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.

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.

Enzymes are gaining increasing importance as catalysts for selective transformations in organic synthetic chemistry. The engineering and design of enzymes is a developing, growing research field that is employed in biocatalysis. In the present thesis, combinatorial protein engineering methods are applied for the development of Candida antarctica lipase A (CALA) variants with broader substrate scope and increased enantioselectivity. Initially, the structure of CALA was deduced by manual modelling and later the structure was established by X-ray crystallography. The elucidation of the structure of CALA revealed several biocatalytically interesting features. With the knowledge derived from the enzyme structure, enzyme variants were produced via iterative saturation mutagenesis (ISM), a powerful protein engineering approach. Several of these variants were highly active and enantioselective towards bulky esters. Furthermore, an extensively combinatorial protein engineering approach was developed and investigated. A CALA variant with a spacious substrate binding pocket that can accommodate an unusually bulky substrate, an ester derivate of the non-steroidal anti-inflammatory drug (S)-ibuprofen, was obtained with this approach. / At the time of the doctoral defence the following paper was unpublished and had a status as follows: Paper nr. 5: Manuscript

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>

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.

<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.</p><p>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.</p><p>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.</p>

The number of monoclonal antibodies approved for therapeutic use has increased rapidlyover the last decade. As a consequence, precise and robust kinetic characterization techniquesare crucial in order to select the best suitable candidates. A kinetic characterization methodwas developed in Gyrolab with automated sample transfers. The characterization wasperformed in solution in a mixing CD, containing an integrated nanoliter mixing chamberwith affinity binding columns. Association rate constants were determined for four anti-TSHantibodies with values ranging from 3x105 M-1s-1 to 10x105 M-1s-1. The antibodies wereranked according to kass. Reproducibility

Millions of years of evolution have resulted in an immense number of different proteins, which participate in virtually every process within cells and thus are of utmost importance for allknown forms of life. In addition, there are several examples of natural proteins which have found use in applications outside their natural environment, such as the use of enzymes infood industry and washing powders or the use of antibodies in diagnostic, bioseparation or therapeutic applications. To improve the performance of proteins in such applications, anumber of techniques, all collectively referred to as ‘protein engineering’, are performed in thelaboratory.Traditionally, methods involving ‘rational design’, where a few alterations are introduced atspecific protein locations to hopefully result in expected improvements have been applied.However, the use of more recent techniques involving a simultaneous construction of a large number of candidate variants (protein libraries) by various diversification principles, fromwhich rare clones showing enhanced properties can be isolated have contributed greatly to thefield of protein engineering.In the present thesis, different protein traits of biotechnological importance have beenaddressed for improvements by the use of such methods, in which there is a crucial need tomaintain a clonal link between the genotype and the phenotype to allow an identification of protein library members isolated by virtue of their functional properties. In all protein library investigations included in this thesis this coupling has been obtained by Escherichia coli bacterialcell-membrane compartmental confinement.In a first study, a combination of error prone PCR and gene-shuffling was applied to the Tobacco Etch Virus (TEV)-protease gene in order to produce collections from which genesencoding variants showing an enhanced soluble expression of the enzyme frequently used inbiotechnology to cleave fusion proteins were identified. Using Green Fluorescence Protein(GFP)-based cell fluorescence analysis, a clone with a five-fold increase in the yield of solubly produced protein was successfully isolated. In a second study, a novel and different GFPbased selection system, in addition also involving targeted in vivo protein degradation principles,was employed for investigations of the substrate sequence space of the same protease. In two additional studies, a selection system denoted Protein Fragment Complementation Assay(PCA), based on the affinity driven structural complementation of a genetically split β-lactamase enzyme was used to identify variants having desired target protein binding abilities,including both specificity and affinity. Using Darwinian principles concerning clonal growth advantages, affibody binding proteins showing sub-nanomolar dissociation constants to thehuman cytokine TNF-α were isolated. Taken together, these studies have shown that the bacterial format is very well suited for use in various aspects of protein library selection. / QC 20100729