Protein Engineering of Pasteurella multocida α2,3-Sialyltransferase with Reduced α2,3-Sialidase Activity and Application in Synthesis of 3′-Sialyllactose

Abstract

Sialyltransferases are key enzymes for the production of sialosides. The versatility of Pasteurella multocida α2,3-sialyltransferase 1 (PmST1) causes difficulties in the efficient synthesis of α2,3-linked sialylatetd compounds, especial its α2,3-sialidase activity. In the current study, the α2,3-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 α2,3-sialyltransferase activity of PMG2 was enhanced by 1.4-fold, while its α2,3-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 α2,3-sialyltransferase activity, but their α2,3-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 α2,3-linked sialosides. This work provides a guide to designing and constructing efficient sialyltransferases.

Determination of 3′-Sialyllactose in Edible Bird’s Nests and the Effect of Stewing Conditions on the 3′-Sialyllactose Content of Edible Bird’s Nest Products

“…The oligosaccharide content change caused by adding sugar during processing has not yet been explained. From this study, it was speculated that monocrystal rock sugar (used in this study) could be hydrolyzed into glucose and fructose [ 28 , 29 , 30 , 31 ], in which the glucose could be dehydrated and condensed with galactose sialate by heat treatment to form 3′-sialyllactose. In sum, it showed that adding sugar (monocrystal rock sugar) could increase the nutritional value of edible bird’s nest products within the tested range.…”