Directed evolution by error-prone PCR of Armillariella tabescens MAN47 β-mannanase gene toward enhanced thermal resistance

Abstract:

Firstly, We used error-prone PCR to induce mutations on Armillariella tabescens MAN47 β-mannanase gene, Secondly, we cloned the mutated fragments into secreted expression vector pYCα, Then the recombinant plasmids were transformed into Saccharomyces cerevisiae BJ5465 after amplified and extracted in DH5α cells. Through three cycles of error-prone PCR we built a mutant database, Then we screened one optimum (named M262) from about 104 mutants. The evoluted MAN47 β-mannanase displayed both higher thermal stability and activity than wide type. The evoluted enzyme M262 retained high activity after treatment at 80oC for 30 min, whereas, the wild type nearly lost activity under this condition. Meanwhile, the activity of M262 can reach to 25?U/mL, which is 4.3 times as wide type under optimum temperature. In addition, pH stability and pH range of evoluted enzyme M262 were both improved compared with wild-type enzyme. The optimum pH was estimated to be similar to that of wild-type enzyme. The sequence comparison illustrated that there were three nucleotide substitutions (T343A/C827T/T1139C) which carried corresponding amino acid changes (Ser115Thr/Thr276Met/Val380Ala). According to homologous modeling by SWISS-MODEL Repository, three mutated amino acids located at the sixth amino acid of the fourth β-sheet, the first amino acid of the sixth a-helix, the turn between the tenth and eleventh β-sheet, respectively.