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l -lactate dehydrogenases (LDHs) has been widely studied for their ability to reduce 2-keto acids for the production of 2-hydroxy acids, whereby 2-hydroxybutyric acids (2-HBA) is among the most important fundamental building blocks for synthesizing pharmaceuticals and biodegradable materials. However, LDHs usually show low activity towards 2-keto acids with longer side chain such as 2-oxobutyric acid (2-OBA). Here rational engineering of the Plasmodium falciparum LDH loop with residue involved in the catalytic proton transfer was initially studied. By combining homology alignment and structure-based design approach, we found that changing the charge characteristics or hydrogen bond network interactions of this loop could improve enzymatic catalytic activities and stabilities towards 2-OBA. Compared with wild type, variant N197D ldh showed 1.15 times higher activity and 2.73 times higher K cat / Km. The half-life of variant N197D ldh at 40 °C increased to 77.9 h compared with 50.4 h of wild type. Furthermore, asymmetric synthesis of (S)-2-HBA with coenzyme regeneration revealed 95.8 g/L production titer within 12 h for variant N197D ldh , 2.05 times higher than using wild type. Our study indicated the importance of loop with residues involved in the catalytic proton transfer process, and the engineered LDH would be more suitable for (S)-2-HBA production. [Display omitted] • Rational engineering the Pf LDH loop with residue His195 involved in the catalytic proton transfer was initially studied. • Variant N197D ldh showed improved activity and stability towards 2-Oxobutyric acid was obtained by this strategy. • Compared with wild type, variant N197D ldh showed 1.15 times higher activities and 2.73 times higher k cat / K m. • Variant N197D ldh showed 95.8 g/L (S)-2-hydroxybutyrate production within 12 h, 2.05 times higher than that of wild type. [ABSTRACT FROM AUTHOR]