Hydroxynitrile lyases in stereoselective catalysis

(R)- as well as (S)-cyanohydrins are now easily available as a result of the excellent accessibility, the relatively high stability and the easy handling of hydroxynitrile lyases (HNLs). The optimization of reaction conditions (solvent, temperature, and using site-directed mutagenesis, etc.) has enabled HNL-catalyzed preparations of optically active cyanohydrins on a technical scale. The enantioselectivity of chiral metal-complex-catalyzed additions of trimethylsilyl cyanide to aldehydes has been improved, but is, by far, not yet competitive with the HNL-catalyzed reactions.     

Hydroxynitrile Lyases from Prunus Seeds in the Preparation of Cyanohydrins

The hydroxynitrile lyase (HNL) activity of nine defatted Prunus seeds was compared for catalyzing the addition of HCN to aromatic, heteroaromatic and α,β-unsaturated aldehydes. Although the conversion and enantiomeric excess (ee) of the corresponding cyanohydrins were both influenced by the HNL source and the chemical structure of the aldehyde, Prunus HNLs were all suitable for the enantioselective preparation of cyanohydrins.     

Hydroxynitrile Lyase Catalysis in Ionic Liquid-containing Systems

The cleavage of mandelonitrile catalysed by hydroxynitrile lyases (HNL) from Prunus amygdalus (PaHNL) and Manihot esculenta (MeHNL) proceeded more rapidly in monophasic aqueous media containing 1-propyl-3-methylimidazolium tetrafluoroborate [C₄MIm][BF₄] than in media containing acetonitrile or THF. Both HNLs were much more thermostable in [C₄MIm][BF₄] than in acetonitrile or THF. The addition of each of the four ionic liquids 1-butyl-, 1-pentyl- and 1-hexyl-3-methylimidazolium tetrafluoroborates at 2–6% (v/v in the aqueous phase) increased both the enzyme activity and the product e.e. in the PaHNL-catalysed transcyanation in an aqueous/DIPE biphasic system. However, MeHNL was inactivated by the ionic liquids, as indicated by the decreased reaction rate, substrate conversion and product e.e.     

Microbial pectate lyases: characterization and enzymological properties

Pectate lyase plays an important role in plant pathogenesis. The enzyme is widely distributed in diverse families of microorganisms. The current knowledge including biochemical studies on microbial pectate lyases, such as isozymes, structure, reaction mechanism, purification and properties like molecular mass, pI, optimum pH and temperature, substrate specificity, metal ion requirement, inhibitors and activators, and kinetic parameters of the enzyme are reviewed.     

Evaluation of the Hydroxynitrile Lyase Activity in Cell Cultures of Capulin （Prunus serotina）

Enzymatic preparations obtained from young plants and cell cultures of capulin were screened for hydroxynitrile lyase activity. The threeweek old plants, grown under sterile conditions, were used to establish a solid cell culture. Crude preparations obtained from this plant material were evaluated for the transformation of benzaldehyde to the corresponding cyanohydrin (mandelonitrile). The results show that the crude material from roots, stalks, and leaves of young plants and calli of roots, stalks, internodes and petioles biocatalyzed the addition of hydrogen cyanide (HCN) to benzaldehyde with a modest to excellent enantioselectivity.     

Structural characterization of Linum usitatissimum hydroxynitrile lyase: A new cyanohydrin decomposition mechanism involving a cyano-zinc complex

Hydroxynitrile lyase from Linum usitatissimum (LuHNL) is an enzyme involved in the catabolism of cyanogenic glycosides to release hydrogen cyanide upon tissue damage. This enzyme strictly conserves the substrate- and NAD(H)-binding domains of Zn²⁺-containing alcohol dehydrogenase (ADH); however, there is no evidence suggesting that LuHNL possesses ADH activity. Herein, we determined the ligand-free 3D structure of LuHNL and its complex with acetone cyanohydrin and (R)-2-butanone cyanohydrin using X-ray crystallography. These structures reveal that an A-form NAD⁺ is tightly but not covalently bound to each subunit of LuHNL. The restricted movement of the NAD+ molecule is due to the “sandwich structure” on the adenine moiety of NAD⁺. Moreover, the structures and mutagenesis analysis reveal a novel reaction mechanism for cyanohydrin decomposition involving the cyano-zinc complex and hydrogen-bonded interaction of the hydroxyl group of cyanohydrin with Glu323/Thr65 and H₂O/Lys162 of LuHNL. The deprotonated Lys162 and protonated Glu323 residues are presumably stabilized by a partially desolvated microenvironment. In summary, the substrate binding geometry of LuHNL provides insights into the differences in activities of LuHNL and ADH, and identifying this novel reaction mechanism is an important contribution to the study of hydroxynitrile lyases.     

Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis.

The 3D structures of complexes between the hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) and several substrate and/or inhibitor molecules, including trichloracetaldehyde, hexafluoracetone, acetone, and rhodanide, were determined by X-ray crystallography. The complex with trichloracetaldehyde showed a covalent linkage between the protein and the inhibitor, which had apparently resulted from nucleophilic attack of the catalytic Ser80-Ogamma. All other complexes showed the substrate or inhibitor molecule merely hydrogen bonded to the protein. In addition, the native crystal structure of Hb-HNL was redetermined at cryo-temperature and at room temperature, eliminating previous uncertainties concerning residual electron density within the active site, and leading to the observation of two conserved water molecules. One of them was found to be conserved in all complex structures and appears to have mainly structural significance. The other water molecule is conserved in all structures except for the complex with rhodanide; it is hydrogen bonded to the imidazole of the catalytic His235 and appears to affect the Hb-HNL catalyzed reaction. The observed 3D structural data suggest implications for the enzyme mechanism. It appears that the enzyme-catalyzed cyanohydrin formation is unlikely to proceed via a hemiacetal or hemiketal intermediate covalently attached to the enzyme, despite the observation of such an intermediate for the complex with trichloracetaldehyde. Instead, the data are consistent with a mechanism where the incoming substrate is activated by hydrogen bonding with its carbonyl oxygen to the Ser80 and Thr11 hydroxy groups. A hydrogen cyanide molecule subsequently replaces a water molecule and is deprotonated presumably by the His235 base. Deprotonation is facilitated by the proximity of the positive charge of the Lys236 side chain.     

Characterization of cold-active pectate lyases from psychrophilic Mrakia frigida

AIMS: This study was conducted to determine optimal conditions for pectate lyase (PL) production by two psychrophilic yeast strains and to compare the properties of the cold-active enzymes using mesophilic PL as reference enzyme. METHODS AND RESULTS: Two psychrophilic yeasts isolated from remote geographical locations (European Alps, north Siberia) produced extracellular cold-active PL. Both strains were identified as Mrakia frigida by analysis of ITS and large subunit (LSU) rRNA sequences. Maximum enzyme production occurred at a cultivation temperature of 1 or 5 degrees C. The apparent optimum for enzyme activity was observed at 30 degrees C and pH 8.5-9. The enzymes were thermolabile, but were resistant to repeated freezing and thawing. CONCLUSION: We describe for the first time alkaline PL-producing representatives of the yeast species M. frigida. The two strains produce cold-active PL with similar properties, but have a different enzyme production pattern. SIGNIFICANCE AND IMPACT OF THE STUDY: The enzymes described in this study could be useful for a wide range of applications, such as low-temperature pretreatment of wastewater containing pectic substances.     

High-throughput in-field bioprospecting for cyanogenic plants and hydroxynitrile lyases

Abstract

Hydroxynitrile lyases (HNLs) are sought-after, stereo-selective biocatalysts used in the agrochemical, pharmaceutical and fine chemical industries to produce cyanohydrin enantiomers. There are several approaches for the discovery of HNLs, most of which are methodologically demanding and not suitable for high-throughput. Bioprospecting studies to date have also been constrained/limited to commercialised plants or botanical gardens, leaving a vast majority of plant species untested for HNL activity or cyanogenesis. To increase the rate of discovery of HCN liberating plants, we devised a Feigl-Anger microfuge tube that is portable and capable of high throughput detection of naturally cyanogenic plants. A workflow suitable for detecting plant candidates containing extractable, novel HNLs was subsequently applied. In this study, we screened over 600 plants for cyanogenic activity as well as the ability to degrade racemic mandelonitrile. We detected 33 plants able to degrade racemic mandelonitrile, of which, 25 were identified to the species level. Six of these plants were found to be naturally cyanogenic. Protein extracts from 5 of the naturally cyanogenic plants retained the ability to degrade racemic mandelonitrile pointing to five yet undescribed enzymes in the species Achyranthes aspera, Davallia trichomonoides, Morus mesozygia, Polypodium aureum “Mandaianum”, and Thelypteris confluens. In contrast, although Acalypha glabrata was found to be naturally cyanogenic, the protein extract did not break down racemic mandelonitrile. Here, we used racemic mandelonitrile as substrate and detected enzymes with mandelonitrile lyase activity, however, any cyanohydrin could be used as part of the approach taken here to detect novel HNLs specific to the substrate utilised.     

A caveat for the use of log P values for the assessment of the biocompatibility of organic solvents

The degree of enzyme deactivation for lipases from Candida rugosa and Pseudomonas sp., hydroxynitrile lyase and mandelate racemase upon exposure to organic solvents can be correlated to their respective partition coefficients (log P values). However, three unexpected results were obtained: (1) the deactivation exerted by protic solvents, e.g., methanol, is severely underestimated; (2) little deactivation by an organic solvent cannot neccessarily be correlated to catalytic activity in this medium, and (3) in contrast to other enzymes, hydroxynitrile lyase is exceptionally stable towards deactivation by DMF.     

Crystallization and preliminary X-ray diffraction studies of mandelonitrile lyase from almonds

Single crystals of three different isoenzymes of (R)?(+) mandelonitrile lyase (hydroxynitrile lyase) from almonds (Prunus amygdalus) have been obtained by hanging drop vapor diffusion using polyethylene glycol 4000 and isopropanol as co-precipitants. The crystals belong to the monoclinic space group P2_l with unit cell parameters a = 69.9, b = 95.1, c = 95.6 Å, and β = 118.5°. A complete set of diffraction data has been collected to 2.6 Å resolution on native crystals of isoenzyme III. © 1994 Wiley-Liss, Inc.     

NAD+ and metal-ion dependent hydrolysis by family 4 glycosidases: structural insight into specificity for phospho-beta-D-glucosides

The import of disaccharides by many bacteria is achieved through their simultaneous translocation and phosphorylation by the phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS). The imported phospho-disaccharides are, in some cases, subsequently hydrolyzed by members of the unusual glycoside hydrolase family GH4. The GH4 enzymes, occasionally found also in bacteria such as Thermotoga maritima that do not utilise a PEP-PTS system, require both NAD(+) and Mn(2+) for catalysis. A further curiosity of this family is that closely related enzymes may show specificity for either alpha-d- or beta-d-glycosides. Here, we present, for the first time, the three-dimensional structure (using single-wavelength anomalous dispersion methods, harnessing extensive non-crystallographic symmetry) of the 6-phospho-beta-glycosidase, BglT, from T.maritima in native and complexed (NAD(+) and Glc6P) forms. Comparison of the active-center structure with that of the 6-phospho-alpha-glucosidase GlvA from Bacillus subtilis reveals a striking degree of structural similarity that, in light of previous kinetic isotope effect data, allows the postulation of a common reaction mechanism for both alpha and beta-glycosidases. Given that the "chemistry" occurs primarily on the glycone sugar and features no nucleophilic attack on the intact disaccharide substrate, modulation of anomeric specificity for alpha and beta-linkages is accommodated through comparatively minor structural changes.     

Kinetics and specificity of alginate lyases

A purified preparation of the extracellular alginate lyase has been used to study kinetics and specificity towards purified, homopolymeric fragments of alginate. The enzyme preparation from Bacillus circulans 1351 degraded both block types, although with different efficiency, and thus appears to be nonspecific. Addition of calcium ions markedly enhanced the reaction rate for the polymannuronate block but had little or no effect on the reaction with polyguluronate. Michaelis-Menten kinetics are not obeyed in the absence of calcium ions and only for the polymannuronate in the presence of calciumThe study of progress curves in response to variation in substrate and enzyme concentrations strongly suggests that the abalone lyase is subject to a reversible product inhibition.     

海藻工具酶——褐藻胶裂解酶研究进展

从海洋生物中筛选提取有价值的酶类,开发海洋多糖降解产物,已成为海洋生物资源开发的一个重要方面。因此,近年来对于海藻工具酶之一的褐藻胶裂解酶及其降解产物——褐藻寡糖的研究日益受到人们的普遍关注。从褐藻胶裂解酶的来源、分类、底物专一性、作用方式及结构与机理研究、酶活力测定和酶学性质等方面,结合本课题组的研究工作综述近十年来有关褐藻胶裂解酶的研究进展。     

A kinetic model for enzyme interfacial activity and stability: pa-hydroxynitrile lyase at the diisopropyl ether/water interface

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.     

Purification,Crystallization and Some Properties of Lipase from Geotrichum candidum Link

Lipase (EC 3.1.1.3) of Geotrichum candidum Link was purified by means of ammonium sulfate fractionation, DEAE-Sephadex column chromatography, gel-filtration on Sephadex G–100 and Sephadex G–200, and was finally crystallized in concentrated aqueous solution. It was confirmed that the crystallized preparation was homogeneous electrophoretically and ultracentrifugally.

It was estimated with the crystalline enzyme that the sedimentation constant (s₂₀, w) was 4.0, the isoelectric point was pH 4.33, and the molecular weight was 53,000~55,000. From the result of amino acid analysis, none of sulfur containing amino acid was detected in the enzyme. It was also recognized that the crystalline preparation contained about 7% of the carbohydrate and very small amount of lipid. It was characterized that the lipase was the most active at pH 5.6~7.0 on olive oil, at 40°C and was stable in the range of pH 4.2 to 9.8 at 30°C for 24 hr, and was stable below 55°C for 15 min.     

Over-expression of hydroxynitrile lyase in transgenic cassava roots accelerates cyanogenesis and food detoxification

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 over-expressing HNL in roots retain the herbivore deterrence of cyanogens while providing a safer food product.     

The C-S lyases of higher plants. Purification and characterization of homogeneous alliin lyase of leek (Allium porrum)

The characteristic odors of freshly macerated tissue of Allium species such as garlic and onion are due to the action of the enzyme alliin lyase (EC 4.4.1.4) on endogenous S-alkyl-I-cysteine sulfoxides which are present as secondary amino acids yielding volatile sulfur-containing products. Purification and characterization of the alliin lyase of leek ( Allium porrum L.) has been carried out for comparison with the analogous enzymes previously characterized from garlic and onion. The purification involved homogenization, followed by ammonium sulfate fractionation, elution from an hydroxylapatite column, concentration of the active fractions and passage through a concanavalin A-Sepharose 4B affinity column. The purified enzyme was found to be a glycoprotein with a pH optimum for activity of 8.0. Sodium dodecylsulfate-urea polyacrylamide gel electrophoresis gels of the homogeneous leek enzyme showed it consisted of 1 subunit with a molecular weight of 48000. By gel filtration, 2 stable forms of the native enzyme with molecular weights of 386000 and 580000 were found.     

Biochemical properties and evaluation of washing performance in commercial detergent compatibility of two collagenolytic serine peptidases secreted by Aspergillus fischeri and Penicillium citrinum

Filamentous fungi secrete diverse peptidases with different biochemical properties, which is of considerable importance for application in various commercial sectors. In this study, we describe the isolation of two fungal species collected from the soil of decayed organic matter: Aspergillus fischeri and Penicillium citrinum. In a submerged bioprocess, we observed better peptidase production with the fungus P. citrinum, which reached a peak production at 168?h with 760?U/mL, in comparison with the fungus A. fischeri, which reached a peak production at 72?h with 460?U/mL. In both situations, the fermentative medium contained 0.5% crushed feathers as a source of nitrogen. On performing biochemical characterization, we detected two alkaline serine peptidases: The one secreted by P. citrinum had optimal activity at pH 7.0 and at 45°C, while the one secreted by A. fischeri had optimal activity in pH 6.5–8 and at 55–60°C. Metallic ions were effective in modulating these peptidases; in particular, Cu²⁺ promoted negative modulation of both peptidases. The peptidases were stable and functional under conditions of nonionic surfactants, temperatures up to 45°C for 1?h, and incubation over a wide pH range. In addition, it was observed that both peptidases had the capacity to hydrolyze collagen and performed well in removing an egg protein stain when supplemented into a commercial powder detergent; this was especially true for the peptidase from P. citrinum.