Addressing the low power generation of anodic biocatalysts is pertinent to the advancement of microbial fuel cell technology. While Propionibacterium freudenreichii ssp. shermanii has shown potential as a biocatalyst, its incomplete consumption of the anodic substrate is a persistent issue. This research aims to optimize substrate consumption to increase power generation using Propionibacterium freudenreichii ssp. shermanii as a biocatalyst. The effect of coculturing Geobacter sulfurreducens with Propionibacterium freudenreichii ssp. shermanii was investigated. The cocatalyst and pure culture performance was tested in an air-cathode microbial fuel cell. Geobacter sulfurreducens produced the highest maximum power density among the experimental cases. Power density produced by Propionibacterium freudenreichii ssp. shermanii was improved in the air-cathode design compared to previous experiments performed in an H-type design. The novel cocatalyst was shown to produce electricity, however a full characterization to elucidate the contribution to power generation by each microbe would be desirable to investigate.

Abstract The aim of this PhD-study was to address some of the overlapping bottlenecks in protein engineering and metagenomics by developing or applying new tools which are useful for both disciplines. Two enzymes were studied as an example: Triosephosphate Isomerase (TIM) and Uridine Phosphorylase (UP). TIM is an important enzyme of the glycolysis pathway and has been investigated via means of protein engineering, while UP is a key enzyme in the pyrimidine-salvage pathway. In this thesis TIM was used to address protein engineering aspects, while UP was used in regards to some metagenomic and bioinformatic aspects. The aspects of a structural driven rational design approach and its implications for further engineering of monomeric TIM variants are discussed. Process development based on a new technology, EnBase®, addresses the relative instability of new variants, compared to its ancestors, for further studies. EnBase® is then applied for the production of 15N isotope labeling of a monomeric TIM variant, A-TIM. Systematical function- and engineering studies on dimeric TIM and monomeric TIM in regards to the hinges of the catalytic loop-6 were conducted to investigate enzyme activity and stability. Both the A178L and P168A were proposed to induce loop-6 closure, a wanted feature for A-TIM variants. The P168A mutants are hardly active, but gave great insight into the catalytic machinery, while the A178L mutants did induce partial loop-6 closure, however in addition, monomeric A178L was destabilized. Homology driven genome mining and subsequent isolation- high throughput (HTP) overexpression of a thermostable UP from the Archaea Aeopyrum pernix was carried out as an example for the production of recombinant proteins. In addition an alternative kinetic method to study the kinetics of UP by means of NMR directly from cell lysate is discussed. The combination of expression libraries and EnBase® in a HTP manner may relieve up the gene-to-product bottleneck. The structural aspects of A. pernix UP are explored by means of simple bioinformatic tools in the last section of this thesis. A thermostable, truncated version of UP was created and its use for protein engineering in the future is explored. The long N-terminal and C-terminal ends of A. pernix UP seem to be involved in stabilizing the dimeric and hexameric structures of UP. However, deletion of the N-terminal end of A. pernix UP yielded a thermostable protein. Overall, the finding in regards to process optimization and HTP expression and optimization and the underlying methods used in the TIM studies and the UP studies are interchangeable.

Content: Application of biocatalyst becomes an imperative due to their eco-friendly advantages. Enzymes in pretanning for unhairing, fiber opening, defleshing and bating are well reported and practiced. However, the role of enzymes as a chemical aids is less explored and consider as a secondary applications. Leather enzymes are known for its hydrolytic behavior which makes it more suitable for pretanning operations. However, typical chemical exhaustive aids acts as a vehicle for the diffusion of chemicals, whereas enzymes aids in the splitting of fibers which facilitate the diffusion of chemicals and create more functional sites for the tanning and post tanning chemicals to interact. In this research, pickled pelts are treated with acid protease and subsequently tanned using chrome tanning agent. Enzymatic treated pelts resulted in better uptake of chromium as compared to conventionally processed leathers. Similarly, after neutralization, chrome tanned leathers are treated with alkaline protease to conventional post tanning has been carried out. Enzymatic treated wet blue leathers showed high uptake of post tanning chemical, uniform dyeing and reduction in the pollution load. From the preliminary research, an interesting finding has augmented that application of enzymes at an optimized concentration, temperature, pH and time would lead to better uptake of chrome which reduces the pollution and minimization pollution load in post tanning. This study, emphasize on the application of enzymes in tanning and post tanning for higher diffusion of chemicals. Take-Away: 1. Replacement of conventional exhaustive aids using biocatalyst 2. Higher exhaustion rate of tanning and post tanning chemicals 3. Futuristic technology for sustainable leather manufacture

11 Abstract Penicillin G acylases (PGAs) belong among enantioselective enzymes catalyzing a hydrolysis of stable amide bond in a broad spectrum of substrates, often having high application potential. PGAEc from Escherichia coli and PGAA from microorganism Achromobacter sp. CCM 4824 were used to catalyze enantioselective hydrolyses of seven selected N-phenylacetylated (N-PhAc) α/β-amino acid racemates. The PGAA showed higher stereoselectivity for three (S) enantiomers: N-PhAc-β-homoleucine, N-PhAc-α-tert- leucine and N-PhAc-β-leucine. We have constructed a homology model of PGAA that was used in molecular docking experiments with the same substrates. In-silico experiments reproduced the data from experimental enzymatic resolutions confirming validity of employed modeling protocol. We employed this protocol to evaluate enantiopreference of PGAA towards seven new substrates with application potential. For five of them, high enantioselectivity of PGAA was predicted for. PGAA was further studied in kinetically controlled syntheses of β-lactam antibiotics (SSBA). The PGAA was significantly more efficient at synthese of ampicillin and amoxicillin (higher S/H ratio and product accumulation) compared with PGAEc . Analogously to prediction of enantioselectivity of PGAA towards new substrates this protocol was applied...

Microbial electrosynthesis (MES) has recently emerged as a novel biotechnology platform for value-added product generation from waste CO2 stream. Integrating MES technology with renewable energy sources for both CO2 valorization and renewable energy storage is regarded as one type of artificial photosynthesis and a perfect example of circular bioeconomy. However, several challenges remain to be addressed to scale-up MES as a feasible process for chemical production, which include enhanced production rate, reduced energy consumption and excellent resistance to external fluctuations. To fill these knowledge gaps, different in-depth approaches were proposed in this dissertation by optimizing the cathode architecture, CO2 flow rates and utilizing efficient photoelectrode to improve MES performance and stability. A novel cathode design, made of conductive hollow fiber membrane, was developed in this dissertation to improve CO2 availability at MES cathode surface via direct CO2 delivery to chemolithoautotrophs through the pores in the hollow fibers. By modifying the hollow fiber surface with carbon nanotubes (CNTs), higher bioproduct formation was achieved with excellent faradaic efficiencies, which could be attributed to the improved surface area for bacterial adhesion and the reduction of cathodic electron transfer resistance. Since CO2 flow rate from industrial facilities typically varies over time, this hollow-fiber architecture was also applied to test the resistance of MES systems to CO2 flow rate fluctuation. Stepwise increase of CO2 flow rates from 0.3 ml/min to 10 ml/min was tested and the effect of CO2 flow rate fluctuations was evaluated in terms of biochemical generation and microbial community. MES was further integrated with renewable energy supply for both energy storage and CO2 transformation into biofuels and biochemicals. Stable MES photoanode, based on molybdenum-doped bismuth vanadate deposited on fluorine-doped tin oxide glass (FTO/BiVO4/Mo), was prepared for efficient solar energy harvesting and overpotential reduction for oxygen evolution reaction (OER), which contributed to one of the highest solar-to-biochemical conversion efficiencies ever reported for photo-assisted MES systems. The applied nature of this dissertation with fundamental insights is of great importance to bring MES one step closer to full-scale applications and enable MES technology to be economically more viable for renewable energy storage and CO2 valorization.

Self-assembly is a fundamental process in the field of nanotechnology, where molecules organize into complex structures spontaneously or induced by environmental factors. Peptides, short chains of amino acids, can self-assemble into many types of nanostructures. The self-assembly of peptides is governed by noncovalent interactions, including electrostatic interactions, hydrogen bonding, hydrophobic interactions, aromatic-aromatic interactions, and van der Waals forces. By varying the amino acid sequences and manipulating environmental parameters, these interactions can be modulated to obtain diverse supramolecular nanostructures, exhibiting a wide range of physical, chemical, and biological properties. Furthermore, the ability to control these properties opens up a world of possibilities in biomedical and biocatalytic applications. From drug delivery systems to enzyme mimics, as well as cancer treatments, the potential of these self-assembling peptides is vast and continues to be a vibrant area of research. Exploiting this potential, this dissertation delves into the design, synthesis, and investigation of self-assembling peptides for a range of applications. The introductory chapters of this document lay the groundwork, providing a comprehensive overview of self-assembly and its potential in biocatalytic and biomedical domains. The focus shifts in the later chapters to drug delivery applications, particularly in the delivery of hydrogen sulfide (H2S), and its implications in cardioprotection and cancer treatment. Finally, this document details an evaluation of self-assembled peptides in the context of biocatalysis using a combined experimental and computational approach. Chapter 3 discusses the design and synthesis of peptide-H2S donor conjugates (PHDCs) with an unusual adamantyl group. Several of PHDCs studied in this chapter self-assembled into novel nanocrescent structures observed under both conventional transmission microscopy (TEM) and cryogenic TEM (cryo-TEM). By varying the C-terminal amino acid with cationic, nonionic, or anionic amino acids, the PHDC morphologies remained unaffected, offering a robust peptide design for crescent-shaped supramolecular nanostructures. Chapter 4 discusses an extension of this project, introducing a cyclohexane in PHDCs instead of an adamantyl group. In this work, we designed and fabricated four constitutional isomeric PHDCs, which self-assembled into nanoribbons with different dimensions and large nanobelts. These morphologies exhibited varying cellular uptake and in vitro H2S release amounts, influencing their protective effects against oxidative stress induced by H2O2. With the knowledge of the impact of subtle changes in PHDC structures, Chapter 5 discusses our further design of three more PHDCs with the variation of side chain capping group, from an aromatic phenyl ring to a cyclohexane unit, to an aliphatic n-hexyl chain. In this chapter, we studied how changes in the hydrocarbon tail can influence the supramolecular nanostructures and their potential ability for colon cancer treatment. A final aspect of H2S delivery in Chapter 6 involves the creation of a stable PHDC with an extended H2S release profile. By integrating the H2S donor into a β-sheet forming peptide sequence with a Newkome-like poly(ethylene glycol) dendron, this PHDC self-assembles into spherical or fibril nanostructures with or without stirring. The H2S release was further studied by triggering release with various charged thiol molecules. Finally, another facet of this document focuses on three constitutional isomeric tetrapeptides containing a catalytic functional amino acid, His. Chapter 7 discusses these tetrapeptides, which self-assembled into nanocoils, nanotoroids, and nanoribbons based on the position of the His residue in the peptide sequence. Computational studies simulating the self-assembling process revealed the distribution of His residues and hydrophobic pockets, reminiscent of natural enzyme binding sites. A tight spatial distribution of His residues and hydrophobic pocket in nanocoils provided a picture for why this morphology exhibited the highest rate enhancement in catalyzing a model ester hydrolysis reaction. This study demonstrated how subtle molecular-level changes impact supramolecular nanostructures and catalytic efficiency. The final chapter details conclusions on all the research in this dissertation and discusses further directions of self-assembling peptides in the application of drug delivery and design of catalyst mimics. / Doctor of Philosophy / Self-assembly is a fascinating process in nanotechnology, where molecular building blocks come together to form complex structures. Peptides, which are short chains made up of amino acids, can play a crucial role in this process. They can organize themselves into various shapes due to different forces acting between their amino acid building blocks. By changing the arrangement of amino acids and adjusting the environment, scientists can create a wide range of nanoscale structures with unique properties from peptides. These self-assembling peptides have enormous potential in fields like medicine and catalysis. This dissertation describes how to design and make self-assembling peptides for various uses. Chapter 1 describes the general structure of the document, and Chapter 2 discusses the basics of self-assembly and how it can be applied in medicine and other areas. Chapters 3-6 focus on using self-assembling peptides to deliver hydrogen sulfide (H2S), a noxious gaseous molecule that is now recognized as a vital signaling molecule involved in various physiological processes. Several classes of peptide-H2S donor conjugates (PHDCs) are discussed in these chapters, including PHDCs that form nanoscale crescents, twisted ribbons, fibers, and other structures. These nanostructures show promise in protecting cells from harmful substances or can act as drugs in cancer treatment. We also investigate how different modifications affect their performance in biomedical applications. The final research chapter, Chapter 7, involves using self-assembling peptides as catalysts, molecules that speed up chemical reactions. By arranging the amino acids in different ways, peptides that form nanoscale coils, toroids, or ribbons-like structures were created. These different shapes influenced how well they catalyzed reactions. Computational modeling studies helped explain how small differences in molecular design led to big impacts on their catalytic abilities. The final chapter discloses conclusions on all the research in this dissertation and discusses the further directions of self-assembling peptides as medicines and catalysts.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23251号 / 農博第2458号 / 新制||農||1085(附属図書館) / 学位論文||R3||N5341(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 阪井 康能, 教授 小川 順, 教授 井上 善晴 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

La Chimie Verte s’engage entre autres à mettre au point des procédés plus respectueux de l’environnement et à émanciper de la pétrochimie les filières industrielles de fabrication de produits. Dans ce contexte, les enzymes représentent des alternatives de choix pour réaliser des réactions de synthèse de molécules écoresponsable à partir de la biomasse végétale. - L’endoglycocéramidase II de Rhodoccoccus sp. M-777, une glycoside-hydrolase, a été la cible d’un travail d'ingénierie du site actif afin de réorienter son activité hydrolytique vers la synthèse de polyglucosides d’alkyles, de potentiels biosurfactants. Une transglycosylase permettant d’atteindre des rendements de production de plus de 70% a été obtenue. La modélisation de la mutation permet de proposer des pistes sur les raisons de cette inversion du ratio hydrolyse/transglycosylation.- Une stratégie d'évolution dirigée a été appliquée à la féruloyle-estérase A d’Aspergillus niger pour la rendre plus résistante aux chocs thermiques et à la présence de solvants, deux propriétés requises pour utiliser cette enzyme pour des réactions de transfert dans des conditions thermodynamiquement favorables. Un catalogue d’enzymes améliorées, pour les deux propriétés, a été obtenu. L'accumulation de ces connaissances permettra de pouvoir plus efficacement rationaliser le design de biocatalyseur pour la synthèse de molécules, en accord avec les attentes de la chimie verte. / Green chemistry promotes the development of more environmentally friendly processes and the ending of polluting petrochemical industries by promoting the use of renewable resources. In this context, enzymes represent interesting alternatives catalysts for chemical transformations. Notably, they constitute tools of choice for synthesis of organic molecules from plant biomass.- Endoglycoceramidase II from Rhodococcus sp. M-777, a retaining glycoside hydrolase, was subjected to active-site remodelling in order to reorient its activity towards the synthesis of alkyl-polyglucosides, molecules with potential biosurfactant properties. Thus, an efficient transglycosylase able to reach production yield of more than 70% of alkyl-cellobiosides was obtained. A modelling study help to identify the determinants of this complete reversion of the transfer / hydrolysis ratio.- A directed evolution strategy was applied to Aspergillus niger feruloyl-esterase A, in order to make it more resistant to heat shocks and to the presence of solvents, two prerequisites to use this enzyme for transfer reactions under thermodynamically favourable conditions. This led to the establishment of a catalog of optimized enzymes for their thermostability, their solvent resistance, or both properties.These results will pave the way towards a more efficient way to rationally design biocatalysts meeting the expectations of green chemistry.

R-phenylacetylcarbinol (PAC) is used as a precursor for production of ephedrine and pseudoephedrine, which are anti-asthmatics and nasal decongestants. PAC is produced from benzaldehyde and pyruvate mediated by pyruvate decarboxylase (PDC). A strain of Rhizopus javanicus was evaluated for its production of PDC. The morphology of R. javanicus was influenced by the degree of aeration/agitation. A relatively high specific PDC activity (328 U decarboxylase g-1 mycelium) was achieved when aeration/agitation were reduced significantly in the latter stages of cultivation. The stability of partially purified PDC and crude extract from R. javanicus were evaluated by examining the enzyme deactivation kinetic in various conditions. R. javanicus PDC was less stable than Candida utilis PDC currently used in our group. A kinetic model for the deactivation of partially purified PDC extracted from C. utilis by benzaldehyde (0?00 mM) in 2.5 M MOPS buffer has been developed. An initial lag period prior to deactivation was found to occur, with first order dependencies of PDC deactivation on exposure time and on benzaldehyde concentration. A mathematical model for the enzymatic biotransformation of PAC and its associated by-products has been developed using a schematic method devised by King and Altman (1956) for deriving the rate equations. The rate equations for substrates, product and by-products have been derived from the patterns for yeast PDC and combined with a deactivation model for PDC from C. utilis. Initial rate and biotransformation studies were applied to refine and validate a mathematical model for PAC production. The rate of PAC formation was directly proportional to the enzyme activity level up to 5.0 U carboligase ml-1. Michaelis-Menten kinetics were determined for the effect of pyruvate concentration on the reaction rate. The effect of benzaldehyde on the rate of PAC production followed the sigmoidal shape of the Monod-Wyman-Changeux (MWC) model. The biotransformation model, which also included a term for PDC inactivation by benzaldehyde, was used to determine the overall rate constants for the formation of PAC, acetaldehyde and acetoin. Implementation of digital pH control for PAC production in a well-stirred organic-aqueous two-phase biotransformation system with 20 mM MOPS and 2.5 M dipropylene glycol (DPG) in aqueous phase resulted in similar level of PAC production [1.01 M (151 g l-1) in an organic phase and 115 mM (17.2 g l-1) in an aqueous phase after 47 h] to the system with a more expensive 2.5 M MOPS buffer.

This project was aimed to use a purple non-sulphur bacterium, Rhodopseudomonas palustris, as a biocatalyst for hydrogen production, from the waste of biodiesel manufacturing, crude glycerol. The goal of this project was to understand the fundamentals relevant to scaling up the process and developing an off the shelf product. The first objective was to determine the ability of R. palustris to generate hydrogen by non-growing cells in comparison to that by growing cells. Similar average hydrogen production rates and energy conversion were found for both processes but a significant difference in the hydrogen yield was observed. Hydrogen production reached ~ 80 % of the theoretical maximum hydrogen yield by non-growing R. palustris, about eight-fold of that reached by growing R. palustris. The high yield suggested that it is economically appealing to use non-growing R. palustris as the biocatalyst for continuous hydrogen production. To accomplish the proposed scale-up systems, understanding its product formation kinetics is the key. It was found that the hydrogen production rate is not growth-associated and depends solely on the dry cell mass with a non-growth associated coefficient of 2.52 (Leudeking–Piret model dP/dt=2.52 X). Light is vital for hydrogen production by non-growing R. palustris, in terms of light intensity and wavelength range. It was found that excessive or insufficient light intensity may constrain the performance. Only photons of light with appropriate wavelengths can excite cytochrome bacteriochlorophyll complexes II in R. palustris to generate hydrogen. Among white LEDs, infrared LEDs, and incandescent light bulbs, at the same light intensity, infrared LEDs gave the best results in the H2 production rate and energy conversion by non-growing cells, 22.0 % ± 1.5 % higher than that with white LEDs and around 25-30 times of that by incandescent light bulbs. It was found that non-growing R. palustris can be immobilised in alginate beads to give similar H2 production rates as that by cells suspended in media. This preliminary result pointed the direction of developing an off the shelf product of immobilised non-growing R. palustris as a biocatalyst for continuous hydrogen production.