Molecular Modification And Thermal Stability Of Cytidine Monophosphate Kinase

Cytidine monophosphate kinase(CMK)is an important member of the nucleoside monophosphate kinase family and plays a key role in nucleotide synthesis and metabolism.CMK catalyzes the conversion of cytidine monophosphate(CMP)to cytidine diphosphate(CDP),and then generates cytidine triphosphate(CTP),which is an important enzyme for CTP synthesis.The research group found that the thermal stability of CMK was poor,which affected the enzymatic reaction efficiency of catalyzing the synthesis of 3’-sialyllactose(3’-SL).In this study,the molecular modification of CMK was carried out by means of rational design modification and the effect on thermal stability was investigated.The main results are as follows:(1)According to the online server Po PMu Si C and the software Py Mo L,the amino acid residue mutation sites of CMK were predicted.Fourteen single point mutants were successfully constructed using site-directed mutagenesis.The results of thermal stability of the enzyme showed that five mutants with improved thermal stability were obtained.The wild-type enzyme(WT)retained 22.3% of the residual enzyme activity.The residual enzyme activities of mutants Q30 G,P62R,E85 I,D148W and K165 L were 35.5%,31.0%,34.9%,34.7% and 36.9%,respectively.Mutants Q30 G,E85I,D148 W and K165 L were subjected to compound mutation to obtain the mutant Q30G/E85I/K165 L with improved thermostability,and its residual enzyme activity was 2.2 times that of the WT.(2)In this study,eleven amino acid residue mutation sites were selected by B-FITTER prediction and analysis.The experiments showed that the thermostability of mutants N77 H,Q79N and R173 S was higher than that of WT.Combined mutation of mutants N77 H,Q79N and R173 S,mutant N77H/R173 S with improved thermostability were obtained.Its residual enzyme activity was 2.1 times that of WT.(3)According to the prediction analysis of Disulfide by Design,combined with sitedirected mutagenesis,seven mutants with disulfide bonds were successfully constructed.Compared with the residual enzyme activity of WT,the residual enzyme activity of mutant V72C/Q79 C was increased by 0.8 times.(4)To further improve the thermal stability of CMK,the above-mentioned dominant mutants with improved thermal stability were subjected to compound mutation.The results showed that the mutant Q30G/E85I/K165 L had the best thermal stability improvement.The cmk-Q30G/E85I/K165 L gene was constructed into the catalytic process of 3’-SL in the previous study.The yield increased by 33.6% in 25 m L of the catalytic system.This study shows that the modification method using rational design can improve the thermal stability of CMK,which has positive implications for the industrial application of CMK.