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Nanna Bjarnholt - One of the best experts on this subject based on the ideXlab platform.
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Diversified glucosinolate metabolism: biosynthesis of hydrogen cyanide and of the Hydroxynitrile glucoside alliarinoside in relation to sinigrin metabolism in Alliaria petiolata
Frontiers in plant science, 2015Co-Authors: Tina Frisch, Mohammed Saddik Motawia, Carl Erik Olsen, Niels Agerbirk, Birger Lindberg Møller, Nanna BjarnholtAbstract:Alliaria petiolata (garlic mustard, Brassicaceae) contains the glucosinolate sinigrin as well as alliarinoside, a γ-Hydroxynitrile glucoside structurally related to cyanogenic glucosides. Sinigrin may defend this plant against a broad range of enemies, while alliarinoside confers resistance to specialized (glucosinolate-adapted) herbivores. Hydroxynitrile glucosides and glucosinolates are two classes of specialized metabolites, which generally do not occur in the same plant species. Administration of [UL-(14)C]-methionine to excised leaves of A. petiolata showed that both alliarinoside and sinigrin were biosynthesized from methionine. The biosynthesis of alliarinoside was shown not to bifurcate from sinigrin biosynthesis at the oxime level in contrast to the general scheme for Hydroxynitrile glucoside biosynthesis. Instead, the aglucon of alliarinoside was formed from metabolism of sinigrin in experiments with crude extracts, suggesting a possible biosynthetic pathway in intact cells. Hence, the alliarinoside pathway may represent a route to Hydroxynitrile glucoside biosynthesis resulting from convergent evolution. Metabolite profiling by LC-MS showed no evidence of the presence of cyanogenic glucosides in A. petiolata. However, we detected hydrogen cyanide (HCN) release from sinigrin and added thiocyanate ion and benzyl thiocyanate in A. petiolata indicating an enzymatic pathway from glucosinolates via allyl thiocyanate and indole glucosinolate derived thiocyanate ion to HCN. Alliarinoside biosynthesis and HCN release from glucosinolate-derived metabolites expand the range of glucosinolate-related defenses and can be viewed as a third line of defense, with glucosinolates and thiocyanate forming protein being the first and second lines, respectively.
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characterization of barley leaf tissue using direct and indirect desorption electrospray ionization imaging mass spectrometry
Journal of Mass Spectrometry, 2011Co-Authors: Nanna Bjarnholt, Steen Honore Hansen, Christian JanfeltAbstract:Chemical profiling of barley (Hordeum vulgare) leaves was demonstrated using direct and indirect desorption electrospray ionization (DESI) imaging mass spectrometry. Direct DESI analysis of the untreated leaves was not possible despite a significant content of Hydroxynitrile glucosides known to reside in the epidermis of the leaves. Instead, the epidermis was stripped off the leaves, thus allowing direct DESI imaging to be performed on the back of the epidermis. Furthermore, indirect DESI imaging was performed by making imprints in porous Teflon of the intact leaves as well as of the stripped epidermis. The DESI images reveal accumulation of Hydroxynitrile glucosides in the leaf epidermis, homogeneously distributed throughout the surface. The indirect DESI approach enables relative quantitation, confirming variations of Hydroxynitrile glucosides content in primary leaves of three different cultivars of barley seedlings. The study presents an example of how to overcome the morphological barriers from the plant surface and perform rapid and repeatable DESI imaging. In addition, a comparison is made of direct and indirect DESI imaging, contributing to the characterization of the recently developed method of indirect DESI imaging of plant material via porous Teflon imprints. Copyright © 2011 John Wiley & Sons, Ltd.
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characterization of barley leaf tissue using direct and indirect desorption electrospray ionization imaging mass spectrometry
Journal of Mass Spectrometry, 2011Co-Authors: Nanna Bjarnholt, Steen Honore Hansen, Christian JanfeltAbstract:Chemical profiling of barley (Hordeum vulgare) leaves was demonstrated using direct and indirect desorption electrospray ionization (DESI) imaging mass spectrometry. Direct DESI analysis of the untreated leaves was not possible despite a significant content of Hydroxynitrile glucosides known to reside in the epidermis of the leaves. Instead, the epidermis was stripped off the leaves, thus allowing direct DESI imaging to be performed on the back of the epidermis. Furthermore, indirect DESI imaging was performed by making imprints in porous Teflon of the intact leaves as well as of the stripped epidermis. The DESI images reveal accumulation of Hydroxynitrile glucosides in the leaf epidermis, homogeneously distributed throughout the surface. The indirect DESI approach enables relative quantitation, confirming variations of Hydroxynitrile glucosides content in primary leaves of three different cultivars of barley seedlings. The study presents an example of how to overcome the morphological barriers from the plant surface and perform rapid and repeatable DESI imaging. In addition, a comparison is made of direct and indirect DESI imaging, contributing to the characterization of the recently developed method of indirect DESI imaging of plant material via porous Teflon imprints.
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the β glucosidases responsible for bioactivation of Hydroxynitrile glucosides in lotus japonicus
Plant Physiology, 2008Co-Authors: Anne Vinther Morant, Carl Erik Olsen, Nanna Bjarnholt, Mads Emil Kragh, Christian Hauge Kjaergaard, Kirsten Jorgensen, Suzanne Michelle Paquette, Markus Piotrowski, Anne Imberty, Birger Lindberg MøllerAbstract:Lotus japonicus accumulates the Hydroxynitrile glucosides lotaustralin, linamarin, and rhodiocyanosides A and D. Upon tissue disruption, the Hydroxynitrile glucosides are bioactivated by hydrolysis by specific β-glucosidases. A mixture of two Hydroxynitrile glucoside-cleaving β-glucosidases was isolated from L. japonicus leaves and identified by protein sequencing as LjBGD2 and LjBGD4. The isolated Hydroxynitrile glucoside-cleaving β-glucosidases preferentially hydrolyzed rhodiocyanoside A and lotaustralin, whereas linamarin was only slowly hydrolyzed, in agreement with measurements of their rate of degradation upon tissue disruption in L. japonicus leaves. Comparative homology modeling predicted that LjBGD2 and LjBGD4 had nearly identical overall topologies and substrate-binding pockets. Heterologous expression of LjBGD2 and LjBGD4 in Arabidopsis (Arabidopsis thaliana) enabled analysis of their individual substrate specificity profiles and confirmed that both LjBGD2 and LjBGD4 preferentially hydrolyze the Hydroxynitrile glucosides present in L. japonicus. Phylogenetic analyses revealed a third L. japonicus putative Hydroxynitrile glucoside-cleaving β-glucosidase, LjBGD7. Reverse transcription-polymerase chain reaction analysis showed that LjBGD2 and LjBGD4 are expressed in aerial parts of young L. japonicus plants, while LjBGD7 is expressed exclusively in roots. The differential expression pattern of LjBGD2, LjBGD4, and LjBGD7 corresponds to the previously observed expression profile for CYP79D3 and CYP79D4, encoding the two cytochromes P450 that catalyze the first committed step in the biosyntheis of Hydroxynitrile glucosides in L. japonicus, with CYP79D3 expression in aerial tissues and CYP79D4 expression in roots.
Christian Janfelt - One of the best experts on this subject based on the ideXlab platform.
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visualizing metabolite distribution and enzymatic conversion in plant tissues by desorption electrospray ionization mass spectrometry imaging
Plant Journal, 2013Co-Authors: Camilla Knudsen, Adam M Takos, Fred Rook, Steen Honore Hansen, Birger Lindberg Møller, Kirsten Jorgensen, Natascha K K Hansen, Rubini Kannangara, Soren Bak, Christian JanfeltAbstract:SUMMARYIn comparison with the technology platforms developed to localize transcripts and proteins, imaging toolsfor visualization of metabolite distributions in plant tissues are less well developed and lack versatility. Thishampers our understanding of plant metabolism and dynamics. In this study, we demonstrate that desorp-tion electrospray ionization mass spectrometry imaging (DESI-MSI) of tissue imprints on porous Teflon maybe used to accurately image the distribution of even labile plant metabolites such as Hydroxynitrile gluco-sides, which normally undergo enzymatic hydrolysis by specific b-glucosidases upon cell disruption. Thisfast and simple sample preparation resulted in no substantial differences in the distribution and ratios of allHydroxynitrile glucosides between leaves from wild-type Lotus japonicus and a b-glucosidase mutant plantthat lacks the ability to hydrolyze certain Hydroxynitrile glucosides. In wild-type, the enzymatic conversionof Hydroxynitrile glucosides and the concomitant release of glucose were easily visualized when a restrictedarea of the leaf tissue was damaged prior to sample preparation. The gene encoding the first enzyme inHydroxynitrile glucoside biosynthesis in L. japonicus leaves, CYP79D3, was found to be highly expressedduring the early stages of leaf development, and the Hydroxynitrile glucoside distribution in mature leavesreflected this early expression pattern. The utility of direct DESI-MSI of plant tissue was demonstrated usingcryo-sections of cassava (Manihot esculenta) tubers. The Hydroxynitrile glucoside levels were highest inthe outer cell layers, as verified by LC–MS analyses. The unexpected discovery of a Hydroxynitrile-deriveddi-glycoside shows the potential of DESI-MSI to discoverand guide investigations into newmetabolic routes.Keywords: metabolite imaging, DESI-MS, cyanogenic glucosides, Hydroxynitrile glucosides, Lotus japoni-cus, Manihot esculenta, Sorghum bicolor, technical advance.INTRODUCTIONAlthough methods for imaging the tissue- and cell-specificlocalization of mRNA and proteins are established tools inplant science for the study of gene expression and proteinfunction and dynamics, techniques for imaging the distri-bution of small molecules in plant tissues are mostly in thedevelopment phase. The metabolite distribution may differfrom the expression pattern of the genes and the localizationof the enzymes responsible for their biosynthesis and subse-quent conversions, and changes in metabolite distributionsmay occur in the absence of changes in gene expression.Therefore, visualizing the distribution and dynamics ofsmall molecules is desirable for understanding the physio-logical role and fate of metabolites in plant growth anddevelopment and in defense reactions as a responseto environmental challenges. The enormous structuraldiversity among the small molecules present in plantsposes a significant challenge for development of imagingtechniques that are applicable to the vast majority of
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characterization of barley leaf tissue using direct and indirect desorption electrospray ionization imaging mass spectrometry
Journal of Mass Spectrometry, 2011Co-Authors: Nanna Bjarnholt, Steen Honore Hansen, Christian JanfeltAbstract:Chemical profiling of barley (Hordeum vulgare) leaves was demonstrated using direct and indirect desorption electrospray ionization (DESI) imaging mass spectrometry. Direct DESI analysis of the untreated leaves was not possible despite a significant content of Hydroxynitrile glucosides known to reside in the epidermis of the leaves. Instead, the epidermis was stripped off the leaves, thus allowing direct DESI imaging to be performed on the back of the epidermis. Furthermore, indirect DESI imaging was performed by making imprints in porous Teflon of the intact leaves as well as of the stripped epidermis. The DESI images reveal accumulation of Hydroxynitrile glucosides in the leaf epidermis, homogeneously distributed throughout the surface. The indirect DESI approach enables relative quantitation, confirming variations of Hydroxynitrile glucosides content in primary leaves of three different cultivars of barley seedlings. The study presents an example of how to overcome the morphological barriers from the plant surface and perform rapid and repeatable DESI imaging. In addition, a comparison is made of direct and indirect DESI imaging, contributing to the characterization of the recently developed method of indirect DESI imaging of plant material via porous Teflon imprints. Copyright © 2011 John Wiley & Sons, Ltd.
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characterization of barley leaf tissue using direct and indirect desorption electrospray ionization imaging mass spectrometry
Journal of Mass Spectrometry, 2011Co-Authors: Nanna Bjarnholt, Steen Honore Hansen, Christian JanfeltAbstract:Chemical profiling of barley (Hordeum vulgare) leaves was demonstrated using direct and indirect desorption electrospray ionization (DESI) imaging mass spectrometry. Direct DESI analysis of the untreated leaves was not possible despite a significant content of Hydroxynitrile glucosides known to reside in the epidermis of the leaves. Instead, the epidermis was stripped off the leaves, thus allowing direct DESI imaging to be performed on the back of the epidermis. Furthermore, indirect DESI imaging was performed by making imprints in porous Teflon of the intact leaves as well as of the stripped epidermis. The DESI images reveal accumulation of Hydroxynitrile glucosides in the leaf epidermis, homogeneously distributed throughout the surface. The indirect DESI approach enables relative quantitation, confirming variations of Hydroxynitrile glucosides content in primary leaves of three different cultivars of barley seedlings. The study presents an example of how to overcome the morphological barriers from the plant surface and perform rapid and repeatable DESI imaging. In addition, a comparison is made of direct and indirect DESI imaging, contributing to the characterization of the recently developed method of indirect DESI imaging of plant material via porous Teflon imprints.
Ulf Hanefeld - One of the best experts on this subject based on the ideXlab platform.
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Hydroxynitrile lyases covalently immobilized in continuous flow microreactors
Catalysis Science & Technology, 2019Co-Authors: Michelle P Van Der Helm, Paula Bracco, Hanna Busch, Katarzyna Szymanska, Andrzej B Jarzebski, Ulf HanefeldAbstract:Enzymes are supreme catalysts when it comes to high enantiopurities and their immobilization will pave the way for continuous operation. In this context, we show the covalent immobilization of Hydroxynitrile lyases HbHNL (from Hevea brasiliensis) and MeHNL (from Manihot esculenta) in a siliceous monolithic microreactor for continuous operation. A thorough characterization of the immobilized HNLs on mesoporous silicates indicated the conditions essential for a successful immobilization. Their application in a continuous flow system enabled a remarkably fast (3.2 min) production of chiral cyanohydrins with high conversion (97%) and high ee (98%) using minimal enzyme loading (STY = 71 g L−1 h−1 mgprotein−1). MeHNL showed increased operational stability, possibly due to a structural difference. The continuous flow microreactor outperformed batch systems, demonstrating the advantage of the mesoporous/macroporous environment for the expression of enzyme activity and the favorable characteristics of the microreactor. Overall, the system shows great potential for future industrial application of biocatalytic asymmetric syntheses.
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Immobilization of Prunus amygdalus Hydroxynitrile Lyase on Celite
'MDPI AG', 2018Co-Authors: Paula Bracco, Guzman Torrelo, Sander Noordam, Glenn De Jong, Ulf HanefeldAbstract:The Hydroxynitrile lyase from Prunus amygdalus was immobilized on Celite R-633. The immobilized enzyme could successfully be utilized in buffer saturated MTBE and excellent conversions of benzaldehyde to R-mandelonitrile were observed. No leaching occurred. To achieve high enantioselectivities, the suppression of the undesired background reaction was essential. This could be achieved by high enzyme loadings and the tight packing of the immobilized enzymes. When the immobilized enzyme is loosely packed, both the enzyme catalysis and the background reaction accelerates and only a modest enantioselectivity is observed. The enzyme was recycled for up to ten times, with some loss of activity and also enantioselectivity after 5 cycles, independent of packing
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enantioselective synthesis of cyanohydrins catalysed by Hydroxynitrile lyases a review
Organic and Biomolecular Chemistry, 2016Co-Authors: Paula Bracco, Jan Von Langermann, Hanna Busch, Ulf HanefeldAbstract:The first enantioselective synthesis was the selective addition of cyanide to benzaldehyde catalysed by a Hydroxynitrile lyase (HNL). Since then these enzymes have been developed into a reliable tool in organic synthesis. HNLs to prepare either the (R)- or the (S)-enantiomer of the desired cyanohydrin are available and a wide variety of reaction conditions can be applied. As a result of this, numerous applications of these enzymes in organic synthesis have been described. Here the examples of the last decade are summarised, the enzyme catalysed step is discussed and the follow-up chemistry is shown. This proves HNLs to be part of main stream organic synthesis. Additionally the newest approaches via immobilisation and reaction engineering are introduced.
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EPR Study of Substrate Binding to Mn(II) in Hydroxynitrile Lyase from Granulicella tundricola
2016Co-Authors: Femke Vertregt, Ulf Hanefeld, Guzman Torrelo, Sarah Trunk, Helmar Wiltsche, Wilfred R. Hagen, Kerstin SteinerAbstract:GtHNL from Granulicella tundricola is a Mn(II) containing Hydroxynitrile lyase with a cupin fold. The quasi-octahedral manganese is pentacoordinated by the enzyme. It catalyzes the enantioselective addition of HCN to aldehydes, yielding R-cyanohydrins. On the Lewis acidic vacant coordination site the Mn binds either substrate or the product, leading to a hexacoordinated 17 electron complex. EPR spectra of the active enzyme are unusually wide with a zero-field splitting approximately equal to the X-band microwave energy. A spectral change is induced by incubation with either one of the substrates/products HCN, benzaldehyde, and/or mandelonitrile. This points toward Mn(II) catalyzed cyanohydrin synthesis
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Immobilisation of Hydroxynitrile lyases
Chemical Society reviews, 2013Co-Authors: Ulf HanefeldAbstract:Hydroxynitrile lyases are a versatile group of enzymes that are applied both in the laboratory and on an industrial scale. What makes them particularly interesting is that to date five structurally unrelated categories of Hydroxynitrile lyases have been discovered. Given their great importance they have often been immobilised utilising many different methodologies. Therefore the Hydroxynitrile lyases are ideally suited to compare different immobilisation methods and their dependence on the structural features of the enzyme in question, since the activity is the same in all cases. This review examines all the different immobilisation methods applied to Hydroxynitrile lyases and draws conclusions on the effect of the approach.
Steen Honore Hansen - One of the best experts on this subject based on the ideXlab platform.
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visualizing metabolite distribution and enzymatic conversion in plant tissues by desorption electrospray ionization mass spectrometry imaging
Plant Journal, 2013Co-Authors: Camilla Knudsen, Adam M Takos, Fred Rook, Steen Honore Hansen, Birger Lindberg Møller, Kirsten Jorgensen, Natascha K K Hansen, Rubini Kannangara, Soren Bak, Christian JanfeltAbstract:SUMMARYIn comparison with the technology platforms developed to localize transcripts and proteins, imaging toolsfor visualization of metabolite distributions in plant tissues are less well developed and lack versatility. Thishampers our understanding of plant metabolism and dynamics. In this study, we demonstrate that desorp-tion electrospray ionization mass spectrometry imaging (DESI-MSI) of tissue imprints on porous Teflon maybe used to accurately image the distribution of even labile plant metabolites such as Hydroxynitrile gluco-sides, which normally undergo enzymatic hydrolysis by specific b-glucosidases upon cell disruption. Thisfast and simple sample preparation resulted in no substantial differences in the distribution and ratios of allHydroxynitrile glucosides between leaves from wild-type Lotus japonicus and a b-glucosidase mutant plantthat lacks the ability to hydrolyze certain Hydroxynitrile glucosides. In wild-type, the enzymatic conversionof Hydroxynitrile glucosides and the concomitant release of glucose were easily visualized when a restrictedarea of the leaf tissue was damaged prior to sample preparation. The gene encoding the first enzyme inHydroxynitrile glucoside biosynthesis in L. japonicus leaves, CYP79D3, was found to be highly expressedduring the early stages of leaf development, and the Hydroxynitrile glucoside distribution in mature leavesreflected this early expression pattern. The utility of direct DESI-MSI of plant tissue was demonstrated usingcryo-sections of cassava (Manihot esculenta) tubers. The Hydroxynitrile glucoside levels were highest inthe outer cell layers, as verified by LC–MS analyses. The unexpected discovery of a Hydroxynitrile-deriveddi-glycoside shows the potential of DESI-MSI to discoverand guide investigations into newmetabolic routes.Keywords: metabolite imaging, DESI-MS, cyanogenic glucosides, Hydroxynitrile glucosides, Lotus japoni-cus, Manihot esculenta, Sorghum bicolor, technical advance.INTRODUCTIONAlthough methods for imaging the tissue- and cell-specificlocalization of mRNA and proteins are established tools inplant science for the study of gene expression and proteinfunction and dynamics, techniques for imaging the distri-bution of small molecules in plant tissues are mostly in thedevelopment phase. The metabolite distribution may differfrom the expression pattern of the genes and the localizationof the enzymes responsible for their biosynthesis and subse-quent conversions, and changes in metabolite distributionsmay occur in the absence of changes in gene expression.Therefore, visualizing the distribution and dynamics ofsmall molecules is desirable for understanding the physio-logical role and fate of metabolites in plant growth anddevelopment and in defense reactions as a responseto environmental challenges. The enormous structuraldiversity among the small molecules present in plantsposes a significant challenge for development of imagingtechniques that are applicable to the vast majority of
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characterization of barley leaf tissue using direct and indirect desorption electrospray ionization imaging mass spectrometry
Journal of Mass Spectrometry, 2011Co-Authors: Nanna Bjarnholt, Steen Honore Hansen, Christian JanfeltAbstract:Chemical profiling of barley (Hordeum vulgare) leaves was demonstrated using direct and indirect desorption electrospray ionization (DESI) imaging mass spectrometry. Direct DESI analysis of the untreated leaves was not possible despite a significant content of Hydroxynitrile glucosides known to reside in the epidermis of the leaves. Instead, the epidermis was stripped off the leaves, thus allowing direct DESI imaging to be performed on the back of the epidermis. Furthermore, indirect DESI imaging was performed by making imprints in porous Teflon of the intact leaves as well as of the stripped epidermis. The DESI images reveal accumulation of Hydroxynitrile glucosides in the leaf epidermis, homogeneously distributed throughout the surface. The indirect DESI approach enables relative quantitation, confirming variations of Hydroxynitrile glucosides content in primary leaves of three different cultivars of barley seedlings. The study presents an example of how to overcome the morphological barriers from the plant surface and perform rapid and repeatable DESI imaging. In addition, a comparison is made of direct and indirect DESI imaging, contributing to the characterization of the recently developed method of indirect DESI imaging of plant material via porous Teflon imprints. Copyright © 2011 John Wiley & Sons, Ltd.
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characterization of barley leaf tissue using direct and indirect desorption electrospray ionization imaging mass spectrometry
Journal of Mass Spectrometry, 2011Co-Authors: Nanna Bjarnholt, Steen Honore Hansen, Christian JanfeltAbstract:Chemical profiling of barley (Hordeum vulgare) leaves was demonstrated using direct and indirect desorption electrospray ionization (DESI) imaging mass spectrometry. Direct DESI analysis of the untreated leaves was not possible despite a significant content of Hydroxynitrile glucosides known to reside in the epidermis of the leaves. Instead, the epidermis was stripped off the leaves, thus allowing direct DESI imaging to be performed on the back of the epidermis. Furthermore, indirect DESI imaging was performed by making imprints in porous Teflon of the intact leaves as well as of the stripped epidermis. The DESI images reveal accumulation of Hydroxynitrile glucosides in the leaf epidermis, homogeneously distributed throughout the surface. The indirect DESI approach enables relative quantitation, confirming variations of Hydroxynitrile glucosides content in primary leaves of three different cultivars of barley seedlings. The study presents an example of how to overcome the morphological barriers from the plant surface and perform rapid and repeatable DESI imaging. In addition, a comparison is made of direct and indirect DESI imaging, contributing to the characterization of the recently developed method of indirect DESI imaging of plant material via porous Teflon imprints.
Roger A. Sheldon - One of the best experts on this subject based on the ideXlab platform.
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9 15 industrial applications of asymmetric synthesis using cross linked enzyme aggregates
Reference Module in Chemistry Molecular Sciences and Chemical Engineering#R##N#Comprehensive Chirality, 2012Co-Authors: Roger A. SheldonAbstract:Biocatalysis has many attractive features for industrial-scale enantioselective synthesis. Enzymes are derived from renewable materials and are biodegradable. Reactions are generally performed in water under mild conditions, in high (enantio)selectivities, and without the need for protection/deprotection and activation steps. For commercial viability, however, it is often necessary to immobilize the enzyme, affording improved stability and facile recovery and reuse. Immobilization as cross-linked enzyme aggregates (CLEAs) has additional advantages compared with alternative methods for immobilization of enzymes. The procedure is simple, does not require highly pure enzymes, has a broad scope and affords robust, recyclable catalysts with good activity retention. The use of CLEAs from various enzymes – proteases, amidases, lipases, nitrilases, nitrile hydratases, Hydroxynitrile lyases – in enantioselective syntheses is described. A combi CLEA containing three enzymes – an ( S )-Hydroxynitrile lyase, a nitrilase, and an amidase – catalyzed the highly enantioselective (>99% enantiomeric excess ( ee ) formation of ( S )-mandelic acid from benzaldehyde and hydrogen cyanide. It is concluded that CLEAs have potential for wide application in enantioselective biocatalysis at industrial scale.
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synthesis of aliphatic s α hydroxycarboxylic amides using a one pot bienzymatic cascade of immobilised oxynitrilase and nitrile hydratase
Advanced Synthesis & Catalysis, 2009Co-Authors: Sander Van Pelt, Fred Van Rantwijk, Roger A. SheldonAbstract:A one-pot bienzymatic cascade combining a Hydroxynitrile lyase (Manihot esculenta, E.C. 4.1.2.10) and a nitrile hydratase (Nitriliruptor alkaliphilus, E.C. 4.2.1.84) for the synthesis of enantiopure aliphatic α-hydroxycarboxylic amides from aldehydes is described. Both enzymes were immobilised as cross-linked enzyme aggregates (CLEAs). Stability tests show that the nitrile hydratase CLEAs are sensitive to water-immiscible organic solvents as well as to aldehydes and hydrogen cyanide (HCN), but are remarkably stable and show useful activity in acidic aqueous environments of pH 4-5. The cascade reactions are consequently carried out by using a portionwise feed of HCN and moderate concentrations of aldehyde in acidic aqueous buffer to suppress the uncatalysed hydrocyanation background reaction. After optimisation, this method was used to synthesise five different kinds of aliphatic α-hydroxycarboxylic amides from the corresponding aldehydes with good yields and with enantiomeric purities comparable to those obtained for the α-Hydroxynitriles in the microaqueous hydrocyanation using hydroxy-nitrile lyase and an excess of HCN.
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Linum usitatissimum Hydroxynitrile Lyase Cross-Linked Enzyme Aggregates: A Recyclable Enantioselective Catalyst
Advanced Synthesis & Catalysis, 2008Co-Authors: Fabien L. Cabirol, Pei Loo Tan, Benson Tay, Shiryn Cheng, Ulf Hanefeld, Roger A. SheldonAbstract:An immobilized form of the Hydroxynitrile lyase from Linum usitatissimum (LuHNL) as cross-linked enzyme aggregate (CLEA) with high specific activity (303.5 U/g) and recovery (33%) was developed. Molecular imprinting using 2-butanone as additive in the immobilization process improved the synthetic activity of the biocatalyst. LuCLEA could be partially recycled for the synthesis of (R)-2-butanone cyanohydrin on a preparative scale over two batches. The enantioenriched cyanohydrin obtained was further hydrolyzed to give (R)-2-hydroxy-2-methylbutyric acid in 85% yield (from 2-butanone) and 87% ee.
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immobilized Hydroxynitrile lyases for enantioselective synthesis of cyanohydrins sol gels and cross linked enzyme aggregates
Advanced Synthesis & Catalysis, 2006Co-Authors: Fabien L. Cabirol, Ulf Hanefeld, Roger A. SheldonAbstract:The Hydroxynitrile lyases (HNLs) from Prunus amygdalus (PaHNL), Manihot esculenta (MeHNL), and Hevea brasiliensis (HbHNL) were successfully immobilized in sol-gels. The cross-linked enzyme aggregate (CLEA) of HbHNL was also prepared. These immobilized enzymes and the commercial PaHNL- and MeHNL-CLEAs were employed for the enantioselective synthesis of cyanohydrins. The sol-gels were highly efficient at low catalyst loading and particularly stable towards the organic solvent (diisopropyl ether) and substrate/product deactivation. The stabilization effect was inconsistent for CLEAs of different HNLs and significant deactivation of PaHNL- and HbHNL-CLEAs in diisopropyl ether was observed. In contrast commercial MeHNL-CLEA proved to be a remarkably robust and efficient biocatalyst in diisopropyl ether.
