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Chemo-enzymatic synthesis of 2,5-furandicarboxylic acid from highly abundant aldaric acids

Bouwknegt, D. (2017) Chemo-enzymatic synthesis of 2,5-furandicarboxylic acid from highly abundant aldaric acids. Master's Thesis / Essay, Industrial Engineering and Management.

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This report documents research on the, glucarate dehydratase (GDH) catalyzed, conversion of d-glucarate into 5-keto-4-deoxy-d-glucarate (KDG). The research was performed in the interest of Syncom B.V., in collaboration with the University of Groningen. The GDH catalyzed conversion was proposed by Syncom B.V. as an alternative route for the production of FDCA, a valuable renewable chemical. Proof that active enzyme could be produced was the first step in developing an alternative route for FDCA. GDH was overexpressed in an E. coli BL21(DE3) culture. Based on SDS, GDH’s size is found to be 48kDa, a revision on earlier published work by (Aghaie et al., 2008). A detection method is developed to determine GDH’s activity on glucarate. A TLC method and a spectrophotometric method, both based on reaction with semicarbazide, were compared. The latter was selected for its higher potential and further developed for use in GDH activity assays. The spectrophotometric method was found convenient since, the method 1) showed high sensitivity, 2) has a high linear correlation between product concentration and absorbance, and 3) shows no disturbing interference of the other compounds present in the activity assays. From the activity assays it was found that the produced GDH is indeed active on the substrate d-glucarate. Active enzyme showed KDG formation over time, where inactivated enzyme did not produce KDG. A linear relation was found between initial substrate concentration and product formation, denoting that the initial substrate concentration is the limiting factor for the amount of product formed over time. The yields obtained where however below 30%, which is probably caused by the impurity of the applied enzyme solution. During current research, a patent was found which already describes the researched enzymatic conversion for use in FDCA production. Possible continuation of this research should therefore focus on application of the enzymatic conversion in production routes different than FDCA. An HPCL procedure is recommend to give further information on enzyme’s activity. Additional research will be needed on the commercial production of GDH in industrially applicable bacteria, which are in general different from E. coli, which is often used for experimental procedures.

Item Type: Thesis (Master's Thesis / Essay)
Degree programme: Industrial Engineering and Management
Thesis type: Master's Thesis / Essay
Language: English
Date Deposited: 15 Feb 2018 08:27
Last Modified: 15 Feb 2018 08:27

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