| 1. |
- Bisagni, Serena, et al.
(författare)
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Cloning and expression of a Baeyer-Villiger monooxygenase oxidizing linear aliphatic ketones from Dietzia sp. D5
- 2014
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Ingår i: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1873-3158 .- 1381-1177. ; 109, s. 161-169
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Tidskriftsartikel (refereegranskat)abstract
- A Baeyer-Villiger monooxygenase has been identified in the genome sequence of Dietzia sp. D5. Sequence similarity search revealed that the enzyme belongs to a group of BVMOs that are closely related to ethionamide monooxygenase from Mycobacterium tuberculosis (EthA). The BVMO was expressed in E. coli BL21-CodonPlus(DE3)-RP and the best expression was achieved when the E. coli cells were cultivated in terrific broth (TB) at 15 degrees C and induced with 0.1 mM of IPTG. Since the purified enzyme did not show any measurable activity, the substrate scope of the BVMO has been determined using whole-cell and crude cell extract systems. The enzyme was most active towards linear aliphatic substrates. However, it has shown a moderate degree of conversion for cyclobutanone, 2-methylcyclohexanone, bicyclo[3.2.0]hept-2-en-6-one, phenylacetone and thioanisole. There was no detectable conversion of ethionamide, cyclohexanone and acetophenone. (C) 2014 Elsevier B.V. All rights reserved.
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| 2. |
- Blikstad, Cecilia, et al.
(författare)
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Functional characterization of a stereospecific diol dehydrogenase, FucO, from Escherichia coli : substrate specificity, pH dependence, kinetic isotope effects and influence of solvent viscosity
- 2010
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Ingår i: Journal of Molecular Catalysis B. - : Elsevier BV. - 1381-1177 .- 1873-3158. ; 66:1-2, s. 148-155
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Tidskriftsartikel (refereegranskat)abstract
- FucO, (S)-1,2-propanediol oxidoreductase, from Escherichia coli is involved in the anaerobic catabolic metabolism of L-fucose and L-rhamnose, catalyzing the interconversion of lactaldehyde to propanediol. The enzyme is specific for the S-enantiomers of the diol and aldehyde suggesting stereospecificity in catalysis. We have studied the enzyme kinetics of FucO with a spectrum of putative alcohol and aldehyde substrates to map the substrate specificity space. Additionally, for a more detailed analysis of the kinetic mechanism, pH dependence of catalysis, stereochemistry in hydride transfer, deuterium kinetic isotope effect of hydride transfer and effect of increasing solvent viscosity were also analyzed. The outcome of this study can be summarized as follows: FucO is highly stereospecific with the highest E-value measured to be 320 for the S-enantiomer of 1,2-propanediol. The enzyme is strictly regiospecific for oxidation of primary alcohols. The enzyme prefers short-chained (2-4 carbons) substrates and does not act on bulkier compounds such as phenyl-substituted alcohols. FucO is an 'A-side' dehydrogenase transferring the pro-R-hydrogen of NADH to the aldehyde substrate. The deuterium KIEs of kcat and kcat/KM were 1.9 and 4.2, respectively, illustrating that hydride transfer is partially rate-limiting but also that other reaction steps contribute to rate limitation of catalysis. Combining the KIE results with the observed effects of increasing medium viscosity proposed a working model for the kinetic mechanism involving slow, rate-limiting, product release and on-pathway conformational changes in the enzyme-nucleotide complexes.
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| 3. |
- Chávez, Georgina, et al.
(författare)
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Baeyer-Villiger oxidation with peracid generated in situ by CaLB-CLEA catalyzed perhydrolysis
- 2013
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Ingår i: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1873-3158 .- 1381-1177. ; 89, s. 67-72
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Tidskriftsartikel (refereegranskat)abstract
- Candida antarctica lipase B, immobilized as cross linked enzyme aggregates (CLEAs) was used to mediate the Baeyer-Villiger oxidation of cyclohexanone to epsilon-caprolactone, and the reaction was compared with the one using Novozym (R) 435 as catalyst. The conversion was dependent on the initial concentration of cyclohexanone, and was about 90% after 48 h at concentrations of up to 0.25 M but was decreased at higher concentrations. Caprolactone concentrations up to 0.6 M had no effect on the reaction efficiency. Among the cyclic ketones tested, the highest degree of conversion was achieved for cyclopentanone (88%) and the lowest for cyclooctanone (about 2%). The effect of methyl substitution and position of substitution on the cycloketone was studied using methylcyclohexanone and it has shown to influence the conversion efficiency. Both hydrogen peroxide and the reaction by-product acetic acid had a deleterious effect on the stability of the biocatalyst. (C) 2012 Elsevier B.V. All rights reserved.
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| 4. |
- Gaffney, Darragh, et al.
(författare)
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Preparation and characterisation of a Ni2+/Co2+-cyclam modified mesoporous cellular foam for the specific immobilisation of His(6)-alanine racemase
- 2014
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Ingår i: Journal of Molecular Catalysis B. - : Elsevier BV. - 1381-1177 .- 1873-3158. ; 109, s. 154-160
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Hamnevik, Emil, et al.
(författare)
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Kinetic characterization of Rhodococcus ruber DSM 44541 alcohol dehydrogenase A
- 2014
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Ingår i: Journal of Molecular Catalysis B. - : Elsevier BV. - 1381-1177 .- 1873-3158. ; 99, s. 68-78
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Tidskriftsartikel (refereegranskat)abstract
- An increasing interest in biocatalysis and the use of stereoselective alcohol dehydrogenases in synthetic asymmetric catalysis motivates detailed studies of potentially useful enzymes such as alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber. This enzyme is capable of catalyzing enantio-, and regioselective production of phenyl-substituted α-hydroxy ketones (acyloins) which are precursors for the synthesis of a range of biologically active compounds. In this study, we have determined the enzyme activity for a selection of phenyl-substituted vicinal diols and other aryl- or alkyl-substituted alcohols and ketones. In addition, the kinetic mechanism for the oxidation of (R)- and (S)-1-phenylethanol and the reduction of acetophenone has been identified as an Iso Theorell-Chance (hit and run) mechanism with conformational changes of the enzyme-coenzyme binary complexes as rate-determining for the oxidation of (S)-1-phenylethanol and the reduction of acetophenone. The underlying cause of the 270-fold enantiopreference for the (S)-enantiomer of 1-phenylethanol has been attributed to non-productive binding of the R-enantiomer. We have also shown that it is possible to tune the direction of the redox chemistry by adjusting pH with the oxidative reaction being favored at pH values above 7.
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| 6. |
- Hara, Piia, et al.
(författare)
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Supported ionic liquids in Burkholderia cepacia lipase-catalyzed asymmetric acylation
- 2010
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Ingår i: Journal of Molecular Catalysis B. - : Elsevier. - 1381-1177 .- 1873-3158. ; 67:1-2, s. 129-134
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Tidskriftsartikel (refereegranskat)abstract
- Lipase PS from Burkholderia cepacia was successfully immobilized on Kynol™ ACC 507-15 active carbon cloth with and without ionic liquids as SILE catalysts. Activity, enantioselectivity and reuse of the catalysts were evaluated in the acylation of 1-phenylethanol with vinyl acetate in toluene and in hexane over the temperature range 25 - 60 °C. The presence of [EMIM][NTf2] clearly stabilized the enzyme against inactivation and preserved enantioselectivity in reuse in a process which is affected by the nature of the IL, solvent and substrate structure.
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| 7. |
- Hedfors, Cecilia, et al.
(författare)
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Lipase chemoselectivity towards alcohol and thiol acyl acceptors in a transacylation reaction
- 2010
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Ingår i: Journal of Molecular Catalysis B. - : Elsevier BV. - 1381-1177 .- 1873-3158. ; 66:1-2, s. 120-123
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Tidskriftsartikel (refereegranskat)abstract
- The lipase chemoselectivity towards an alcohol and a thiol was investigated for the two lipases Candida antarctica lipase B (CalB) and Rhizomucor miehei lipase (Rml). Hexanol and hexanethiol were used as acyl acceptors in a transacylation reaction with ethyl octanoate in cyclohexane. CalB showed the highest chemoselectivity ratio (k(cat)/K-M)(OH)/(k(cat)/K-M)(SH), of 88,000 while the ratio for Rml was 1200. That could be compared with the ratio, k(OH)/k(SH), of 120 for the non-catalyzed reaction. Thus, the enzyme contribution to the chemoselectivity between hexanol and hexanethiol was 730 for CalB and 10 for Rml. High K-M values displayed towards hexanethiol (above 1.8 M) were the largest contribution to the selectivity. No saturation was achieved. The K-M values were more than two orders of magnitude higher than those of hexanol.
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| 8. |
- Henschel, Henning, et al.
(författare)
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Effect of the cross-linker on the general performance and temperature dependent behaviour of a molecularly imprinted polymer catalyst of a Diels-Alder reaction
- 2011
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Ingår i: Journal of Molecular Catalysis B. - : Elsevier BV. - 1381-1177 .- 1873-3158. ; 72:3-4, s. 199-205
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Tidskriftsartikel (refereegranskat)abstract
- Here we present a series of molecularly imprinted polymers capable of catalysing the Diels-Alder reaction between benzyl 1,3-butadienylcarbamate (1) and N,N-dimethyl acrylamide (2). The polymer systems studied here demonstrated an unusual cross-linker and temperature dependent behaviour, namely that polymer catalysis of the Diels-Alder reaction was lower at elevated temperature, in contrast to the solution reaction. Furthermore, not only was the catalytic activity significantly influenced by the choice of cross-linker, but in a similar fashion also the extent of the temperature effect, indicating a close relationship between catalysis and the observed inhibition. Molecular dynamics simulations of both the polymer systems studied were used to provide insight into the molecular background of transition state stabilisation, and differences in properties of the systems based on different cross-linkers.
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| 9. |
- Ibrahim, Victor, et al.
(författare)
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Laccase catalysed modification of lignin subunits and coupling to p-aminobenzoic acid
- 2013
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Ingår i: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1873-3158 .- 1381-1177. ; 97, s. 45-53
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Tidskriftsartikel (refereegranskat)abstract
- Laccase catalysed oxidation of syringyl and guaiacyl subunits of lignin and their modification with an aromatic amine, p-aminobenzoic acid (PABA) were investigated. Laccase from Galerina sp. HC1 isolated earlier by us was used as the main catalyst, and Trametes versicolor laccase was used for comparison. Among the syringyl compounds, syringic acid and syringaldehyde were oxidised to 2,6-dimethoxy-1,4-benzoquinone, and in the presence of PABA yielded a cross-coupling imine product. The reaction with methyl syringol resulted in several products whose structures were determined. The possible oxidative coupling pathways were proposed for the formation of the identified products. Oxidation of syringol and the guaiacyl compounds resulted mainly in homooligomers by free radical mechanism, with a negligible tendency of reaction with the nucleophilic group of PABA. Similar treatment of Eucalyptus Kraft lignin, which is rich in syringyl moieties, showed the presence of identical products obtained with syringic acid and syringaldehyde. (C) 2013 Elsevier B.V. All rights reserved.
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| 10. |
- Khan, Sami, et al.
(författare)
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Immobilization of thermostable beta-glucosidase variants on acrylic supports for biocatalytic processes in hot water
- 2012
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Ingår i: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1873-3158 .- 1381-1177. ; 80, s. 28-38
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Tidskriftsartikel (refereegranskat)abstract
- Two variants of the thermostable beta-glucosidase TnBgl1A (wt and N221S/P342L) from Thermotoga neapolitana were immobilized on acrylic supports (Eupergit (R) C, Eupergit (R) C250L, and cryogel) and evaluated at conditions close to the boiling point of water. Thermo-gravimetric analysis showed the supports to be stable <250 degrees C. Both wt and N221S/P342L were covalently bound to oxirane-groups respectively via glutaraldehyde spacers, and for coupling reactions 26 Lys and 20 Ser/Thr were surface-located. Immobilized enzymes were active on all supports in the temperature range 80-95 degrees C, but the observed specific activity was low (<= 19 U mg(-1)) using the cryogel. More than 91% of the initial activity was maintained after ten times recycling, and the same was recovered after 3 months storage at 4 degrees C for Eupergit (R) supports by simply incubating the preparation with bovine serum albumin. No storage loss was detectable on cryogels. The glutaraldehyde spacer improved activity on cryogels, but not on Eupergie supports. Immobilization on Eupergit (R) C250L yielded the highest observed specific activity (254 U mg(-1) for N221S/P342L) in a procedure including blocking of free oxirane-groups by BSA. This biocatalyst was used for on-line hydrolysis of quercetin-glucosides in a yellow onion extract at 80 degrees C, proving the immobilized biocatalyst to be promising in on-line systems for extraction and hydrolysis using hot pressurized water. (C) 2012 Elsevier B.V. All rights reserved.
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