Production of nitrogen containing chemicals from cyanophycin

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. </p

Site-Selective Aliphatic C–H Chlorination Using N -Chloroamides Enables a Synthesis of Chlorolissoclimide

Methods for the practical, intermolecular functionalization of aliphatic C-H bonds remain a paramount goal of organic synthesis. Free radical alkane chlorination is an important industrial process for the production of small molecule chloroalkanes from simple hydrocarbons, yet applications to fine chemical synthesis are rare. Herein, we report a site-selective chlorination of aliphatic C-H bonds using readily available N-chloroamides and apply this transformation to a synthesis of chlorolissoclimide, a potently cytotoxic labdane diterpenoid. These reactions deliver alkyl chlorides in useful chemical yields with substrate as the limiting reagent. Notably, this approach tolerates substrate unsaturation that normally poses major challenges in chemoselective, aliphatic C-H functionalization. The sterically and electronically dictated site selectivities of the C-H chlorination are among the most selective alkane functionalizations known, providing a unique tool for chemical synthesis. The short synthesis of chlorolissoclimide features a high yielding, gram-scale radical C-H chlorination of sclareolide and a three-step/two-pot process for the introduction of the β-hydroxysuccinimide that is salient to all the lissoclimides and haterumaimides. Preliminary assays indicate that chlorolissoclimide and analogues are moderately active against aggressive melanoma and prostate cancer cell lines

Production of cyanophycin in Rhizopus oryzae through the expression of a cyanophycin synthetase encoding gene

Cyanophycin or cyanophycin granule peptide is a protein that results from non-ribosomal protein synthesis in microorganisms such as cyanobacteria. The amino acids in cyanophycin can be used as a feedstock in the production of a wide range of chemicals such as acrylonitrile, polyacrylic acid, 1,4-butanediamine, and urea. In this study, an auxotrophic mutant (Rhizopus oryzae M16) of the filamentous fungus R. oryzae 99-880 was selected to express cyanophycin synthetase encoding genes. These genes originated from Synechocystis sp. strain PCC6803, Anabaena sp. strain PCC7120, and a codon optimized version of latter gene. The genes were under control of the pyruvate decarboxylase promoter and terminator elements of R. oryzae. Transformants were generated by the biolistic transformation method. In only two transformants both expressing the cyanophycin synthetase encoding gene from Synechocystis sp. strain PCC6803 was a specific enzyme activity detected of 1.5 mU/mg protein. In one of these transformants was both water-soluble and insoluble cyanophycin detected. The water-soluble fraction formed the major fraction and accounted for 0.5% of the dry weight. The water-insoluble CGP was produced in trace amounts. The amino acid composition of the water-soluble form was determined and constitutes of equimolar amounts of arginine and aspartic acid

The bioinspired construction of an ordered carbon nitride array for photocatalytic mediated enzymatic reduction

A carbon nitride array (CNA) material has been constructed using a sacrificial diatom template. A regular carbon nitride nanorod array could be replicated from the periodic and regular nanochannel array of the template. The directional charge transport properties and high light harvesting capability of the CNA gives much better performance in splitting water to give hydrogen than its bulk counterpart. Furthermore, by combining with a rhodium complex as a mediator, the nicotinamide adenine dinucleotide (NADH) cofactor of many enzymes could be photocatalytically regenerated by the CNA. The rate of the in situ NADH regeneration is high enough to reverse the biological pathway of the three dehydrogenase enzymes, which then leads to the sustainable conversion of formaldehyde to methanol and also the reduction of carbon dioxide into methanol

Evolving biocatalysis to meet bioeconomy challenges and opportunities

4siThe unique selectivity of enzymes, along with their remarkable catalytic activity, constitute powerful tools for transforming renewable feedstock and also for adding value to an array of building blocks and monomers produced by the emerging bio-based chemistry sector. Although some relevant biotransformations run at the ton scale demonstrate the success of biocatalysis in industry, there is still a huge untapped potential of catalytic activities available for targeted valorization of new raw materials, such as waste streams and CO2. For decades, the needs of the pharmaceutical and fine chemistry sectors have driven scientific research in the field of biocatalysis. Nowadays, such consolidated advances have the potential to translate into effective innovation for the benefit of bio-based chemistry. However, the new scenario of bioeconomy requires a stringent integration between scientific advances and economics, and environmental as well as technological constraints. Computational methods and tools for effective big-data analysis are expected to boost the use of enzymes for the transformation of a new array of renewable feedstock and, ultimately, to enlarge the scope of biocatalysis.partially_openopenPellis, Alessandro; Cantone, Sara; Ebert, Cynthia; Gardossi, LuciaPellis, Alessandro; Cantone, Sara; Ebert, Cynthia; Gardossi, Luci

Photobiocatalysis: The Power of Combining Photocatalysis and Enzymes

Photobiocatalysts are constituted by a semiconductor with or without a light harvester that activates an enzyme. A logical source of inspiration for the development of photobiocatalysts has been natural photosynthetic centers. In photobiocatalysis, the coupling of the semiconductor and the enzyme frequently requires a natural cofactor and a relay transferring charge carriers from the semiconductor. The most widely studied photobiocatalysts so far make use of conduction band electrons of excited semiconductors to promote enzymatic reductions mediated by NAD(+)/NADH and an electron relay. The present review presents the state of the art in the field and has been organized based on the semiconductor and the reaction type including oxidations, hydrogen generation, and CO2 reduction. The possibility of direct enzyme activation by the semiconductor and the influence of the nature of mediator are also discussed as well as the use of mimics of the enzyme active center in combination with the semiconductor. The final section summarizes the state of the art of photobiocatalysis and comments on our view on future developments of the field.Financial support by the Spanish Ministry of Economy and Competitiveness (Severo Ochoa and CTQ2012-32315) is gratefully acknowledged. J.A.M.-A. acknowledges the assistance of the CSIC for the award of a Postdoctoral JAE-Doc contract co-financed by the European Social Fund.Maciá Agulló, JA.; Corma Canós, A.; García Gómez, H. (2015). Photobiocatalysis: The Power of Combining Photocatalysis and Enzymes. Chemistry - A European Journal. 21(31):10940-10959. https://doi.org/10.1002/chem.201406437S1094010959213

Production of nitrogen containing chemicals from cyanophycin

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