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  • 151.
    Takwa, Mohamad
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Xiao, Yan
    Simpson, Neil
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Malmström, Eva M.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Koning, Cor.
    Heise, Andreas
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lipase Catalyzed HEMA Initiated Ring-Opening Polymerization: In Situ Formation of Mixed Polyester Methacrylates by Transesterification2008In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 9, no 2, p. 704-710Article in journal (Refereed)
    Abstract [en]

    2-Hydroxyethyl methacrylate (HEMA) was used as initiator for the enzymatic ring-opening polymerization (ROP) of ω-pentadecalactone (PDL) and ∈-caprolactone (CL). The lipase B from Candida antarctica was found to catalyze the cleavage of the ester bond in the HEMA end group of the formed polyesters, resulting in two major transesterification processes, methacrylate transfer and polyester transfer. This resulted in a number of different polyester methacrylate structures, such as polymers without, with one, and with two methacrylate end groups. Furthermore, the 1,2-ethanediol moiety (from HEMA) was found in the polyester products as an integral part of HEMA, as an end group (with one hydroxyl group) and incorporated within the polyester (polyester chains acylated on both hydroxyl groups). After 72 h, as a result of the methacrylate transfer, 79% (48%) of the initial amount of the methacrylate moiety (from HEMA) was situated (acylated) on the end hydroxyl group of the PPDL (PCL) polyester. In order to prepare materials for polymer networks, fully dimethacrylated polymers were synthesized in a one-pot procedure by combining HEMA-initiated ROP with end-capping using vinyl methacrylate. The novel PPDL dimethacrylate (>95% incorporated methacrylate end groups) is currently in use for polymer network formation. Our results show that initiators with cleavable ester groups are of limited use to obtain well-defined monomethacrylated macromonomers due to the enzyme-based transesterification processes. On the other hand, when combined with end-capping, well-defined dimethacrylated polymers (PPDL, PCL) were prepared.

  • 152.
    Tan, Tien Chye
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience.
    Wongnate, T.
    Sucharitakul, J.
    Krondorfer, I.
    Sygmund, C.
    Haltrich, D.
    Chaiyen, P.
    Peterbauer, C. K.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    The 1.6 Å Crystal Structure of Pyranose Dehydrogenase from Agaricus meleagris Rationalizes Substrate Specificity and Reveals a Flavin Intermediate2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 1Article in journal (Refereed)
    Abstract [en]

    Pyranose dehydrogenases (PDHs) are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a) position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O) activates oxygen by a mechanism that proceeds via a covalent flavin C(4a)-hydroperoxide intermediate. Although the flavin C(4a) adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2 position accessible for oxidation, whereas 2-fluorinated glucose performed poorly (C3 accessible), indicating that the glucose C2 position is the primary site of attack.

  • 153.
    Tan, Tien-Chye
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Regioselective Control of beta-D-Glucose Oxidation by Pyranose 2-Oxidase Is Intimately Coupled to Conformational Degeneracy2011In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 409, no 4, p. 588-600Article in journal (Refereed)
    Abstract [en]

    Trametes multicolor pyranose 2-oxidase (P2O) is a flavoprotein oxidase that oxidizes D-glucose at C2 to 2-keto-D-glucose by a highly regioselective mechanism. In this work, fluorinated sugar substrates were used as mechanistic probes to investigate the basis of regioselectivity in P2O. Although frequently used to study the mechanisms of glycoside hydrolases, our work provides the first example of applying these probes to sugar oxidoreductases. Our previous structure of the P2O mutant H167A in complex with the slow substrate 2-deoxy-2-fluoro-D-glucose showed a substrate-binding mode compatible with oxidation at C3. To accommodate the sugar, a gating segment, (FSY456)-F-454, in the substrate recognition loop partly unfolded to create a spacious and more polar active site that is distinct from the closed state of P2O. The crystal structure presented here shows that the preferred C2 oxidation where an ordered complex of P2O H167A with 3-deoxy-3-fluoro-D-glucose at 1.35 angstrom resolution was successfully trapped. In this semi-open C2-oxidation complex, the substrate recognition loop tightens to form an optimized substrate complex stabilized by interactions between Asp452 and glucose O4, as well as Tyr456 and the glucose O6 group, interactions that are not possible when glucose is positioned for oxidation at C3. The different conformations of the (FSY456)-F-454 gating segment in the semiopen and closed states induce backbone and side-chain movements of Thr169 and Asp452 that add further differential stabilization to the individual states. We expect the semi-open state (C2-oxidation state) and closed state to be good approximations of the active-site structure during the reductive half-reaction (sugar oxidation) and oxidative half-reaction (O-2 reduction).

  • 154.
    Tan, Tien-Chye
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Pitsawong, Warintra
    Wongnate, Thanyaporn
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Biochemistry. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Haltrich, Dietmar
    Chaiyen, Pimchai
    Divne, Christina
    KTH, School of Biotechnology (BIO), Biochemistry. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    H-Bonding and Positive Charge at the N(5)/O(4) Locus Are Critical for Covalent Flavin Attachment in Trametes Pyranose 2-Oxidase2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 402, no 3, p. 578-594Article in journal (Refereed)
    Abstract [en]

    Flavoenzymes perform a wide range of redox reactions in nature, and a subclass of flavoenzymes carry covalently bound cofactor. The enzyme-flavin bond helps to increase the flavin's redox potential to facilitate substrate oxidation in several oxidases. The formation of the enzyme-flavin covalent bond-the flavinylation reaction-has been studied for the past 40 years. For the most advocated mechanism of autocatalytic flavinylation, the quinone methide mechanism, appropriate stabilization of developing negative charges at the flavin N(1) and N(5) loci is crucial. Whereas the structural basis for stabilization at N(1) is relatively well studied, the structural requisites for charge stabilization at N(5) remain less clear. Here, we show that flavinylation of histidine 167 of pyranose 2-oxidase from Trametes multicolor requires hydrogen bonding at the flavin N(5)/O(4) locus, which is offered by the side chain of Thr169 when the enzyme is in its closed, but not open, state. Moreover, our data show that additional stabilization at N(5) by histidine 548 is required to ensure high occupancy of the histidyl flavin bond. The combination of structural and spectral data on pyranose 2-oxidase mutants supports the quinone methide mechanism. Our results demonstrate an elaborate structural fine-tuning of the active site to complete its own formation that couples efficient holoenzyme synthesis to conformational substates of the substrate-recognition loop and concerted movements of side chains near the flavinylation ligand. (c) 2010 Elsevier Ltd. All rights reserved.

  • 155. Vaida, Cristian
    et al.
    Takwa, Mohamad
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Keul, Helmut
    Möller, Martin
    gamma-Acyloxy-epsilon-Caprolactones: Synthesis, Ring-Opening Polymerization vs. Rearrangement by Means of Chemical and Enzymatic Catalysis2008In: Macromolecular Symposia, ISSN 1022-1360, E-ISSN 1521-3900, Vol. 272, p. 28-38Article in journal (Refereed)
    Abstract [en]

    gamma-Acyloxy-epsilon-caprolactones (3a-d) were prepared in two steps starting from 4-hydroxy-cyclohexanone (1). in the first step acylation of the hydroxyl group occurs and in the second step ring enlargement by Baeyer-Villiger oxidation. if this order of reaction is inverted rearrangement occurs in the Baeyer-Villiger oxidation Of 4-hydroxy-cyclohexanone leading to gamma-hydroxyethyl-gamma-butyrolactone. Using the first procedure gamma-acetyloxy- (33), gamma-benzoyloxy-(3B), gamma-acryloyloxy-(3c), and gamma-methacryloyloxy-epsilon-caprolactone (3d) were prepared. These monomers and for comparison reasons epsilon-caprolactone and gamma-methyl-epsilon-caprolactone were polymerized by means of chemical and enzymatic catalysis. The results were different depending on the monomer structure and catalyst used. in the presence of a chemical catalyst, all the monomers, except gamma-acetyloxy-epsilon-caprolactone, undergo controlled ring-opening polymerization. gamma-Acetyloxy-epsilon-caprolactone (3a), however, rearranges to a large extent under polymerization conditions to give gamma-acetyloxyethyl-gamma-butyrolactone (6a). in the presence of an enzyme (Novozyme 435, Lipase B from Candida antarctica (CALB) immobilized on a macroporous resin) all gamma-acyloxy-epsilon-caprolactones partly rearrange to result the corresponding gamma-acyloxy-gamma-butyrolactones, while epsilon-caproiactone and gamma-methyl-epsilon-caprolactone yield the corresponding polymers, the latter even in a stereoselective manner as reported earlier in the literature. A molecular dynamic study was performed with 3a and 3b as substrates to gain information on the substrate recognition displayed by CALB. A mechanism for the chemically and enzymatically catalyzed reactions of gamma-acyloxy-epsilon-caprolactones is proposed.

  • 156. Vallikivi, I.
    et al.
    Fransson, Linda
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Järving, I.
    Pehk, T.
    Samel, N.
    Tougu, V.
    Villo, L.
    Parve, O.
    The modelling and kinetic investigation of the lipase-catalysed acetylation of stereoisomeric prostaglandins2005In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 35, no 1-3, p. 62-69Article in journal (Refereed)
    Abstract [en]

    The lipase-catalysed acetylation of the hydroxyl groups of five stereoisomeric prostaglandins of type F was investigated by means of molecular dynamics simulations and the results compared with experimental observations. An NMR spectroscopic monitoring was performed to estimate reaction velocities and the regioselectivity. A molecular modelling protocol that could qualitatively differentiate between the OH groups of prostaglandins being either accessible or unaccessible to the Candida antarctica lipase B (CALB) catalysed acetylation was developed. The protocol developed analysed the protein structure deformation, the content of essential hydrogen bonds and the function-based subset energy of tetrahedral intermediates along the molecular dynamics simulations trajectory. The tetrahedral intermediates displaying a deformation RMS value lower than 3.0 angstrom, an essential hydrogen bond content over 50% and a subset energy less than -95 kJ/mol were classified active. In total, the accessibility of 16 out of 17 different prostaglandin OH groups was correctly predicted.

  • 157.
    Vallin, Michaela
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Mutant Lipase-Catalyzed Kinetic Resolution of Bulky Phenyl Alkyl sec-Alcohols: A Thermodynamic Analysis of Enantioselectivity2010In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 3, p. 411-416Article in journal (Refereed)
    Abstract [en]

    The size of the stereoselectivity pocket of Candida antarctica lipase B limits the range of alcohols that can be resolved with this enzyme. These steric constrains have been changed by increasing the size of the pocket by the mutation W104A. The mutated enzyme has good activity and enantioselectivity toward bulky secondary alcohols, such as 1-phenylalkanols, with alkyl chains up to eight carbon atoms. The S enantiomer was preferred in contrast to the wild-type enzyme, which has R selectivity. The magnitude of the enantioselectivity changes in an interesting way with the chain length of the alkyl moiety. It is governed by interplay between entropic and enthalpic contributions and substrates with long alkyl chains are resolved best with E values higher than 100. The enantioselectivity increases with temperature for the small substrates, but decreases for the long ones.

  • 158. Veld, Martijn A. J.
    et al.
    Fransson, Linda
    KTH, School of Biotechnology (BIO), Biochemistry.
    Palmans, Ania R. A.
    Meijer, E. W.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lactone Size Dependent Reactivity in Candida Antarctica Lipase B: A Molecular Dynamics and Docking Study2009In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 10, no 8, p. 1330-1334Article in journal (Refereed)
  • 159. Veld, Martijn A. J.
    et al.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Palmans, Anja R. A.
    Meijer, E. W.
    Fast DKR of amines using isopropyl 2-methoxyacetate as acyl donor2007In: European Journal of Organic Chemistry, ISSN 1434-193X, E-ISSN 1099-0690, no 32, p. 5416-5421Article in journal (Refereed)
    Abstract [en]

    The dynamic kinetic resolution (DKR) of various primary amine substrates was performed using a modified version of the Backvall system. A single equivalent of isopropyl 2-methoxyacetate was used as acyl donor in combination with p-MeO Shvo complex as the racemization catalyst and Novozym 435 as the acylation catalyst. A reaction temperature of 100 degrees C was employed to ensure a high racemization rate. Adding 2,4-dimethyl-3-pentanol (DMP) as hydrogen donor at a concentration of 0.5 m successfully suppressed side product formation. Under these modified DKR conditions, complete conversion was observed for most substrates within 26 It showing both high ee values and good chemoselectivity, whereas the original system required a reaction time of 72 h. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007).

  • 160.
    Vongvilai, Pornrapee
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Linder, Mats
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry (closed 20110630).
    Sakulsombat, Morakot
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Humble, Maria Svedendahl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Brinck, Tore
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry (closed 20110630).
    Ramström, Olof
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Racemase Activity of B. cepacia Lipase Leads to Dual-Function Asymmetric Dynamic Kinetic Resolution of alpha-Aminonitriles2011In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 50, no 29, p. 6592-6595Article in journal (Refereed)
    Abstract [en]

    Applaudable promiscuity: Racemase-type activity discovered for B. cepacia lipase with N-substituted α-aminonitriles is proposed to involve a C-C bond-breaking/forming mechanism in the hydrolase site of the enzyme, as supported by experimental data and calculations. This promiscuous activity in combination with the transacylation activity of the enzyme enabled the asymmetric synthesis of N-methyl α-aminonitrile amides in high yield (see scheme).

  • 161.
    Wang, Bo
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Land, Henrik
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    An efficient single-enzymatic cascade for asymmetric synthesis of chiral amines catalyzed by omega-transaminase2013In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 49, no 2, p. 161-163Article in journal (Refereed)
    Abstract [en]

    An efficient single-enzymatic cascade approach for the asymmetric synthesis of chiral amines has been developed, which applies the amino donor 3-aminocyclohexa-1,5-dienecarboxylic acid spontaneously tautomerizing to reach reaction completion with excellent ee values.

  • 162.
    Wang, Bo
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Liu, Y.
    Zhang, D.
    Feng, Y.
    Li, J.
    Efficient kinetic resolution of amino acids catalyzed by lipase AS 'Amano' via cleavage of an amide bond2012In: Tetrahedron: asymmetry, ISSN 0957-4166, E-ISSN 1362-511X, Vol. 23, no 18-19, p. 1338-1342Article in journal (Refereed)
    Abstract [en]

    Herein the efficient kinetic resolution of non-natural alpha-amino acids catalyzed by lipase AS 'Amano' via cleaving the amide bond is reported. The starting materials were the corresponding amino acid amides and the amino acids were generated with ees of up to 99% with E values of >600. These results indicated that the lipase AS 'Amano' could be a powerful amide hydrolase for the kinetic resolution of amino acid starting from the corresponding amino acid amides.

  • 163. Wang, Yi-Qiang
    et al.
    Shen, Ji-Kang
    Berglund, Torkel
    KTH, School of Biotechnology (BIO), Biochemistry.
    Ohlsson, Anna B.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Tang, Xiao-Feng
    Zhou, Zhai-Kui
    Wu, Ruo-Yan
    Zhou, Xiao-Hui
    Chen, Jie-Nan
    Analysis of expressed sequence tags from Ginkgo mature foliage in China2010In: TREE GENET GENOMES, ISSN 1614-2942, Vol. 6, no 3, p. 357-365Article in journal (Refereed)
    Abstract [en]

    Ginkgo biloba L. is a tree native to China, which has large importance within medicine and horticulture. The extracts from Ginkgo mature leaves with rich flavonoids and terpenoids are commonly used for a variety of folk remedies. We constructed a cDNA library derived from mature leaves of Ginkgo, which consisted of 8.12 x 10(5) clones with the insert length of 500-2,000 bp. We performed an analysis of expressed sequence tags (ESTs) and obtained partial sequences from 2,039 clones, which represented 1,437 unigenes consisting of 249 contigs and 1,188 singletons. The 2,039 ESTs were submitted to GenBank (dbEST) at NCBI and were assigned GenBank accession numbers from GE647881 to GE649919. The 1,235 cDNA clones out of 2,039 (60.1%) were assigned putative functions, and the remaining 804 clones were not similar to any known gene sequences in the databases. The five largest categories of Ginkgo clones were: "energy" (19.4%), "disease/defense" (16%), "metabolism" (11.3%), "unclassified proteins" (12.5%), and "secondary metabolism" (9%). The highly expressed transcripts in the cDNA library were some genes related to photosynthesis, disease/defense, and flavonoid biosynthesis, including ribulose-bisphosphate carboxylase small-chain gene, pathogenesis-related protein gene, light-harvesting chlorophyll a/b binding protein of photosystem gene, catalase gene, and phenylcoumaran benzylic ether reductase gene et al. Many genes with ESTs similar to photosynthesis, secondary metabolism, and stress-response genes were characterized. The analysis of ESTs indicates that it is a useful approach for isolating Ginkgo genes homologous to known genes. Our results provide new information about mature leaf-specific transcripts of Ginkgo.

  • 164.
    Wingstrand, Erica
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Lundgren, Stina
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Hamberg, Anders
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Moberg, Christina
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    655-ORGN - Synthesis of highly enantioenriched cyanohydrins by dual Lewis acid - Lewis base activation2007In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 234, p. 655-ORGN-Article in journal (Other academic)
    Abstract [en]

    Enantioenriched acylated cyanohydrins serve as versatile synthons and are themselves important synthetic targets.  By using our efficient catalytic dual Lewis acid - Lewis base activation system, a range of α-ketonitriles were added to both arom. and aliph. aldehydes affording highly enantioenriched O-acylated cyanohydrins in excellent yields.  The reactions proceeded smoothly in only one step with perfect atom economy.  Our recent results as well as the scope and limitations of the system will be presented together with mechanistic aspects.

  • 165.
    Wittrup Larsen, Marianne
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Expression of a lipase in prokaryote and eukaryote host systems allowing engineering2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Pseudozyma (Candida) antarctica lipase B (PalB) was expressed in Escherichia coli facilitating protein engineering. The lack of glycosylation was evaluated for a deeper understanding of the difficulties in expressing PalB in E. coli. Different systems were tested: periplasmic expression in Rosetta (DE3), cytosolic expression in Rosetta-gami 2(DE3), Origami 2(DE3), and coexpression of groES and groEL. Periplasmic expression resulted 5.2 mg/L active PalB at 16 °C in shake flasks. This expression level was improved by using the EnBase technology, enabling fed-batch cultivation in 24-deep well scale. The feed rate was titrated with the addition of α-amylase, which slowly releases glucose as energy source. Different media were evaluated where the EnBase mineral salt medium resulted in 7.0 mg/L of active PalB.

    Protein secreted directly into the media was obtained using the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter for screening and production of PalB in P. pastoris. A protease sensitive fusion protein CBM-PalB (cellulose-binding module) was used as a model system. When optimised, the expression system resulted in 46 mg/L lipase in 72 hours in shake flask, 37 mg/L lipase in 28 hours in 96-deep-well plate format, and 2.9 g PalB per 10 L bioreactor cultivation.

    The E. coli expression system was used to express a small focused library of PalB variants, designed to prevent water from entering the active site through a hypothesised tunnel. Screening of the library was performed with a developed assay, allowing for simultaneous detection of both transacylation and hydrolytic activity. From the library a mutant S47L, in which the inner part of the tunnel was blocked, was found to catalyse transacylation of vinyl butyrate in 20 mM butanol 14 times faster than hydrolysis. Water tunnels, assisting water in reaching the active sites, were furthermore found by molecular modelling in many hydrolases. Molecular modelling showed a specific water tunnel in PalB. This was supported by experimental data, where the double mutant Q46A S47L catalysed transacylation faster than hydrolysis compared to the wild type PalB.

  • 166.
    Wittrup Larsen, Marianne
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Bornscheuer, Uwe T.
    Expression of Candida antarctica lipase B in Pichia pastoris and various Escherichia coli systems2008In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 62, p. 90-97Article in journal (Refereed)
    Abstract [en]

    Candida antarctica lipase B (CALB) carrying a point mutation, N74S, resulting in a non-glycosylated protein was actively expressed in Pichia pastoris yielding 44 mg/L which was similar to that of the glycosylated CALB wild type expressed in A pastoris. Hence, the major obstacle in the Escherichia coli expression of CALB is not the lack of glycosylation. To understand and improve the expression of CALB in E. coli, a comprehensive investigation of four different systems were tested: periplasmic expression in Rosetta (DE3), cytosolic expression in Rosetta-gami 2(DE3) and Origami 2(DE3) as well as co-expression with chaperones groES and groEL in Origami B(DE3), all using the pET-22b(+) vector and the T7lac promoter.

    Furthermore the E. coli expression was carried out at three different temperatures (16, 25 and 37 C) to optimise the expression. Periplasmic expression resulted in highest amount of active CALB of the four systems, yielding a maximum of 5.2 mg/L culture at 16 degrees C, which is an improvement to previous reports.

    The specific activity of CALB towards tributyrin in E. coli was found to be the same for periplasmic and cytosolic expression. Active site titration showed that the CALB mutant N74S had a lower specific activity in comparison to wild type CALB regardless of expression host. The expected protein identity was confirmed by LC-ESI-MS analysis in E. coli, whereas in P. pastoris produced CALB carried four additional amino acids from an incomplete protein processing.

  • 167. Xiao, Yan
    et al.
    Takwa, Mohamad
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Koning, Cor E.
    Heise, Andreas
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    Systematic Comparison of HEA and HEMA as Initiators in Enzymatic Ring-Opening Polymerizations2009In: Macromolecular Bioscience, ISSN 1616-5187, E-ISSN 1616-5195, Vol. 9, no 7, p. 713-720Article in journal (Refereed)
    Abstract [en]

    Two initiators (HEA and HEMA) containing a cleavable ester bond were compared in the lipase-catalyzed ROP of CL and PDL. HEA and HEMA displayed similar reaction efficiencies as initiators (acyl acceptors) in the enzymatic ROP. However, transacylation reactions were found to be 15 times faster on the HEA-initiated polyesters as compared with the HEAAA initiated polyesters (HEA/HEMA moieties as acyl donors). While in both cases the amount of HEA- and HEMA-initiated polymers could be maximized by H short reaction times, a well-defined (meth)acrylation by this approach was not possible. Our results show that trans esterification reactions are present at high rates throughout the enzyme-catalyzed ROP.

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