High-throughput screening of B factor saturation mutated Rhizomucor miehei lipase thermostability based on synthetic reaction

Conventional lipase screening methods are mostly based on hydrolytic activity, which may not always be the best method to assess the enzyme activity, especially for evaluating synthetic activity. Here we developed a high throughput and visual method to screen clones with high synthetic activity and used it to assess lipases thermostability. All mutants’ lipase synthetic activity were identified through esterification of caprylic acid and ethanol with methyl red as the pH indicator adding in the substrates on according to the color change halo around the colony on culture plates since synthetic reaction was often accompanied with a rise in pH. After two rounds operation with the pH indicator screening method, we obtained a double mutant Asn120Lys/Lys131Phe from the Rhizomucor miehei lipase saturation mutated library based on amino acid residue B factors. The mutant's initial synthetic activity was a little higher than wild type and its thermostability in synthetic reaction was enhanced, which remained 63.1% residual activity after being heated at 70 °C for 5 h comparing to 51.0% of wild type. The double mutant with the two residue replacements balanced well between stability and activity. Yeast surface display technology and the pH indicator method, combined with colony screening were shown to facilitate high-throughput screening for lipase synthetic activity.

► Lipase hydrolytic activity cannot represent its synthetic activity.
► pH indicator was used for screening lipase synthetic activity on colony plate.
► B factor saturation mutagenesis increased lipase thermostability in synthetic reaction.
► Good variant balanced well between flexibility and rigidity.
► Yeast surface display facilitate lipase evolution.