Finding alternatives to petroleum-based energy sources is of interest since it could reduce the emissions of net carbon dioxide to the atmosphere by increasing the usage of renewable energy sources. To do so improvements are needed in the renewable energy production sector. Biogas production is of interest since the anaerobic digestion process can degrade many different biomolecules and is, contrary to e.g. bioethanol and biodiesel, not dependent on specific molecules. Thus, many wastes such as slaughterhouse waste, sludge from waste water treatment and lignocellulose residual material etc. can be used as substrates for biogas production. However, there are limitations in the degradation process depending on the composition of the selected substrate. To overcome these limitations such as inhibition of different microorganisms, or recalcitrant substrate, different methods can be used to increase the biogas production.  In this study different substrates were selected and analyzed/treated for remedies of early stage rate limiting problems of the anaerobic digestion process. Different analyzes and techniques were selected depending on the limitations correlated to the main problematic component of the specific substrate.  Improvements could be reached for the degradation of slaughterhouse waste by augmentation with the clay mineral zeolite. Addition of different enzymes to the substrate environment of different waste water treatment plant sludges resulted in limited life time of the selected enzymes. However, certain enzymes proved to be promising candidates with an effect of increased biogas production rate and yield for the time that the enzyme remained active. In an additional experiment, cellulolytic enzymes, naturally produced by a biogas producing microbial community, were induced, collected and added to a biogas experiment of ensiled forage ley, by which it was shown that these cellulases led to an increase in biogas production rate and yield. Thus, the studies demonstrate different techniques for improving the anaerobic digestion process of different types of substrates. / <p>Handledare saknas</p>

No description available.

Biokatalys medför ett alternativt tillvägagångsätt för att kunna utforska och utveckla kemiskt syntetiserade vägar för produktionen av eftertraktade kemikalier, där hållbarhet och miljön tas till beaktan. I denna studie undersöktes potentialen av en enzymatisk kaskad för produktion av 5-hydroximetylfurfurylamin (HMFA). HMFA är en förening med tillämpningar inom flera industrier som till exempel jordbruks- och läkemedelsindustrin. Den enzymatiska kaskaden består av två reaktioner, varav den första involverar dekarboxylering av lysin med användning av lysindekarboxylas för att producera en så kallad ”smart amindonator” kadaverin. Den andra reaktionen i kaskaden består utav ett transaminas från Silicibacter pomeroyi (SpTA) som konverterarar 5-hydroximetylfurfuryl (HMF) till HMFA med hjälp av det framkallade kadaverinet från den första reaktionen i kaskaden. En enzymatisk kaskad tillåter mildare reaktionsbetingelser, mindre avfall och energisnål användning som därmed minskar miljöpåverkan samtidigt som det beaktar några av dem 12 principerna av grön kemi. Det uppstod utmaningar som hindrade slutförandet av den enzymatiska kaskaden, men trots detta erhölls värdefulla insikter. Denna studie belyser de invecklade reaktionsmekanismerna och några av de svårigheterna med immobilisering av enzym på EziG bärare. Trots att den avsedda kaskaden inte slutfördes, gav lärdomarna nya perspektiv samt potentiella områden att fortsätta undersöka för framtida framsteg inom biokatalys. / Biocatalysis is a promising alternative to chemical synthesis routes for high value chemicals which considers the sustainability and environmental aspect. In this study the feasibility of utilizing an enzymatic cascade for the production of 5-hydroxymethylfurfurylamine (HMFA) was explored. HMFA is a compound with diverse applications in industries such as agriculture and pharmaceuticals. The cascade consists of two main reactions, the first of which involves the decarboxylation of lysine using a lysine decarboxylase to produce cadaverine. The cadaverine produced will then be utilized as an amine donor in the second reaction, which involves the use of a transaminase derived from Silicibacter pomeroyi (SpTA) together with 5-hydroxymethylfurfural (HMF). This cascade considers the principals of green chemistry such as milder reaction conditions and less waste, hence aiming to reduce the environmental impact. Although there were challenges preventing the completion of the enzymatic cascade, valuable insights were gained. The contribution of this study sheds light on the intricate reaction mechanisms and some of the key difficulties with enzyme immobilisation. While the intended cascade was not finalized, the lessons learned will provide for new perspectives and potential future advancements in biocatalysis.

Den nära förestående bristen på fossila resurser i kombination med deras associerade miljöfarlighet betonar behovet av utveckling av alternativa, mer hållbara kemikalier. I denna studie utvecklades en enzymatisk kaskad för förädling av 5-(hydroxymetyl)furfural (HMF) till 5-(aminometyl)-2-furfuraldehyd (AMFA). Kaskaden omfattar transaminering av HMF till 5-(hydroxymetyl)furfurylamin (HMFA) följt av oxidation av HMFA till AMFA. Transaminas från Silicibacter pomeroyi (SpATA) immobiliserades via his6-taggar på EziG-proteinbärare från EnginZyme AB. Proteinbärarna placerades i spin-kolonner under transamineringen vilket möjliggjorde omhändertagande av SpATA efter transamineringen av HMF. För oxidationen utvärderades alkoholdyhydrogenas från Thermoanaerobacter brockii och hästlever samt galaktosoxidas från Dactylium dendroides (GOase). Omsättning och produktbildning analyserades med HPLC. Resultaten indikerar att SpATA effektivt katalyserar transamineringen av HMF, att alkohol dehydrogenasen inte förmår katalysera oxidationen av HMF till HMFA och att galaktosoxidaset kan oxidera HMFA med hög omsättning vilket leder oss att tro att den föreslagna kaskaden för förädling av HMF till AMFA är möjlig. / The imminent shortage of fossil resources coupled with their associated environmental hazards stresses the need for the development of alternative, more sustainable chemicals. In this study an enzymatic cascade was developed for the valorisation of 5-(hydroxymethyl)furfural (HMF) into 5-(aminomethyl)-2-furfuraldehyde (AMFA). The cascade involves the transamination of HMF into 5-(hydroxymethyl)furfurylamine (HMFA) followed by the oxidation of HMFA into AMFA. Transaminases from Silicibacter pomeroyi (SpATA) was immobilised via his6-tags onto EziG-protein carriers from EnginZyme AB. The protein carriers were placed in spin-columns during the transamination which allowed for salvaging of the SpATA after the transamination of HMF. For the oxidation, alcohol dehydrogenases from Thermoanaerobacter brockii and horse liver as well as galactose oxidase from Dactylium dendroides (GOase) were evaluated. The conversion and product formation were analysed by HPLC. The results indicate that the SpATA efficiently catalyses the transamination of HMF, that the alcohol dehydrogenases are not able to catalyse the oxidation of HMF nor HMFA and that the GOase can oxidize HMFA with high conversion which leads us to believe that the proposed cascade for the valorisation of HMF to AMFA is feasible.

Användningen av biokatalysatorer, särskilt enzymer, inom kemikalie- och läkemedelsindustrin erbjuder betydande fördelar jämfört med de traditionella kemo-katalytiska metoderna som historiskt har dominerat industrin. En viktig klass av enzymer, transaminaser, spelar en central roll i tillverkningen av kirala aminer, som utgör grundläggande byggstenar i dessa industriella sektorer. Denna studie är inriktad på ett specifikt amin transaminas från Silicibacter pomeroyi. Tidigare har detta enzym visat förmåga att katalysera en mängd olika reaktioner för produktion av kirala aminer, men för att realisera dess fulla potential inom industriella tillämpningar krävs förbättrad stabilitet vid högre temperaturer. I motsats till de vanligt förekommande metoderna för proteinteknik, såsom rationell design och riktad evolution, används i denna studie släktsekvensrekonstruktion för att skapa mer temperaturtåliga varianter av detta enzym. Tidigare användning av denna metod har visat sig kunna generera proteiner med högre temperaturtålighet. Genom denna metod, där förfäder till detta enzym återskapas utifrån bevarade sekvenser, förväntas generering av varianter som kan bibehålla sin funktion vid högre temperaturer under en längre tid. Genom att utforska denna alternativa strategi för proteinteknik strävar studien efter att ge mer robusta biokatalysatorer för industriella tillämpningar. Utfallet från denna studie visade att två förfäder hade ökad termostabilitet. Detta visade sig dels genom analys av T5015 som påvisade en 3.9 och 6 C° förbättring för respektive förfader. Vidare påvisade t1/2 mätningar att dessa enzymer kunde utstå 2.06 till 3.72 gånger så lång tid vid 55 C° innan de inaktiverades. De påvisade dock lägre specifik aktivitet vid rumstemperatur, där en bidragande faktor till detta var att enbart en liten fraktion av förfäderna är korrekt veckade. Detta visar att det är nödvändigt med fortsatta förbättringar och fortsatta studier kring substratacceptans och stabilitet i olika lösningsmedel. Sammanfattningsvis påvisar resultaten att släktsekvensrekonstruktion är en proteinteknik som fungerar för att skapa proteiner med ökad termostabilitet och bör ses som ett mer självklart alternativ till riktad evolution och rationell design. / The utilisation of biocatalysts, particularly enzymes, in chemical and pharmaceutical industries presents significant advantages over the traditional chemocatalytic methods that historically dominated the industry. A crucial class of enzymes, transaminases, play a central role in the production of chiral amines, fundamental building blocks in these industrial sectors. This study focuses on a specific amine transaminase from Silicibacter pomeroyi. While this enzyme has previously demonstrated the ability to catalyse a variety of reactions for chiral amine production, realising its full potential in industrial applications requires enhanced stability at higher temperatures. In contrast to commonly employed protein engineering methods such as rational design and directed evolution, this study utilises ancestral sequence reconstruction to generate more temperature-resistant variants of this enzyme. Previous applications of this method have shown promising results in generating proteins with increased thermal stability. Through this approach, wherein ancestors of this enzyme are recreated from extant sequences, it is expected that variants capable of maintaining function at higher temperatures will be produced. By exploring this alternative strategy for protein engineering, the study aims to provide more robust biocatalysts for industrial applications. The outcome of this study is that two ancestors exhibited increased thermostability. This was evidenced by the analysis of T5015, which showed an improvement of 3.9 and 6 °C for each respective ancestor. Furthermore, t1/2 measurements indicated that they remained active for 2.06 to 3.72 fold longer at 55 °C before becoming inactive. However, they exhibited lower specific activity at room temperature, partially due to only a small fraction of the ancestral protein samples being properly folded. This suggests further improvements and continued investigations into substrate acceptance and stability in different solvents are required. In conclusion, this study demonstrates that ancestral sequence reconstruction is a protein engineering technique effective in enhancing protein thermostability and should be considered a more viable alternative to directed evolution and rational design.

Conjugates of soybean trypsin inhibitor (SBTI) and potato serine protease inhibitor (PSPI) immobilized on metal oxide particles of ~100nm diameter were prepared. Inhibition of trypsin hydrolysis of BAPA by these conjugates was measured and enzyme kinetics constants kcat, KM, kcat/KM and ki were determined. Metal oxide particles presented an inhibitory effect similar to that of a competitive inhibitor, noticed through the increase value of the K M constant. Furthermore, PSPI conjugates had the highest inhibition of trypsin, illustrated by the significantly higher value of KM relative to the value for particles only.

This thesis describes the rational design of a novel enzyme, a thiolester hydrolase, derived from human glutathione transferase (GST) A1-1 by the introduction of a single histidine residue. The first section of the thesis describes the design and the determination of the reaction mechanism. The design was based on the crystal structure of human GST A1-1 complexed with S-benzylglutathione. The resulting enzyme, A216H, catalyzed the hydrolysis of the non-natural substrate GSB, a thiolester of glutathione and benzoic acid. The reaction followed saturation kinetics with a kcat of 0.00078 min-1 and KM of 5 μM. The rate constant ratio, (kcat/KM)/kuncat, was found to be more than 107 M-1. The introduction of a single His residue in position 216 opened up a novel reaction pathway in human GST A1-1 and is a nice example of catalytic promiscuity. The substrate requirements were investigated and A216H was found to be selective since only two out of 18 GS-thiolesters tested were substrates for A216H. The reaction mechanism of the A216H-catalyzed hydrolysis of GSB was determined and found to proceed via an acyl intermediate at Y9. The hydrolysis was catalyzed by H216 that acts as a general base and the deacylation was found to be the rate-determining step. The Y9-intermediate could be selectively trapped by oxygen nucleophiles and primary alcohols, in particular 1-propanol and trifluoroethanol, were the most efficient. In addition, saturation kinetics was obtained in the acyl transfer reaction with 1-propanol indicating the presence of a second binding site in A216H. The second section of this thesis describes the site-specific covalent modification of human GST A1-1. The addition of GSB to the wild-type protein results in a site-specific benzoylation of only one tyrosine residue, Y9, out of ten present in the protein (one out of totally 51 nucleophiles). The reaction was tested with five GST classes (Alpha, Mu, Pi, Theta and Omega) and found to be specific for the Alpha class isoenzymes. The covalent modification reaction was further refined to target a single lysine residue, K216, providing a more stable linkage in the form of an amide bond. The reaction was found to be versatile and approximately 50% of the GS-thiolesters tested acylated K216, including a fluorophore. / <p>On the day of the public defence the status of article II was: Submitted and article IV was: In press.</p>

The search for alternatives to oil-based monomers has sparked interest for scientists to focus on the use of renewable resources for energy production, for the synthesis of polymeric materials and in other areas. With the use of renewable resources, scientists face new challenges to first isolate interesting molecules and then to process them. Enzymes are nature’s own powerful catalysts and display a variety of activities. They regulate important functions in life. They can also be used for chemical synthesis due to their efficiency, selectivity and mild reaction conditions. The selectivity of the enzyme allows specific reactions enabling the design of building blocks for polymers. In the work presented here, a lipase (Candida antarctica lipase B (CalB)) was used to produce building blocks for polymers. An efficient route was developed to selectively process epoxy-functional fatty acids into resins with a variety of functional groups (maleimide, oxetane, thiol, methacrylate). These oligoester structures, based on epoxy fatty acids from birch bark and vegetable oils, could be selectively cured to form thermosets with tailored properties. The specificity of an esterase with acyl transfer activity from Mycobacterium smegmatis (MsAcT) was altered by rational design. The produced variants increased the substrate scope and were then used to synthesize amides in water, where the wild type showed no conversion. A synthetic procedure was developed to form mixed dicarboxylic esters by selectively reacting only one side of divinyl adipate in order to introduce additional functional groups. / <p>QC 20170823</p>

Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose. Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns. Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.

Thiopurine S-methyltransferase (TPMT) belongs to the Class I S-adenosylmethionine-dependent methyltransferase (SAM-MT) super family of structurally related proteins. Common to the members of this large protein family is the catalysis of methylation reactions using S-adenosylmethionine (SAM) as a methyl group donor, although SAM-MTs act on a wide range of different substrates and carry out numerous biologically important functions. While the natural function of TPMT is unknown, this enzyme is involved in the metabolism of thiopurines, a class of pharmaceutical substances administered in treatment of immune-related disorders. Specifically, methylation by TPMT inactivates thiopurines and their metabolic intermediates, which reduces the efficacy of clinical treatment and increases the risk of adverse side effects. To further complicate matters, TPMT is a polymorphic enzyme with over 40 naturally occurring variants known to date, most of which exhibit lowered methylation activity towards thiopurines. Consequently, there are individual variations in TPMTmediated thiopurine inactivation, and the administered dose has to be adjusted prior to clinical treatment to avoid harmful side effects. Although the clinical relevance of TPMT is well established, few studies have investigated the molecular causes of the reduced methylation activity of variant proteins. In this thesis, the results of biophysical characterization of two variant proteins, TPMT*6 (Y180F) and TPMT*8 (R215H), are presented. While the properties of TPMT*8 were indistinguishable from those of the wild-type protein, TPMT*6 was found to be somewhat destabilized. Interestingly, the TPMT*6 amino acid substitution did not affect the functionality or folding pattern of the variant protein. Therefore, the decreased in vivo functionality reported for TPMT*6 is probably caused by increased proteolytic degradation in response to the reduced stability of this protein variant, rather than loss of function. Also presented herein are novel methodological approaches for studies of TPMT and its variants. Firstly, the advantages of using 8-anilinonaphthalene-1-sulfonic acid (ANS) to probe TPMT tertiary structure and active site integrity are presented. ANS binds exclusively to the native state of TPMT with high affinity (KD ~ 0.2 μm) and a 1:1 ratio. The stability of TPMT was dramatically increased by binding of ANS, which was shown to co-localize with the structurally similar adenine moiety of the cofactor SAM. Secondly, an enzyme activity assay based on isothermal titration calorimetry (ITC) is presented. Using this approach, the kinetics of 6-MP and 6-TG methylation by TPMT has been characterized.