关键词: 易错PCR, 定向进化, 褐藻胶裂解酶, 氨基酸序列

Abstract: In order to improve the enzymatic activity of alginate lyase Alg-2, directed evolution based on error prone PCR was carried out on the marine bacterium Tamlana sp. S12. After two rounds of error prone PCR and 96-well plate fermentation, two positive and two negative mutants were obtained with 162%, 241%, 53% and 11% higher enzymatic activity as compared with the control, respectively. According to kinetic analysis, Km value of the positive strain P2-81 was reduced by 50% while that of the negative strain N2-47 was increased by 106% as compared with the parental one, implying that the substrate affinity of the enzyme in the mutant strains is greatly increased or reduced. The results of gene sequencing and amino acid sequence analysis showed that Glu, Thr, Ser, Asp and Tyr played a positive role in the increased activity of alginate lyase whereas mutation of Lys in the conserved region had a negative effect. The role of Asp and Lys was further confirmed by directed mutations. The results of this study can be helpful to gain a deep understanding of the catalytic mechanism of alginate lyases and to provide a theoretical basis for alginate lyases reconstruction using rational design methods.

Key words: error prone PCR, directed evolution, alginate lyases, amino acid sequence