Directed evolution improves the catalytic activity of laccase in papermaking.

Hong NI; Fan YANG; Lei WANG; Bianxia LI; Huanan LI; Jiashu LIU; Zhengbing JIANG; Wanli CHENG

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Directed evolution improves the catalytic activity of laccase in papermaking.

Author: Hong NI ¹ ; Fan YANG ¹ ; Lei WANG ² ; Bianxia LI ¹ ; Huanan LI ¹ ; Jiashu LIU ¹ ; Zhengbing JIANG ¹ ; Wanli CHENG ¹
Author Information

1. State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei University, Wuhan 430062, Hubei, China.
2. School of Material Science and Chemical Engineering, Hubei University of Technology, Wuhan 430072, Hubei, China.
Publication Type:Journal Article
Keywords: biobleaching; directed evolution; enzymatic characteristics; laccase; paper pulp
MeSH: Laccase/chemistry*; Directed Molecular Evolution; Enzyme Stability; Bacillus pumilus/genetics*; Mutation; Biocatalysis; Catalysis
From: Chinese Journal of Biotechnology 2025;41(1):308-320
CountryChina
Language:Chinese
Abstract: As a biocatalyst, laccase has been widely studied and applied in the papermaking industry. However, the low catalytic efficiency and poor stability of natural laccase limit its application in the pulping process. To develop the laccase with high activity and strong tolerance, we carried out directed evolution for modification of the laccase derived from Bacillus pumilus and screened out the mutants F282L/F306L and Q275P from the random mutant library by high-throughput screening. The specific activities of F282L/F306L and Q275P were 280.87 U/mg and 453.94 U/mg, respectively, which were 1.42 times and 2.30 times that of the wild-type laccase. Q275P demonstrated significantly improved thermal stability, with the relative activity 20% higher than that of the wild-type laccase after incubation at 40 ℃, 50 ℃, and 70 ℃ for 4 h. F282L/F306L and Q275P showed greater tolerance to metal ions and organic solvents than the wild-type laccase. The K_m value of the wild-type laccase was 374.97 μmo/L, and those of F282L/F306L and Q275P were reduced to 318.96 μmo/L and 360.71 μmo/L, respectively, which suggested that the substrate affinity of laccase was improved after mutation. The k_cat values of F282L/F306L and Q275P for the substrate ABTS were 574.00 s^-1 and 898.03 s^-1, respectively, which were 1.1 times and 1.7 times that of the wild-type laccase, indicating the improved catalytic efficiency. Q275P demonstrated better performance than the wild-type laccase in pulping, as manifested by the reduction of 0.82 in the Kappa number and the increases of 2.00% ISO, 7.8%, and 7.2% in whiteness, tensile index, and breaking length, respectively. This work lays a foundation for improving the adaptation of laccase to the environment of the papermaking industry.