Refolding of Recombinant N-acetyl-D-glucosamine 2-epimerase by a fed-batch Process

N-乙醯－D-葡糖胺2-差向異構酶是以酵素法合成抗病毒藥物的重要前驅物N-乙醯神經氨酸的重要酵素之一。利用大腸桿菌生産N-乙醯－D-葡糖胺2-差向異構酶時通常會在細胞質中形成不具活性之內含體。研究發現利用稀釋法進行N-乙醯－D-葡糖胺2-差向異構酶內含體之重折疊時，會産生藉由疏水性作用力而形成的寡聚物。利用饋料批次稀釋法可顯著降低寡聚物之形成並因而提昇重折疊之效率，且可溶性蛋白質的回收率隨饋料速率的增加而降低。在特定重折壘階段中添加谷胱甘太(glutathione)進行變硫鍵之重組可提高可溶性蛋白質之産率逹一倍。此外，添加精酸與甘油亦可增加可溶性蛋白質産率與酵素之比活性。在最適條件下，重折疊酵素之比活性可逹0.47IU/mg，約為原酵素之30%。

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並列摘要

N-Acetyl-D-glucosamine 2-epimerase is one of the key enzymes for the enzymeatic synthesis of the enzymatic synthesis of N-acetylneuraminic acid, a sialic acid and a critical precursor for the synthesis of some antiviral agents. Overexpression of the recombinant epimerase in Escherichia coli led to the formation of protein inclusion bodies. Refolding of guanidine HCl-solubilized protein by direct dilution resulted in the formation of soluble oligomers, mediated probably by hydrophobic interactions. The extent of aggregateion of protein subunits into inactive oligomers could be efficiently reduced by employing fed-batch refolding process, in which the solubilized proteins were added continuously at a pre-determined rate. The yields of soluble proteins decreased with the feeding rates. The addition of glutathione into refolding buffer at certain stage of the refolding process could enhance the yield of soluble proteins more than two-fold, possibly by resolving the inadvertently formed disulfide bridges among the protein subunits that contain 10 cysteine residues each. Folding aids such as L-arginine and glycerol were found effective in increasing the yield of soluble proteins and the specific activeity of the refolded proteins. Under the optimal condition, a specific activity of 0.47IU/mg was obtained with an activity recovery yield of ca. 30%. The specific activity of the refolded proteins was significantly lower than that of the native protein, 1.23IU/mg, indicating that more information concerning the 3D structure of native N-acetyl-D-glucosamine 2-epimerase and the role of its cofactor, ATP, for catalytic activity is needed for the development of a more efficient refolding process.

並列關鍵字

Refolding ； Fed-batch ； Epimerase ； L-Arginine ； Sialic acid

國際替代計量

Refolding of Recombinant N-acetyl-D-glucosamine 2-epimerase by a fed-batch Process

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