Establishment and Application of High-Throughput Screening Method for Pullulanase Mutant Library

Abstract:

Pullulanase gene （GenBank Accession No：AX203843） was heterologous expression in BL21 （DE3） in shake flask and scale-down to the microtiter plate scale successfully. Based on microtiter plate，a high-throughput culture method and an efficient high-throughput detection method for pullulanase activity were established. Random mutagenesis on pullulanase gene was performed through error-prone PCR strategy. The error-prone PCR products were recombinated in expression vector pET-28a（+）-pelB and then introducted into BL21 （DE3） to construct mutant library. The High-throughput screening method for Pullulanase in study was used to screen the mutant library efficiently and optimum mutant 4A4 was screened，the pullulanase activity in supernatant improved 1.465 folds. This method not only is also applicable to high-throughput screening of amylase and cellulase，but also provides a new way for high-throughput screening of strain library and directed evolution of proteins.