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Richard T. Sayre - One of the best experts on this subject based on the ideXlab platform.
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Blackwell Publishing Ltd.
2013Co-Authors: Dimuth Siritunga, Diana Arias-garzon, A White, Richard T. SayreAbstract:Over-expression of Hydroxynitrile Lyase in transgeni
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Overexpression of Hydroxynitrile Lyase in Cassava Roots Elevates Protein and Free Amino Acids while Reducing Residual Cyanogen Levels
2013Co-Authors: Narayanan N Narayanan, Claire Ellery, Uzoma Ihemere, Richard T. SayreAbstract:Cassava is the major source of calories for more than 250 million Sub-Saharan Africans, however, it has the lowest proteinto-energy ratio of any major staple food crop in the world. A cassava-based diet provides less than 30 % of the minimum daily requirement for protein. Moreover, both leaves and roots contain potentially toxic levels of cyanogenic glucosides. The major cyanogen in cassava is linamarin which is stored in the vacuole. Upon tissue disruption linamarin is deglycosylated by the apolplastic enzyme, linamarase, producing acetone cyanohydrin. Acetone cyanohydrin can spontaneously decompose at pHs.5.0 or temperatures.35uC, or is enzymatically broken down by Hydroxynitrile Lyase (HNL) to produce acetone and free cyanide which is then volatilized. Unlike leaves, cassava roots have little HNL activity. The lack of HNL activity in roots is associated with the accumulation of potentially toxic levels of acetone cyanohydrin in poorly processed roots. We hypothesized that the over-expression of HNL in cassava roots under the control of a root-specific, patatin promoter would not only accelerate cyanogenesis during food processing, resulting in a safer food product, but lead to increased root protein levels since HNL is sequestered in the cell wall. Transgenic lines expressing a patatin-driven HNL gene construct exhibited a 2–20 fold increase in relative HNL mRNA levels in roots when compared with wild type resulting in a threefold increase in total root protein in 7 month old plants. After food processing, HNL overexpressing lines had substantially reduced acetone cyanohydrin and cyanide levels in roots relative to wild-type roots. Furthermore, steady state linamari
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Overexpression of Hydroxynitrile Lyase in cassava roots elevates protein and free amino acids while reducing residual cyanogen levels
PLoS ONE, 2011Co-Authors: Narayanan N Narayanan, Claire Ellery, Uzoma Ihemere, Richard T. SayreAbstract:Cassava is the major source of calories for more than 250 million Sub-Saharan Africans, however, it has the lowest protein-to-energy ratio of any major staple food crop in the world. A cassava-based diet provides less than 30% of the minimum daily requirement for protein. Moreover, both leaves and roots contain potentially toxic levels of cyanogenic glucosides. The major cyanogen in cassava is linamarin which is stored in the vacuole. Upon tissue disruption linamarin is deglycosylated by the apolplastic enzyme, linamarase, producing acetone cyanohydrin. Acetone cyanohydrin can spontaneously decompose at pHs >5.0 or temperatures >35°C, or is enzymatically broken down by Hydroxynitrile Lyase (HNL) to produce acetone and free cyanide which is then volatilized. Unlike leaves, cassava roots have little HNL activity. The lack of HNL activity in roots is associated with the accumulation of potentially toxic levels of acetone cyanohydrin in poorly processed roots. We hypothesized that the over-expression of HNL in cassava roots under the control of a root-specific, patatin promoter would not only accelerate cyanogenesis during food processing, resulting in a safer food product, but lead to increased root protein levels since HNL is sequestered in the cell wall. Transgenic lines expressing a patatin-driven HNL gene construct exhibited a 2-20 fold increase in relative HNL mRNA levels in roots when compared with wild type resulting in a threefold increase in total root protein in 7 month old plants. After food processing, HNL overexpressing lines had substantially reduced acetone cyanohydrin and cyanide levels in roots relative to wild-type roots. Furthermore, steady state linamarin levels in intact tissues were reduced by 80% in transgenic cassava roots. These results suggest that enhanced linamarin metabolism contributed to the elevated root protein levels.
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over expression of Hydroxynitrile Lyase in transgenic cassava roots accelerates cyanogenesis and food detoxification
Plant Biotechnology Journal, 2004Co-Authors: Dimuth Siritunga, Wanda L B White, Diana I Ariasgarzon, Richard T. SayreAbstract:Summary Cassava ( Manihot esculenta , Crantz) roots are the primary source of calories for more than 500 million people, the majority of whom live in the developing countries of Africa. Cassava leaves and roots contain potentially toxic levels of cyanogenic glycosides. Consumption of residual cyanogens (linamarin or acetone cyanohydrin) in incompletely processed cassava roots can cause cyanide poisoning. Hydroxynitrile Lyase (HNL), which catalyses the conversion of acetone cyanohydrin to cyanide, is expressed predominantly in the cell walls and laticifers of leaves. In contrast, roots have very low levels of HNL expression. We have over-expressed HNL in transgenic cassava plants under the control of a double 35S CaMV promoter. We show that HNL activity increased more than twofold in leaves and 13-fold in roots of transgenic plants relative to wild-type plants. Elevated HNL levels were correlated with substantially reduced acetone cyanohydrin levels and increased cyanide volatilization in processed or homogenized roots. Unlike acyanogenic cassava, transgenic plants overexpressing HNL in roots retain the herbivore deterrence of cyanogens while providing a safer food product.
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cyanogenesis in cassava the role of Hydroxynitrile Lyase in root cyanide production
Plant Physiology, 1998Co-Authors: Wanda L B White, Jennifer M Mcmahon, Diana I Ariasgarzon, Richard T. SayreAbstract:In the cyanogenic crop cassava (Manihot esculenta, Crantz), the final step in cyanide production is the conversion of acetone cyanohydrin, the deglycosylation product of linamarin, to cyanide plus acetone. This process occurs spontaneously at pH greater than 5.0 or enzymatically and is catalyzed by Hydroxynitrile Lyase (HNL). Recently, it has been demonstrated that acetone cyanohydrin is present in poorly processed cassava root food products. Since it has generally been assumed that HNL is present in all cassava tissues, we reinvestigated the enzymatic properties and tissue-specific distribution of HNL in cassava. We report the development of a rapid two-step purification protocol for cassava HNL, which yields an enzyme that is catalytically more efficient than previously reported (Hughes, J., Carvalho, F., and Hughes, M. [1994] Arch Biochem Biophys 311: 496–502). Analyses of the distribution of HNL activity and protein indicate that the accumulation of acetone cyanohydrin in roots is due to the absence of HNL, not to inhibition of the enzyme. Furthermore, the absence of HNL in roots and stems is associated with very low steady-state HNL transcript levels. It is proposed that the lack of HNL in cassava roots accounts for the high acetone cyanohydrin levels in poorly processed cassava food products.
Franz Effenberger - One of the best experts on this subject based on the ideXlab platform.
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addition of hydrocyanic acid to carbonyl compounds
Future Directions in Biocatalysis, 2007Co-Authors: Franz Effenberger, Anja Bohrer, Siegfried ForsterAbstract:Abstract The Hydroxynitrile Lyase (HNL)-catalyzed addition of HCN to aldehydes is the most important synthesis of non-racemic cyanohydrins. Since now not only ( R )-PaHNL from almonds is available in unlimited amounts, but the recombinant ( S )-HNLs from cassava (MeHNL) and rubber tree (HbHNL) are also available in giga units, the large-scale productions of non-racemic cyanohydrins have become possible. The synthetic potential of chiral cyanohydrins for the stereoselective preparation of biologically active compounds has been developed during the last 15 years.
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Hydroxynitrile Lyase catalyzed addition of hcn to 2 substituted cyclopentanones
Tetrahedron-asymmetry, 2004Co-Authors: Christoph Kobler, Franz EffenbergerAbstract:Abstract A systematic investigation of the stereoselectivity of Hydroxynitrile Lyases (HNLs) catalyzed addition of HCN to a variety of monosubstituted cyclopentanones yielding the corresponding cyanohydrins is presented. With PaHNL from bitter almond as catalyst, the HCN addition to 2-alkyl cyclopentanones 1b–d is highly (R)-selective, leading to the cis-(1R,2S)- and trans-(1R,2R)-diastereomers. The addition to the sterically less demanding methyl compound 1a is far less selective. With MeHNL from cassava, the expected (S)-selectivity of the HCN addition is very high for the cyclopentanones 1c–d with larger substituents, but only for the cis-(1S,2R)- and not the trans-(1S,2S)-diastereomers. Dynamic kinetic resolution was observed for the MeHNL-catalyzed addition of HCN to the racemic alkyl 2-oxocyclopentane carboxylates 4a and 4b. Continuous equilibration via keto-enol tautomerism and the preferred enzymatic conversion of the (R)-enantiomers of the ketones 4 results in the formation of the cis-(1R,2S)-diastereomers in >50% yield. The absolute configurations of the synthesized cyanohydrins were determined by X-ray crystallography of O-p-bromobenzoyl derivatives.
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crystal structure of Hydroxynitrile Lyase from sorghum bicolor in complex with the inhibitor benzoic acid a novel cyanogenic enzyme
Biochemistry, 2002Co-Authors: Hanspeter Lauble, Burkhard Miehlich, Harald Wajant, Siegfried Forster, Franz EffenbergerAbstract:The crystal structure of the Hydroxynitrile Lyase from Sorghum bicolor (SbHNL) in complex with the inhibitor benzoic acid has been determined at 2.3 A resolution and refined to a crystallographic R-factor of 16.5%. The SbHNL sequence places the enzyme in the α/β hydrolase family where the active site nucleophile is predicted to be organized in a characteristic pentapeptide motif which is part of the active site strand−turn−helix motif. In SbHNL, however, a unique two-amino acid deletion is next to the putative active site Ser158, removing thereby the putative oxyanion hole-forming Tyr residue. The presented X-ray structure shows that the overall folding pattern of SbHNL is similar to that of the closely related wheat serine carboxypeptidase (CPD-WII); however, the deletion in SbHNL is forcing the putative active site residues away from the expected hydrolase binding site toward a small hydrophobic cleft, which also contains the inhibitor benzoic acid, defining thereby a completely different SbHNL active s...
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mechanistic aspects of cyanogenesis from active site mutant ser80ala of Hydroxynitrile Lyase from manihot esculenta in complex with acetone cyanohydrin
Protein Science, 2001Co-Authors: Hanspeter Lauble, Burkhard Miehlich, Harald Wajant, Siegfried Forster, Franz EffenbergerAbstract:The structure and function of Hydroxynitrile Lyase from Manihot esculenta (MeHNL) have been analyzed by X-ray crystallography and site-directed mutagenesis. The crystal structure of the MeHNL–S80A mutant enzyme has been refined to an R-factor of 18.0% against diffraction data to 2.1-A resolution. The three-dimensional structure of the MeHNL–S80A–acetone cyanohydrin complex was determined at 2.2-A resolution and refined to an R-factor of 18.7%. Thr11 and Cys81 involved in substrate binding have been substituted by Ala in site-directed mutagenesis. The kinetic measurements of these mutant enzymes are presented. Combined with structural data, the results support a mechanism for cyanogenesis in which His236 as a general base abstracts a proton from Ser80, thereby allowing proton transfer from the hydroxyl group of acetone cyanohydrin to Ser80. The His236 imidazolium cation then facilitates the leaving of the nitrile group by proton donating.
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structure of Hydroxynitrile Lyase from manihot esculenta in complex with substrates acetone and chloroacetone implications for the mechanism of cyanogenesis
Acta Crystallographica Section D-biological Crystallography, 2000Co-Authors: Hanspeter Lauble, Burkhard Miehlich, Harald Wajant, Siegfried Forster, Franz EffenbergerAbstract:The crystal structures of Hydroxynitrile Lyase from Manihot esculenta (MeHNL) complexed with the native substrate acetone and substrate analogue chloroacetone have been determined and refined at 2.2 A resolution. The substrates are positioned in the active site by hydrogen-bond interactions of the carbonyl O atom with Thr11 OG, Ser80 OG and, to a lesser extent, Cys81 SG. These studies support a mechanism for cyanogenesis as well as for the stereospecific MeHNL-catalyzed formation of (S)-cyanohydrins, which closely resembles the base-catalyzed chemical reaction of HCN with carbonyl compounds.
Wanda L B White - One of the best experts on this subject based on the ideXlab platform.
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over expression of Hydroxynitrile Lyase in transgenic cassava roots accelerates cyanogenesis and food detoxification
Plant Biotechnology Journal, 2004Co-Authors: Dimuth Siritunga, Wanda L B White, Diana I Ariasgarzon, Richard T. SayreAbstract:Summary Cassava ( Manihot esculenta , Crantz) roots are the primary source of calories for more than 500 million people, the majority of whom live in the developing countries of Africa. Cassava leaves and roots contain potentially toxic levels of cyanogenic glycosides. Consumption of residual cyanogens (linamarin or acetone cyanohydrin) in incompletely processed cassava roots can cause cyanide poisoning. Hydroxynitrile Lyase (HNL), which catalyses the conversion of acetone cyanohydrin to cyanide, is expressed predominantly in the cell walls and laticifers of leaves. In contrast, roots have very low levels of HNL expression. We have over-expressed HNL in transgenic cassava plants under the control of a double 35S CaMV promoter. We show that HNL activity increased more than twofold in leaves and 13-fold in roots of transgenic plants relative to wild-type plants. Elevated HNL levels were correlated with substantially reduced acetone cyanohydrin levels and increased cyanide volatilization in processed or homogenized roots. Unlike acyanogenic cassava, transgenic plants overexpressing HNL in roots retain the herbivore deterrence of cyanogens while providing a safer food product.
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cyanogenesis in cassava the role of Hydroxynitrile Lyase in root cyanide production
Plant Physiology, 1998Co-Authors: Wanda L B White, Jennifer M Mcmahon, Diana I Ariasgarzon, Richard T. SayreAbstract:In the cyanogenic crop cassava (Manihot esculenta, Crantz), the final step in cyanide production is the conversion of acetone cyanohydrin, the deglycosylation product of linamarin, to cyanide plus acetone. This process occurs spontaneously at pH greater than 5.0 or enzymatically and is catalyzed by Hydroxynitrile Lyase (HNL). Recently, it has been demonstrated that acetone cyanohydrin is present in poorly processed cassava root food products. Since it has generally been assumed that HNL is present in all cassava tissues, we reinvestigated the enzymatic properties and tissue-specific distribution of HNL in cassava. We report the development of a rapid two-step purification protocol for cassava HNL, which yields an enzyme that is catalytically more efficient than previously reported (Hughes, J., Carvalho, F., and Hughes, M. [1994] Arch Biochem Biophys 311: 496–502). Analyses of the distribution of HNL activity and protein indicate that the accumulation of acetone cyanohydrin in roots is due to the absence of HNL, not to inhibition of the enzyme. Furthermore, the absence of HNL in roots and stems is associated with very low steady-state HNL transcript levels. It is proposed that the lack of HNL in cassava roots accounts for the high acetone cyanohydrin levels in poorly processed cassava food products.
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cyanogenesis in cassava manihot esculenta crantz
Journal of Experimental Botany, 1995Co-Authors: Jennifer M Mcmahon, Wanda L B White, Richard T. SayreAbstract:Cassava is the most agronomically important of the cyanogenic crops. Linamarin, the predominant cyanogenic glycoside in cassava, can accumulate to concentrations as high as 500 mg kg -1 fresh weight in roots and to higher levels in leaves. Recently, the pathway of linamarin synthesis and the cellular site of linamarin storage have been determined. In addition, the cyanogenic enzymes, linamarase and Hydroxynitrile Lyase, have been characterized and their genes cloned. These results, as well as studies on the organ- and tissue-specific localization of linamarase and Hydroxynitrile Lyase, allow us to propose models for the regulation of cyanogenesis in cassava. There remain, however, many unanswered questions regarding the tissue-specific synthesis, transport, and accumulation of cyanogenic glycosides. The resolution of these questions will facilitate the development of food processing, biochemical and transgenic plant approaches to reducing the cyanogen content of cassava foods.
Christoph Kratky - One of the best experts on this subject based on the ideXlab platform.
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substrate binding in the fad dependent Hydroxynitrile Lyase from almond provides insight into the mechanism of cyanohydrin formation and explains the absence of dehydrogenation activity
Biochemistry, 2009Co-Authors: Ingrid Dreveny, Aleksandra Andryushkova, Anton Glieder, Karl Gruber, Christoph KratkyAbstract:In a large number of plant species Hydroxynitrile Lyases catalyze the decomposition of cyanohydrins in order to generate hydrogen cyanide upon tissue damage. Hydrogen cyanide serves as a deterrent against herbivores and fungi. In vitro Hydroxynitrile Lyases are proficient biocatalysts for the stereospecific synthesis of cyanohydrins. Curiously, Hydroxynitrile Lyases from different species are completely unrelated in structure and substrate specificity despite catalyzing the same reaction. The Hydroxynitrile Lyase from almond shows close resemblance to flavoproteins of the glucose−methanol−choline oxidoreductase family. We report here 3D structural data of this Lyase with the reaction product benzaldehyde bound within the active site, which allow unambiguous assignment of the location of substrate binding. Based on the binding geometry, a reaction mechanism is proposed that involves one of the two conserved active site histidine residues acting as a general base abstracting the proton from the cyanohydrin ...
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the active site of Hydroxynitrile Lyase from prunus amygdalus modeling studies provide new insights into the mechanism of cyanogenesis
Protein Science, 2009Co-Authors: Ingrid Dreveny, Christoph Kratky, Karl GruberAbstract:The FAD-dependent Hydroxynitrile Lyase from almond (Prunus amygdalus, PaHNL) catalyzes the cleavage of R-mandelonitrile into benzaldehyde and hydrocyanic acid. Catalysis of the reverse reaction—the enantiospecific formation of α-Hydroxynitriles—is now widely utilized in organic syntheses as one of the few industrially relevant examples of enzyme-mediated C–C bond formation. Starting from the recently determined X-ray crystal structure, systematic docking calculations with the natural substrate were used to locate the active site of the enzyme and to identify amino acid residues involved in substrate binding and catalysis. Analysis of the modeled substrate complexes supports an enzymatic mechanism that includes the flavin cofactor as a mere "spectator" of the reaction and relies on general acid/base catalysis by the conserved His-497. Stabilization of the negative charge of the cyanide ion is accomplished by a pronounced positive electrostatic potential at the binding site. PaHNL activity requires the FAD cofactor to be bound in its oxidized form, and calculations of the pKa of enzyme-bound HCN showed that the observed inactivation upon cofactor reduction is largely caused by the reversal of the electrostatic potential within the active site. The suggested mechanism closely resembles the one proposed for the FAD-independent, and structurally unrelated HNL from Hevea brasiliensis. Although the actual amino acid residues involved in the catalytic cycle are completely different in the two enzymes, a common motif for the mechanism of cyanogenesis (general acid/base catalysis plus electrostatic stabilization of the cyanide ion) becomes evident.
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The Hydroxynitrile Lyase from almond: a Lyase that looks like an oxidoreductase.
Structure (London England : 1993), 2001Co-Authors: Ingrid Dreveny, Anton Glieder, Karl Gruber, Andrew Thompson, Christoph KratkyAbstract:Abstract Background: Cyanogenesis is a defense process of several thousand plant species. Hydroxynitrile Lyase, a key enzyme of this process, cleaves a cyanohydrin into hydrocyanic acid and the corresponding aldehyde or ketone. The reverse reaction constitutes an important tool in biocatalysis. Different classes of Hydroxynitrile Lyases have convergently evolved from FAD-dependent oxidoreductases, α/β hydrolases, and alcohol dehydrogenases. The FAD-dependent Hydroxynitrile Lyases (FAD-HNLs) carry a flavin cofactor whose redox properties appear to be unimportant for catalysis. Results: We have determined the crystal structure of a 61 kDa Hydroxynitrile Lyase isoenzyme from Prunus amygdalus (PaHNL1) to 1.5 A resolution. Clear electron density originating from four glycosylation sites could be observed. As concerns the overall protein fold including the FAD cofactor, PaHNL1 belongs to the family of GMC oxidoreductases. The active site for the HNL reaction is probably at a very similar position as the active sites in homologous oxidases. Conclusions: There is strong evidence from the structure and the reaction product that FAD-dependent Hydroxynitrile Lyases have evolved from an aryl alcohol oxidizing precursor. Since key residues implicated in oxidoreductase activity are also present in PaHNL1, it is not obvious why this enzyme shows no oxidase activity. Similarly, features proposed to be relevant for hydroxy-nitrile Lyase activity in other Hydroxynitrile Lyases, i.e., a general base and a positive charge to stabilize the cyanide, are not obviously present in the putative active site of PaHNL1. Therefore, the reason for its HNL activity is far from being well understood at this point.
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mechanism of cyanogenesis the crystal structure of Hydroxynitrile Lyase from hevea brasiliensis
Structure, 1996Co-Authors: Ulrike Wagner, Herfried Griengl, Meinhard Hasslacher, Helmut Schwab, Christoph KratkyAbstract:Abstract Background: Over three thousand species of plants, including important food crops such as cassava, use cyanogenesis, the liberation of HCN upon tissue damage, as a defense against predation. Detoxification of cyanogenic food crops requires disruption of the cyanogenic pathway. Hydroxynitrile Lyase is one of the key enzymes in cyanogenesis, catalyzing the decomposition of an acyanohydrin to form HCN plus the corresponding aldehyde or ketone. These enzymes are also of potential utility for industrial syntheses of optically pure chiral cyanohydrins, being used to catalyze the reverse reaction. We set out to gain insight into the catalytic mechanism of this important class of enzymes by determining the three-dimensional structure of Hydroxynitrile Lyase from the rubber tree, Hevea brasiliensis . Results The crystal structure of the enzyme has been determined to 1.9 A resolution. It belongs to the α / β hydrolase superfamily, with an active site that is deeply buried within the protein and connected to the outside by a narrow tunnel. The catalytic triad is made up of Ser80, His235 and Asp207. By analogy with known mechanisms of other members of this superfamily, catalysis should involve an oxyanion hole formed by the main chain NH of Cys81 and the side chains of Cys81 and Thr11. Density attributed to a histidine molecule or ion is found in the active site. Conclusion By analogy with other α / β hydrolases, the reaction catalyzed by Hydroxynitrile Lyase involves a tetrahedral hemiketal or hemiacetal intermediate formed by nucleophilic attack of Ser80 on the substrate, stabilized by the oxyanion hole. The SH group of Cys81 is probably involved in proton transfer between the HCN and the Hydroxynitrile OH. This mechanism is significantly different from the corresponding uncatalyzed solution reaction.
Harvey W Blanch - One of the best experts on this subject based on the ideXlab platform.
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a kinetic model for enzyme interfacial activity and stability pa Hydroxynitrile Lyase at the diisopropyl ether water interface
Biotechnology and Bioengineering, 2002Co-Authors: Luis Cascao G Pereira, Andrea Hickel, C J Radke, Harvey W BlanchAbstract:A kinetic framework is developed to describe enzyme activity and stability in two-phase liquid-liquid systems. In particular, the model is applied to the enzymatic production of benzaldehyde from mandelonitrile by Prunus amygdalus Hydroxynitrile Lyase (pa-Hnl) adsorbed at the diisopropyl ether (DIPE)/aqueous buffer interface (pH = 5.5). We quantitatively describe our previously obtained experimental kinetic results (Hickel et al., 1999; 2001), and we successfully account for the aqueous-phase enzyme concentration dependence of product formation rates and the observed reaction rates at early times. Multilayer growth explains the early time reversibility of enzyme adsorption at the DIPE/buffer interface observed by both enzyme-activity and dynamic-interfacial-tension washout experiments that replace the aqueous enzyme solution with a buffer solution. The postulated explanation for the unusual stability of pa-Hnl adsorbed at the DIPE/buffer interface is attributed to a two-layer adsorption mechanism. In the first layer, slow conformational change from the native state leads to irreversible attachment and partial loss of catalytic activity. In the second layer, pa-Hnl is reversibly adsorbed without loss in catalytic activity. The measured catalytic activity is the combined effect of the deactivation kinetics of the first layer and of the adsorption kinetics of each layer. For the specific case of pa-Hnl adsorbed at the DIPE/buffer interface, this combined effect is nearly constant for several hours resulting in no apparent loss of catalytic activity. Our proposed kinetic model can be extended to other interfacially active enzymes and other organic solvents. Finally, we indicate how interfacial-tension lag times provide a powerful tool for rational solvent selection and enzyme engineering.
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Hydroxynitrile Lyase at the diisopropyl ether water interface evidence for interfacial enzyme activity
Biotechnology and Bioengineering, 1999Co-Authors: Andrea Hickel, C J Radke, Harvey W BlanchAbstract:A novel recycle reactor has been designed to determine the interfacial activity of Hydroxynitrile Lyase in a diisopropyl ether (DIPE)/water two-phase system. The reactor provides a known interfacial area. Enzyme activity toward mandelonitrile cleavage is continuously measured in the reactor by following benzaldehyde product formation in the DIPE organic phase with an optical flow cell. For the first time, we establish that this enzymatic reaction is carried out by the Hydroxynitrile Lyase residing at the organic solvent/water interface and not in the aqueous bulk phase. Hydroxynitrile Lyase adsorbs at the interface and exhibits extraordinary stability. Denaturation does not occur over several hours, although the surface pressure increases under the same conditions over this time span. Increases in surface pressure indicate enzyme penetration through the interface although no loss of enzyme activity is observed. Adsorption of p-Hnl at the interface is fit by the Langmuir equilibrium adsorption model with an adsorption equilibrium constant of 0.032 L mg−1. For the mandelonitrile-cleavage reaction at ambient temperature, p-Hnl follows Michaelis–Menten kinetics at the interface with a Michaelis constant of 14.4 mM and a specific activity close that for the bulk aqueous phase. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 425–436, 1999.
