| Summary: | 碩士 === 國立中正大學 === 化學工程研究所 === 92 === Abstract
Sialic acids are belonging to a family of nine carbon 2-keto-3-deoxy sugars, which are components of animal oligosaccharide and exist in viruses, bacteria, and many higher animals including human being. N-acetyl-D-neuraminic acid (Neu5Ac) is a main structure of the sialic acids; while 2-keto-3-deoxy-D-glycero-D-galacto-nonopyranulosonic acid (KDN) is the deaminated form of N-acetylneuraminic acid. Sialic acids can be used for building up our immunization, as inhibitors of influenza viruses, and immunoregulator and effector in tumor antimetastasis and anti-Alzheimer’s disease. On the other hand, KDN might also be used as a substitute of Neu5Ac, and can play an important role of selection antagonists and be used as an additive of the health food. For this reason, it’s a very worth for the study of KDN synthesis, based on the development in enzymatic reaction engineering.
In this study, the synthesis of KDN from the condensation of D-mannose with pyruvic acid using the fusion protein of N-acetylneuraminic acid aldolase (NANA aldolase) with a double tag designed for purification and immobilization as the biocatalyst. First, the fusion proteins of N-Acetylneuraminic acid aldolase were induced by IPTG to over-express in the recombinant bacteria E.coli BL21. The target proteins were purified and subject to study their enzymatic kinetics. Finally, fusion proteins were used for the enzymatic production of KDN.
In the culture of recombinant bacteria E.coli BL21 for the expression of fusion protein, the temperature of 28℃ was better than 37℃, because that the fusion protein can be broken when the cells were culture at 37℃. Based on the GST tag, an efficient and rapid purification strategy was applied for the isolation of fusion protein from the crude cell extracts by using a GST affinity column. After the affinity purification, two fusion proteins, GST-NANA aldolase-(Arg)5, and NANA aldolase-(Arg)5, were obtained with molecular weights of 59 & 33.2 kDa, respectively. In the study of the enzyme kinetics, we obtained the Km and Vmax values for fusion proteins to be 14 mM and 222 U/mg for GST-NANA aldolase-(Arg)5, and 2.4 mM and 203 U/mg for NANA aldolase-(Arg)5. The activities for both fusion proteins are higher than NANA aldolase.
Finally, in the production of KDN, experimental results suggested that using fusion protein (GST-NANA aldolase-(Arg)5 or NANA aldolase-(Arg)5) as a biocatalyst, the reaction for 5 hr could result in the same level of KDN production. The concentration of KDN in final reaction mixture concentration was about 16 ~ 18 mM, which was of commercially potential. Therefore, increasing the conversion of KDN production, creating a purification technology for KDN and so on would be of great important in the future.
Key words: KDN, sialic acid, N-acetyl-D-neuraminic acid aldolase, fusion protein, immobilization, protein purification, clone, affinity tag, large-scale production, E.coli BL21, enzymatic kinetics.
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