Submitted to: Appl Biochem Biotechnol

Protein Engineering and Homologous Expression of Serratia marcescens Lipase for Efficientsynthesis of a Pharmaceutically Relevant Chiral Epoxyester

Supplementary material

Table S1.Specific activity of S. marcescenslipase and its mutants towards pNPB.

Variant / Spec. activity(U/mgprot) / Folds
LipAWT / 50.3 ± 2.7 / 1.0
LipAL315S / 111 ± 7.0 / 2.6
LipAS271F / 131 ± 9.6 / 2.2
LipAL315S/S271F / 231 ± 6.8 / 4.6

Table S2. The kinetic parameters of native LipA and LipAS271F for (±)-MPGM hydrolysis.

Variant / Km
(mM ) / Vmax (µmol/min/mg) / kcat
(s-1) / kcat/Km
(mM-1s-1) / Improv.
(folds) / E
LipA / 263 ± 86 / 121 ± 26 / 131 / 0.5 / 1.0 / >100
LipAS271F / 91 ± 27 / 183 ± 21 / 198 / 2.2 / 4.4 / >100

E:enantiomeric ratio.

Table S3.The concentration of lipase expressed in E. coli and Serratia marcescens

Variant / Concentration of lipase (mg/ml)
LipAWTinE. coli / 0.1
The wild Serratiamarcescens / 0.4
LipAWTin Serratiamarcescens / 4.2

Fig. S1.(a)Effect of temperature on the activity of mutant LipAS271F. (b)Effect of pH on the activity of mutant LipAS271F. (b) Data represent the mean of three replicates; Error bar represents standard deviation.

Fig. S2.The SDS-PAGE analysis of lipase expressed in E. coli (a) and Serratia marcescens (b).

Symbols: (a)Lane M, Protein marker; Lane 1, cell lysate supernatant of LipA; Lane 2, cell lysate precipitate of LipA; Lane 3, cell lysate supernatant of LipAS271F; Lane 4, cell lysate precipitate of LipAS271F. (b)Lane M, Protein marker;Lane 1,wild-typeSerratia strain; Lane 2, recombinant Serratia strain pUC-LipA; Lane 3, recombinant Serratia strain pUC-LipAS271F.

Fig. S3. Structural model of LipA based on the modeling of SM6 (PDB ID: 2 QUA).

Fig. S4. 18 amino acid residues in the range of 8 Å around the substrate