Kinetic Characterization of a Porcine Intestinal Alkaline Phosphatase Isomer over-Expressed in the E. coli BL21 (λDE3)

Intestinal alkaline phosphatases (IAP), one group of the most abundant apical membrane-bound enzymes in the gut, play a pivotal role in protecting intestinal health and preserving mutually beneficial host-microbial relationships against pathogenic bacterial toxins by reducing their toxicity through dephosphorylation of the endotoxin lipopolysaccharides (LPS) lipid moiety and other emblematic members of pathogen-associated-molecular patterns (PAMPs) such as ATP, therefore preventing gut dysbiosis and enteric diseases. There are 6 alkaline phosphatase genes identified in the porcine genome with possible 4 IAP and 1 tissue-non-specific alkaline phosphatase isomers that are all likely expressed in the small intestine. To elucidate roles of post-translational glycosylation on IAP isomer affinity, this study was conducted to characterize a porcine IAP isomer (protein product ID: XP_003133777.1-X1) that had been over-expressed in the prekarotic E. Coli BL21 (λDE3) without glycosylation. Both negative and X1-IAP over-expressed E. coli BL21 (λDE3) cell samples were sonicated to be homogenous and diluted for further enzyme kinetic analyses. The kinetic experiments were carried out by using the chromogenic synthetic substrate p-nitrophenyl phosphate (PNPP) with 16 gradient concentrations of PNPP, ranging 0 - 6 mM in incubation media in 4 replicates at pH 7.4 and 37⁰C for 30 minutes. The kinetics (parameter estimates ± SE, P < 0.05, R² = 0.91 – 0.94, n = 64) of the X1-IAP isomer specific and the intrinsic E. coli BL21 (λDE3) alkaline phosphatase activities were obtained, including Vmax values of 11.59 ± 1.58 vs. 19.02 ± 3.39 nmol/(mg protein·min); and K_m values of 4.72 ± 1.16 vs. 7.56 ± 2.09 mM. The X1-IAP isomer Km value measured at 4.72 ± 1.16 mM in this study is substantially higher than the range of the IAP Km values (0.1 - 0.3 mM) determined in the weanling porcine jejunum under the same conditions (using PNPP at pH 7.4 and 37⁰C) from our previous studies. It is known that alkaline phosphatase isomers expressed in vivo in the porcine gut would be highly glycosylated. Results of this study show that the porcine X1-IAP isomer over-expressed in the E. coli BL21 (λDE3) without glycosylation was associated with a very low enzyme affinity. Therefor, factors affecting post-translational glycosylation of the alkaline phosphatase isomers in the porcine gut would greatly influence their enzyme affinity towards detoxifying relatively low levels of pathogenic bacterial LPS and other emblematic PAMPs members in the gut lumen for protecting gut health.