| Summary: | 碩士 === 國立中正大學 === 化學工程研究所 === 105 === The purpose of this study was to co-express two enzymes that cooperatively synthesize the sialic acid. These two enzymes for the cooperative synthesis of sialic acid were N-acetylneuraminic acid aldolase (Neu5Ac aldolase or nanA) and N-acetyl glucosamine 2-epimerase, (GlcNAc 2-epimerase or 2-epimerase). The N-terminus and C-terminus of Neu5Ac aldolase were connected with glutathione S-transferase (GST) and five arginine residues (5R), respectively. The C-terminus of GlcNAc 2-epimerase was connected with five aspartic acid residues (5D) followed by six His resiaues. Neu5Ac aldolase and GlcNAc 2-epimerase were sub-cloned from Escherichia coli K12 and Synechocystis sp. (PCC6803) by PCR, respectively. I have constructed five expression vectors, including pETDuet_GST-nanA-5R, pETDuet_ GST-nanA-5R_2-epimerase-5D-6His, pGEX-2TK_nanA_5R-SD-GST-2- epimerase, pGEX-2TK_nanA-5R, pGEX-2TK_nanA-5R_T7 promoter-2 epimerase-5D-6His-terminator. Each vector was tansformed into E. coli BL21(DE3) for protein expression. After induction by 1 mM IPTG, only bacteria encoding the plasmid, pGEX-2TK_nanA-5R_T7 promoter-2 epimerase-5D-6His-terminator, can simultaneously express GST-nanA-5R and 2-epimerase-5D-6His. However, most of expressed enzymes were present in the inclusion body. The SEM images (5000x magnification) indicated that the average size of an inclusion body was 0.59µm±0.13µm, N=60. In the presence of the substrate, Neu5Ac (20 mM), the specific activities of Neu5AC aldolase in the pellet and supernatant are (U/mg=3.98±0.72, N=3) and (U/mg=13.0±1.60, N=3), respectively. The specific activities of GlaNAc 2-epimerase in the pellet and supernatant are (U/mg=0.97±0.10, N=3) and (U/mg=8.65±0.60, N=3), respectively, when GlcNAc (100 mM) was used as the substrate in the activity assay.
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