细菌中的一种重要酶。它在脂多糖（LPS）和核苷酸的去磷酸化过程中起着关键作用，这些物质会过度刺激细胞信号通路，导致动物和人类组织炎症。ALP活性和表达水平不足与多种疾病有关。本研究旨在利用海洋细菌KMM 296（CmAP）在烟草的转化叶片和愈伤组织中生产重组ALP，并阐明植物宿主对其物理和化学性质的影响。烟草已被证明具有通用性，在分子农业中被广泛用作异源宿主。将编码CmAP的基因克隆到二元载体pEff和pHREAC中，并通过农杆菌浸润和叶盘法，使用EHA105菌株诱导愈伤组织，将其转化到烟草叶片中。通过酶活性和蛋白质电泳筛选转化植株中重组CmAP（rCmAP）的产生，其对应于成熟CmAP的55 kDa。在携带pEFF-CmAP构建体的叶片匀浆中检测到较高的rCmAP活性（14.6 U/mg），使用金属亲和层析进一步纯化150倍，随后进行阴离子交换层析。在不同温度（15-75°C）、暴露时间（≤1小时）、不同缓冲液、pH值、二价金属离子和盐浓度下评估酶活性和稳定性。结果表明，rCmAP相对耐热，在15-45°C下保持活性长达1小时。其活性在35°C、Tris HCl（pH 9.0-11.0）中40分钟时最高。rCmAP显示出比在烟草中获得的更高的耐盐性和二价金属依赖性。这可以进一步探索用于无LPS的CmAP的经济高效且大规模可扩展生产，以用于可能的生物医学和农业应用。
