Abstract: In order to improve protein yield, laccase gene LAC1 was optimized by codon, and pET-32a(+)-LAC1 recombinant vector was constructed and expressed in Escherichia coli BL21 (DE3). The enzyme properties showed that the molecular weight of the target laccase protein was about 65 ku, and its activity was stable below 55 ℃ and pH 3.5-5.0. 5 mmol/L Ca2+, Cu2+, Mg2+ promoted the activity of the recombinant laccase, and the specific activity of the recombinant laccase was 32.3 U/mg after purification by nickel column. By analyzing the structure and active center of recombinant LAC1 laccase, the gene sequence was compared with that of other fungal laccase sequences, and the difference section was found. For this section, random mutation was introduced during the chemical synthesis of DNA fragments, and the random mutation library was constructed and the library capacity was measured. A mutant with enzyme activity enhancement was obtained by batch screening, and its three-dimensional structure was predicted by homologous modeling. The results showed that four amino acids of the mutated enzyme were changed and the enzyme activity was greatly increased by 2.3 times.

Key words: laccase, enzyme properties, random mutation, mass screening , homology modeling