Study on directed evolution of β-mannanase in vitro by error-prone PCR.

Error-prone PCR was used to directed evolution β-mannanase in vitro, random introduction of mutations into β-mannanase by adjusting dNTP ratio and setting Mn2+ of different concentrations, a mutant library was constructed, and the enzymatic properties of the mutant enzymes were studied. The results showed that during the construction of the mutation library, the positive conversion rate could reach the highest 80% and the number of amino acid mutations was 2.0 with the concentration of Mn2+ 0.01 mmol/L, which was in accordance with the general principles of error-prone PCR mutation libraries; after two rounds of error-prone PCR, a storage capacity of 4000 was constructed, the mutant enzymes GwMan26A-27 of mutant Pichia pastoris X-33/pGAPZαA-GwMan26A-27 with acid resistance, high temperature resistance and high enzyme activity was finally selected; compared with before mutation, GwMan26A-27 has a wider enzymatic reaction temperature before the mutation and a suitable pH range, the thermal stability and pH stability also had been greatly improved; in the pepsin solution and trypsin solution for water bath 2 h, the digestive enzymes stability of GwMan26A-27 had also been improved. [ABSTRACT FROM AUTHOR]