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  • 51.
    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)
  • 52.
    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)
  • 53.
    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)
  • 54.
    Engelmark Cassimjee, Karim
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Kourist, Robert
    University of Greifswald, Germany.
    Lindberg, Diana
    Uppsala university, SE.
    Wittrup Larsen, Marianne
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Widersten, Mikael
    Uppsala university, SE.
    Bornscheuer, Uwe T
    University of Greifswald, DE.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    A One Step General Enzyme Immobilization Method for Organic and Aqueous Solvents2008In: Book of Abstracts, 2008Conference paper (Refereed)
  • 55.
    Engelmark Cassimjee, Karim
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Marí­n, Sílvia Rodríguez
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Synthesis of cyclic polyamines by enzymatic generation of an amino aldehyde in situ2012In: Macromolecular rapid communications, ISSN 1022-1336, E-ISSN 1521-3927, Vol. 33, no 18, p. 1580-1583Article in journal (Refereed)
    Abstract [en]

    Multifunctional polycationic polyamines, for example, used in drug and gene delivery, have product range limitations in their synthesis methods. Here, we synthesize a polyamine by forming a self-assembling amino aldehyde from the corresponding amino alcohol with horse liver alcohol dehydrogenase (HLADH), followed by reduction. Circular polyamines were synthesized from 3-amino-propan-1-ol as starting material, analogous to cyclic polyamines formed from azetidin. The product had an isolated yield of 89.7% or 15.3 g L -1. The predicted range of possible polyamine products by this method is broad since many amino alcohols are putative substrates for HLADH. The enzyme also had activity for 2-amino-propan-1-ol and 2-amino-2-phenyl-ethanol, for which the enantioselectivity was 330 (S) and 32 (R), respectively.

  • 56.
    Engelmark Cassimjee, Karim
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Svedendahl Humble, Maria
    KTH, School of Biotechnology (BIO), Biochemistry.
    Land, Henrik
    KTH, School of Biotechnology (BIO), Biochemistry.
    Abedi, Vahak
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Chromobacterium violaceum ω-Transaminase VariantTrp60Cys Shows Increased Specificity for (S)-1-Phenylethylamine and 4’-Substituted Acetophenones, andFollows Swain-Lupton ParameterisationManuscript (preprint) (Other academic)
  • 57.
    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)
  • 58. Gaffney, Darragh
    et al.
    Abdallah, Noreldeen H.
    Cooney, Jakki C.
    Laffir, Fathima R.
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Hanefeld, Ulf
    Magner, Edmond
    Preparation and characterisation of a Ni2+/Co2+-cyclam modified mesoporous cellular foam for the specific immobilisation of His(6)-alanine racemase2014In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 109, p. 154-160Article in journal (Refereed)
    Abstract [en]

    Nickel and cobalt cyclam modified mesocellular foam (MCF) materials were prepared and characterised. The metal cyclam modified materials displayed reduced surface areas and pore diameters in comparison to MCF. The modified materials were used to specifically anchor a histidine tagged form of the enzyme, alanine racemase (HT-AlaR). Non-specific adsorption was predominantly hydrophobic//hydrophilic in nature and could be significantly reduced in the presence of 2% polyethylene glycol. The activity of HT-AlaR immobilised on Ni and Co-MCF was essentially the same as that of the free enzyme, demonstrating that enzymes can be specifically immobilised within the pores of mesoporous materials in a stable and catalytically active manner.

  • 59.
    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.

  • 60. Hauer, Bernhard
    et al.
    Engelmark Cassimjee, David Karim
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Process for producing polyamines2009Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    The present invention relates to a process for the production of a polyamine involving the use of enzymes; in particular to a process performed in aqueous environment; to the polyamines produced by said method; as well as the use of said polyamines for manufacturing paper, for immobilizing enzymes, or for preparing pharmaceutical or cosmetical compositions. The invention also relates to a novel method for in situ regeneration of cofactors NAD(P)+.

  • 61.
    Hedin, Eva. M. K.
    et al.
    KTH, Superseded Departments, Biotechnology.
    Patkar, S. A.
    KTH, Superseded Departments, Biotechnology.
    Vind, J.
    KTH, Superseded Departments, Biotechnology.
    Svendsen, A.
    KTH, Superseded Departments, Biotechnology.
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Berglund, Per
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Selective reduction and chemical modification of oxidized lipase cysteine mutants2002In: Canadian journal of chemistry (Print), ISSN 0008-4042, E-ISSN 1480-3291, Vol. 80, no 6, p. 529-539Article in journal (Refereed)
    Abstract [en]

    Thirteen single-cysteine mutants of the 33 kDa fungal triacylglycerol lipase Thermomyces (formerly Humicola) lanuginosa lipase (TLL, EC 3.1.1.3) Were produced and characterized for the purpose of site-directed chemical modification with spectroscopic reporter groups. All cysteine mutants were found to be predominantly blocked by oxidation to disulfides with endogenous cysteine during production. The fraction of lipase molecules with free sulfhydryl groups was analyzed by labeling with N-biotinylaminoethyl methanethiosulfonate, followed by a novel dot-blot method based on biotin-streptavidin interactions. A non-invasive method for the reduction of the introduced cysteine was elaborated for this protein containing three native disulfide bridges. The site-specifically reduced TLL mutants were then labeled with the sulfhydryl-specific reagents 2-(5-dimethylaminonaphth-1-ylsulfonamido)ethyl methanethiosulfonate or (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate, and studied by fluorescence and electron spin resonance (ESR) spectroscopy.

  • 62. Holmberg, E.
    et al.
    Holmquist, M.
    Hedenstrom, E.
    Berglund, P.
    Mid Sweden University.
    Norin, T.
    Hogberg, H. -E
    Hult, K.
    Reaction conditions for the resolution of 2-methylalkanoic acids in esterification and hydrolysis with lipase from Candida cylindracea1991In: Applied Microbiology and Biotechnology, ISSN 01757598 (ISSN), Vol. 35, no 5, p. 572-578Article in journal (Refereed)
    Abstract [en]

    We have demonstrated resolution of 2-methylalkanoic acids using lipase from Candida cylindracea as a catalyst. The resolution of 2-methyldecanoic acid was more successful than that of 2-methylbutyric acid both by esterification and hydrolysis. This indicates that the resolution of the acid is dependent on the chain length of the acid moiety. The chain length of the alcohol moiety of the ester affected the resolution of the long-chain acid only. Using esterification, (R)-2-methyldecanoic acid was produced in an enantiomeric excess (e.e.) of 95% (E = 40). If the enantiomeric ratio is low (E = 3.6), as in the resolution of 2-methylbutyric acid, esterification combined with a high equilibrium conversion could be used to yield the remaining acid in a high e.e. In the hydrolytic reactions, the e.e. and the equilibrium conversion were dependent on the pH and the presence of CaCl2. When octyl 2-methyldecanoate was hydrolysed at pH 8.0 in the presence of CaCl2, the (S)-acid was formed with an e.e. of 80% (E = 9), but when the hydrolysis was carried out at pH 7.5 without CaCl2, a very low e.e. and a low equilibrium conversion were observed. The latter conditions allowed the esterification of 2-methyldecanoic acid with 1-octanol even in aqueous medium.

  • 63. Holmquist, M.
    et al.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    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.

  • 64. Holmquist, M.
    et al.
    Martinelle, Mats
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Berglund, P.
    Mid Sweden University.
    Clausen, I. G.
    Patkar, S.
    Svendsen, A.
    Hult, K.
    Lipases from Rhizomucor miehei and Humicola lanuginosa: Modification of the lid covering the active site alters enantioselectivity1993In: Journal of Protein Chemistry, ISSN 02778033 (ISSN), Vol. 12, no 6, p. 749-757Article in journal (Refereed)
    Abstract [en]

    The homologous lipases from Rhizomucor miehei and Humicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed the S- enantiomer of 1-heptyl 2-methyldecanoate (R. miehei: E(S) = 8.5; H. lanuginosa: E(S) = 10.5), but the R-enantiomer of phenyl 2-methyldecanoate (E(R) = 2.9). Chemical arginine specific modification of the R. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (E(R) = 2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (E(S) = 2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (E(S) = 1.9) and increased the enantioselectivity with the aromatic ester (E(R) = 4.4) as substrates. The mutation, Glu 87 Ala, in the lid of the H. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (E(S) = 17.4) and a decreased enantioselectivity with the phenyl ester (E(R) = 2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.

  • 65.
    Holmquist, Mats
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Berglund, Per
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Improved lipase enantioselectivity by combinatorial and rational redesign.2000In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 219, no 1, p. U163-U163Article in journal (Refereed)
  • 66.
    Hult, Karl
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Berglund, Per
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Engineered enzymes for improved organic synthesis2003In: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 14, no 4, p. 395-400Article, review/survey (Refereed)
    Abstract [en]

    Recent developments to modify enzymes for use in organic synthesis have targeted several areas. These include altering the reaction mechanism of the enzyme to catalyse new reactions, switching substrate specificity, expanding substrate specificity, and improving substrate specificity, such as enantioselectivity in kinetic resolutions. Such modifications can be achieved either by rational redesign, which requires knowledge of the enzyme structure, or by random mutagenesis methods followed by screening. Both strategies of enzyme engineering can be successful and are very useful for improving the utility of enzymes for applied catalysis. Several examples illustrating these concepts in a variety of enzyme classes have appeared recently.

  • 67.
    Hult, Karl
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Enzyme promiscuity: mechanism and applications2007In: Trends in Biotechnology, ISSN 0167-7799, E-ISSN 1879-3096, Vol. 25, no 5, p. 231-238Article, review/survey (Refereed)
    Abstract [en]

    Introductory courses in biochemistry teach that enzymes are specific for their substrates and the reactions they catalyze. Enzymes diverging from this statement are sometimes called promiscuous. It has been suggested that relaxed substrate and reaction specificities can have an important role in enzyme evolution; however, enzyme promiscuity also has an applied aspect. Enzyme condition promiscuity has, for a long time, been used to run reactions under conditions of low water activity that favor ester synthesis instead of hydrolysis. Together with enzyme substrate promiscuity, it is exploited in numerous synthetic applications, from the laboratory to industrial scale. Furthermore, enzyme catalytic promiscuity, where enzymes catalyze accidental or induced new reactions, has begun to be recognized as a valuable research and synthesis tool. Exploiting enzyme catalytic promiscuity might lead to improvements in existing catalysts and provide novel synthesis pathways that are currently not available.

  • 68. Högberg, H. -E
    et al.
    Berglund, P.
    Mid Sweden University.
    Edlund, H.
    Fägerhag, J.
    Hedenström, E.
    Lundh, M.
    Nordin, O.
    Servi, S.
    Vörde, C.
    Biocatalysis as a useful tool in pheromone synthesis. Enantiomerically pure building blocks from baker's yeast reductions and enzyme catalysed resoluti1994In: Catalysis Today, ISSN 09205861 (ISSN), Vol. 22, no 3, p. 591-606Article in journal (Refereed)
    Abstract [en]

    Biocatalytical methods are presented which provide useful building blocks for pheromone synthesis. Examples of the utility of this approach are the preparation of building blocks for the synthesis of stereochemically pure isomers of pine sawfly pheromones and some other pheromones. Enantiom- erically pure ( 98% ee) 2-methyl-1-alkanols 2 were obtained via baker's yeast reduction of suitable α,β-unsaturated aldehydes, and by using lipases from Pseudomonas to effect resolution by transesterification of suitable racemic precursors to 2-methyl-1-alkanols 2 which gave high enantiomeric ratios E > 100. The resolution by esterification mediated by lipase from Candida rugosa of racemic 2-methylalkanoic acids also gave high enantiomeric ratios E> 100 after having improved the reaction conditions by regulating water activity, by choice of the appropriate complimentary substrate alcohol and by adjusting the initial equivalents of the latter present at the start. Also discussed is the separation of diastereomers of diprionol 1, which is naturally occurring in the pine sawfly Neodiprion sertifer, where it is the direct precursor of its pheromone. © 1994.

  • 69. Högberg, Hans-Erik
    et al.
    Edlund, Helen
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Hedenström, Erik
    WATER ACTIVITY INFLUENCES ENANTIOSELECTIVITY IN A LIPASE-CATALYZED RESOLUTION BY ESTERIFICATION IN AN ORGANIC-SOLVENT1993In: Tetrahedron: asymmetry, ISSN 0957-4166, E-ISSN 1362-511X, Vol. 4, no 10, p. 2123-2126Article in journal (Refereed)
    Abstract [en]

    The enantioselectivity of Candida rugosa lipase-mediated esterification of 2-methylalkanoic acids with n-alcohols in cyclohexane is dependent on water activity.

  • 70.
    Karlsson, Sara
    et al.
    KTH, School of Education and Communication in Engineering Science (ECE), Learning.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Education Assessment Exercise (EAE) - A path to quality enhancement in engineering education?2012In: Proceedings of the 40th SEFI Annual Conference 2012 - Engineering Education 2020: Meet the Future, European Society for Engineering Education (SEFI) , 2012Conference paper (Refereed)
    Abstract [en]

    In Sweden, as elsewhere, higher education institutions (HEIs) face a complex reality. Government, students, research councils and industry expect HEIs to deliver ever higher quality education and research, and HEIs need to have strategies to deal with such expectations. This paper explores one of the strategies chosen by KTH Royal Institute of Technology: self-initiated evaluation, exemplified by an Education Assessment Exercise (EAE) undertaken in 2011. In the paper, the context in which the project came about is discussed. Further, the project process, with emphasis on the self-evaluation phase, is described and analysed. This is in order to make an assessment on the benefits of the project so far, and to identify the issues that merit further consideration in future. THE CONTEXT In the paper, it is argued that the EAE project is best understood in the context of Swedish and European higher education politics and the on-going transformation thereof. The autonomy movement, in tandem with the expansion of quality assurance in higher education, are key characteristics of this transformation. The EAE is described as a strategic attempt by KTH management at channelling internal development needs and external demands into one project. Table 1 describes the EAE in relation to the scope and aims covered by the Standards and Guidelines for Quality Assurance in the European Higher Education Area (ESG) and by the Swedish National Agency for Higher Education (HSV) evaluations. THE EAE PROJECT The EAE project was a comprehensive internal evaluation of 90 education programmes at KTH, undertaken in 2011. It primarily had a formative aim. In the project process, it was hoped that new arenas for discussion and reflection would emerge which in turn would foster quality enhancement. In part, the EAE also had a summative aim. The intention was to assess whether the intended learning outcomes had been achieved by KTH students, and thereby provide a status report in advance of the external HSV evaluation scheduled to take place in the latter half of 2012. In the EAE, an internationally recognised methodology was used, comprising self-evaluation at programme level, followed by peer review including site visit and report by an external, internationally composed panel of assessors. The self-evaluation format included questions on the prerequisites, processes and outcomes of education delivery. The external panel of assessors comprised 50 subject area experts, students and industry representatives. LESSONS LEARNED AND ISSUES FOR FURTHER EXPLORATION The most welcome aspect of the EAE, and the clearest benefit of the project so far, was the opportunity to discuss educational quality matters, to form new networks and to identify strengths, weaknesses and ways forward. The project confirmed the strength of the enhancement approach as well as the importance of adapting evaluation methodology to local circumstances. Another lesson learned from the EAE was the importance of clarity regarding project aims. Above all, participants must have a clear picture of what the potential consequences, positive or negative, of the evaluation might be. Regardless of which approach is chosen, it needs to be well communicated and substantiated. A third lesson learned was that comprehensive evaluation projects of this type require a lot of time, energy and resources. In order to avoid evaluation fatigue and to get long-term benefits, it is important that the project dynamics are fed into a regular quality management system afterwards. The EAE project made clear reference to the national and the international policy context. Given the on-going transformation of the higher education sector, it will be an imperative task for KTH management to explore the new boundaries of autonomy and to work out future quality assurance strategies that suit internal needs as well as external expectations. In doing so, experiences from completing the EAE project will be of great value. Hopefully, sharing the experiences will be of value to other engineering education providers as well.

  • 71.
    Karlsson, Sara
    et al.
    KTH.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Malmström, Eva
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Ledarskap i kravfylld tid: utveckling genom utvärdering vid KTH2011Conference paper (Other academic)
  • 72. Kohls, Hannes
    et al.
    Anderson, Mattias
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Dickerhoff, Jonathan
    Weisz, Klaus
    Cordova, Armando
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Brundiek, Henrike
    Bornscheuer, Uwe T.
    Hoehne, Matthias
    Selective Access to All Four Diastereomers of a 1,3-Amino Alcohol by Combination of a Keto Reductase- and an Amine Transaminase-Catalysed Reaction2015In: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. 357, no 8, p. 1808-1814Article in journal (Refereed)
    Abstract [en]

    The biocatalytic synthesis of chiral amines has become a valuable addition to the chemists' tool-box. However, the efficient asymmetric synthesis of functionalised amines bearing more than one stereocentre, such as 1,3-amino alcohols, remains challenging. By employing a keto reductase (KRED) and two enantiocomplementary amine transaminases (ATA), we developed a biocatalytic route towards all four diastereomers of 4-amino-1-phenylpentane-2-ol as a representative molecule bearing the 1,3-amino alcohol functionality. Starting from a racemic hydroxy ketone, a kinetic resolution using an (S)-selective KRED provided optically active hydroxy ketone (86% ee) and the corresponding diketone. Further transamination of the hydroxy ketone was performed by either an (R)- or an (S)-selective ATA, yielding the (2R,4R)- and (2R,4S)-1,3-amino alcohol diastereomers. The remaining two diastereomers were accessible in two subsequent asymmetric steps: the diketone was reduced regio- and enantioselectively by the same KRED, which yielded the (S)-configured hydroxy ketone. Eventually, the subsequent transamination of the crude product with (R)- and (S)-selective ATAs yielded the remaining (2S,4R)and (2S,4S)-diastereomers, respectively.

  • 73.
    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.

  • 74.
    Liu, Rong
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Högberg, Hans-Erik
    Mittuniversitetet.
    Chemoenzymatic preparation of isomerically pure 3-bromo-2-butyl ethyl malonate esterManuscript (Other academic)
  • 75.
    Liu, Rong
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Högberg, Hans-Erik
    Mittuniversitetet.
    Preparation of the four stereoisomers of 3-bromo-2-butanol or their acetates via lipase-catalysed resolutions of the racemates derived from dl- or meso-2,3-butanediol.2005In: Tetrahedron: asymmetry, ISSN 0957-4166, E-ISSN 1362-511X, Vol. 16, p. 2607-2611Article in journal (Refereed)
    Abstract [en]

    The four stereoisomeric 3-bromo-2-butanols and/or their acetates were prepared via lipase-catalysed kinetic resolution by hydrolyses of the acetates of the (+/-)-syn- and (+/-)-anti-3-bromo-2-butanols, or via esterifications of the alc hols. The diastereomeric bromoacetates were obtained by syntheses from the dl- and meso-2,3-butanediols, respectively. On a preparative scale, the four stereoisomers, either as the free alcohols or as their acetates, were obtained in > 95% ee, and in 35-40% yield (based on the starting racemates).

  • 76.
    Marx, Lisa
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Ríos-Lombardía, Nicolás
    EntreChem SL.
    Süss, Philipp
    Enzymicals AG.
    Höhne, Matthias
    University of Greifswald.
    Morís, Francisco
    EntreChem SL.
    González-Sabín, Javier
    EntreChem SL.
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Chemoenzymatic Synthesis of SertralineManuscript (preprint) (Other academic)
  • 77.
    Marx, Lisa
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Süss, Philipp
    Enzymicals AG.
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Fractional Factorial Experimental Design and Scale-Up of a Transaminase ProcessManuscript (preprint) (Other academic)
  • 78.
    Marx, Lisa
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Süss, Philipp
    Enzymicals AG.
    Morís, Francisco
    EntreChem SL.
    González-Sabín, Javier
    EntreChem SL.
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Enzymatic one-pot two-step cascade for the synthesis of VyvanseManuscript (preprint) (Other academic)
  • 79.
    Palo-Nieto, Carlos
    et al.
    Mid Sweden University.
    Afewerki, Samson
    Anderson, Mattias
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Tai, Cheuk-Wai
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Córdova, Armando
    Integrated Heterogeneous Metal/Enzymatic Multiple Relay Catalysis for Eco-Friendly and Asymmetric Synthesis2016In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 6, p. 3932-3940Article in journal (Refereed)
    Abstract [en]

    Organic synthesis is in general performed using stepwise transformations where isolation and purification of key intermediates is often required prior to further reactions. Herein we disclose the concept of integrated heterogeneous metal/enzymatic multiple relay catalysis for eco-friendly and asymmetric synthesis of valuable molecules (e.g., amines and amides) in one-pot using a combination of heterogeneous metal and enzyme catalysts. Here reagents, catalysts, and different conditions can be introduced throughout the one-pot procedure involving multistep catalytic tandem operations. Several novel cocatalytic relay sequences (reductive amination/amidation, aerobic oxidation/reductive amination/amidation, reductive amination/kinetic resolution and reductive amination/dynamic kinetic resolution) were developed. They were next applied to the direct synthesis of various biologically and optically active amines or amides in one-pot from simple aldehydes, ketones, or alcohols, respectively.

  • 80.
    Ruggieri, Federica
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Campillo-Brocal, Jonatan C.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Chen, Shan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Humble, Maria S.
    Pharem Biotech AB.
    Walse, Björn
    SARomics Biostructures AB.
    Logan, Derek D. T.
    SARomics Biostructures AB.
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Insight into the dimer dissociation process of the Chromobacterium violaceum (S)-selective amine transaminase2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322Article in journal (Refereed)
    Abstract [en]

    One of the main factors hampering the implementation in industry of transaminase-based processes for the synthesis of enantiopure amines is their often low storage and operational stability. Our still limited understanding of the inactivation processes undermining the stability of wild-type transaminases represents an obstacle to improving their stability through enzyme engineering. In this paper we present a model describing the inactivation process of the well-characterized (S)-selective amine transaminase from Chromobacterium violaceum. The cornerstone of the model, supported by structural, computational, mutagenesis and biophysical data, is the central role of the catalytic lysine as a conformational switch. Upon breakage of the lysine-PLP Schiff base, the strain associated with the catalytically-active lysine conformation is dissipated in a slow relaxation process capable of triggering the known structural rearrangements occurring in the holo-to-apo transition and ultimately promoting dimer dissociation. Due to the occurrence in the literature of similar PLP-dependent inactivation models valid for other non-transaminase enzymes belonging to the same fold-class, the role of the catalytic lysine as conformational switch might extend beyond the transaminase enzyme group and offer new insight to drive future non-trivial engineering strategies.

  • 81.
    Ruggieri, Federica
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Campillo-Brocal, Jonatan C.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Chen, Shan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Humble, Maria S.
    Pharem Biotech AB.
    Walse, Björn
    SARomics Biostructures AB.
    Logan, Derek D. T.
    SARomics Biostructures AB.
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Insight into the dimer dissociation process of the Chromobacterium violaceum (S)-selective amine transaminase2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 16946Article in journal (Refereed)
    Abstract [en]

    One of the main factors hampering the implementation in industry of transaminase-based processes for the synthesis of enantiopure amines is their often low storage and operational stability. Our still limited understanding of the inactivation processes undermining the stability of wild-type transaminases represents an obstacle to improving their stability through enzyme engineering. In this paper we present a model describing the inactivation process of the well-characterized (S)-selective amine transaminase from Chromobacterium violaceum. The cornerstone of the model, supported by structural, computational, mutagenesis and biophysical data, is the central role of the catalytic lysine as a conformational switch. Upon breakage of the lysine-PLP Schiff base, the strain associated with the catalytically-active lysine conformation is dissipated in a slow relaxation process capable of triggering the known structural rearrangements occurring in the holo-to-apo transition and ultimately promoting dimer dissociation. Due to the occurrence in the literature of similar PLP-dependent inactivation models valid for other non-transaminase enzymes belonging to the same fold-class, the role of the catalytic lysine as conformational switch might extend beyond the transaminase enzyme group and offer new insight to drive future non-trivial engineering strategies.

  • 82. Scheidt, Thomas
    et al.
    Land, Henrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Anderson, Mattias
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Chen, Yujie
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Yi, Dong
    Fessner, Wolf-Dieter
    Fluorescence-Based Kinetic Assay for High-Throughput Discovery and Engineering of Stereoselective omega-Transaminases2015In: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. 357, no 8, p. 1721-1731Article in journal (Refereed)
    Abstract [en]

    omega-Transaminases are a valuable class of enzymes for the production of chiral amines with either (R)- or (S)-configuration in high optical purity and 100% yield by the biocatalytic reductive amination of prochiral ketones. A versatile new assay was developed to quantify omega-transaminase activity for the kinetic characterization and enantioselectivity typing of novel or engineered enzymes based on the conversion of 1-(6-methoxynaphth-2-yl)alkylamines. The associated release of the acetonaphthone product can be monitored by the development of its bright fluorescence at 450 nm with very high sensitivity and selectivity. The assay principle can be used to quantify omega-transaminase catalysis over a very broad range of enzyme activity. Because of its simplicity and low substrate consumption in microtiter plate format the assay seems suitable for liquid screening campaigns with large library sizes in the directed evolution of optimized transaminases. For assay substrates that incorporate structural variations, an efficient modular synthetic route was developed. This includes racemate resolution by lipase-catalyzed transacylation to furnish enantiomerically pure (R)and (S)-configured amines. The latter are instrumental for the rapid enantioselectivity typing of omega-transaminases. This method was used to characterize two novel (S)-selective taurine-pyruvate transaminases of the subtype 6a from thermophilic Geobacillus thermodenitrificans and G. thermoleovorans.

  • 83. Steffen-Munsberg, F.
    et al.
    Vickers, C.
    Thontowi, A.
    Schätzle, S.
    Meinhardt, T.
    Svedendahl Humble, M.
    Land, Henrik
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Bornscheuer, U. T.
    Höhne, M.
    Revealing the Structural Basis of Promiscuous Amine Transaminase Activity2013In: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 5, no 1, p. 154-157Article in journal (Refereed)
  • 84. Steffen-Munsberg, F.
    et al.
    Vickers, C.
    Thontowi, A.
    Schätzle, S.
    Tumlirsch, T.
    Svedendahl Humble, M.
    Land, Henrik
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Bornscheuer, U. T.
    Höhne, M.
    Connecting Unexplored Protein Crystal Structures to Enzymatic Function2013In: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 5, no 1, p. 150-153Article in journal (Refereed)
  • 85.
    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.

  • 86.
    Steffen-Munsberg, Fabian
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Greifswald University, Germany.
    Vickers, Clare
    Kohls, Hannes
    Land, Henrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Mallin, Hendrik
    Nobili, Alberto
    Skalden, Lilly
    van den Bergh, Tom
    Joosten, Henk-Jan
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Höhne, Matthias
    Bornscheuer, Uwe T.
    Bioinformatic analysis of a PLP-dependent enzyme superfamily suitable for biocatalytic applications2015In: Biotechnology Advances, ISSN 0734-9750, E-ISSN 1873-1899, Vol. 33, no 5, p. 566-604Article in journal (Refereed)
    Abstract [en]

    In this review we analyse structure/sequence-function relationships for the superfamily of PLP-dependent enzymes with special emphasis on class III transaminases. Amine transaminases are highly important for applications in biocatalysis in the synthesis of chiral amines. In addition, other enzyme activities such as racemases or decarboxylases are also discussed. The substrate scope and the ability to accept chemically different types of substrates are shown to be reflected in conserved patterns of amino acids around the active site. These findings are condensed in a sequence-function matrix, which facilitates annotation and identification of biocatalytically relevant enzymes and protein engineering thereof.

  • 87.
    Svedendahl Humble, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Biocatalytic Promiscuity2011In: European Journal of Organic Chemistry, ISSN 1434-193X, E-ISSN 1099-0690, no 19, p. 3391-3401Article, review/survey (Refereed)
    Abstract [en]

    Enzymes are attractive catalysts because of their promiscuity and their ability to perform highly regio-, chemo- and stereo-selective transformations. Enzyme promiscuity allows optimisation of industrial processes that require reaction conditions different from those in nature. Many enzymes can be used in reactions completely different from the reaction the enzyme originally evolved to perform. Such catalytically promiscuous reactions can be secondary activities hidden behind a native activity and might be discovered either in screening for that particular activity or, alternatively, by chance. Recently, researchers have designed enzymes to show catalytic promiscuity. It is also possible to design new enzymes from scratch by computer modelling (de novo design), but most work published to date starts from a known enzyme backbone. Promiscuous activity might also be induced or enhanced by rational design or directed evolution (or combinations thereof). Enzyme catalytic promiscuity provides fundamental knowledge about enzyme/substrate interactions and the evolution of new enzymes. New enzymes are required by industry, which needs to optimise chemical processes in an environmentally sustainable way. In this review various aspects of enzyme catalytic promiscuity are considered from a biocatalytic perspective.

  • 88.
    Svedendahl Humble, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry.
    Abedu, Vahak
    Federsel, Hans-Jürgen
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Key Amino Acid Residues for Reversed or Improved Enantiospecificity of an omega-Transaminase2012In: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 4, no 8, p. 1167-1172Article in journal (Refereed)
    Abstract [en]

    Transaminases inherently possess high enantiospecificity and are valuable tools for stereoselective synthesis of chiral amines in high yield from a ketone and a simple amino donor such as 2-propylamine. Most known ?-transaminases are (S)-selective and there is, therefore, a need of (R)-selective enzymes. We report the successful rational design of an (S)-selective ?-transaminase for reversed and improved enantioselectivity. Previously, engineering performed on this enzyme group was mainly based on directed evolution, with few exceptions. One reason for this is the current lack of 3D structures. We have explored the ?-transaminase from Chromobacterium violaceum and have used a homology modeling/rational design approach to create enzyme variants for which the activity was increased and the enantioselectivity reversed. This work led to the identification of key amino acid residues that control the activity and enantiomeric preference. To increase the enantiospecificity of the C. violaceum ?-transaminase, a possible single point mutation (W60C) in the active site was identified by homology modeling. By site-directed mutagenesis this enzyme variant was created and it displayed an E value improved up to 15-fold. In addition, to reverse the enantiomeric preference of the enzyme, two other point mutations (F88A/A231F) were identified. This double mutation created an enzyme variant, which displayed substrate dependent reversed enantiomeric preference with an E value shifted from 3.9 (S) to 63 (R) for 2-aminotetralin.

  • 89.
    Svedendahl Humble, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry.
    Håkansson, Maria
    Kimbung, Yengo R
    Walse, Björn
    Abedi, Vahak
    Federsel, Hans-Jürgen
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Logan, Derek T
    Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.2012In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, no 5, p. 779-792Article in journal (Refereed)
    Abstract [en]

    The bacterial ω-transaminase from Chromobacterium violaceum (Cv-ωTA, EC2.6.1.18) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5'-phosphate (PLP). Here, we present four crystal structures of Cv-ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular mass of ∼ 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo-Cv-ωTA structure reveals unique 'relaxed' conformations of three critical loops involved in structuring the active site that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered 'roof ' over the PLP-binding site, whereas in the other apo form and the holo form the 'roof' is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the 'roof' structure may be associated with substrate binding in Cv-ωTA and some other transaminases. DATABASE: The atomic coordinates and structure factors for the Chromobacterium violaceumω-transaminase crystal structures can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession codes 4A6U for the holoenzyme, 4A6R for the apo1 form, 4A6T for the apo2 form and 4A72 for the mixed form STRUCTURED DIGITAL ABSTRACT: •  -transaminases and -transaminases bind by dynamic light scattering (View interaction) •  -transaminase and -transaminase bind by x-ray crystallography (View interaction) •  -transaminase and -transaminase bind by x-ray crystallography (View interaction).

  • 90.
    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)
  • 91.
    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)
  • 92.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Branneby, Cecilia
    Lindberg, Lina
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Reversed Enantiopreference of an omega-Transaminase by a Single-Point Mutation2010In: CHEMCATCHEM, ISSN 1867-3880, Vol. 2, no 8, p. 976-980Article in journal (Refereed)
    Abstract [en]

    Altering the characteristics of an active-site loop in an (S)-selective omega-transaminase from Arthrobacter citreus (variant CNB05-01) influences the enantioselectivity. This active-site loop belongs to the second subunit of the dimeric enzyme structure that participates in the coordination of pyridoxal-5'-phosphate (PLP) in the so called "phosphate group binding cup". Three amino acid residues (E326, V328, and Y331) in this loop are selected by homology modeling for site-directed mutagenesis aiming to increase the enzyme enantioselectivity for 4-fluorophenylacetone. By combining these mutations, five enzyme variants are created. The performance of these variants is explored using a model system consisting of isopropylamine and 4-fluorophenylacetone or 4-nitroacetophenone in asymmetric synthesis using a whole-cell system approach. Three of the five variants show increased enantioselectivity for 4-fluorophenylacetone compared to CNB05-01. Variant CNB05-01/Y331C increases the enantioselectivity from 98% ee to over 99.5% ee. A single-point mutation, V328A, turn the (S)-selective omega-transaminase into an (R)-selective enzyme. This switch in enantioselectivity is substrate dependent, exhibiting (R) selectivity for 4-fluorophenylacetone and retaining (S) selectivity for 4-nitroacetophenone. The shift in enantiopreference is further confirmed by molecular docking simulations. Homology modeling is shown to be a powerful tool to target important amino acid residues in this enzyme in order to improve enantioselectivity by rational design.

  • 93.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Branneby, Cecilia
    Lindberg, Lina
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Reversed enantiopreference of an ω-transaminase by a single-point mutationManuscript (preprint) (Other academic)
  • 94.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Carlqvist, Peter
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Branneby, Cecilia
    KTH, School of Biotechnology (BIO), Biochemistry.
    Allnér, Olof
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Frise, Anton
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Brinck, Tore
    Direct Epoxidation in Candida antarctica Lipase B Studied by Experiment and Theory2008In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 9, no 15, p. 2443-2451Article in journal (Refereed)
    Abstract [en]

    Candida antarctica lipase B (CALB) is a promiscuous serine hydrolase that, besides its native function, catalyzes different side reactions, such as direct epoxidation. A single-point mutant of CALB demonstrated a direct epoxidation reaction mechanism for the epoxidation of alpha,beta-unsaturated aldehydes by hydrogen peroxide in aqueous and organic solution. Mutation of the catalytically active Ser105 to alanine made the previously assumed indirect epoxidation reaction mechanism impossible. Gibbs free energies, activation parameters, and substrate selectivities were determined both computationally and experimentally. The energetics and mechanism for the direct epoxidation in CALB Ser105Ala were investigated that the reaction proceeds through a two step-mechanism with formation of an oxyanionic intermediate. The active-site residue His224 functions as a general acid-base catalyst with support from Asp187. Oxyanion stabilization is facilitated by two hydrogen bonds from Thr40.

  • 95.
    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)
  • 96.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Branneby, C.
    Abedi, V.
    Wells, A.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    CASCAT: Redesign of omega-Transaminases for Synthesis of Chiral Amines2010In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 150, p. S123-S124Article in journal (Other academic)
    Abstract [en]

    Transaminases (EC 2.6.1.18) are attractive biocatalysts for synthesis of chiral amines and alpha-amino acids. These enzymes catalyze transfer of an amine group from a donor substrate to an acceptor compound using the cofactor pyridoxal-5′-phoshate (PLP). omega-Transaminases are a versatile subgroup of the transaminases that does not require a carboxylic acid group in alpha-position (in contradiction toalpha-transaminases) and hence accept a wider spectrum of ketones or amines. The omega-transaminases are employed industrially for production of both R- and S-enantiopure amines.

    One bottleneck is the unfavourable equilibrium in such reactions run in the synthesis mode. We have developed a one-pot multi-enzyme system in a cascade fashion for equilibrium displacement by removing formed acetone.

    Another issue is the fact that most omega-transaminases show S-selectivity, however a few R-selective strains do exist. We have used an S-selective omega-transaminase variant from Arthrobacter citreus and created an R-selective variant by rational redesign using a homology enzyme model. This homology modelling/rational design approach was further explored on an omega-transaminase from Chromobacterium violaceum.

  • 97.
    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)
  • 98.
    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)
  • 99.
    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.
    Fast carbon-carbon bond formation by a promiscuous lipase2005In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 127, no 51, p. 17988-17989Article in journal (Refereed)
  • 100.
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