Currently nitrogen containing bulk chemicals are produced from naphtha. However, as explained in Chapter 1 it would be more energy efficient, less capital intensive and eventually more economical to start from functionalized compounds that already have nitrogen incorporated, such as amino acids. Recent developments have made it possible to specifically fix and thus isolate L-aspartic acid and L-arginine from agricultural waste streams in the form of the polypeptide cyanophycin (CGP). The aim of the research presented in this thesis was to explore and optimize the different (bio)conversion steps involved in the envisioned route from CGP towards nitrogen containing chemicals.
In Chapter 2 a study is presented on the acid and base catalyzed hydrolysis of CGP. Acid catalyzed hydrolysis of CGP yields both L-aspartic acid and L-arginine at comparable rates and is therefore suitable for complete hydrolysis. It was observed that upon base catalyzed hydrolysis of CGP the rate of L-arginine liberation is overall significantly higher than that of L-aspartic acid, which enables selective hydrolysis of CGP. Over time L-aspartic acid liberation and thus hydrolysis of the polyaspartic acid backbone of CGP will occur and therefore a choice needs to be made between the degree of polyaspartic acid functionality and the polymer length of the CGP residue.
The results of a study on the applicability of Escherichia coli L-aspartate α-decarboxylase (ADC) for the production of -alanine from L-aspartic acid are presented in Chapter 3. The α-decarboxylation of L-aspartic acid using ADC has several advantages, such as its high selectivity, ease of production, lack of product inhibition and high thermostability under storage conditions. In addition, covalent immobilization of ADC on Sepabeads EC-EP epoxy supports is straightforward and makes the enzyme slightly more stable. However, ADC’s low operational stability, probably caused by irreversible transamination of its catalytically essential pyruvoyl group, needs to be addressed before large scale applications become feasible.
The results of a study on stabilization and immobilization of Bacillus subtilis arginase are presented in Chapter 4. In view of its application in the hydrolysis of L-arginine to L-ornithine and urea, B. subtilis arginase was successfully stabilized and immobilized. Initial pH of the substrate solution, addition of aspartic acid and reducing agents all had an effect on the operational stability of B. subtilis arginase.
A remarkably good operational stability (total turnover number, TTN = 1.13•108) was observed at the pH of arginine free base (pH 11.0), which was further improved with the addition of sodium dithionite (TTN > 1•109). Furthermore, B. subtilis arginase was successfully immobilized on three commercially available epoxy-activated supports. Immobilization on Sepabeads EC-EP was most successful resulting in a recovered activity of 75% and enhanced thermostability.
In Chapter 5 a study on Trypanosoma brucei ornithine decarboxylase (TbODC) is described. The stabilization and immobilization of TbODC were investigated for its application in the conversion of L-ornithine to 1,4-diaminobutane. The stability of TbODC is substantially improved upon addition of dithiothreitol (DTT), which not only has a stabilizing, but also an activating effect. For optimal performance of TbODC, the pH should be controlled at pH 8 and the ionic strength should be kept
to a minimum. Furthermore, TbODC shows an optimum in productivity at 40°C with respect to its temperature dependent activity and stability. Although TbODC’s immobilization on Sepabeads EC-HFA leads to an almost threefold improvement in operational stability, additional research to improve the operational stability of TbODC is recommended.
The impact of the results described in the previous chapters on the overall route from CGP to nitrogen containing chemicals is discussed in Chapter 6. Of the three enzymes studied in this thesis only reuse of B. subtilis arginase by immobilization on epoxy supports would be feasible in terms of material costs. In view of the overall process design, three subjects for further study were identified: (i) minimization of pH adjustments, (ii) isolation and reuse of additives and (iii) recycling of process heat. In view of the biorefinery approach in general, investigation of additional methods to selectively isolate amino acids from complex biomass mixtures and methods to isolate amino compounds from aqueous environments is encouraged.
In conclusion, CGP appears to be a valuable molecule in the production of nitrogen containing chemicals from residual biomass streams. This thesis provides routes from CGP towards nitrogen containing chemicals, indicating the strengths of these routes and emphasizing where further optimization is required.