Over-expression, characterization, and modification of highly active alkaline phosphatase from a Shewanella genus bacterium

Figures & data

Fig. 1. SDS-PAGE analyses of crude and purified recombinant T3-3AP.

Notes: Lane 1, crude extract following sonication; lane 2, heat treated solution; lane 3, ammonium sulfate precipitation; lane 4, eluate from Phenyl Sepharose column; lane 5, elute from DEAE Sepharose column.

Table 1. Purification of recombinant T3-3AP.

Process	AP activity (KU)	Protein^a (mg)	Specific activity (U/mg)	Purification (fold)
Crude homogenate	5380	152000	35.4	1.0
Heat treatment	4640	76300	60.8	1.7
(NH4)2SO4 ppt.	4000	15500	258	7.3
Phenyl-Sepharose	2530	420	6030	170
DEAE-Sepharose	2440	283	8620	244

Notes:

Detailed conditions of each process are described in the Materials and methods.

^a Amount of protein was determined by absorbance at 280 nm.

Table 2. Comparison of activities toward luminescent substrates.

Substrate	Relative light unit (×10^5)
	CIAP	T3-3 WT	T3-3 K161S + K184S
PPD	1.56	3.48	1.82
APS-5	23.7	32.5	28.0

Fig. 2. Biophysical properties of recombinant T3-3AP Notes: (A) effect of pH on the stability of recombinant T3-3AP; enzymes were incubated at 25 °C for 24 h in acetate buffer (○), MES buffer (◆), triethanolamine buffer (□), and glycine buffer (▲). Residual activity was measured at pH 8.0 at 60 °C. Initial activity before incubation is defined as 100%. (B) Effect of temperature on the stability of recombinant T3-3AP (◆) and CIAP (□); APase solutions were incubated at various temperatures for 60 min and were then cooled, and residual activity was measured at pH 9.8 at 37 °C. Initial activities before incubation are defined as 100%. (C) Effect of pH on the activity of recombinant T3-3AP; measurements were performed in diethanolamine buffer at 37 °C. Activities under optimal pH conditions are defined as 100.

Fig. 3. Residual specific activity of wild type and mutant T3-3AP enzymes after maleimide activation.

Notes: Maleimide activation was performed in the presence of various concentrations of EMCS (0.25, 0.5, and 1.0 mM) at 30 °C for 30 min, and residual activity with the substrate pNPP at pH 9.8 was measured at 37 °C; wild type enzyme (◆), K184S (□), and K161S + K184S (▲). Higher concentration of EMCS results in higher amount of maleimides introduced into the APases. Quantification of maleimide was performed as previously described.¹²⁾

Fig. 4. Enzyme linked immunosorbent assays (ELISA) using APase-conjugated IgG.

Notes: Sandwich ELISA were performed using anti-KOD DNA polymerase IgG labeled with T3-3AP (K161S + K184S) (▲) or CIAP (○). APS-5 (A) and PPD (B) were used as substrates.

Fig. 5. Effects of Mg²⁺ and Zn²⁺ on ELISA reactivity.

Notes: Sandwich ELISA 0.1 μg/ml antigen samples were performed using wash solution containing various concentration of Mg²⁺ (0–3 mM) and Zn²⁺ (0–1 mM). (A) APS-5 and (B) PPD were used as luminescent substrates.

Table 3. Conservation of amino acid residues that contribute to metal binding in ECAP.

	Metal binding site
	M1			M2			M3
ECAP*	327	331	412	51	369	370	153	155	322	328
	Asp	His	His	Asp	Asp	His	Asp	Thr	Glu	Lys
T3-3AP	265	269	400	47	307	308	147	149	260	266
	Asp	His	His	Asp	Asp	His	His	Thr	Glu	Trp
SCAP*	227	231	362	9	269	270	109	111	222	228
	Asp	His	His	Asp	Asp	His	His	Thr	Glu	Trp
HPAP*	316	320	432	42	357	358	153	155	311	317
	Asp	His	His	Asp	Asp	His	His	Ser	Glu	His
CIAP II	316	320	432	42	357	358	153	155	311	317
	Asp	His	His	Asp	Asp	His	His	Ser	Glu	His

Notes:

M1, M2, and M3 are metal binding sites that are occupied by Zn1, Zn2, and Mg, respectively, in native ECAP.²⁾ Asterisks (*) indicate APases for which 3D structures have been solved.

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