Enhanced catalytic efficiency of CotA-laccase by DNA shuffling

Figures & data

Table 1. Sequences of specific primer pairs used for cloning procedures.

Genes	Primer name	Primer sequences (from 5ʹto 3ʹ)
B. subtilis LS03	Forward (CotA-3F)	CGGGGATCCGACACTTGAAAAATTTG
	Reverse (CotA-3R)	CCGCTCGAGTTTATGGGGATCAG
B. amyloliquefaciens LS05	Forward (CotA-5F)	CGGGAATTCGGCACTGGAAAAATTTGCAGATG
	Reverse (CotA-5R)	CCGCTCGAGCTGCTTATCCGTGACGTCCAT

Figure 1. SDS-PAGE analysis of CotA-laccase. Protein samples were loaded in each lane as follows: cell extract wild-type CotA (lane 1), cell extract 5E29 CotA (lane 2), molecular weight marker (M), purified wild-type CotA (lane 3), purified 5E29 CotA (lane 4).

Table 2. Kinetic constants for the wild-type CotA and 5E29 mutant using SGZ as substrate.

Laccase	Km (µM)	kcat (s^−1)	kcat/Km (s^−1 µM^−1)
WT	24.8 ± 3.4	6.3 ± 0.9	0.254
5E29	20.3 ± 1.3	7.6 ± 2.7	0.374

Figure 2. Effect of pH on the activity (a) optimal pH of wild-type CotA and 5E29 mutant for catalyzing ABTS and SGZ; (b) pH stability of wild-type CotA and 5E29 mutant.

Figure 3. Effect of temperature on the activity (a) optimal temperature of wild-type CotA and 5E29 mutant for catalyzing SGZ; The thermostability of wild-type CotA and 5E29 mutant (b) at 60°C, (c) 70°C, (d) 80°C.

Figure 4. Local structure of the wild-type laccase and its variants. (a) The position of Gln367 and neighboring residues of the wild-type laccase (b) Gln-367-Arg.

Figure 5. Decolorization of synthetic dyes by 5E29 and the wild-type CotA-laccases. (a) IC: Indigo carmine (b) CR: Congo Red.