Study on engineering aspect of gene cloning of N-acetyl-D-glucosamine 2-epimerase and N-acety-D-neuraminic acid aldolase for the production of sialic acids

• Main Authors: Tzu-Hsien Wang, 王子賢
• Other Authors: Wen-Chien Lee
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/82045059292024122315

博士 === 國立中正大學 === 化學工程所 === 93 === A variety of biological functions has been attributed to sialic acid and its derivatives. They become much valuable since several new functions and applications have been discovered recently. Two enzymes, N-acetylgucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetylneuraminic acid aldolase (Neu5Ac aldolase), are required for the enzymatic production of sialic acid. GlcNAc 2-epimerase catalyzes the inter-conversion of N-acetyl-D-glucosamine (GlcNAc) to N-acetyl-D-mannosamine (ManNAc), while Neu5Ac aldolase catalyzes the production of N-acetylneuraminic acid (Neu5Ac) from ManNAc and pyruvate. Both enzymatic reactions are reversible. In this study, the engineering consideration was emphasized on the cloning of GlcNAc 2-epimerase and Neu5Ac aldolase genes, amplified from Synechocystis sp. PCC6803 and Escherichia coli K12, respectively by the PCR method. Using the pGEX-series vectors, recombinant vectors were constructed to overproduce tagged fusion proteins in E. coli BL21. In addition to the conventional gene cloning method, a novel method called solid-phase gene recombination was proposed and used for the recombination of a GST-containing plasmid and Neu5Ac aldolase gene. It provided an alternative approach for gene cloning although the efficiency was very low. By the affinity of GST tag in the N terminus, the fusion proteins could be purified to homogeneity using a GSH-resin column and immobilized on the particles bound with glutathione (GSH). Five successive aspartic acid residues (5D) were attached to the C-terminus of GlcNAc 2-epimerase tagged with in the N-terminus to form a double-tagged fusion protein. The introduction of 5D tag to GlcNAc 2-epimerase could increase the protein solubility and provide the strongly negatively-charged groups for its immobilization on the ion-exchange resins by the electrostatic interaction or association with positively-charged Neu5Ac aldolase fusion protein. Results indicate that the double-tagged GlcNAc 2-epimerase had a higher soluble protein fraction and specific enzymatic activity. However, the double-tagged fusion protein showed no superiority on the immobilization over the single-tagged fusion. Three recombinant strains, E. coli BL21 (pGEX-1λT-2EP), E. coli BL21 (pGEX-2TK-2EP-5D), and E. coli BL21 (pGEX-nanA), were successfully established. Results from sequencing the reconstruction of each gene fusion suggested that the insertion of DNA was in the proper reading frame. After a culture for 8-10 h, the recombinant strains E. coli BL21 (pGEX-1λT-2EP) and E. coli BL21 (pGEX-2TK-2EP-5D) entered the stationary phase. IPTG (0.01 mM) was then added to the cultures for a 2.5-h-induction of overexpression of fusion proteins for 2.5 h. The following cell lysis led to crude protein extracts respectively containing 0.3 and 0.4% of single- and double-tagged fusion proteins. By affinity chromatography on the GSH column, the fusion proteins, GST-GlcNAc 2-epimerase and GST-GlcNAc 2-epimerase-5D could be purified to homogeneity using a GST-resin column. On the other hand, the recombinant strain E. coli BL21 (pGEX-nanA) was cultured for 3 h to reach the log phase, at which IPTG (1 mM) was IPTG added for an induction for 7 h. A 2.15% of fusion protein GST-Neu5Ac aldolase was obtained in the crude protein extract, which could be purified to homogeneity. Whatever the recombinant strain was induced, the over-expressed fusion proteins were largely present in the form of inclusion body or aggregate. Increasing the volume of lysis buffer and concentration of Trition X-100 could be helpful for resolublizing the fusion protein from inclusion body or aggregate into the protein extract. Results from activity assay indicate that the overexpressed protein in either single- or double-tagged form was enzymatic active. Using the ManNAc (40 mM) as the substrate, the enzyme activities for purified single- and double-tagged GlcNAc 2-epimerase were determined to be 8.7 and 11.54 U/mg, respectively. These two fusion proteins respectively showed Km values of 8.9 and 7.7 mM, both are close to those reported in the literature for GlcNAc 2-epimerase, suggesting that the addition of GST and 5D tags could marginally change the affinity of this enzyme for substrate. The optimal pH for the highest enzyme activity by either the single- or double-tagged fusion protein was determined to be 8.0. When the enzymatic activity was estimated at pH 7, the optimal temperature was 50°C. When the enzymatic activity was estimated at pH 7, the optimal temperature was 50°C. On the other hand, using N-acetyl-D-neuraminic acid (Neu5Ac) as the substrate, the purified fusion protein of Neu5Ac aldolase showed a specific activity of 3.8 U/mg and a Km value of 2.8 mM that is close to those reported in the literature for Neu5Ac aldolase, indicating that the addition of GST tag in the N terminus did not change the affinity of Neu5Ac aldolase for substrate. Both single- and double-tagged GlcNAc 2-epimerase were able to bound onto the surface of anionic ion-exchangers. The single-tagged fusion protein exhibited a better binding capacity, but the double-tagged fusion protein was better in the recovery of activity. When 0.04 g/L single-tagged GST-GlcNAc 2-epimerase were applied to the ManNAc solution for an isomeration for 12 h, a conversion of 38% could be achieved. The conversion could even approach to 77.8% by using 0.12 g/L double tagged GST-GlcNAc 2-epimerase-5D for an isomeration in 24 h. As the double tagged GST-GlcNAc 2-epimerase-5D which was immobilized on the anionic ion-exchanger (Q Sepharose) was used for the inter-conversion of ManAc and GlcNAc, a conversion of 57 % could be achieved in 35 h. Also, the single-tagged GST-Neu5Ac aldolase fusion protein, which was applied even in a very small dosage, could effectively catalyze the production of 2-keto-3-deoxy-D-glycero-D-galacto-nonopyranulosonic acid (KDN) from D-mannose and pyruvic acid. When the dosage of fusion protein increased from 0.035 to 0.06 g/L, the conversion increased from 10 to 20%. For the production of sialic acid (N-acetyl-D-neuraminic acid) in a single stage, single-tagged fusion proteins of GST-Neu5Ac aldolase and GST-GlcNAc 2-epimerase were immobilized on particles of PS-GMA containing bound GSH on the surface with a density of 73.01 ± 9.5 μmol GSH/g particles. Both immobilized fusion proteins were mixed together and added to the reaction mixture of GlcNAc and pyruvic acid for the production of Neu5Ac. Preliminary results indicate that an overall conversion of 7 % could be achieved. In the future, an ideal process including enzyme production, purification and immobilization for the production of sialic acids could be established based on the data obtained in the present work.