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  • 51.
    Håkansson Hederos, Sofia
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Catalysis and Site-Specific Modification of Glutathione Transferases Enabled by Rational Design2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    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.

  • 52.
    Johansson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Microscale measurement of kinetic binding properties of monoclonal antibodies in solution using Gyrolab2011Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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

  • 53.
    Johansson, Mikaela
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Metaproteogenomics-guided enzyme discovery: Targeted identification of novel proteases in microbial communities2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Industrial biotechnology is a large and growing industry as it is part of establishing a “greener” and more sustainable bioeconomy-based society. Using enzymes as biocatalysts is a viable alternative to chemicals and energy intense industrial processes and is en route to a more sustainable industry. Enzymes have been used in different areas for ages and are today used in many industrial processes such as biofuels production, food industry, tanning, chemical synthesis, pharmaceuticals etc. Enzymes are today a billion-dollar industry in itself and the demand for novel catalysts for various present and future processes of renewable resources are high and perfectly in line with converting to a more sustainable society.

    Most enzymes used in industry today have been identified from isolated and pure cultured microorganisms with identified desirable traits and enzymatic capacities. However, it is known that less than 1% of all microorganisms can be can be obtained in pure cultures. Thus, if we were to rely solely on pure culturing, this would leave the 99% of the microorganisms that constitutes the “microbial dark matter” uninvestigated for their potential in coding for and producing valuable novel enzymes. Therefore, to investigate these “unculturable” microorganisms for novel and valuable enzymes, pure-culture independent methods are needed.

    During the last two decades there has been a fast and extensive development in techniques and methods applicable for this purpose. Especially important has been the advancements made in mass spectrometry for protein identification and next generation sequencing of DNA. With these technical developments new research fields of proteomics and genomics have been developed, by which the complete protein complement of cells (the proteome) and all genes (the genome) of organisms can be investigated. When these techniques are applied to microbial communities these fields of research are known as meta-proteomics and meta-genomics.

    However, when applied to complex microbial communities, difficulties different from those encountered in their original usage for analysis of single multicellular organisms or cell linages arises, and when used independently both methods have their own limitations and bottlenecks. In addition, both metaproteomics and metagenomics are largely non-targeting techniques. Thus, if the purpose is still to - somewhat contradictory – use these non-targeting methods for targeted identification of novel enzymes with certain desired activities and properties from within microbial communities, special measures need to be taken.

    The work presented in this thesis describes the development of a method that combines

    metaproteomics and metagenomics (i.e. metaproteogenomics) for the targeted discovery of novel enzymes with desired activities, and their correct coding genes, from within microbial communities. Thus, what is described is a method that can be used to circumvent the pure-culturing problem so that a much larger fraction of the microbial dark matter can be specifically investigated for the identification of novel valuable enzymes.

  • 54.
    Jönsson, Leif J.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Alriksson, Björn
    Nilvebrant, Nils-Olof
    Bioconversion of lignocellulose: inhibitors and detoxification2013In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 6, article id 16Article in journal (Refereed)
    Abstract [en]

    Bioconversion of lignocellulose by microbial fermentation is typically preceded by an acidic thermochemical pretreatment step designed to facilitate enzymatic hydrolysis of cellulose. Substances formed during the pretreatment of the lignocellulosic feedstock inhibit enzymatic hydrolysis as well as microbial fermentation steps. This review focuses on inhibitors from lignocellulosic feedstocks and how conditioning of slurries and hydrolysates can be used to alleviate inhibition problems. Novel developments in the area include chemical in-situ detoxification by using reducing agents, and methods that improve the performance of both enzymatic and microbial biocatalysts.

  • 55. Kang, Min-Kyoung
    et al.
    Zhou, Yongjin J.
    Buijs, Nicolaas A.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Functional screening of aldehyde decarbonylases for long-chain alkane production by Saccharomyces cerevisiae2017In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 16, article id 74Article in journal (Refereed)
    Abstract [en]

    Background: Low catalytic activities of pathway enzymes are often a limitation when using microbial based chemical production. Recent studies indicated that the enzyme activity of aldehyde decarbonylase (AD) is a critical bottleneck for alkane biosynthesis in Saccharomyces cerevisiae. We therefore performed functional screening to identify efficient ADs that can improve alkane production by S. cerevisiae. Results: A comparative study of ADs originated from a plant, insects, and cyanobacteria were conducted in S. cerevisiae. As a result, expression of aldehyde deformylating oxygenases (ADOs), which are cyanobacterial ADs, from Synechococcus elongatus and Crocosphaera watsonii converted fatty aldehydes to corresponding Cn-1 alkanes and alkenes. The CwADO showed the highest alkane titer (0.13 mg/L/OD600) and the lowest fatty alcohol production (0.55 mg/L/OD600). However, no measurable alkanes and alkenes were detected in other AD expressed yeast strains. Dynamic expression of SeADO and CwADO under GAL promoters increased alkane production to 0.20 mg/L/OD600 and no fatty alcohols, with even number chain lengths from C8 to C14, were detected in the cells. Conclusions: We demonstrated in vivo enzyme activities of ADs by displaying profiles of alkanes and fatty alcohols in S. cerevisiae. Among the AD enzymes evaluated, cyanobacteria ADOs were found to be suitable for alkane biosynthesis in S. cerevisiae. This work will be helpful to decide an AD candidate for alkane biosynthesis in S. cerevisiae and it will provide useful information for further investigation of AD enzymes with improved activities.

  • 56. Kocík, Jaroslav
    et al.
    Samikannu, Ajaikumar
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bourajoini, Hasna
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pham, Tung Ngoc
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Mikkola, Jyri-Pekka
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Faculty of Science and Engineering, Industrial Chemistry & Reaction Engineering, Johan Gadolin Process Chemistry Centre, Åbo Akademi University, Åbo-Turku, Finland.
    Hájek, Martin
    Čapek, Libor
    Screening of active solid catalysts for esterification of tall oil fatty acids with methanol2017In: Journal of Cleaner Production, ISSN 0959-6526, E-ISSN 1879-1786, Vol. 155, no 1, p. 34-38Article in journal (Refereed)
    Abstract [en]

    The paper is focused on the description of the activity/selectivity of mesoporous silica based materials loaded with various types of active species in the esterification of tall oil free fatty acids. The metals such as aluminium, molybdenum, gallium and zinc, including their combinations were impregnated on the mesoporous silica, which was tested in esterification reaction. All these catalysts preserved its tall oil free fatty conversion in the first and the second catalytic cycles. However, while only insignificant amount of gallium or molybdenum was lost from the solid catalyst into the liquid phases, zinc leached from every studied solid catalyst. In contrast to impregnated gallium on mesoporous silica, which exhibited higher acidity and higher tall oil free fatty acids conversion in the first catalytic cycle, but its value was not preserved in the second catalytic test.

  • 57.
    Kurtovic, Sanela
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Modén, Olof
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Shokeer, Abeer
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Structural determinants of glutathione transferases with azathioprine activity identified by DNA shuffling of alpha class members2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 375, no 5, p. 1365-1379Article in journal (Refereed)
    Abstract [en]

    A library of alpha class glutathione transferases (GSTs), composed of chimeric enzymes derived from human (A1-1, A2-2 and A3-3), bovine (A1-1) and rat (A2-2 and A3-3) cDNA sequences was constructed by the method of DNA shuffling. The GST variants were screened in bacterial lysates for activity with the immunosuppressive agent azathioprine, a prodrug that is transformed into its active form, 6-mercaptopurine, by reaction with the tripeptide glutathione catalyzed by GSTs. Important structural determinants for activity with azathioprine were recognized by means of primary structure analysis and activities of purified enzymes chosen from the screening. The amino acid sequences could be divided into 23 exchangeable segments on the basis of the primary structures of 45 chosen clones. Segments 2, 20, 21, and 22 were identified as primary determinants of the azathioprine activity representing two of the regions forming the substrate-binding H-site. Segments 21 and 22 are situated in the C-terminal helix characterizing alpha class GSTs, which is instrumental in their catalytic function. The study demonstrates the power of DNA shuffling in identifying segments of primary structure that are important for catalytic activity with a targeted substrate. GSTs in combination with azathioprine have potential as selectable markers for use in gene therapy. Knowledge of activity-determining segments in the structure is valuable in the protein engineering of glutathione transferase for enhanced or suppressed activity.

  • 58.
    Kürten, Charlotte
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    On Catalytic Mechanisms for Rational Enzyme Design Strategies2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Enzymes enable life by promoting chemical reactions that govern the metabolism of all living organisms. As green catalysts, they have been extensively used in industry. However, to reach their full potential, engineering is often required, which can benefit from a detailed understanding of the underlying reaction mechanism.

    In Paper I, we screened for an esterase with promiscuous amidase activity capitalizing on a key hydrogen bond acceptor that is able to stabilize the rate limiting nitrogen inversion. In silicoanalyses revealed the esterase patatin as promising target that indeed catalyzed amide hydrolysis when tested in vitro. While key transition state stabilizers for amide hydrolysis are known, we were interested in increasing our fundamental understanding of terpene cyclase catalysis (Paper II-V). In Paper II, kinetic studies in D2O-enriched buffers using a soluble diterpene cyclase suggested that hydrogen tunneling is part of the rate-limiting protonation step. In Paper III, we performed intense computational analyses on a bacterial triterpene cyclase to show the influence of water flow on catalysis. Water movement in the active site and in specific water channels, influencing transition state formation, was detected using streamline analysis. In Paper IV and V, we focused on the human membrane-bound triterpene cyclase oxidosqualene cyclase. We first established a bacterial expression and purification protocol in Paper IV, before performing detailed in vitroand in silicoanalyses in Paper V. Our analyses showed an entropy-driven reaction mechanism and the existence of a tunnel network in the structure of the human enzyme. The influence of water network rearrangements on the thermodynamics of the transition state formation were confirmed. Introducing mutations in the tunnel lining residues severely affected the temperature dependence of the reaction by changing the water flow and network rearrangements in the tunnels and concomitant the active site.

  • 59.
    Kürten, Charlotte
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Eriksson, Adam
    Maddalo, Gianluca
    Edfors, Fredrik
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Syrén, Per-Olof
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Engineering of water networks in class II terpene cyclases underscores the importance of amino acid hydration and entropy in biocatalysis and enzyme designManuscript (preprint) (Other academic)
  • 60.
    Land, Henrik
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Hendil-Forssell, Peter
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    One-pot biocatalytic amine transaminase/acyl transferase cascade for aqueous formation of amides from aldehydes or ketones2016In: catalysis science & technology, ISSN 2044-4753, Vol. 6, p. 2897-2900Article in journal (Refereed)
    Abstract [en]

    An efficient one-pot one-step biocatalytic amine transaminase/acyl transferase cascade for the formation of amides from the corresponding aldehydes and ketones in aqueous solution has been developed. N-benzyl-2-methoxyacetamide has been synthesized utlilizing the developed cascade in conversions up to 97%. The cascade was also evaluated for the synthesis of chiral amides.

  • 61.
    Larsson, Caroline
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Bacterial Sortase A as a drug target2012Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Sortase A is a housekeeping enzyme of Gram-positive bacteria that catalyses the anchoring of surface proteins to the bacterial peptidoglycan. The enzyme works to establish an interaction between bacteria and host cells and is essential for pathogenesis. This makes Sortase A a potential suitable target for inhibition, in order to treat bacterial infections.

    In this degree project Sortase A from Staphylococcus aureus was explored and potential inhibitors were investigated by performing enzyme activity and bacterial binding assays. A robust FRET assay was developed and optimized for a recombinant version of the enzyme and serves as a good starting point for studying inhibition.

  • 62.
    Löfdahl, Per-Åke
    KTH, School of Engineering Sciences (SCI), Physics.
    Improved solubility of TEV protease by directed evolution2006In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 121, no 3, p. 291-298Article in journal (Refereed)
    Abstract [en]

    The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV–GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEVSH, in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.

  • 63.
    Löfdahl, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    On bacterial formats in protein library technology2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    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.

  • 64.
    Löfdahl, Per-Åke
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Nord, Olof
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Janzon, Lars
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Selection of TNF-alpha binding affibody molecules using a beta-lactamase protein fragment complementation assay2009In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 26, no 5, p. 251-259Article in journal (Refereed)
    Abstract [en]

    Protein fragment complementation assays (PCAs) based on different reporter proteins have been described as powerful tools for monitoring dynamic protein-protein interactions in living cells. The present study describes the construction of a PCA system based on genetic splitting of TEM-1 beta-lactamase for the selection of proteins specifically interacting in the periplasm of Escherichia coli bacterial cells, and its application for the selection of affibody molecules binding human tumour necrosis factor-alpha (TNF-alpha) from a combinatorial library. Vectors encoding individual members of a naive 10(9) affibody protein library fused to a C-terminal fragment of the beta-lactamase reporter were distributed via phage infection to a culture of cells harbouring a common construct encoding a fusion protein between a non-membrane anchored version of a human TNF-alpha target and the N-terminal segment of the reporter. An initial binding analysis of 29 library variants derived from surviving colonies using selection plates containing ampicillin and in some cases also the P-lactamase inhibitor tazobactam, indicated a stringent selection for target binding variants. Subsequent analyses showed that the binding affinities (K(D)) for three selected variants studied in more detail were in the range 14-27 nm. The selectivity in binding to TNF-alpha for these variants was further demonstrated in both a cross-target PCA-based challenge and the specific detection of a low nm concentration of TNF-alpha spiked into a complex cell lysate sample. Further, in a biosensor-based competition assay, the binding to TNF-alpha of three investigated affibody variants could be completely blocked by premixing the target with the therapeutic monoclonal antibody adalimumab (Humira (R)), indicating overlapping epitopes between the two classes of reagents. The data indicate that beta-lactamase PICA is a promising methodology for stringent selection of binders from complex naive libraries to yield high affinity reagents with selective target binding characteristics.

  • 65.
    Löfdahl, Per-Åke
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Affinity maturation of a TNF-α binding affibodymolecule by Darwinian survival selection2010In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 55, p. 111-120Article in journal (Refereed)
    Abstract [en]

    The introduction of different methodologies for construction and screening ofcomplex protein libraries has provided powerful means in protein engineeringfor development of molecules with desired traits. A challenge faced in manysituations is to adapt a given methodology for efficient and rapid identification ofthe most interesting variants present in a library. In the present study, theconcept of Darwinian selection based on a growth advantage for clones havingthe desired trait has been investigated. Using a β-lactamase-based ProteinFragment Complementation Assay (PCA), an affinity maturation of a TNF-αbinding affibody molecule of an initial 2 nM affinity for the target has beenperformed. Initial characterization of the PCA system, based on the affinitydriven reconstitution of β-lactamase activity in the periplasm of cells harbouringa library member showing affinity for a co-expressed target protein, showed thatthe system was responsive to promoter induction level, interaction affinity andapplied selection pressure. Using combinatorial protein engineering principles, a107 library of second generation affibody molecules was constructed andsubjected to selection of improved variants by library growth in liquid culture.The results showed that after a pre-selection step on semi-solid media toeliminate non-binding variants, present in majority, two rounds of selection inliquid culture resulted in an enrichment for binders showing up ten-fold higheraffinity to the TNF-α target than the ancestral variant. Biosensor analysesshowed that the major factor for the improved affinity could be attributed toreduced off-rate constants.

  • 66. Löfgren, J.
    et al.
    Görbe, T.
    Oschmann, M.
    Svedendahl, Maria
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Bäckvall, J. -E
    Transesterification of a Tertiary Alcohol by Engineered Candida antarctica Lipase A2019In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633Article in journal (Refereed)
    Abstract [en]

    Tertiary alcohols are known to be challenging substrates for applications in asymmetric synthesis due to their complexity and steric hinderance. The occurrence of tertiary alcohols and their esters in nature indicates the presence of natural biocatalytic synthetic routes for their preparation. Lipase A from Candida antarctica (CalA) is a hydrolase that has previously been shown to catalyze the transesterification of racemic 2-phenylbut-3-yn-2-ol at a low rate. In this work, the activity of that enzyme was improved by protein engineering through a semi-rational design strategy. An enzyme library was created and screened for transesterification activity towards racemic 2-phenylbut-3-yn-2-ol in an organic solvent. One successful enzyme variant (L367G) showed a tenfold increased reaction rate compared to the wild-type enzyme, while maintaining a high enantioselectivity.

  • 67. Maciejewska, Barbara
    et al.
    Zrubek, Karol
    Espaillat, Akbar
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wisniewska, Magdalena
    Rembacz, Krzysztof P.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Dubin, Grzegorz
    Drulis-Kawa, Zuzanna
    Modular endolysin of Burkholderia AP3 phage has the largest lysozyme-like catalytic subunit discovered to date and no catalytic aspartate residue2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 14501Article in journal (Refereed)
    Abstract [en]

    Endolysins are peptidoglycan-degrading enzymes utilized by bacteriophages to release the progeny from bacterial cells. The lytic properties of phage endolysins make them potential antibacterial agents for medical and industrial applications. Here, we present a comprehensive characterization of phage AP3 modular endolysin (AP3gp15) containing cell wall binding domain and an enzymatic domain (DUF3380 by BLASTP), both widespread and conservative. Our structural analysis demonstrates the low similarity of an enzymatic domain to known lysozymes and an unusual catalytic centre characterized by only a single glutamic acid residue and no aspartic acid. Thus, our findings suggest distinguishing a novel class of muralytic enzymes having the activity and catalytic centre organization of DUF3380. The lack of amino acid sequence homology between AP3gp15 and other known muralytic enzymes may reflect the evolutionary convergence of analogous glycosidases. Moreover, the broad antibacterial spectrum, lack of cytotoxic effect on human cells and the stability characteristics of AP3 endolysin advocate for its future application development.

  • 68. Makes, Frantisek
    et al.
    Brunskog, Maria
    Gotland University, School of Culture, Energy and Environment.
    Enzymatic restoration and authentication of Guiseppe Arcimboldo’s “Vertumnus”2011Book (Other academic)
    Abstract [en]

    Court painter Guiseppe Arcimboldo is one of the most illustrious artists, and his allegoric portrait of the Emperor Rudolf II as the God Vertumnus is of world renown. This art work landed in the collection at Skokloster Castle, north of Stockholm, Sweden, after a series of dramatic events and under partly unclear circumstances. Due to its remarkable artistic qualities the painting is quite often temporarily exhibited at other venues in Sweden or abroad. Environmental changes in combination with the indoor climate at Skokloster have caused stress in the panel, and subsequent decay of the ground and the paint layers. This publication is on the restoration of the painting in 1988 and again in 2010 by enzymatic means. Enzymes also have been used for authentication of another painting attributed to Arcimboldo, with Vertumnus as reference, a hypothesis that could not be supported. Frantisek Makes has worked as conservator-scientist for Swedish museums, private collectors and stakeholders, and he has been guest lecturer at the Dept. for Integrated Conservation at Gotland University.

  • 69.
    Marx, Lisa
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Rios-Lombardia, Nicolas
    Farnberger, Judith F.
    Kroutil, Wolfgang
    Benitez-Mateos, Ana I.
    Lopez-Gallego, Fernando
    Moris, Francisco
    Gonzalez-Sabin, Javier
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Chemoenzymatic Approaches to the Synthesis of the Calcimimetic Agent Cinacalcet Employing Transaminases and Ketoreductases2018In: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. 360, no 11, p. 2157-2165Article in journal (Refereed)
    Abstract [en]

    Abstract Several chemoenzymatic routes have been explored for the preparation of cinacalcet, a calcimimetic agent. Transaminases (TAs) and ketoreductases (KREDs) turned out to be useful biocatalysts for the preparation of key optically active precursors. Thus, the asymmetric amination of 1‐acetonaphthone yielded an enantiopure (R)‐amine, which can be alkylated in one step to yield cinacalcet. Alternatively, the bioreduction of the same ketone resulted in an enantiopure (S)‐alcohol, which was easily converted into the previous (R)‐amine. In addition, the reduction was efficiently performed with the KRED and its cofactor co‐immobilized on the same porous surface. This self‐sufficient heterogeneous biocatalyst presented an accumulated total turnover number (TTN) for the cofactor of 675 after 5 consecutive operational cycles. Finally, in a preparative scale synthesis the TA‐based approach was performed in aqueous medium and led to enantiopure cinacalcet in two steps and 50% overall yield.

  • 70.
    Modén, Olof
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Mutational Analysis and Redesign of Alpha-class Glutathione Transferases for Enhanced Azathioprine Activity2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Glutathione transferase (GST) A2-2 is the human enzyme most efficient in catalyzing azathioprine activation. Structure-function relationships were sought explaining the higher catalytic efficiency compared to other alpha class GSTs. By screening a DNA shuffling library, five recombined segments were identified that were conserved among the most active mutants. Mutational analysis confirmed the importance of these short segments as their insertion into low-active GSTs introduced higher azathioprine activity. Besides, H-site mutagenesis led to decreased azathioprine activity when the targeted positions belonged to these conserved segments and mainly enhanced activity when other positions were targeted. Hydrophobic residues were preferred in positions 208 and 213.

    The prodrug azathioprine is today primarily used for maintaining remission in inflammatory bowel disease. Therapy leads to adverse effects for 30 % of the patients and genotyping of the metabolic genes involved can explain some of these incidences. Five genotypes of human A2-2 were characterized and variant A2*E had 3–4-fold higher catalytic efficiency with azathioprine, due to a proline mutated close to the H-site. Faster activation might lead to different metabolite distributions and possibly more adverse effects. Genotyping of GSTs is recommended for further studies.

    Molecular docking of azathioprine into a modeled structure of A2*E suggested three positions for mutagenesis. The most active mutants had small or polar residues in the mutated positions. Mutant L107G/L108D/F222H displayed a 70-fold improved catalytic efficiency with azathioprine. Determination of its structure by X-ray crystallography showed a widened H-site, suggesting that the transition state could be accommodated in a mode better suited for catalysis.

    The mutational analysis increased our understanding of the azathioprine activation in alpha class GSTs and highlighted A2*E as one factor possibly behind the adverse drug-effects. A successfully redesigned GST, with 200-fold enhanced catalytic efficiency towards azathioprine compared to the starting point A2*C, might find use in targeted enzyme-prodrug therapies.

  • 71.
    Modén, Olof
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Zhang, Wei
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Mutational analysis of human glutathione transferase A2-2 identifies structural elements supporting high activity with the prodrug azathioprine2012In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 25, no 4, p. 189-197Article in journal (Refereed)
    Abstract [en]

    Glutathione transferase (GST) A2-2 is the human enzyme displaying the highest catalytic activity with the prodrug azathioprine (Aza). The reaction releases pharmacologically active 6-mercaptopurine by displacing the imidazole moiety from the Aza molecule. The GST-catalyzed reaction is of medical significance, since high rates of Aza activation may lead to adverse side effects in treated patients. The present study involves structureactivity relationships in GST A2-2 variants. Chimeric GSTs were previously generated by DNA shuffling and two peptide segments, one N-terminal and one C-terminal, were identified as primary determinants of Aza activity. The segments contain several residues of the substrate-binding H-site and their significance for supporting high Aza activity was investigated. Substitution of the corresponding two small regions in the low-activity human GST A3-3 or rat GST A3-3 by the human GST A2-2 segments generated chimeras with approximate to 10-fold enhanced Aza activity. The H-site residues Met208 and Leu213 in the C-terminal segment of GST A2-2 were mutated to produce a library with all possible residue combinations. At a calculated 93 library coverage, all of the 1880 mutants examined showed wild-type or decreased Aza activity, even though some retained activities with alternative substrates, further emphasizing the importance of this region for the targeted activity.

  • 72.
    Nameer, Samer
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Semlitsch, Stefan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Johansson, Mats
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    One-pot enzyme-catalyzed synthesis of dual-functional polyester macromers towards surface active hydrophobic filmsManuscript (preprint) (Other academic)
    Abstract [en]

    Selective enzyme catalysis is a valuable tool for the processing of monomers into value-added materials. In the present study natural resources were used to retrieve an ω-hydroxy fatty acid monomer containing an epoxide functionality. A procedure was developed for the synthesis of dual-functional oligomers by utilizing lipase catalysis in a one-pot synthesis route. The chemoselectivity of the enzyme allowed addition of thiol monomers to the retrieved epoxy monomers, without harming the epoxides, achieving a thiol-epoxy functional polyester resin. The synthesis reached full conversion (>99 %) after 8 h. It was possible to selectively crosslink the resin, through UV-initiated cationic polymerization of the epoxides into thiol-functional thermosets. The curing performance was followed in situ by Real-Time FTIR. The thiol groups on the surface of the film were accessible for post-modification.

  • 73.
    Nierstrasz, Vincent
    University of Borås, Faculty of Textiles, Engineering and Business.
    Enzymatic Processing of Textiles2015Conference paper (Other (popular science, discussion, etc.))
  • 74.
    Nierstrasz, Vincent
    University of Borås, Swedish School of Textiles.
    Surface modification and functionalisation of textile materials using digital inkjet2014Conference paper (Refereed)
  • 75.
    Odnell, Anna
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Influencing anaerobic digestion early stage processes for increased biomethane production from different substrate components2018Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    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.

  • 76.
    Olsen, Elisabeth K.
    et al.
    UiT The Arctic University of Norway, Norway.
    Hansen, Espen
    UiT The Arctic University of Norway, Norway.
    Moodie, Lindon W. K.
    University of Umeå, Sweden.
    Isaksson, Johan
    UiT The Arctic University of Norway, Norway.
    Sepčić, Kristina
    University of Ljubljana, Slovenia.
    Cergolj, Marija
    University of Ljubljana, Slovenia; University of Rijeka, Croatia.
    Svenson, Johan
    RISE, SP – Sveriges Tekniska Forskningsinstitut, SP Kemi Material och Ytor, Medicinteknik.
    Andersen, Jeanette H.
    UiT The Arctic University of Norway, Norway.
    Marine AChE inhibitors isolated from Geodia barretti: Natural compounds and their synthetic analogs2016In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 5, p. 1629-1640Article in journal (Refereed)
    Abstract [en]

    Barettin, 8,9-dihydrobarettin, bromoconicamin and a novel brominated marine indole were isolated from the boreal sponge Geodia barretti collected off the Norwegian coast. The compounds were evaluated as inhibitors of electric eel acetylcholinesterase. Barettin and 8,9-dihydrobarettin displayed significant inhibition of the enzyme, with inhibition constants (Ki) of 29 and 19 μM respectively towards acetylcholinesterase via a reversible noncompetitive mechanism. These activities are comparable to those of several other natural acetylcholinesterase inhibitors of marine origin. Bromoconicamin was less potent against acetylcholinesterase, and the novel compound was inactive. Based on the inhibitory activity, a library of 22 simplified synthetic analogs was designed and prepared to probe the role of the brominated indole, common to all the isolated compounds. From the structure-activity investigation it was shown that the brominated indole motif is not sufficient to generate a high acetylcholinesterase inhibitory activity, even when combined with natural cationic ligands for the acetylcholinesterase active site. The four natural compounds were also analysed for their butyrylcholinesterase inhibitory activity in addition and shown to display comparable activities. The study illustrates how both barettin and 8,9-dihydrobarettin display additional bioactivities which may help to explain their biological role in the producing organism. The findings also provide new insights into the structure-activity relationship of both natural and synthetic acetylcholinesterase inhibitors.

  • 77. Pavankumar, A. R.
    et al.
    Zelenin, Sergey
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundin, A.
    Schulte, T.
    Rajarathinam, K.
    Rebellato, P.
    Ardabili, Sahar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Salas, J.
    Achour, A.
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bioanalytical advantages of a novel recombinant apyrase enzyme in ATP-based bioluminescence methods2018In: Analytica Chimica Acta, ISSN 0003-2670, E-ISSN 1873-4324, Vol. 1025, p. 118-123Article in journal (Refereed)
    Abstract [en]

    Ultrasensitive measurements of intracellular ATP (intATP) based on the firefly luciferase reactions are frequently used to enumerate bacterial or mammalian cells. During clinical applications, extracellular ATP (extATP) should be depleted in biological samples since it interferes with intATP and affects the quantification of bacteria. The extATP can be eliminated by ATP-degrading enzymes but complete hydrolysis of extATP remains a challenge for today's commercial enzymes. The catalytic efficiency of ATP-degrading enzymes depends on enzyme characteristics, sample composition and the ability to deplete diphosphates, triphosphates and their complexes generated during the reaction. This phenomenon restricts the usage of bioluminescence-based ATP methods in clinical diagnostics. In light of this, we have developed a recombinant Shigella flexneri apyrase (RSFA) enzyme and analysed its ATP depletion potential with five commercial biochemical sources including potato apyrase, acid phosphatase, alkaline phosphatase, hexokinase and glycerol kinase. The RSFA revealed superior activity by completely eliminating the extracellular ATP and ATP-complexes, even in biological samples like urine and serum. Therefore, our results can potentially unwrap the chemical and bio-analytical applications of ATP-based bioluminescence tests to develop highly sensitive point-of-care diagnostics.

  • 78.
    Quehenberger, Julian
    et al.
    Research Division Biochemical Engineering, Institute of Chemical, Environmental and Bioscience Engineering, Faculty of Technical Chemistry, TU Wien, Vienna, 1060, Austria.
    Reichenbach, Tom
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Baumann, Niklas
    Research Division Biochemical Engineering, Institute of Chemical, Environmental and Bioscience Engineering, Faculty of Technical Chemistry, TU Wien, Vienna, 1060, Austria.
    Rettenbacher, Lukas
    Research Division Biochemical Engineering, Institute of Chemical, Environmental and Bioscience Engineering, Faculty of Technical Chemistry, TU Wien, Vienna, 1060, Austria.
    Divne, Christina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Spadiut, Oliver
    Research Division Biochemical Engineering, Institute of Chemical, Environmental and Bioscience Engineering, Faculty of Technical Chemistry, TU Wien, Vienna, 1060, Austria.
    Kinetics and Predicted Structure of a Novel Xylose Reductase from Chaetomium thermophilum.2019In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, no 1, article id E185Article in journal (Refereed)
    Abstract [en]

    While in search of an enzyme for the conversion of xylose to xylitol at elevated temperatures, a xylose reductase (XR) gene was identified in the genome of the thermophilic fungus Chaetomium thermophilum. The gene was heterologously expressed in Escherichia coli as a His6-tagged fusion protein and characterized for function and structure. The enzyme exhibits dual cofactor specificity for NADPH and NADH and prefers D-xylose over other pentoses and investigated hexoses. A homology model based on a XR from Candida tenuis was generated and the architecture of the cofactor binding site was investigated in detail. Despite the outstanding thermophilicity of its host the enzyme is, however, not thermostable.

  • 79.
    Rodrigues, Diogo
    et al.
    Novartis Pharma AG, 4002 Basel, Switzerland.
    Kittelmann, M.
    Eggimann, F.
    Bachler, T.
    Abad, S.
    Camattari, A.
    Glieder, A.
    Winkler, M.
    Lütz, S.
    Production of recombinant human aldehyde oxidase in Escherichia coli and optimization of its application for the preparative synthesis of oxidized drug metabolites2014In: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 6, no 4, p. 1028-1042Article in journal (Refereed)
    Abstract [en]

    Recombinant human aldehyde oxidase (AO) was expressed in Escherichia coli. Different cell disruption methods and conditions of cell culture in shake flasks and bioreactors and of biotransformation on an analytical scale were tested to optimize the synthesis of oxidized AO drug metabolites. The volumetric productivity was increased 24-fold by optimizing the cell culture conditions. The highest yield was achieved in a 25 L stirred tank bioreactor under non-oxygen-limited conditions and high lactose feed rate. Suspensions of highly concentrated and well-aerated whole cells at neutral pH and relatively low temperatures led to the best conversion. The solvent for the substrate and the buffering agent for the biotransformation had an important effect. In a biotransformation with AO, 210 mg of famciclovir was converted to diacetyl penciclovir a yield of 82 %. The optimized protocol represents a viable method for the preparative synthesis of oxidized AO metabolites of drugs. Drug metabolites: Recombinant human aldehyde oxidase is expressed in Escherichia coli. The highest volumetric productivity is achieved in a 25 L stirred tank bioreactor under non-oxygen-limited conditions and high lactose feed rate. In a biotransformation with concentrated whole cells at pH 7.4 and 30 °C, 210 mg of famciclovir is converted to diacetyl penciclovir in a yield of 82 %. The optimized protocol enables the preparative synthesis of oxidized aldehyde oxidase metabolites of drugs.

  • 80.
    Ruggieri, Federica
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. SARom Biostruct AB, SE-22381 Lund, Sweden.
    van Langen, Luuk M.
    Logan, Derek T.
    Walse, Björn
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Transaminase-catalyzed racemization with potential for dynamic kinetic resolutions2018In: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 10, no 21, p. 5026-5032Article in journal (Refereed)
    Abstract [en]

    Dynamic kinetic resolution (DKR) reactions in which a stereoselective enzyme and a racemization step are coupled in one‐pot would represent powerful tools for the production of enantiopure amines through enantioconvergence of racemates. The exploitation of DKR strategies is currently hampered by the lack of effective, enzyme‐compatible and scalable racemization strategies for amines. In the present work, the proof of concept of a fully biocatalytic method for amine racemization is presented. Both enantiomers of the model compound 1‐methyl‐3‐phenylpropylamine could be racemized in water and at room temperature using a couple of wild‐type, non‐proprietary, enantiocomplementary amine transaminases and a minimum amount of pyruvate/alanine as a co‐substrate couple. The biocatalytic simultaneous parallel cascade reaction presented here poses itself as a customizable amine racemization system with potential for the chemical industry in competition with traditional transition‐metal catalysis.

  • 81.
    Sandström, Anders G.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Highly Combinatorial Reshaping of the Candida antarctica lipase A Substrate Pocket Using an Extremely Condensed LibraryManuscript (preprint) (Other academic)
    Abstract [en]

    A highly combinatorial structure based protein engineering method is demonstrated resulting in a thorough modification of the binding pocket of Candida antarctica lipase A (CALA). Nine amino acid sites surrounding the entire pocket were simultaneously mutated, contributing to a sculpting of the substrate pocket toward a sterically demanding substrate, an ibuprofen ester. The best variant was highly active and displayed remarkable increase in enantioselectivity toward the substrate, with an E-value of 101, compared to the wild type CALA that poor activity and possesses an E-value of 3.4. The potential mutations introduced were a highly reduced set of amino acids, containing only the wild type residue and an alternative residue, preferably a smaller one with similar properties. These minimal ‘binary’ sets allow for extremely condensed protein libraries. The choice of amino acid sites were based on a computer model, with the substrate forcibly bound in the active site. This highly combinatorial method can be used to obtain tailor-made enzymes that are active toward substrates that are not normally accepted by the enzyme. When multiple sites are altered simultaneously, there is a higher possibility of obtaining positive synergistic effects, and the protein fitness landscape is explored efficiently.

  • 82.
    Sandström, Anders G.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Protein Engineering of Candida antarctica Lipase A: Enhancing Enzyme Properties by Evolutionary and Semi-Rational Methods2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    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.

  • 83.
    Sandén, Camilla
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Nanostructures on a Vector: Enzymatic Oligo Production for DNA Nanotechnology2012Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The technique of DNA origami utilizes the specific and limited bonding properties of DNA to fold single stranded DNA sequences of various lengths to form a predesigned structure. One longer sequence is used as a scaffold and numerous shorter sequences called staples, which are all complementary to the scaffold sequence, are used to fold the scaffold into intricate shapes. The most commonly used scaffold is derived by extracting the genome of the M13 phage and the staples are usually chemically synthesized oligonucleotides. Longer single stranded sequences are difficult to synthesize with high specificity, which limits the choices of scaffold sequences available. In this project two main methods of single stranded amplification, Rolling Circle Amplification (RCA) and the usage of helper phages, were explored with the goal to produce both a 378 nt scaffold and staple sequences needed for folding a DNA origami structure. To facilitate imaging by Transmission Electron Microscopy (TEM) of this small structure, the DNA origami structure was created to form a polymer structure. Production of the scaffold sequence in high yield was unsuccessful and no well-defined polymers were found in the folded samples, though a few results showed promise for further studies and optimizations. Due to time constraints of this project, only production of the scaffold sequence was tested. Unfortunately the scaffold produced by the helper phages was of the complementary strand to that used to design the DNA origami structure, and could therefore not be used for folding. The correct strand was produced by the RCA where the yield was too low when using Phi29 DNA polymerase for proper folding to take place, though small scale RCA by Bst DNA polymerase on the other hand showed promising results. These results indicate that the scaffold production may not be far off but still more experience in producing intermediate size oligonucleotides may be necessary before succeeding in high yield production of this 378 nt long sequence. The promise given by this production is to enable high yield, high purity, low cost and also an easily scalable process set-up. This would be an important step in future DNA nanotechnology research when moving from small scale laboratory research to large scale applications such as targeted drug delivery systems.

  • 84.
    Schimpf, Ulrike
    et al.
    Berlin Humboldt Univ IASP, Inst Agr & Urban Ecol Projects, Philippstr 13, D-10115 Berlin, Germany..
    Schulz, Ronald
    PILZHOF Dr Schulz, Dorfstr 16, D-16356 Krummensee, Germany..
    Industrial by-products from white-rot fungi production. Part II: Application in anaerobic digestion for enzymatic treatment of hay and straw2019In: Process Biochemistry, ISSN 1359-5113, E-ISSN 1873-3298, Vol. 76, p. 142-154Article in journal (Refereed)
    Abstract [en]

    By-products of white-rot fungi cultivations are valuable resources for the production of useful enzyme cocktails. These enzymes, which act synergistically to deconstruct lignocellulose polymers, can be recovered and potentially applied in industrial processes. This study investigated the application of processed by-products from Lentinula edodes cultivations in mesophilic and thermophilic anaerobic digestions of hay and straw. Untreated and mechanically treated hay and straw were investigated in biochemical methane potential assays with or without application of enzyme-containing materials. Biomasses, inocula and processed by-product were analyzed chemically and the degradation rate of lignocellulose polymers determined. In mesophilic conditions, all of the fungus-derived enzyme treatments increased the methane yield. A newly generated enzyme preparation significantly enhanced the methane yield of chopped hay and straw, and accelerated the rate of hemicellulose degradation. In general, the degree of cellulose degradation correlated with the methane yield. The novel enzyme preparation contains a larger variety of enzymes than is commonly found in biogas enzyme preparations and is thus an attractive candidate for significant process improvement. Our new investigation further shows that enzyme preparations of L. edodes have a high potential for catalytic activity in lignocellulose-rich systems without manure as co-substrate.

  • 85. Sehl, Torsten
    et al.
    Bock, Saskia
    Marx, Lisa
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Maugeri, Zaira
    Walter, Lydia
    Westphal, Robert
    Vogel, Constantin
    Menyes, Ulf
    Erhardt, Martin
    Mueller, Michael
    Pohl, Martina
    Rother, Doerte
    Asymmetric synthesis of (S)-phenylacetylcarbinol - closing a gap in C-C bond formation2017In: Green Chemistry, ISSN 1463-9262, E-ISSN 1463-9270, Vol. 19, no 2, p. 380-384Article in journal (Refereed)
    Abstract [en]

    (S)-Phenylacetylcarbinol [(S)-PAC] and its derivatives are valuable intermediates for the synthesis of various active pharmaceutical ingredients (APIs), but their selective synthesis is challenging. As no highly selective enzymes or chemical catalysts were available, we used semi-rational enzyme engineering to tailor a potent biocatalyst to be >97% stereoselective for the synthesis of (S)-PAC. By optimizing the reaction and process used, industrially relevant product concentrations of >48 g L-1 (up to 320 mM) were achieved. In addition, the best enzyme variant gave access to a broad range of ring-substituted (S)-PAC derivatives with high stereoselectivity, especially for meta-substituted products.

  • 86.
    Semlitsch, Stefan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Building blocks for polymer synthesis by enzymatic catalysis2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    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.

  • 87.
    Semlitsch, Stefan
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Mixed vinyl adipate esters through selective synthesis using a designed esterase/acyltransferaseManuscript (preprint) (Other academic)
    Abstract [en]

    Methods for selective syntheses of mixed dicarboxylic esters from symmetrical diesters are of great interest. An esterase from Mycobacterium smegmatis, MsAcT, has shown to be an efficient catalyst for acyl transfer reactions using both alcohols and amines. It has a restricted active site resulting in a narrow acyl donor specificity. This limitation was used for the development of a selective synthesis of mixed vinyl adipate esters from divinyl adipate. The single mutant, L12A, achieved over 90% conversion of divinyl adipate with three different alcohols leading to the corresponding mixed vinyl adipate esters. This method allows the synthesis of vinyladipoyl esters at mild reaction conditions.

  • 88. Shafiei, Marzieh
    et al.
    Karimi, Keikhosro
    University of Borås, School of Engineering.
    Taherzadeh, Mohammad J.
    University of Borås, School of Engineering.
    Palm Date Fibers: Analysis and Enzymatic Hydrolysis2010In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 11, no 11, p. 4285-4296Article in journal (Refereed)
    Abstract [en]

    Waste palm dates were subjected to analysis for composition and enzymatic hydrolysis of their flesh fibers. The fruit contained 32% glucose and 30% fructose, while the water-insoluble fibers of its flesh consisted of 49.9% lignin and 20.9% polysaccharides. Water-insoluble fibers were settled to 55% of its initial volume in 12 h. The presence of skin and flesh colloidal fibers results in high viscosity and clogging problems during industrial processes. The settling velocity of the fibers was improved by enzymatic hydrolysis. Hydrolysis resulted in 84.3% conversion of the cellulosic part of the fibers as well as reducing the settling time to 10 minutes and the final settled volume to 4% of the initial volume. It implies easier separation of the fibers and facilitates fermentation processes in the corresponding industries. Two kinds of high- and low-lignin fibers were identified from the water-insoluble fibers. The high-lignin fibers (75% lignin) settled easily, while the low-lignin fibers (41.4% lignin) formed a slurry suspension which settled very slowly. The hydrophilicity of these low-lignin fibers is the major challenge of the industrial processes.

  • 89. Siedler, S.
    et al.
    Kumar Khatri, Narendar
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zsohár, A.
    Kjærbølling, I.
    Vogt, M.
    Hammar, Petter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nielsen, C. F.
    Marienhagen, J.
    Sommer, M. O. A.
    Jönsson, Håkan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Development of a Bacterial Biosensor for Rapid Screening of Yeast p-Coumaric Acid Production2017In: ACS Synthetic Biology, E-ISSN 2161-5063, Vol. 6, no 10, p. 1860-1869Article in journal (Refereed)
    Abstract [en]

    Transcription factor-based biosensors are used to identify producer strains, a critical bottleneck in cell factory engineering. Here, we address two challenges with this methodology: transplantation of heterologous transcriptional regulators into new hosts to generate functional biosensors and biosensing of the extracellular product concentration that accurately reflects the effective cell factory production capacity. We describe the effects of different translation initiation rates on the dynamic range of a p-coumaric acid biosensor based on the Bacillus subtilis transcriptional repressor PadR by varying its ribosomal binding site. Furthermore, we demonstrate the functionality of this p-coumaric acid biosensor in Escherichia coli and Corynebacterium glutamicum. Finally, we encapsulate yeast p-coumaric acid-producing cells with E. coli-biosensing cells in picoliter droplets and, in a microfluidic device, rapidly sort droplets containing yeast cells producing high amounts of extracellular p-coumaric acid using the fluorescent E. coli biosensor signal. As additional biosensors become available, such approaches will find broad applications for screening of an extracellular product.

  • 90.
    Sjöström, Staffan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Droplet microfluidics for directed evolution of biocatalysts2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Biocatalysts, biologically derived catalysts, are of great importance for a wide range of industrial applications. They are used in the production of for example foods, pharmaceuticals and biofuels. Improving biocatalysts commonly relies on directed evolution, i.e. mutagenesis to form diverse variants followed by functional screening in an iterative fashion.

    Droplet microfluidics is an emerging technology that can be applied for high throughput screening. A key feature of droplet microfluidics is the ability to encapsulate discrete objects, such as single cells, in picoliter-sized droplets at rates of over 1000 cells per second. Each droplet serves as a reaction vessel, analogous to a microwell, where a single clone can be screened.

    In this thesis, droplet microfluidics is employed for directed evolution of biocatalysts. In paper I, a multiplexed droplet microfluidic method for characterization of enzyme variants is presented and validated by measuring the kinetics of β-galactosidase inhibited by IPTG. In paper II-III, a method for directed evolution of cells with improved production of industrially important enzymes is presented. Two rounds of directed evolution yielded improved strains. The strains had up to 6 times increased enzyme expression levels and whole-genome sequencing revealed 300 mutations, many of which mapped to the protein secretory pathway. In Paper IV, a method for directed evolution of enzyme variants under conditions lethal to host cells is developed. The method is used to screen for α-amylase variants with improved activity or stability at pH4. In Paper V, a method to screen cyanobacteria cell factories is developed and we show that the method can enrich for a strain with high production of L-lactate. In Paper VI, the metabolism of yeast cells encapsulated in microfluidic droplets is studied and found to depend on the choice of emulsion incubation device.

    Taken together, droplet microfluidics is a promising technology for directed evolution of biocatalysts with the potential to vastly increase throughput and cut costs. The technology could pave the way for process customized biocatalysts and help replace polluting processes with sustainable green chemistry.

  • 91.
    Speda, Jutta
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Johansson, Mikaela A.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. InZymes Biotech AB, Linköping, Sweden.
    Assessment of sample preparation methods for metaproteomics of extracellular proteins2017In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 516, p. 23-36Article in journal (Refereed)
    Abstract [en]

    Enzyme discovery in individual strains of microorganisms is compromised by the limitations of pure culturing. In principle, metaproteomics allows for fractionation and study of different parts of the protein complement but has hitherto mainly been used to identify intracellular proteins. However, the extracellular environment is also expected to comprise a wealth of information regarding important proteins. An absolute requirement for metaproteomic studies of protein expression, and irrespective of downstream methods for analysis, is that sample preparation methods provide clean, concentrated and representative samples of the protein complement. A battery of methods for concentration, extraction, precipitation and resolubilization of proteins in the extracellular environment of a constructed microbial community was assessed by means of 2D gel electrophoresis and image analysis to elucidate whether it is possible to make the extracellular protein complement available for metaproteomic analysis. Most methods failed to provide pure samples and therefore negatively influenced protein gel migration and gel background clarity. However, one direct precipitation method (TCA-DOC/acetone) and one extraction/precipitation method (phenol/methanol) provided complementary high quality 2D gels that allowed for high spot detection ability and thereby also spot detection of less abundant extracellular proteins.

  • 92.
    Speda, Jutta
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology. Rational Enzyme Mining AB, Linköping, Sweden.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology. Rational Enzyme Mining AB, Linköping, Sweden.
    Induced differential metaproteomics for the identification of cellulases in a methanogenic microbial community2013In: BioMicroWorld 2013: Book of Abstracts, 2013, p. 679-Conference paper (Other academic)
    Abstract [en]

    The identification of novel enzymes for use in industrial biotechnology is an important goal in enzyme discovery. The need for novel biocatalysts for sustainable and efficient bioenergy production and the development of new biomaterials especially gives rise to new strategic opportunities of proteomic research. Most industrially relevant enzymes to date have been isolated from pure cultured microorganisms. It is however well established that only a small fraction of all existing microorganisms can be obtained in pure cultures, thus limiting the potential of finding novel enzymes. The possibility to identify valuable enzymes directly from complete microbial communities would therefore potentially give access to a huge number of novel enzyme candidates.

    Metaproteomics, or “the large-scale characterization of the entire protein complement of environmental microbiota at a given point in time” has hitherto mainly been used to understand ecosystem function. In order to reach our goals we have instead used the dynamics of metaproteomics to develop a method based on “induced differential metaproteomics”, in which a desired enzyme activity is induced in a microbial population and compared to a non-induced reference of the very same population. In a first example the goal was to induce, select and identify cellulases from a methanogenic community, maintained in a biogas reactor at a metabolic steady-state in a chemically defined medium.

    Two aliquots were subtracted from the reactor, of which one was treated to induce cellulase activity. At the peak of cellulase activity and biogas production in the induced sample, proteins from the liquid phase of the two samples were prepared for 2D-DIGE of the extra-cellular proteins. Out of several hundred protein spots generated by the microbial community and visible in the 2D-DIGE experiment, 95 could be identified as up-regulated in the induced sample by image analysis, as compared to the references (thus representing potential cellulases). In-gel digestion and tandem mass-spectrometry of located and selected up-regulated proteins revealed that 18 out of 30 proteins could be assigned as cellulases or associated to cellulolytic activity giving a remarkable hit-rate of 60 % and thus demonstrating the feasibility of the approach.

    These cellulases found can be expected to be highly active and stable at the conditions in which they are naturally produced (pH, temp., salinity etc.). A strategic objective of research, both in academia and in thebiotechnology industry, is to identify novel, highly active microbial enzymes that are stable at the different conditions of various industrial applications. Thus, one of our future prospects includes to further employ the described methodology to identify novel enzymes from microbial communities originating from more extreme environments.

  • 93.
    Speda, Jutta
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Induced differential metaproteomics: identification of cellulases in a methanogenic microbial community at mesophilic conditions2014Conference paper (Other academic)
  • 94.
    Speda, Jutta
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Induced differential metaproteomics: identification of thermostable cellulases in a methanogenic microbial community2014Conference paper (Other academic)
    Abstract [en]

    The identification of novel enzymes for use in industrial biotechnology is an important goal in enzyme discovery. Most industrially relevant enzymes to date have been isolated from pure cultured microorganisms. For future discovery of novel enzymes this is however a major bottleneck since it is well established that only a small fraction of all microorganisms can be obtained in pure cultures. The possibility to identify enzymes directly from complete microbial communities would therefore give access to a huge number of novel enzyme candidates.

    Metaproteomics has hitherto mainly been used to understand ecosystem functions. We have instead used the dynamics of proteomics to develop a method based on “induced differential metaproteomics”, by which a desired enzyme activity is induced in a full microbial population and compared to a non-induced reference of the very same population. In a first example the goal was to induce, select and identify cellulases from a thermophilic methanogenic community.

    Out of several hundred detectable proteins in a 2D-DIGE experiment, 24 proteins could be identified as at least two-fold up-regulated upon induction. For some proteins spots, the cellulolytic activity was further validated by activity staining using 2D-zymography. Mass spectrometry analysis revealed that 21 out of the 24 up-regulated proteins are cellulases or associated to cellulolytic activity giving a remarkable hit-rate of 88%. This demonstrates the high efficiency and precision of the method, by which a much wider span of the microbial world can be scanned for novel and targeted enzymes.

  • 95.
    Steffen-Munsberg, Fabian
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Greifswald University.
    Matzel, Philipp
    Sowa, Miriam A.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Bornscheuer, Uwe T.
    Höhne, Matthias
    Bacillus anthracis ω-amino acid:pyruvate transaminase employs a different mechanism for dual substrate recognition than other amine transaminases2016In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 100, p. 4511-4521Article in journal (Refereed)
    Abstract [en]

    Understanding the metabolic potential of organisms or a bacterial community based on their (meta) genome requires the reliable prediction of an enzyme’s function from its amino acid sequence. Besides a remarkable development in prediction algorithms, the substrate scope of sequences with low identity to well-characterized enzymes remains often very elusive. From a recently conducted structure function analysis study of PLP-dependent enzymes, we identified a putative transaminase from Bacillus anthracis (Ban-TA) with the crystal structure 3N5M (deposited in the protein data bank in 2011, but not yet published). The active site residues of Ban-TA differ from those in related (class III) transaminases, which thereby have prevented function predictions. By investigating 50 substrate combinations its amine and ω-amino acid:pyruvate transaminase activity was revealed. Even though Ban-TA showed a relatively narrow amine substrate scope within the tested substrates, it accepts 2-propylamine, which is a prerequisite for industrial asymmetric amine synthesis. Structural information implied that the so-called dual substrate recognition of chemically different substrates (i.e. amines and amino acids) differs from that in formerly known enzymes. It lacks the normally conserved ‘flipping’ arginine, which enables dual substrate recognition by its side chain flexibility in other ω-amino acid:pyruvate transaminases. Molecular dynamics studies suggested that another arginine (R162) binds ω-amino acids in Ban-TA, but no side chain movements are required for amine and amino acid binding. These results, supported by mutagenesis studies, provide functional insights for the B. anthracis enzyme, enable function predictions of related proteins, and broadened the knowledge regarding ω-amino acid and amine converting transaminases.

  • 96.
    Svedendahl, Maria
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Branneby, Cecilia
    Cambrex Karlskoga AB.
    Carlqvist, Peter
    KTH, Superseded Departments, Chemistry.
    Brinck, Tore
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry (closed 20110630).
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Berglund, Per
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Michael-type additions catalyzed by a rationally redesigned lipase2004Conference paper (Refereed)
  • 97.
    Svedendahl, Maria
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Branneby, Cecilia
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Carlqvist, Peter
    KTH, Superseded Departments, Chemistry.
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Brinck, Tore
    KTH, Superseded Departments, Chemistry.
    Berglund, Per
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Expanding the Synthetic Scope of Hydrolytic Enzymes: Catalysis of Aldol- and Michael-Type Additions2004Conference paper (Refereed)
  • 98.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry.
    Abedi, Vahak
    AstraZeneca.
    Federsel, Hans-Jürgen
    AstraZeneca.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    From S to R: Key Residues Controlling Enantiomer Preference and Activity in omega-Transaminase2011Conference paper (Refereed)
  • 99.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry.
    Branneby, Cecilia
    Cambrex Karlskoga AB.
    Sjöstrand, Ulf
    Cambrex Karlskoga AB.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Rational Redesign of ω-Transaminases2010Conference paper (Refereed)
  • 100.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Exploring Promiscuous Activities in a Lipase2009Conference paper (Refereed)
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