Heterologous Expression and Characterization of Cobetia amphilecti KMM 296 Alkaline Phosphatase in Nicotiana spp.

Alkaline phosphatase (ALP) of the PhoA family is an important enzyme in eukaryotes and certain gut and marine bacteria; it plays a crucial role in the detoxification of intestinal microbiome lipopolysaccharides (LPS) and excess of extracellular nucleotides, which overstimulate cell signaling and cause tissue inflammation in a host. Insufficient levels of this enzyme have been linked to several disorders in humans. This study aims at producing a recombinant PhoA (rec-PhoA) from the marine bacterium C. amphilecti KMM 296 in transiently transformed leaves of N. benthamiana and stably transformed N. tabacum, as well as elucidating its physical and chemical properties and anti-inflammatory potential. Nicotiana spp. have proven to be versatile and are extensively used as heterologous hosts in molecular farming. The alp gene was cloned into binary vectors pEff and pHREAC restricted with AscI, SmaI, and BsaI enzymes, respectively, and then incorporated into N. benthamiana leaves by means of agro-infiltration and into N. tabacum following the leaf disc method using the Agrobacterium tumefaciens EHA105 strain. The enzymatic activity of rec-PhoA from the transformed plants was confirmed in the leaves' lysate after purification using a metal-affinity column (Ni²⁺-IMAC-Sepharose) and, subsequently, Source-15Q ion-exchange sorbent (GE Healthcare). The purified rec-PhoA was visualized on SDS-PAGE as a 55 kDa band corresponding to the mature protein. The enzyme rec-PhoA was relatively thermostable and retained its activity at 15 - 45 ℃ up to 1 hr, with the highest activity in Tris HCl (pH 9.0–11.0) at 35 ℃, 40 min. It was salt-tolerant and divalent metal-dependent; Mn²⁺ and Mg²⁺ enhanced its activity. This study established that the plant-based expression of C. amphilecti PhoA in Nicotiana spp. is an appropriate method for the production of a pharma-grade LPS-free recombinant enzyme. This can be further explored for the cost-effective and massively scalable production of LPS-detoxifying ALP, for possible applications in human and animal therapy, as well as in plant growth promotion and biocontrol.