TABLE 1.

pH optima and activity ratios of PhyA mutants at different pH pairs

Mutant	Optimal pHa		Ratio of activityb
	First	Second	pH 3.5/5.5	pH 3.5/2.5
WT	5-5.5	2.5 (67)	0.38 ± 0.01	0.55 ± 0.03
Q50L	5.0		0.68 ± 0.01*	1.51 ± 0.03*
Q50P	5.5	3.0 (61)	0.50 ± 0.01*	0.94 ± 0.02*
K91A	5-5.5	2.5 (60)	0.33 ± 0.00*	0.55 ± 0.00
K91E	5-5.5		0.46 ± 0.03*	1.76 ± 0.11*
K94E	5-5.5	2.0 (69)	0.34 ± 0.01*	0.56 ± 0.01
E228Q	5-5.5	2.5 (66)	0.34 ± 0.01*	0.52 ± 0.02
E228K	3.8	3.0 (83)	1.72 ± 0.01*	1.08 ± 0.01*
D262H	5.0		0.87 ± 0.02*	1.51 ± 0.03*
K301E	2.5	4-4.5 (93)	1.40 ± 0.01*	0.75 ± 0.02*
E228K-K300D	5.5		0.52 ± 0.01*	1.07 ± 0.03*
E228K-K300T	NA		NA	NA
E228K-K300R	4.0	3.0 (81)	1.44 ± 0.23*	0.91 ± 0.14*
E228K-K300R-K301E	4.0		4.00 ± 0.05*	1.11 ± 0.02*
E228K-K300T-K301E	3.0-4.0		2.25 ± 0.03*	1.06 ± 0.05*
E228K-K300D-K301E	4.0	3.0 (95)	2.28 ± 0.03*	0.93 ± 0.03*
K94E-E228K	4.5	3.0 (78)	0.69 ± 0.04*	0.79 ± 0.04*
K94E-E228K-K301E	2.5-4.5		1.38 ± 0.07*	0.81 ± 0.04*
K94E-K300E-K301E	3.0-4.0		12.68 ± 0.14*	1.07 ± 0.01*
K300E-K301E	3-4.5		6.62 ± 0.11*	1.11 ± 0.03*
K91A-E228Q-K300E	4.5-5		0.70 ± 0.05*	1.53 ± 0.11*
K94A-E228A-D262A-K300D	5.5		0.55 ± 0.02*	0.94 ± 0.04*

^aRelative activity at the second optimal pH, as at the first optimal pH, is expressed as a percentage in parentheses.

^bThe ratios of phytase activity at different pH pairs are expressed as mean ± standard error (n = 3 to 6). The buffer conditions were as follows: pH 5.5, 0.2 M citrate; pH 3.5, 0.2 M glycine-HCl; pH 2.5, 0.2 M glycine-HCl. An asterisk indicates a difference (P < 0.05) from the WT. NA, not applicable.