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  • 1. Abahazi, Emese
    et al.
    Satorhelyi, Peter
    Erdelyi, Balazs
    Vertessy, Beata G.
    Land, Henrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Paizs, Csaba
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Poppe, Laszlo
    Covalently immobilized Trp60Cys mutant of omega‰-transaminase from Chromobacterium violaceum for kinetic resolution of racemic amines in batch and continuous-flow modes2018In: Biochemical engineering journal, ISSN 1369-703X, E-ISSN 1873-295X, Vol. 132, p. 270-278Article in journal (Refereed)
    Abstract [en]

    Covalent immobilization of an engineered omega-transaminase mutant Trp60Cys from Chromobacterium violaceum (CvTAW60C) was performed on bisepoxide-activated aminoalkyl resins. Activity of the various CvTAW60C preparations was evaluated in kinetic resolution of four racemic amines (rac-1a–d). The most active EA-G-CvTAW60C preparation (CvTAW60C attached to polymeric resin with ethylamine function activated with glycerol diglycidyl ether—EA-G) could perform the kinetic resolution of racemic 4-phenylbutan-2-amine (rac-1a) over 49% conversion up to 19 consecutive reaction cycles or in media containing up to 50% v/v DMSO as cosolvent in batch mode reactions. The immobilization process of CvTAW60C onto the EA-G resin filled in stainless steel bioreactors was also tested in flow-through mode. Kinetic resolution of three racemic amines containing aromatic moieties (rac-1a-c) was performed in continuous-flow mode resulting in easy-to-separate mixture of the corresponding ketone (2a–c) and the non-converted (R)-amine in high enantiopurity (ee(R)-1a-c ≥ 96%).

  • 2.
    Ahmed, Ajaj
    et al.
    Department of Microbiology, M.G.S. University, Bikaner, India.
    Dabi, Narendra Kumar
    Department of Microbiology, M.G.S. University, Bikaner, India.
    Verma, Swati
    Department of Microbiology, M.G.S. University, Bikaner, India.
    Gehlot, Praveen
    Department of Botany, J.N.V. University, Jodhpur, India.
    Purohit, Praveen
    Department of Chemistry, Engineering College, Bikaner, 334001, India.
    Kumar, Rajender
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Meghwanshi, Gautam Kumar
    Department of Microbiology, M.G.S. University, Bikaner, India.
    Evaluation of Thar Desert bacterial lipases for catalytic efficiencies and biodiesel production potentials2023In: Biologia, ISSN 0006-3088, E-ISSN 1336-9563, Vol. 78, no 4, p. 1187-1197Article in journal (Refereed)
    Abstract [en]

    The present work describes the screening of thermotolerant bacteria isolated from Thar Desert environmental samples for lipase activity and their catalytic efficiencies, such as tolerance to extreme pHs, temperatures, and organic solvents, and efficiency to synthesize biodiesel from waste cooking oils. The selected lipases were thermos-alkaliphilic in nature showing good activity at higher temperatures and in the alkaline pH range with optimal activity at 45 °C and pH 8 or 9. The lipases efficiently converted oils to biodiesel (fatty acid methyl ester), giving up to 78% conversion under specific reaction conditions. The enzyme (lipase) mediated biodiesel production will soon offer an eco-friendly and sustainable energy source for automobiles and industrial applications. The thermos-alkaliphilic properties of these lipases along with their efficiency to produce fatty acid methyl ester from waste cooking oil and methanol as well as other prospective applications, make them potential candidates for biodiesel production and other prospective applications such as the synthesis of flavor and fragrance esters and remediation of various environmental pollutants.

  • 3.
    Berglund, P.
    et al.
    University of Toronto, Canada.
    Stabile, M. R.
    Gold, M.
    Jones, J. B.
    Mitchinson, C.
    Bott, R. R.
    Graycar, T. P.
    Altering the specificity of subtilisin B. lentus by combining site-directed mutagenesis and chemical modification1996In: Bioorganic & Medicinal Chemistry Letters, ISSN 0960-894X, E-ISSN 1464-3405, Vol. 6, no 21, p. 2507-2512Article in journal (Refereed)
    Abstract [en]

    The thiol side chain of the M222C mutant of the subtilisin from Bacillus lentus (SBL) has been chemically modified by methyl-, aminoethyl-, and sulfonatoethylthiosulfonate reagents. Introduction of charged residues into the active site of the enzyme reduced the catalytic efficiency with Suc-AAPF-pNA as the substrate, but resulted in better binding of sterically demanding boronic acid inhibitors.

  • 4.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    BIO-AMINES: Searching for a Novel Approach to Biocatalytic Transaminations – a Vinnova Sponsored Project2009In: Book of abstracts, 2009Conference paper (Other academic)
  • 5.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Biocatalysis with Promiscuous Enzymes2007In: 2007 European BioPerspectives / [ed] Dechema, 2007Conference paper (Refereed)
  • 6.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Enzyme Catalytic Promiscuity and Rational Design2006In: Book of abstracts, 2006Conference paper (Refereed)
  • 7.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    omega-Transaminases: Tailored for Chiral Amine Synthesis2010In: Biocat2010 / [ed] Ralf Grote, Garabed Antranikian, Hamburg, Germany: TuTech Verlag , 2010Conference paper (Refereed)
  • 8.
    Berglund, Per
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Christiernin, M.
    Hedenström, E.
    Enantiorecognition of chiral acids by Candida rugosa lipase: Two substrate binding modes evidenced in an organic medium2001In: American Chemical Society Symposium Series (ACS), ISSN 0097-6156, E-ISSN 1947-5918, Vol. 776, p. 263-273Article in journal (Refereed)
    Abstract [en]

    We have identified the existence of different modes of binding the enantiomers of 2-methyl-branched carboxylic acids to a lipase active site by rational substrate engineering. Similar to hydrolysis, previously investigated, we have now evidence for differential binding modes in the Candida rugosa lipase-catalyzed esterifications in cyclohexane. The relevance of considering two different binding modes to understand lipase enantiorecognition is demonstrated by introducing bulky substituents on a chiral carboxylic acid which impose a different orientation of the substrate acyl chain in the active site of Candida rugosa lipase. With this substrate engineering approach based on molecular modeling it is thus possible to markedly alter the enantioselectivity of the lipase. Examples from hydrolysis and new results from esterifications in an organic solvent are presented and discussed.

  • 9.
    Berglund, Per
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hedenström, Erik
    Mid Sweden university.
    Preparation of 2-, 3-, and 4-methylcarboxylic acids and the corresponding alcohols of high enantiopurity by lipase-catalyzed esterification2001In: Enzymes in Nonaqueous Solvents: Methods and Protocols / [ed] Vulfson, E. N.; Halling, P. J.; Holland, H. L., Totowa: Humana Press , 2001, p. 307-317Chapter in book (Refereed)
  • 10.
    Berglund, Per
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Holmquist, Mats Torsten
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Hult, Karl
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Reversed enantiopreference of Candida rugosa lipase supports different modes of binding enantiomers of a chiral acyl donor1998In: Journal of Molecular Catalysis - B Enzymatic, ISSN 1381-1177, Vol. 5, no 1-4, p. 283-287Article in journal (Refereed)
    Abstract [en]

    Molecular modelling identifies two different productive modes of binding the enantiomers of a 2-methyldecanoic acid ester to the active site of Candida rugosa lipase (CRL). The fast reacting S-enantiomer occupies the previously identified acyl-binding tunnel of the enzyme, whereas the R- enantiomer leaves the tunnel empty. The modelling suggested that if both enantiomers were forced to bind to the active site leaving the tunnel empty, the enzyme would reverse its enantiopreference to become R-enantioselective. To test this hypothesis, we designed a structural analogue to 2- methyldecanoic acid, 2-methyl-6-(2-thienyl)hexanoic acid, which was expected to be too bulky to fit its acyl moiety into the acyl-binding tunnel. The CRL- catalysed hydrolysis of the ethyl ester of this substrate resulted in the preferential conversion of the R-enantiomer as predicted by molecular modelling. This represents the first kinetic evidence supporting the existence of two different modes of binding the enantiomers of a 2- methyldecanoic acid ester to the active site of CRL. We have shown that a rational 3D based approach in combination with substrate engineering can be used to predict and control the stereochemical outcome of a lipase catalysed reaction.

  • 11.
    Berglund, Per
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Biocatalytic synthesis of enantiopure compounds using lipases: Chapter 212000In: Stereoselective Biocatalysis / [ed] Patel, R. N., New York: Marcel Dekker, 2000, p. 633-657Chapter in book (Refereed)
  • 12.
    Berglund, Per
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Svedendahl, Maria
    KTH, School of Biotechnology (BIO), Biochemistry.
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry.
    Branneby, Cecilia
    Cambrex Karlskoga AB.
    Abedi, Vahak
    AstraZeneca.
    Wells, Andrew
    AstraZeneca.
    Federsel, Hans-Jürgen
    AstraZeneca.
    Omega-Transaminases Redesigned for Chiral Amine Synthesis2011In: BIT Life Sciences’ 2nd Symposium on Enzymes & Biocatalysis, Dalian, China: BIT Life Sciences , 2011Conference paper (Refereed)
  • 13.
    Berglund, Per
    et al.
    Mid Sweden University.
    Vörde, Carin
    Hogberg, Hans-Erik
    Esterification of 2-methylalkanoic acids Catalysed by Lipase from Candida rugosa: Enantioselectivity as a Function of water Activity and Alcohol Chain Length1994In: Biocatalysis and Biotransformation, ISSN 1024-2422, E-ISSN 1029-2446, Vol. 9, no 1-4, p. 123-130Article in journal (Refereed)
  • 14.
    Bi, Ran
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Lignocellulose Degradation by Soil Micro-organisms2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Lignocellulosic biomass is a sustainable resource with abundant reserves. Compared to petroleum ‐ based products, the biomass ‐ derived polymers and chemicals give better environmental profiles. A lot of research interest is focused on understanding the lignocellulose structures.

    Lignin, among the three major wood components, represents most difficulty for microbial degradation because of its complex structure and because cross ‐ linking to hemicellulose makes wood such a compact structure. Nevertheless, wood is naturally degraded by wood ‐ degrading micro ‐ organisms and modified and partly degraded residual of lignin goes into soil. Therefore soil serves as a good environment in which to search for special lignin ‐ degraders. In this thesis, different types of lignin have been used as sole carbon sources to screen for lignin ‐ degrading soil micro ‐ organisms. Eleven aerobic and three anaerobic microbe strains have been isolated and identified as able to grow on lignin. The lignin degradation patterns of selected strains have been studied and these partly include an endwise cleavage of  β‐ O ‐ 4 bonds in lignin and is more complex than simple hydrolytic degradation.

    As lignin exists in wood covalently bonded to hemicellulose, one isolated microbe strain, Phoma herbarum, has also been studied with regards to its ability to degrade covalent lignin polysaccharide networks (LCC). The results show that its culture filtrate can attack lignin ‐ polysaccharide networks in a manner different from that of the commercial enzyme product, Gammanase, possibly by selective cleavage of phenyl glucoside bonds. The effects on LCC of Phoma herbarum also enhance polymer extractability. Hot ‐ water extraction of a culture filtrate of Phoma herbarum ‐ treated fiberized spruce wood material gave an amount of extracted galactoglucomannan more than that given by the Gammanase ‐ treated material and non ‐ enzyme ‐ treated material.

    Over millions of years of natural evolution, micro ‐ organisms on the one hand develop so that they can degrade all wood components to get energy for growth, while plants on the other hand also continuously develop to defend from microbial attack. Compared with lignin and cellulose, hemicelluloses as major components of plant cell walls, are much more easily degraded, but hemicelluloses differ from cellulose in that they are acetylated to different extents. The biological functions of acetylation are not completely understood, but it is suggested is that one function is to decrease the microbial degradability of cell walls. By cultivation of soil micro ‐ organisms using mannans acetylated to deffernent degrees as sole carbon source on agar plates, we were able to see significant trends where the resistance towards microbial degradation of glucomannan and galactomannan increased with increasing degree of acetylation. Possible mechanisms and the technological significance of this are discussed. Tailoring the degree of acetylation of polysaccharide materials might slow down the biodegradation, making it possible to design a material with a degradation rate suited to its application.

    Download full text (pdf)
    Thesis
  • 15.
    Biundo, Antonino
    et al.
    Univ Nat Resources & Life Sci, Vienna, Austria..
    Ribitsch, Doris
    Austrian Ctr Ind Biotechnol, Graz, Austria..
    Syrén, Per-Olof
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. Vienna, Austria..
    Guebitz, Georg M.
    Univ Nat Resources & Life Sci, Vienna, Austria..
    Increasing amide acceptance on a polyester-hydrolyzing enzyme2016In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 33, p. S105-S105Article in journal (Other academic)
  • 16.
    Biundo, Antonino
    et al.
    University of Bari, Department of Biosciences, Biotechnology and Biopharmaceutics, via Orabona, 4, Bari, 70125, Italy, via Orabona, 4; REWOW srl, Via Ciasca 9, Bari, 70124, Italy, Via Ciasca 9.
    Saénz Méndez, Patricia
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology. Karlstads Universitet, Faculty of Health, Science and Technology, Universitetsgatan 2, 65188 Karlstad, Sweden.
    Görbe, Tamas
    Menten AI, 2225 E Bayshore Road, Suite 200, Palo Alto, CA, 94303, USA.
    Enzyme Modification2021In: Biocatalysis for Practitioners: Techniques, Reactions and Applications, Wiley , 2021, p. 33-62Chapter in book (Other academic)
    Abstract [en]

    The chapter on protein engineering has introduced and discussed an overview of the available methods for the modification of enzymes. Starting with the classical directed evolution (DE) technique, which has been applied extensively throughout several different biocatalytic processes, the reader moves toward the semi-rational, rational, and de novo design of enzymes. Meanwhile, DE is clearly the current industry-leading technology; depending on the understanding of the particular enzymatic system, and on the available structural information, other techniques such as rational design are also becoming fast and efficient solutions for the development of new catalysts. This success would not be possible without the constant improvements of the computational techniques and the newly developed modeling systems for enzyme engineering.

  • 17.
    Branneby, Cecilia
    et al.
    Cambrex Karlskoga AB.
    Svedendahl, Maria
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lipase-Catalyzed Aldol and Michael-Type Reactions2005In: Book of abstracts, 2005Conference paper (Refereed)
  • 18.
    Branneby, Cecilia
    et al.
    Cambrex Karlskoga AB.
    Svedendahl, Maria
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lipase-Catalyzed Aldol and Michael-Type Reactions2006Conference paper (Refereed)
  • 19.
    Chandrakumaran, Sajitha
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Investigation of an enzymatic cascade for the production of 5- hydroxymethylfurfurylamine2023Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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. 

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    fulltext
  • 20.
    Chawachart, Niwat
    et al.
    Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand.
    Anbarasan, Sasikala
    Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, P.O. Box 16100, 00076, Aalto, Finland.
    Turunen, Samuel
    Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, P.O. Box 16100, 00076, Aalto, Finland.
    Li, He
    Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, P.O. Box 16100, Aalto, 00076, Finland.
    Khanongnuch, Chartchai
    Department of Biotechnology, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai, 50200, Thailand.
    Hummel, Michael
    Department of Forest Products Technology, School of Chemical Technology, Aalto University, P.O. Box 16300, 00076, Aalto, Finland.
    Sixta, Herbert
    Department of Forest Products Technology, School of Chemical Technology, Aalto University, P.O. Box 16300, 00076, Aalto, Finland.
    Granström, Tom
    Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, P.O. Box 16100, 00076, Aalto, Finland.
    Lumyong, Saisamorn
    Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand.
    Turunen, Ossi
    Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, P.O. Box 16100, 00076, Aalto, Finland.
    Thermal behaviour and tolerance to ionic liquid [emim] OAc in GH10 xylanase from Thermoascus aurantiacus SL16W2014In: Extremophiles, ISSN 1431-0651, E-ISSN 1433-4909, Vol. 18, no 6, p. 1023-1034Article in journal (Refereed)
    Abstract [en]

    GH10 xylanase from Thermoascus aurantiacus strain SL16W (TasXyn10A) showed high stability and activity up to 70–75 C. The enzyme’s half-lives were 101 h, 65 h, 63 min and 6 min at 60, 70, 75 and 80 C, respectively. The melting point (Tm), as measured by DSC, was 78.5 C, which is in line with a strong activity decrease at 75–80 C. The biomass-dissolving ionic liquid 1-ethyl-3-methylimidazolium acetate ([emim]OAc) in 30 % concentration had a small effect on the stability of TasXyn10A; Tm decreased by only 5 C. It was also observed that [emim]OAc inhibited much less GH10 xylanase (TasXyn10A) than the studied GH11 xylanases. The Km of TasXyn10A increased 3.5-fold in 15 % [emim]OAc with xylan as the substrate, whereas the approximate level of Vmax was not altered. The inhibition of enzyme activity by [emim]OAc was lesser at higher substrate concentrations. Therefore, high solid concentrations in industrial conditions may mitigate the inhibition of enzyme activity by ionic liquids. Molecular docking experiments indicated that the [emim] cation has major binding sites near the catalytic residues but in lower amounts in GH10 than in GH11 xylanases. Therefore, [emim] cation likely competes with the substrate when binding to the active site. The docking results indicated why the effect is lower in GH10.

  • 21.
    Chen, Shan
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Land, Henrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Svedendahl Humble, Maria
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Stabilization of an amine transaminase for biocatalysis2016In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 124, p. 20-28Article in journal (Refereed)
    Abstract [en]

    The amine transaminase from Chromobacterium violaceum (Cv-ATA) is a well-known enzyme to achievechiral amines of high enantiomeric excess in laboratory scales. However, the low operational stabilityof Cv-ATA limits the enzyme applicability on larger scales. In order to improve the operational stabilityof Cv-ATA, and thereby extending its applicability, factors (additives, co-solvents, organic solvents anddifferent temperatures) targeting enzyme stability and activity were explored in order to find out how tostore and apply the enzyme. The present investigation shows that the melting point of Cv-ATA is improvedby adding sucrose or glycerol, separately. Further, by storing the enzyme at higher concentrations and inco-solvents, such as; 50% glycerol, 20% methanol or 10% DMSO, the active dimeric structure of Cv-ATAis retained. Enzyme stored in 50% glycerol at −20◦C was e.g., still fully active after 6 months. Finally,the enzyme performance was improved 5-fold by a co-lyophilization with surfactants prior to usage inisooctane.

  • 22. Christou, Nina Eleni
    et al.
    Lane, Thomas J.
    et al.,
    Time-resolved crystallography captures light-driven DNA repair2023In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 382, no 6674, p. 1015-1020Article in journal (Refereed)
    Abstract [en]

    Photolyase is an enzyme that uses light to catalyze DNA repair. To capture the reaction intermediates involved in the enzyme's catalytic cycle, we conducted a time-resolved crystallography experiment. We found that photolyase traps the excited state of the active cofactor, flavin adenine dinucleotide (FAD), in a highly bent geometry. This excited state performs electron transfer to damaged DNA, inducing repair. We show that the repair reaction, which involves the lysis of two covalent bonds, occurs through a single-bond intermediate. The transformation of the substrate into product crowds the active site and disrupts hydrogen bonds with the enzyme, resulting in stepwise product release, with the 3' thymine ejected first, followed by the 5' base.

  • 23. Dan, Meiling
    et al.
    Zheng, Yuting
    Zhao, Guohua
    Hsieh, Yves S. Y.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Wang, Damao
    Current insights of factors interfering the stability of lytic polysaccharide monooxygenases2023In: Biotechnology Advances, ISSN 0734-9750, E-ISSN 1873-1899, Vol. 67, p. 108216-108216, article id 108216Article in journal (Refereed)
    Abstract [en]

    Cellulose and chitin are two of the most abundant biopolymers in nature, but they cannot be effectively utilized in industry due to their recalcitrance. This limitation was overcome by the advent of lytic polysaccharide monooxygenases (LPMOs), which promote the disruption of biopolymers through oxidative mechanism and provide a breakthrough in the action of hydrolytic enzymes. In the application of LPMOs to biomass degradation, the key to consistent and effective functioning lies in their stability. The efficient transformation of biomass resources using LPMOs depends on factors that interfere with their stability. This review discussed three aspects that affect LPMO stability: general external factors, structural factors, and factors in the enzyme-substrate reaction. It explains how these factors impact LPMO stability, discusses the resulting effects, and finally presents relevant measures and considerations, including potential resolutions. The review also provides suggestions for the application of LPMOs in polysaccharide degradation. 

  • 24.
    Engelmark Cassimjee, Karim
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Affinity Tag Purification Method and Immobilization of the Promiscuous Enzyme Alanine Racemase2006Conference paper (Refereed)
  • 25.
    Engelmark Cassimjee, Karim
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Affinity Tag Purification Method of the Promiscuous Enzyme Alanine Racemase2006In: Book of abstracts, 2006Conference paper (Other academic)
  • 26.
    Engelmark Cassimjee, Karim
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Immobilization Method for the Promiscuous Enzyme Alanine Racemase2007In: BIOTRANS Oviedo 2007 / [ed] Vicente Gotor, 2007Conference paper (Refereed)
  • 27.
    Engelmark Cassimjee, Karim
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Branneby, Cecilia
    Cambrex Karlskoga AB.
    Sjöstrand, Ulf
    Cambrex Karlskoga AB.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    High Yield Transamination with Isopropyl Amine as Donor, by Employment of YADH and in situ Cofactor Regeneration2009In: Book of abstracts, 2009Conference paper (Refereed)
  • 28.
    Engelmark Cassimjee, Karim
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Branneby, Cecilia
    Cambrex Karlskoga AB.
    Sjöstrand, Ulf
    Cambrex Karlskoga AB.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    High Yield Transamination with Isopropyl Amine as Donor, by Employment of YADH and in situ Cofactor Regeneration2009Conference paper (Refereed)
  • 29.
    Engelmark Cassimjee, Karim
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Kourist, Robert
    University of Greifswald, Germany.
    Lindberg, Diana
    Uppsala university, SE.
    Wittrup Larsen, Marianne
    KTH, School of Biotechnology (BIO), Biochemistry.
    Widersten, Mikael
    Uppsala university, SE.
    Bornscheuer, Uwe T
    University of Greifswald, DE.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    A One Step Enzyme Extraction and Immobilization Method for Organic and Aqueous Solvents2008In: Biocat2008 / [ed] Ralf Grote, Garabed Antranikian, Hamburg, Germany: TuTech Innovation GmbH , 2008Conference paper (Refereed)
  • 30.
    Engelmark Cassimjee, Karim
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Kourist, Robert
    University of Greifswald, Germany.
    Lindberg, Diana
    Uppsala university, SE.
    Wittrup Larsen, Marianne
    KTH, School of Biotechnology (BIO), Biochemistry.
    Widersten, Mikael
    Uppsala university, SE.
    Bornscheuer, Uwe T
    University of Greifswald, DE.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    A One Step General Enzyme Immobilization Method for Organic and Aqueous Solvents2008In: Book of Abstracts, 2008Conference paper (Refereed)
  • 31.
    Engelmark Cassimjee, Karim
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Svedendahl, Maria
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Rational Redesign of omega-Transaminase2010In: Biocat2010, Hamburg, Germany: TuTech Verlag , 2010Conference paper (Refereed)
  • 32.
    Finnveden, Maja
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Enzyme catalysis towards bio-based UV-curable buildingblocks2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Polymeric materials are found in virtually all areas of daily life; they are found in everything from packages keeping our food safe to the buildings where we spend our days, and the production is a worldwide industry. Although polymeric materials play a big part in sustainable solution’s, a lot can be done to develop more environmental methods for producing them. Both the process conditions and the resources that go in are important to consider. As more people understand that we need to manage our planet’s resources and ecosystem differently the demand for sustainable materials is increasing.

    Catalysis is a key for designing chemistry for the environment and an interesting alternative is enzyme catalysis. Enzymes are proteins working as catalysts in biochemical reactions. One of the most prominent features of enzymes’ is their selectivity, which means that they have preferences towards forming one product over others. Using enzymes’ as catalysts in synthetic chemical reactions the selectivity can be used to produce a wide range of products without side reaction occurring. Further benefits of using enzyme catalysis include high rate acceleration and working under mild reaction conditions.

    In the work presented here the selectivity and efficiency of enzymes have been combined with photochemistry in new efficient methods for the synthesis ofpolymeric materials. The enzymes used were the well-known lipase B form Candida antarctica and an esterase/acyltransferase from Mycobacterium smegmatis.

    The thesis divides into three parts in which three kinds of components were synthesized by enzyme catalysis: (i) unsaturated polyesters; (ii) vinyl ether building-blocks; and (iii) bio-based polyamides. In the first two parts the efficiency and selectivity of enzyme catalysis at low temperatures were utilized to synthesize building-blocks that can be further used for photopolymerization. By using enzyme catalysis structures that can be difficult or even impossible to access with conventional chemistry have been made. In part (iii) photochemistry was used to synthesize a monomer that was polymerized by enzyme catalysis to produce polyamides.

    All three parts presented in this thesis show the potential of the combination of enzymes and photochemistry to give access to polymeric materials under benign conditions. The work thus advances the capacity to manufacture building-blocks to create new sustainable polymeric materials.

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    Enzyme catalysis towards bio-based UV-curable buildingblocks
  • 33.
    Finnveden, Maja
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. KTH Royal Institute of Technology.
    Hendil-Forssell, Peter
    Claudino, Mauro
    Johansson, Mats
    KTH, Superseded Departments (pre-2005), Fibre and Polymer Technology.
    Martinelle, Mats
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Lipase Catalyzed Synthesis of renewable plant oil-based polyamidesManuscript (preprint) (Other academic)
    Abstract [en]

    Enzyme catalyzed synthesis towards renewable polyamides was investigated using Candida antarctica lipase B. A fatty acid-derived AB-type functional monomer, having one amine and one methyl ester functionality was homopolymerized at 80 and 140°C. Additionally, the organobase 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) was used as catalyst. The results from the two catalysts were comparable. However, the amount of lipase added was 1200 times lower showing that the lipase was a more efficient catalyst for this system as compared to TBD. Moreover, the AB type monomer was copolymerized with 1,12-diaminododecan to synthesize oligoamides of two different lengths.

  • 34.
    Finnveden, Maja
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Hendil-Forssell, Peter
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Claudino, Mauro
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Johansson, Mats
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Martinelle, Mats
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Lipase-Catalyzed Synthesis of Renewable Plant Oil-Based Polyamides.2019In: Polymers, E-ISSN 2073-4360, Vol. 11, no 11, article id 1730Article in journal (Refereed)
    Abstract [en]

    Enzyme catalyzed synthesis of renewable polyamides was investigated using Candida antarctica lipase B. A fatty acid-derived AB-type functional monomer, having one amine and one methyl ester functionality, was homopolymerized at 80 and 140 °C. Additionally, the organobase 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) was used as a catalyst. The results from the two catalysts were comparable. However, the amount of lipase added was 1.2 × 103 times lower, showing that the lipase was a more efficient catalyst for this system as compared to TBD. Moreover, the AB-type monomer was copolymerized with 1,12-diaminododecane to synthesize oligoamides of two different lengths.

  • 35. Fiorati, Andrea
    et al.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Svedendahl, Maria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. Pharem Biotech AB, Biovation Park, Forskargatan 20 J, SE-15136 Södertälje, Sweden.
    Tessaro, Davide
    Application of Transaminases in a Disperse System for the Bioamination of Hydrophobic Substrates2020In: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. 362, no 5, p. 1156-1166Article in journal (Refereed)
    Abstract [en]

    Abstract The challenging bioamination of hydrophobic substrates has been attained through the employment of a disperse system consisting of a combination of a low polarity solvent (e. g. isooctane or methyl-tert-butylether), a non-ionic surfactant and a minimal amount of water. In these conditions, amine transaminases (ATA) were shown to efficiently carry out the reductive amination of variously substituted cyclohexanones, providing good conversions often coupled with a superior stereoselectivity if compared with the corresponding chemical reductive amination. An array of synthetically useful 4-substituted aminocyclohexanes was consequentially synthesized through biocatalysis, analyzed and stereochemically characterized.

  • 36.
    Gullfot, Fredrika
    KTH, School of Biotechnology (BIO), Glycoscience.
    Synthesis of xyloglucan oligo- and polysaccharides with glycosynthase technology2009Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose.

    Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns.

    Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.

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    FULLTEXT01
  • 37.
    Guo, Fei
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Transaminase biocatalysis: optimization and application2017In: Green Chemistry, ISSN 1463-9262, E-ISSN 1463-9270, Vol. 19, no 2, p. 333-360Article in journal (Refereed)
    Abstract [en]

    Transaminases (TAs) are one of the most promising biocatalysts in organic synthesis for the preparation of chiral amino compounds. The concise reaction, excellent enantioselectivity, environmental friendliness and compatibility with other enzymatic or chemical systems have brought TAs to the attention of scientists working in the area of biocatalysis. However, to utilize TAs in a more efficient and economical way, attempts have to be made to optimize their performance. The demand for various substrate specificities, stability under non-physiological conditions and higher conversions in reversible reactions have been targeted and investigated thoroughly. A number of both protein- and process-based strategies have been developed to improve TAs and systems involving TAs. Moreover, by combination with other enzymes in cascade reactions or even in more complex systems, so called synthetic biology and systems biocatalysis, TAs can be biocatalysts with immense potential in the industrial production of high-value chemical products. This review will highlight strategies for optimization of TAs and will discuss a number of elegant systems for improving their performance. Transaminase biocatalysis has been, and will continue to be, one of the most interesting topics in green organic synthesis.

  • 38.
    Gustafsson, Camilla
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry.
    Vassiliev, Serguei
    Department of Biological Sciences, Brock University, Ontario, Canada.
    Kürten, Charlotte
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Syrén, Per-Olof
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Brinck, Tore
    MD Simulations Reveal Complex Water Paths in Squalene–Hopene Cyclase: Tunnel-Obstructing Mutations Increase the Flow of Water in the Active Site2017In: ACS Omega, E-ISSN 2470-1343, Vol. 2, no 11, p. 8495-8506Article in journal (Refereed)
    Abstract [en]

    Squalene–hopene cyclase catalyzes the cyclization of squalene to hopanoids. A previous study has identified a network of tunnels in the protein, where water molecules have been indicated to move. Blocking these tunnels by site-directed mutagenesis was found to change the activation entropy of the catalytic reaction from positive to negative with a concomitant lowering of the activation enthalpy. As a consequence, some variants are faster and others are slower than the wild type (wt) in vitro under optimal reaction conditions for the wt. In this study, molecular dynamics (MD) simulations have been performed for the wt and the variants to investigate how the mutations affect the protein structure and the water flow in the enzyme, hypothetically influencing the activation parameters. Interestingly, the tunnel-obstructing variants are associated with an increased flow of water in the active site, particularly close to the catalytic residue Asp376. MD simulations with the substrate present in the active site indicate that the distance for the rate-determining proton transfer between Asp376 and the substrate is longer in the tunnel-obstructing protein variants than in the wt. On the basis of the previous experimental results and the current MD results, we propose that the tunnel-obstructing variants, at least partly, could operate by a different catalytic mechanism, where the proton transfer may have contributions from a Grotthuss-like mechanism.

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    MD Simulations Reveal Complex Water Paths in Squalene–Hopene Cyclase: Tunnel-Obstructing Mutations Increase the Flow of Water in the Active Site
  • 39. Gustafsson, Kerstin
    et al.
    Blidberg, Eva
    Elfgren, I. K.
    Hellström, Anna
    Kylin, Henrik
    Gorokhova, E.
    Direct and indirect effects of the fungicide azoxystrobin in outdoor brackish water microcosms2010In: Ecotoxicology, ISSN 0963-9292, E-ISSN 1573-3017, Vol. 19, no 2, p. 431-444Article in journal (Refereed)
    Abstract [en]

    The effects of the strobilurin fungicide azoxystrobin were studied in brackish water microcosms, with natural plankton communities and sediment. Two experiments were conducted: Experiment 1 (nominal conc. 0, 15 and 60 mu g/L, 24-L outdoor microcosms for 21 days) and a second, follow-up, Experiment 2 (nominal conc. 0, 3, 7.5, 15 mu g/L, 4-L indoor microcosms for 12 days). The microcosms represent a simplified brackish water community found in shallow semi-enclosed coastal areas in agricultural districts in the Baltic Sea region. Measured water concentrations of the fungicide (Experiment 1) were, on average, 83 and 62% of nominal concentrations directly after application, and 25 and 30% after 21 days, for the low and high dose treatments, respectively, corresponding to mean DT50-values of 15.1 and 25.8 days, for low and high dose treatments, respectively. In Experiment 1, direct toxic effects on calanoid copepods at both test concentrations were observed. Similarly, in Experiment 2, the copepod abundance was significantly reduced at all tested concentrations. There were also significant secondary effects on zooplankton and phytoplankton community structure, standing stocks and primary production. Very few ecotoxicological studies have investigated effects of plant protection products on Baltic organisms in general and effects on community structure and function specifically. Our results show that azoxystrobin is toxic to brackish water copepods at considerably lower concentrations than previously reported from single species tests on freshwater crustaceans, and that direct toxic effects on this ecologically important group may lead to cascade effects altering lower food webs and ecosystem functioning.

  • 40.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Muraleedharan, Madhu Nair
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Surface Expression of omega-Transaminase in Escherichia coli2014In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 7, p. 2293-2298Article in journal (Refereed)
    Abstract [en]

    Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus omega-transaminase in Escherichia coli using a surface display vector based on the autotransporter adhesin involved in diffuse adherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity.

  • 41.
    Hassan, Noor
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Geiger, Barbara
    Gandini, Rosaria
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Patel, Bharat K. C.
    Kittl, Roman
    Haltrich, Dietmar
    Nguyen, Thu-Ha
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Tan, Tien Chye
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Engineering a thermostable Halothermothrix orenii beta-glucosidase for improved galacto-oligosaccharide synthesis2016In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 100, no 8, p. 3533-3543Article in journal (Refereed)
    Abstract [en]

    Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining beta-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a beta-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (-1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent k(cat), but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower k(cat)). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with similar to 10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs.

  • 42.
    Hendil-Forssell, Peter
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Rational engineering of esterases for improved amidase specificity in amide synthesis and hydrolysis2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Biocatalysis is an ever evolving field that uses enzymes or microorganisms for chemical synthesis. By utilizing enzymes that generally have evolved for specific reactions under mild conditions and temperatures, biocatalysis can be a more environmentally friendly option compared to traditional chemistry.

    Amide-type chemistries are important and bond formation avoiding poor atom economy is of high priority in organic chemistry. Biocatalysis could potentially be a solution but restricted substrate scope is a limitation. Esterases/lipases usually display broad substrate scope and catalytic promiscuity but are poor at hydrolyzing amides compared to amidases/proteases. The difference between the two enzyme classes is hypothesized to reside in one key hydrogen bond present in amidases, which facilitates the transition state for nitrogen inversion during catalysis.

    In this thesis the work has been focused on introducing a stabilizing hydrogen bond acceptor in esterases, mimicking that found in amidases, to develop better enzymatic catalysts for amide-based chemistries.

    By two strategies, side-chain or water interaction, variants were created in three esterases that displayed up to 210-times increased relative amidase specificity compared to the wild type. The best variant displayed reduced activation enthalpy corresponding to a weak hydrogen bond. The results show an estimated lower limit on how much the hydrogen bond can be worth to catalysis.

    MsAcT catalyze kinetically controlled N-acylations in water. An enzymatic one-pot one-step cascade was developed for the formation of amides from aldehydes in water that gave 97% conversion. In addition, engineered variants of MsAcT with increased substrate scope could synthesize an amide in water with 81% conversion, where the wild type gave no conversion. Moreover, variants of MsAcT displayed up to 32-fold change in specificity towards amide synthesis and a switch in reaction preference favoring amide over ester synthesis.

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  • 43.
    Hendil-Forssell, Peter
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Semlitsch, Stefan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Engineering the esterase/acyltransferase from Mycobacterium smegmatis: extended substrate scope for amide synthesis in waterManuscript (preprint) (Other academic)
    Abstract [en]

    Some esterases/lipases display high acyl transfer activity, favoring alcoholysis over hydrolysis, which make them valuable catalysts for synthesis reactions in aqueous media. An esterase from Mycobacterium smegmatis, MsAcT, has been characterized as an efficient catalyst for ester synthesis in water. The acyl donor specificity for MsAcT was however found to be very narrow and the enzyme displayed no activity towards esters with larger acyl group than butyrate. With rational engineering, the narrow acyl donor specificity of wild type MsAcT enzyme was altered and variants displaying extended substrate scope were generated. A double mutant, T93A/F154A, could accommodate methyl nonanoate as substrate, i.e. five carbons longer acyl group as compared to wild type, without compromising the acyl transfer capabilities. With similar selectivity towards a broad range of acyl donors (propionate to nonanoate) this is a more applicable catalyst than the wild type. Furthermore, the T93A/F154A variant was an efficient catalyst for synthesis of N-benzylhexanamide in water using methyl hexanoate as acyl donor, which is not a substrate for the wild type enzyme. The conversion reached 81% and the enzyme variant could potentially be used to produce amides in water with a wide variety of acyl donors.

  • 44.
    Hendil-Forssell, Peter
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Semlitsch, Stefan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Rational engineering of an esterase/acyltransferase for improved amidase specificity in amide synthesis and hydrolysisManuscript (preprint) (Other academic)
    Abstract [en]

    The esterase/acyltransferase from Mycobacterium smegmatis, MsAcT, display high acyltransfer capacity in water media with demonstrations found for both ester and amide syntheses. However, it has recently been discovered that esterases in contrast to amidases lack a key hydrogen bond in the transition state, donated by the scissile NH-group of the substrate. Esterases with improved amidase performance have been achieved with the introduction of amino-acid side chains or water network as hydrogen bond acceptors. Using the esterase from Mycobacterium smegmatis, MsAcT, the influence of this hydrogen bond was studied in both amide hydrolysis and synthesis, using a rational engineering approach. Two positions were selected for mutagenesis and enzyme variants with improved performance in amide synthesis and hydrolysis were generated. Compared to the wild-type, variant F154A had the highest absolute increase in amidase specificity (11-fold) and I194Q had the greatest change in relative amidase versus esterase reaction specificity (160-fold). The relative reaction specificities for amide over ester synthesis followed a similar trend as that of hydrolysis and the best variant was I194Q with a 32-fold increase compared to wt. Based on MD-simulations water seems to play an important role in the transition state as a hydrogen bond bridge between the NH-group of the amide substrate and the enzyme.

  • 45. Hernández-Ibáñez, N.
    et al.
    Montiel, V.
    Gomis-Berenguer, Alicia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry. CEMHTI, CNRS (UPR 3079) University of Orléans, 45071, Orléans, France.
    Ania, C.
    Iniesta, J.
    Effect of confinement of horse heart cytochrome c and formate dehydrogenase from Candida boidinii on mesoporous carbons on their catalytic activity2021In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 44, no 8, p. 1699-1710Article in journal (Refereed)
    Abstract [en]

    This study reports the immobilization of two biocatalysts (e.g., cytochrome c—Cyt c—and the non-metalloenzyme formate dehydrogenase from Candida boidinii–cbFDH) on a series of mesoporous carbons with controlled pore sizes. The catalytic activity of the nanoconfined proteins was correlated with the pore size distribution of the carbon materials used as supports. The electrochemical behaviour of nanoconfined Cyt c showed direct electron transfer electroactivity in pore sizes matching tightly the protein dimension. The pseudo-peroxidase activity towards H2O2 reduction was enhanced at pH 4.0, due to the protein conformational changes. For cbFDH, the reduction of CO2 towards formic acid was evaluated for the nanoconfined protein, in the presence of nicotinamide adenine dinucleotide (NADH). The carbons displayed different cbFDH uptake capacity, governed by the dimensions of the main mesopore cavities and their accessibility through narrow pore necks. The catalytic activity of nanoconfined cbFDH was largely improved, compared to its performance in free solution. Regardless of the carbon support used, the production of formic acid was higher upon immobilization with lower nominal cbFDH:NADH ratios. 

  • 46. Holmquist, M.
    et al.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Creation of a synthetically useful lipase with higher than wild-type enantioselectivity and maintained catalytic activity1999In: Organic Letters, ISSN 1523-7060, E-ISSN 1523-7052, Vol. 1, no 5, p. 763-765Article in journal (Refereed)
    Abstract [en]

    Formula presented Wild type I: 89.9% ee (E=32) Wild type II: 79.8% ee (E=10) Lipase hybrid: 95.4% ee (E=54) We have found that two Geotrichum candidum lipase isozymes have remarkably different abilities to differentiate between enantiomers of ethyl 2-methyldecanoate. By rational recombination of selected portions of the two isozymes, we have created a novel lipase with an enantioselectivity superior to that of the best wild-type parent isozyme. Site-directed mutagenesis identified two key amino acid residues responsible for the improved enantioselectivity without compromised total activity of the reengineered enzyme.

  • 47.
    Holmquist, Mats
    et al.
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Berglund, Per
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Improved lipase enantioselectivity by combinatorial and rational redesign.2000In: Abstracts of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 219, no 1, p. U163-U163Article in journal (Refereed)
  • 48. Hu, Y.
    et al.
    Zhu, Z.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden.
    Siewers, V.
    Heterologous transporter expression for improved fatty alcohol secretion in yeast2018In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 45, p. 51-58Article in journal (Refereed)
    Abstract [en]

    The yeast Saccharomyces cerevisiae is an attractive host for industrial scale production of biofuels including fatty alcohols due to its robustness and tolerance towards harsh fermentation conditions. Many metabolic engineering strategies have been applied to generate high fatty alcohol production strains. However, impaired growth caused by fatty alcohol accumulation and high cost of extraction are factors limiting large-scale production. Here, we demonstrate that the use of heterologous transporters is a promising strategy to increase fatty alcohol production. Among several plant and mammalian transporters tested, human FATP1 was shown to mediate fatty alcohol export in a high fatty alcohol production yeast strain. An approximately five-fold increase of fatty alcohol secretion was achieved. The results indicate that the overall cell fitness benefited from fatty alcohol secretion and that the acyl-CoA synthase activity of FATP1 contributed to increased cell growth as well. This is the first study that enabled an increased cell fitness for fatty alcohol production by heterologous transporter expression in yeast, and this investigation indicates a new potential function of FATP1, which has been known as a free fatty acid importer to date. We furthermore successfully identified the functional domain of FATP1 involved in fatty alcohol export through domain exchange between FATP1 and another transporter, FATP4. This study may facilitate a successful commercialization of fatty alcohol production in yeast and inspire the design of novel cell factories.

  • 49.
    Johansson, Johannes
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Development of a Novel Biocatalytic Cascade for the Valorisation of 5-(Hydroxymethyl)furfural2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The imminent shortage of fossil resources coupled with their associated environmental hazards stresses the need for the development of alternative, more sustainable chemicals. In this study an enzymatic cascade was developed for the valorisation of 5-(hydroxymethyl)furfural (HMF) into 5-(aminomethyl)-2-furfuraldehyde (AMFA). The cascade involves the transamination of HMF into 5-(hydroxymethyl)furfurylamine (HMFA) followed by the oxidation of HMFA into AMFA. Transaminases from Silicibacter pomeroyi (SpATA) was immobilised via his6-tags onto EziG-protein carriers from EnginZyme AB. The protein carriers were placed in spin-columns during the transamination which allowed for salvaging of the SpATA after the transamination of HMF. For the oxidation, alcohol dehydrogenases from Thermoanaerobacter brockii and horse liver as well as galactose oxidase from Dactylium dendroides (GOase) were evaluated. The conversion and product formation were analysed by HPLC. The results indicate that the SpATA efficiently catalyses the transamination of HMF, that the alcohol dehydrogenases are not able to catalyse the oxidation of HMF nor HMFA and that the GOase can oxidize HMFA with high conversion which leads us to believe that the proposed cascade for the valorisation of HMF to AMFA is feasible.

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  • 50. 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.

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