. 2012 Mar;78(5):1370–1384. doi: 10.1128/AEM.07530-11

Table 1.

Kinetic constants at pH 5.0 of the purified PcL variants obtained by directed evolution and TAI breakdown

Laccase	Substrate	K_m (mM)	k_cat (s⁻¹)	k_cat/K_m (mM⁻¹ s⁻¹)	TAI^a (fold) vs α-PcL	Improvement vs α-PcL^b
Laccase	Substrate	K_m (mM)	k_cat (s⁻¹)	k_cat/K_m (mM⁻¹ s⁻¹)	TAI^a (fold) vs α-PcL	k_cat	Expression
Wild-type PcL	ABTS	0.035 ± 0.002	38.0 ± 0.4	1,085
	Sinapic acid	0.013 ± 0.000	21.6 ± 0.2	1,662
	DMP	0.027 ± 0.002	16.3 ± 0.3	581
α*-PcL	ABTS	0.020 ± 0.001	35.1 ± 0.9	1,755	40		40
	Sinapic acid	0.020 ± 0.002	19.9 ± 0.5	948
	DMP	0.012 ± 0.002	10.0 ± 0.3	833
7A9 (3rd generation)	ABTS	0.013 ± 0.001	205.1 ± 4.5	15,777	900	5.8	155
	Sinapic acid	0.020 ± 0.003	124.4 ± 5.6	6,193		6.1
	DMP	0.106 ± 0.004	90.6 ± 0.8	858		9
α*-3PO (6th generation)	ABTS	0.024 ± 0.002	482.6 ± 10.2	19,944	8,000	13.7	584
	Sinapic acid	0.023 ± 0.002	197.7 ± 4.7	8,457		10.4
	DMP	0.213 ± 0.013	196.9 ± 3.1	923		19.6

The total activity improvement (TAI) was measured using 3 mM ABTS as the substrate in supernatants of cultures grown in 96-well plates. Values represent the average of five measurements.

The improvement in expression is defined as the ratio of the total increase in activity and k_cat with ABTS as the substrate. α-PcL, parent type used in the directed evolution in S. cerevisiae (formed by the native α-factor preproleader and the native PcL); wild-type PcL, original laccase homologously expressed in P. cinnabarinus containing the native secretion leader; α*-PcL, native PcL fused to the evolved α-factor preproleader for secretion in S. cerevisiae; α*-3PO, ultimate variant of the whole evolution process (containing the evolved α-factor preproleader plus the evolved PcL).