Error-Prone PCR

Error-prone PCR of SDH was performed by the GeneMorph II Random Mutagenesis Kit (Agilent, Santa Clara, CA) with pMD19-cspA-SDH as a template by using primer pair 5′-TTCCAGAGATTATTGCTGTTTACGG-3′/5′-GTGAAAAGTTCTTCTCCCTTACCCA-3′. The amplified fragment carrying A tail was directly cloned with the vector pMD19-Simple transformed into E. coli BL21 (DE3) derivative strains mentioned above. For primary screening, the colonies appeared on the LB plates with ampicillin were picked up into 96-deep well plates by QPix420 (MD, Genetix, UK). These cultured cells in the deep-well plate were cultured at 37°C for 2–3 h, then moved to culture at 30°C for 20–24 h. For high-throughput screening, 40 uL of supernatant was transferred into another 96-well plate, then mixed with buffer containing 2-KLG reductase and NADH to a total volume 200 uL. The last one of each 96-well plate was set as a control. The absorbance at 340 nm, which is the optimum absorbance for NADH, was detected by a microplate reader (BioTek, Winooski, VT).