Application Of Recombinant Sialidase On The Conversion Of Gangliosides

In this work, the sialidase gene of Brevibacterium casei was cloned and recombinant Escherichia coli BL21（DE3）-pET-28a（+）-Sia for efficient production of sialidase was constructed, the enzymatic properties of recombinant sialidase were studied and gangliosides （GLS） were converted into monosialoganglioside（GMl） using whole cells, free enzyme and immobilized enzyme respectively.The conditions for sialidase production by the recombinant strain were optimized. The yield of sialidase on shaking flask level reached1.6X10₄U/mL under the optimal conditions: concentration of the revulsant0.1mM, induction time12h, temperature25℃. The yield is120times higher than that of the sialidase produced by Brevibacterium casei. Moreover, the yield on3.7L fermenter level reached2.4X10₅U/mL.Epoxide material LH305-HY03was selected as the immobilization materials. The conditions for immobilization of sialidase after optimization were listed as following:load of sialidase1.0X10₅U/g support, temperature15℃, pH6.5, immobilization time18h. The activity of the immobilized sialidase reached8.8X10₄U/g under the above conditions.The optimal reaction temperature of sialidase was increased from37℃to47℃after immobilization. The optimal reaction pH for both immobilized sialidase and free sialidase was5.5. However, the immobilized sialidase kept stable in a wider pH range of5.0-8.0. The storage stability of the immobilized enzyme was improved. Half of the immobilized sialidase activity was lost after6consecutive batches of reaction. The Km and Vmax of immobilized sialidase was3.88x10"3mmol/L and576mmol/min, respectively. Km is2.3times of that of free sialidase, indicating a decreased affinity to the substrate.The relative content of GM1after the conversion of GLS reached75.4%、72.1%and68.7%, using whole cell, free sialidase and immobilized sialidase respectively. In whole cell biocatalysis, product inhibition was alleviated because the product was metabolized by the cells; in another hand, purification became more complicated for the production of byproducts. In contrast, free sialidase catalysis simplified purification process with the problem of product inhibition remaining. Despite that the conversion rate of immobilized sialidase was lower than that of whole cell and free enzyme, it was more reliable to apply immobilized enzyme in industrialization of GM1for its tolerance to environment and reusability.