Purification and characterization of formaldehyde dismutases of Methylobacterium sp. FD1

Figures & data

Table 1. Primers for PCR amplification of formaldehyde dismutase genes of FD1.

Primer	Sequence (5ʹ-3ʹ)	Objective DNA fragment
FD1-fdm1-F	GAAGGAGATATACATATGGCCGCAAATGGAAACCGCGTC	His-tagged Fdm1 gene
FD1-fdm1-R6His	GTGATGGTGGTGATGATGCGAGGCCTTTTTCACGCTGCC
FD1-fdm2-F	GAAGGAGATATACATATGGCATCCAGCAATCGGGTCATCACG	His-tagged Fdm2 gene
FD1-fdm2-R6His	GTGATGGTGGTGATGATGGGCCACCAGTTTCGTCGTGTT
FD1-fdm3-F	GAAGGAGATATACATATGGCTACAGGCAACCGGATCGTA	His-tagged Fdm3 gene
FD1-fdm3-R6His	GTGATGGTGGTGATGATGGGCCGCCACAGAGCCGTGCGG

Figure 1. Amino acid alignment of the three Fdms of FD1, the Fdm of P. putida F61, and the glutathione-independent Fdh of P. putida C-83.

Figure 2. SDS-PAGE of Fdm1 (a), Fdm2 (b), and Fdm3 (c) of FD1 purified from each recombinant E. coli by IMAC.

Table 2. Purification of Fdm1, Fdm2, and Fdm3 of FD1.

Enzyme	Purification step	Total protein (mg)	Total activity (U)	Specific activity (U/mg)	yield (%)	Purification (fold)
Fdm1	Soluble protein extract	110	14,000	130	100	1
	Elution (pH 4.5)	5.3	3,100	590	22	4.5
Fdm2	Soluble protein extract	110	3,900	35	100	1
	Elution (pH 4.5)	1.0	520	520	13	15
Fdm3	Soluble protein extract	180	17,000	96	100	1
	Elution (pH 4.5)	23	5,500	240	32	2.5

Enzyme activity was determined as follows. Fdm was added in 0.5 mL of 100 mM sodium phosphate buffer (pH 7.0) containing 50 mM formaldehyde (containing methanol), and the mixture was incubated at 30°C. Subsequently, 0.2 mL of 2 M HCl was added to the mixture to stop the reaction. Fdm activity was estimated by measuring the formic acid generated from formaldehyde using HPLC. One unit corresponded to the amount enzyme that degrades 1 µmol of formaldehyde in 1 min. Other experimental conditions are specified in materials and methods.

Figure 3. Effect of pH (a) and temperature (b) on the formaldehyde dismutase activity of the Fdms of FD1.

Figure 4. Effect of formaldehyde concentration on the formaldehyde dismutase activity of the Fdms of FD1 and the Lineweaver-Burk plots (inset).

Table 3. Enzymatic kinetic parameters of Fdm1, Fdm2, and Fdm3 of FD1.

	kinetic parameter
enzyme	Km (mM)	Vmax (µmol-formaldehyde/mg/min)	kcat (1/s)	kcat/Km (1/s/M)
Fdm1	620	6,000	4,300	6,900
Fdm2	870	10,000	7,500	8,600
Fdm3	410	3,000	2,300	5,600

Abbreviations: K_m: Michaelis constant; V_max: maximum reaction velocity; k_cat: turnover number; k_cat/K_m: catalytic efficiency. Experimental conditions are specified in materials and methods.

Figure 5. Substrate specificity of Fdm1, Fdm2, and Fdm3 of FD1.

Table 4. Alcohol-NDMA oxidoreductase activities of Fdm1, Fdm2, and Fdm3 of FD1.

Enzyme	Activity (nmol-NDMA/mg/min)
	methanol	ethanol
Fdm1	97 ± 0.8	270 ± 25
Fdm2	80 ± 0.2	260 ± 50
Fdm3	410 ± 4.0	3100 ± 130

Experimental conditions are specified in materials and methods.