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Protein engineering is an effective method to improve the thermal stability of biological macromolecules. Cytidine monophosphate kinase (CMK) is a key enzyme involved in nucleotide metabolism and participates in the production of cytidine triphosphate. It has previously been confirmed that CMK shows low thermal stability. This study aimed to enhance the thermal stability of CMK. Herein, we first analyzed native CMK using PoPMuSiC. Thereafter, we constructed single point mutants (Q30G, P62R, E85I, D148W and K165L) and multipoint mutants of CMK with higher thermal stability using site-directed mutagenesis. The thermal stability of the mutant Q30G/E85I/K165L was observed to be 1.2-fold higher than that of the wild type. The mutants were then allowed to catalyse 3′-sialyllactose (3′-SL) synthesis. After 51.72 mmol/L N-acetylneuraminic acid (sialic acid) was consumed in a 25 mL reaction system and 50.67 mmol/L 3′-SL was obtained, a decrease of 2 h was recorded in the reaction time, whereas the yield rate increased by 33.6%. The results suggest that the mutant Q30G/E85I/K165L can improve the thermal stability of CMK and its catalytic efficiency for 3′-SL synthesis. These findings are crucial for various applications involving enzyme conversion. [ABSTRACT FROM AUTHOR]