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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. [ABSTRACT FROM AUTHOR]

K-promoted Mo-based catalysts showed great promise for the hydrogenation of CS2 to methyl mercaptan (CH3SH). However, the research on the synergistic effect of K and Mo, and the active site of CS2 hydrogenation to CH3SH were unexplored widely. To solve this problem, the synergistic effect of K and Mo in the K-promoted Mo-based catalysts for CS2 hydrogenation to prepare CH3SH was investigated. The mesoporous alumina was the support and loaded the active components potassium and molybdenum to prepare the catalyst. The results suggested that the active components K and Mo can not only cooperatively regulate the acid-base sites on the catalyst surface, but also stabilize the molybdate species at +5 valence during the reduction process and increase the Mo unsaturated coordination sites. Combined with the results of the catalytic activity evaluation, indicating that the main active site of the catalysts is the weak Lewis acid-base site, and the strong acidic site and strong alkaline site are not conducive to the formation of CH3SH. Moreover, the possible catalytic mechanism of CS2 hydrogenation to CH3SH on the weak Lewis acid-base sites of the catalysts was proposed. The research results of this paper can provide an experimental basis and theoretical guidance for the design of high-performance CH3SH synthesis catalyst and further mechanism research. [ABSTRACT FROM AUTHOR]