[Background] Optically pure L-phenyllactic acid (L-PLA) is a natural antimicrobial agent and also a highly value-added chiral compound with potential applications in food, pharmaceutical and biomaterial areas. Although L-PLA can be produced by asymmetric reduction of phenylpyruvic acid (PPA) by L-LcLDH, an L-lactate dehydrogenase from Lactobacillus casei CICIM B1192, it displays low activity. To improve the activity of a wild-type L-LcLDH towards PPA, an L-LcLDH mutant, L-LcLDHQ88R, was successfully constructed by our lab. Its catalytic efficiency (kcat/Km) was 4.9-fold higher than that of L-LcLDH. [Objective] To further improve the catalytic efficiency of L-LcLDHQ88R towards PPA, the amino acid Ile229 located in the substrate-binding pocket of L-LcLDHQ88R or L-LcLDH was randomly substituted with any one of 20 amino acids. [Methods] Using a recombinant plasmid pET-22b-LcldhQ88R as the template, the Ile229-encoding codon in LcldhQ88R was subjected to site-saturated mutagenesis by whole-plasmid PCR technique. Then, the mutant library of L-LcLDHQ88R was constructed by transforming pET-22b-LcldhQ88R variants into E. coli BL21(DE3), respectively. Finally, an E. coli transformant expressing the highest activity towards PPA was screened from this library. [Results] The selected transformant, E. coli/LcldhQ88R/I229Q, contains a double-mutant gene in which the Gln88- and Ile229-encoding codons in Lcldh was substituted with Arg- and Gln-encoding ones, respectively. The specific activity of purified L-LcLDHQ88R/I229Q towards PPA was 18.5- and 2.3-fold those of L-LcLDH and L-LcLDHQ88R, while its catalytic efficiency was 6.8- and 1.4-folds those of the latter two, respectively. Additionally, the pH and temperature properties of L-LcLDHQ88R/I229Q did not obviously changes compared to L-LcLDH. The analysis of catalytic mechanism by molecular docking (MD) simulation showed that the double-mutation of Q88R/I229Q enlarges the inlet of substrate-binding pocket and alters the steric structure of active centre, which may contribute to the increase of activity. [Conclusion] The double-mutation of Q88R/I229Q in L-LcLDH primary structure greatly enhanced its specific activity and catalytic efficiency, making L-LcLDHQ88R/I229Q a promising candidate for asymmetric reduction of PPA.