3.3 Construction of α-L-rhamnosidase r-Rha1 mutants
Site-directed mutagenesis to replace the specific residue as identified above was achieved by the KOD-Plus-Mutagenesis Kit (Toyobo, Japan) using the specific primer pairs and the DH5α/19-9k plasmid as a template for polymerase chain reaction (PCR) [42]. Then the product of PCR was transformed into freshly prepared E. coliDH5α by thermal shock at 42ºC for 90 s. The cell suspension was cultivated at 37 °C in luria-bertani medium containing 100 μg/mL ampicillin.
The genes were transformed into P. pastoris SMD1168 cells for protein expression. E. coli transformants were pooled, and the plasmid DNA was extracted using a TIANprep Mini Plasmid Kit (Tiangen, Beijing, China). The resulting recombinant vectors were separately linearized with Sal I, and transformed into P. pastoris SMD1168 using a Gene Pulser Apparatus (Bio-Rad, Hercules, USA). The transformants were scanned after growing the clones on minimal dextrose medium plates at 30ºC for 3 d. Transformed yeast cells were selected on yeast extract peptone dextrose agar-plates containing 2.5 mg/mL G418 (Transgen Biotech, China). Colony PCR was carried out to confirm that whether or not the α-L-rhamnosidase gene has integrated into the genome. The General (5’AOX, 3’AOX) and Specified primers (Q9K-F, Q9K-R) as shown Table S3 were used for the selection of clones.