Protein Engineering of Pasteurella multocida alpha 2,3-Sialyltransferase with Reduced alpha 2,3-Sialidase Activity and Application in Synthesis of 3 '-Sialyllactose

Sialyltransferases are key enzymes for the production of sialosides. The versatility of Pasteurella multocida alpha 2,3-sialyltransferase 1 (PmST1) causes difficulties in the efficient synthesis of alpha 2,3-linked sialylatetd compounds, especial its alpha 2,3-sialidase activity. In the current study, the alpha 2,3-sialidase activity of PmST1 was further reduced by rational design-based protein engineering. Three double mutants PMG1 (M144D/R313Y), PMG2 (M144D/R313H) and PMG3 (M144D/R313N) were designed and constructed using M144D as the template and kinetically investigated. In comparison with M144D, the alpha 2,3-sialyltransferase activity of PMG2 was enhanced by 1.4-fold, while its alpha 2,3-sialidase activity was reduced by 4-fold. Two PMG2-based triple mutants PMG2-1 (M144D/R313H/T265S) and PMG2-2 (M144D/R313H/E271F) were then designed, generated and characterized. Compared with PMG2, triple mutants showed slightly improved alpha 2,3-sialyltransferase activity, but their alpha 2,3-sialidase activities were increased by 2.1-2.9 fold. In summary, PMG2 was used for preparative-scale production of 3 '-SL (3 '-sialyllactose) with a yield of >95%. These new PmST1 mutants could be potentially utilized for efficient synthesis of alpha 2,3-linked sialosides. This work provides a guide to designing and constructing efficient sialyltransferases.