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.     

Improving catalytic performance of an arylacetonitrilase by semirational engineering

Arylacetonitrilases have been widely acknowledged as important alternatives to chemical catalysts for synthesizing optically pure 2-hydroxyphenylacetic acids from nitriles. In this work, two residues (Thr132 and Ser190) located at the catalytic tunnel in the active site of an arylacetonitrilase nitA from uncultured organisms were mutated separately by site-directed mutagenesis. Ser190 was demonstrated to be the critical position which has a greater influence on arylacetonitrilase nitA activity than Thr132. The replacement of serine at position 190 with glycine increases its activity toward mandelonitrile and (o, m, p)-chloromandelonitrile, whereas replacing it with leucine abolished its activity. The best mutant S190G exhibited threefold higher specific activity toward mandelonitrile compared with that of wild-type nitA, which rendered it promising for industrial application. Homology modeling and molecular docking experiments were in agreement with the kinetic assays and support the improved catalytic performance.

     

Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida

The highly enantioselective arylacetonitrilase of Pseudomonas putida was purified to homogeneity using a combination of (NH₄)₂SO₄ fractionation and different chromatographic techniques. The enzyme has a molecular weight of 412 kDa and consisted of approximately nine to ten identical subunits (43 kDa). The purified enzyme exhibited a pH optimum of 7.0 and temperature optimum of 40°C. The nitrilase was highly susceptible to thiol-specific reagents and metal ions and also required a reducing environment for its activity. These reflected the presence of a catalytically essential thiol group for enzyme activity which is in accordance with the proposed mechanism for nitrilase-catalyzed reaction. The enzyme was highly specific for arylacetonitriles with phenylacetonitrile and its derivatives being the most preferred substrates. Higher specificity constant (k _cat/K _m) values for phenylacetonitrile compared to mandelonitrile also revealed the same. Faster reaction rate achieved with this nitrilase for mandelonitrile hydrolysis was possibly due to the low activation energy required by the protein. Incorporation of low concentration (<5%) of organic solvent increased the enzyme activity by increasing the availability of the substrate. Higher stability of the enzyme at slightly alkaline pH and ambient temperature provides an excellent opportunity to establish a dynamic kinetic resolution process for the production of (R)-(−)-mandelic acid from readily available mandelonitrile.     

Biocatalytic synthesis of (<Emphasis Type="Italic">R</Emphasis>)-(−)-mandelic acid from racemic mandelonitrile by cetyltrimethylammonium bromide-permeabilized cells of <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis> ECU0401

The nitrilase from Alcaligenes faecalis ECU0401 belongs to the category of arylacetonitrilase, which could hydrolyze 2-chloromandelonitrile, 3,4-dimethoxyphenylacetonitrile, mandelonitrile, and phenylacetonitrile into the corresponding arylacetic acids. To overcome the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilization of Alcaligenes faecalis ECU0401 in relation to nitrilase activity was optimized by using cetyltrimethylammonium bromide (CTAB) as permeabilizing agent. The nitrilase activity from Alcaligenes faecalis ECU0401 increased 4.5-fold when the cells were permeabilized with 0.3% (w/v) CTAB for 20 min at 25°C and pH 6.5. Consequently, almost all the mandelonitrile was consumed and converted to (R)-(−)-mandelic acid with greater than 99.9% enantiomeric excess (e.e.) by the CTAB-permeabilized cells. The permeability barrier has been significantly reduced in the hydrolysis of mandelonitrile by using CTAB-permeabilized cells and a dynamic resolution was successfully achieved, giving a 100% theoretical yield of (R)-(−)-mandelic acid. Efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 3.82 g (R)-(−)-mandelic acid g⁻¹ dry cell weight (dcw) cell after 20 cycles of repeated use.     

Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis

(R)-mandelic acid was produced from racemic mandelonitrile using free and immobilized cells of Pseudomonas putida MTCC 5110 harbouring a stereoselective nitrilase. In addition to the optimization of culture conditions and medium components, an inducer feeding approach is suggested to achieve enhanced enzyme production and therefore higher degree of conversion of mandelonitrile. The relationship between cell growth periodicity and enzyme accumulation was also studied, and the addition of the inducer was delayed by 6 h to achieve maximum nitrilase activity. The nitrilase expression was also authenticated by the sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis. P. putida MTCC 5110 cells were further immobilized in calcium alginate, and the immobilized biocatalyst preparation was used for the enantioselective hydrolysis of mandelonitrile. The immobilized system was characterized based on the Thiele modulus (ϕ). Efficient biocatalyst recycling was achieved as a result of immobilization with immobilized cells exhibiting 88% conversion even after 20 batch recycles. Finally, a fed batch reaction was set up on a preparative scale to produce 1.95 g of (R)-(-)-mandelic acid with an enantiomeric excess of 98.8%.     

The toxicity of peach tree roots

Summary The HCN content of peach plants and the effect of this and other substances formed from peach root residues on growth of peach trees and on soil microorganisms were investigated. Peach root bark contained appreciable amounts of HCN. HCN was released from live roots after mechanical injury. Benzaldehyde and KCN were toxic to rooted peach trees in the greenhouse and they inhibited respiration of peach root tips. A similar suppression of respiration was caused by benzoic acid, mandelonitrile, and water extracts of peach root bark incubated in peach and non-peach soils. Extracts from peach soil caused greater inhibition to respiration of peach root tips than extracts from non-peach soil.KCN, mandelonitrile, benzaldehyde, peach root bark, and amygdalin reduced the total micro-organism, actinomycete, pythium, and pathogenic nematode population of an old peach soil.     

Production of R-(-)-mandelic acid from mandelonitrile by Alcaligenes faecalis ATCC 8750.

R-(-)-Mandelic acid was produced from racemic mandelonitrile by Alcaligenes faecalis ATCC 8750. Ammonium acetate or L-glutamic acid as the carbon source and n-butyronitrile as the inducer in the culture medium were effective for bacterial growth and the induction of R-(-)-mandelic acid-producing activity. The R-(-)-mandelic acid formed from mandelonitrile by resting cells was present in a 100% enantiomeric excess. A. faecalis ATCC 8750 has an R-enantioselective nitrilase for mandelonitrile and an amidase for mandelamide. As R-(-)-mandelic acid was produced from racemic mandelonitrile in a yield of 91%, whereas no S-mandelonitrile was left, the S-mandelonitrile remaining in the reaction is spontaneously racemized because of the chemical equilibrium and is used as the substrate. Consequently, almost all the mandelonitrile is consumed and converted to R-(-)-mandelic acid. R-(-)-Mandelic acid was also produced when benzaldehyde plus HCN was used as the substrate.     

Production of R-(-)-mandelic acid from mandelonitrile by Alcaligenes faecalis ATCC 8750.

R-(-)-Mandelic acid was produced from racemic mandelonitrile by Alcaligenes faecalis ATCC 8750. Ammonium acetate or L-glutamic acid as the carbon source and n-butyronitrile as the inducer in the culture medium were effective for bacterial growth and the induction of R-(-)-mandelic acid-producing activity. The R-(-)-mandelic acid formed from mandelonitrile by resting cells was present in a 100% enantiomeric excess. A. faecalis ATCC 8750 has an R-enantioselective nitrilase for mandelonitrile and an amidase for mandelamide. As R-(-)-mandelic acid was produced from racemic mandelonitrile in a yield of 91%, whereas no S-mandelonitrile was left, the S-mandelonitrile remaining in the reaction is spontaneously racemized because of the chemical equilibrium and is used as the substrate. Consequently, almost all the mandelonitrile is consumed and converted to R-(-)-mandelic acid. R-(-)-Mandelic acid was also produced when benzaldehyde plus HCN was used as the substrate.     

Efficient production of (R)-(−)-mandelic acid in biphasic system by immobilized recombinant E. coli

The recombinant Escherichia coli M15/BCJ2315 which harbored a mandelonitrilase from Burkholderia cenocepacia J2315 was immobilized via catecholic chitosan and functionalized with magnetism by iron oxide nanoparticles. The immobilized cells showed high activity recovery, enhanced stability and good operability in the enantioselective hydrolysis of mandelonitrile to (R)-(−)-mandelic acid. Furthermore, the immobilized cells were reused up to 15 cycles without any activity loss in completely hydrolyzing mandelonitrile (100 mM) within 1 h in aqueous solution. The ethyl acetate–water biphasic system was built and optimized. Under the optimal conditions, as high as 1 M mandelonitrile could be hydrolyzed within 4 h with a final yield and ee value of 99% and 95%, respectively. Moreover, the successive hydrolysis of mandelonitrile was performed by repeated use of the immobilized cells for 6 batches, giving a final productivity (g L⁻¹ h⁻¹) and relative production (g g⁻¹) of 40.9 and 38.9, respectively.     

Enantioselective Nitrilase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis>: Cloning,Heterologous Expression,and Bioreactor Studies

Nitrilases have attracted tremendous attention for the preparation of optically pure carboxylic acids. This article aims to address the production and utilization of a highly enantioselective nitrilase from Pseudomonas putida MTCC 5110 for the hydrolysis of racemic mandelonitrile to (R)-mandelic acid. The nitrilase gene from P. putida was cloned in pET 21b(+) and over-expressed as histidine-tagged protein in Escherichia coli. The histidine-tagged enzyme was purified from crude cell extracts of IPTG-induced cells of E. coli BL21 (DE3). Inducer replacement studies led to the identification of lactose as a suitable and cheap alternative to the costly IPTG. Effects of medium components, various physico-chemical, and process parameters (pH, temperature, aeration, and agitation) for the production of nitrilase by engineered E. coli were optimized and scaled up to a laboratory scale bioreactor (6.6 l). Finally, the recombinant E. coli whole-cells were utilized for the production of (R)-(−)-mandelic acid.     

Cloning and biochemical properties of a highly thermostable and enantioselective nitrilase from Alcaligenes sp. ECU0401 and its potential for (R)-(?)-mandelic acid production

A nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli BL21 (DE3) in a soluble form. The encoded protein with a His₆-tag was purified to nearly homogeneity as revealed by SDS-PAGE with a molecular weight of approximately 38.5 kDa, and the holoenzyme was estimated to be composed of 10 subunits of identical size by size exclusion chromatography. The V _max and K _m parameters were determined to be 27.9 μmol min⁻¹ mg⁻¹ protein and 21.8 mM, respectively, with mandelonitrile as the substrate. The purified enzyme was highly thermostable with a half life of 155 h at 30 °C and 94 h at 40 °C. Racemic mandelonitrile (50 mM) could be enantioselectively hydrolyzed to (R)-(−)-mandelic acid by the purified nitrilase with an enantiomeric excess of 97%. The extreme stability, high activity and enantioselectivity of this nitrilase provide a solid base for its practical application in the production of (R)-(−)-mandelic acid.     

1-Phenyl-2-pentanone and methyl salicylate: new defense allomone components and their content shift during ontogenetic development of the millipede <Emphasis Type="Italic">Nedyopus tambanus mangaesinus</Emphasis> (Polydesmida: Paradoxosomatidae)

Twelve components were identified in hexane extracts of the polydesmid millipede Nedyopus tambanus mangaesinus (Attems, 1909) and their contents were examined at all stadia of ontogenetic development including two adult conditions (before and after hibernation). Two compounds, 1-phenyl-2-pentanone and methyl salicylate, were newly identified as components of the millipede together with six well-known polydesmoid compounds (benzaldehyde, benzyl alcohol, benzoic acid, benzoyl cyanide, mandelonitrile, and mandelonitrile benzoate) and four phenols (phenol, p-cresol, 2-methoxyphenol, and 2-methoxy-4-methylphenol). Benzaldehyde and benzoyl cyanide were distributed from nymphs at stadium I, and other components started to become detectable at more advanced stadia. The largest content (35.1%) of methyl salicylate was detected in nymphs at stadium I, together with benzaldehyde and benzoyl cyanide, implying the reinforcement of defensive functions during or after egg emergence. The content (18.8%) of 1-phenyl-2-pentanone reached a maximum in hibernated females among all stadia and conditions. Its female-biased distribution at the mating season (ca. 5.7-fold more than in males) implies its sexually related functions.     

Characterization of a novel hydroxynitrile lyase from Nandina domestica Thunb

The leaves of Nandina domestica Thunb. exhibited high hydroxynitrile lyase (HNL) activity in (R)-mandelonitrile synthesis. The specific activity of young leaves was significantly higher than that of mature leaves. We isolated two HNLs with molecular mass of 24.9 kDa (NdHNL-S) and 28.0 kDa (NdHNL-L) from the young leaves. Both NdHNLs were composed of two identical subunits, without FAD and carbohydrates. We purified NdHNL-L and revealed its enzymatic properties. The whole deduced amino acid sequence of NdHNL-L was not homologous to any other HNLs, and the specific activity for mandelonitrile synthesis by NdHNL-L was higher than that by other plant HNLs. The enzyme catalyzed enantioselective synthesis of (R)-cyanohydrins, exhibited high activity at pH 4.0, and high stability in the pH range of 3.5–8.0 and below 55°C. Thus, NdHNL-L is a novel HNL with novel amino acid sequence and has a potential for the efficient production of (R)-cyanohydrins.     

Decarboxylation of Oxalic Acid during Ripening of Banana Fruit (Musa sapientum L.)

ABSTRACT

Japanese apricot, Prunus mume Sieb. et Zucc., biosynthesizes the l-phenylalanine-derived cyanogenic glucosides prunasin and amygdalin. Prunasin has biological properties such as anti-inflammation, but plant extraction and chemical synthesis are impractical. In this study, we identified and characterized UGT85A47 from Japanese apricot. Further, UGT85A47 was utilized for prunasin microbial production. Full-length cDNA encoding UGT85A47 was isolated from Japanese apricot after 5?- and 3?-RACE. Recombinant UGT85A47 stoichiometrically catalyzed UDP-glucose consumption and synthesis of prunasin and UDP from mandelonitrile. Escherichia coli C41(DE3) cells expressing UGT85A47 produced prunasin (0.64 g/L) from racemic mandelonitrile and glucose. In addition, co-expression of genes encoding UDP-glucose biosynthetic enzymes (phosphoglucomutase and UTP-glucose 1-phosphate uridiltransferase) and polyphosphate kinase clearly improved prunasin production up to 2.3 g/L. These results showed that our whole-cell biocatalytic system is significantly more efficient than the existing prunasin production systems, such as chemical synthesis.     

Random mutagenesis of the arylacetonitrilase from Pseudomonas fluorescens EBC191 and identification of variants,which form increased amounts of mandeloamide from mandelonitrile

The nitrilase from Pseudomonas fluorescens EBC191 was modified by introducing random mutations via error-prone PCR techniques in order to obtain nitrilase variants, which form increased amounts of mandeloamide from racemic mandelonitrile. A screening system was established and experimentally optimized, which allowed the screening of nitrilase variants with the intended phenotype. This system was based on the simultaneous expression of nitrilase variants and the mandeloamide converting amidase from Rhodococcus rhodochrous MP50 in recombinant Escherichia coli cells. The formation of increased amounts of mandeloamide from mandelonitrile by the nitrilase variants was detected after the addition of hydroxylamine and ferric iron ions by taking advantage of the acyltransferase activity of the amidase, which resulted in the formation of coloured iron(III)–hydroxamate complexes from mandeloamide. The system was applied for the screening of libraries of nitrilase variants and 30 enzyme variants identified, which formed increased amounts of mandeloamide from racemic mandelonitrile. The increase in amide formation was quantified by high-performance liquid chromatography and the genes encoding the relevant nitrilase variants sequenced. Thus, different types of mutations were identified. One group of mutants carried different deletions at the carboxy-terminus. The other types of variants carried amino acid exchanges in positions that had not been related previously to an increased amide formation. Finally, a nitrilase variant was created by combining two independently obtained point mutations. This enzyme variant demonstrated a true nitrile hydratase activity as it formed mandeloamide and mandelic acid in a ratio of about 19:1 from racemic mandelonitrile.     

Inhibitory Effects of Some Spice and Herb Extracts Against Arcobacter butzleri, A. cryaerophilus, and A. skirrowii

Seventeen spice and medicinal plant extracts (methanol and chloroform) were assayed for their antimicrobial activity against Arcobacter butzleri, A. cryaerophilus, and A. skirrowii. In general, all of the tested extracts were able, to a different extent, to inhibit the growth of the selected Arcobacter species. Cinnamon, bearberry, chamomile, sage and rosemary extracts showed strong antimicrobial activity toward arcobacter strains tested. Overall, the methanol extracts showed better activity than the chloroform extracts (P < 0.05); however, enhanced antibacterial activity of chloroform extracts of cinnamon and rosemary has been observed in comparison with their methanol counterparts. The inhibitory dose of the most active extracts (the diameter of zone of inhibition ≥ 20 mm) was determined using the disc-diffusion method as well.     

Crosslinked enzyme aggregates of hydroxynitrile lyase partially purified from Prunus dulcis seeds and its application for the synthesis of enantiopure cyanohydrins

Hydroxynitrile lyases are powerful catalysts in the synthesis of enantiopure cyanohydrins which are key synthons in the preparations of a variety of important chemicals. The response surface methodology including three‐factor and three‐level Box–Behnken design was applied to optimize immobilization of hydroxynitrile lyase purified partially from Prunus dulcis seeds as crosslinked enzyme aggregates (PdHNL‐CLEAs). The quadratic model was developed for predicting the response and its adequacy was validated with the analysis of variance test. The optimized immobilization parameters were initial glutaraldehyde concentration, ammonium sulfate saturation concentration, and crosslinking time, and the response was relative activity of PdHNL‐CLEA. The optimal conditions were determined as initial glutaraldehyde concentration of 25% w/v, ammonium sulfate saturation concentration of 43% w/v, and crosslinking time of 18 h. The preparations of PdHNL‐CLEA were examined for the synthesis of (R)‐mandelonitrile, (R)‐2‐chloromandelonitrile, (R)‐3,4‐dihydroxymandelonitrile, (R)‐2‐hydroxy‐4‐phenyl butyronitrile, (R)‐4‐bromomandelonitrile, (R)‐4‐fluoromandelonitrile, and (R)‐4‐nitromandelonitrile from their corresponding aldehydes and hydrocyanic acid. After 96‐h reaction time, the yield–enantiomeric excess values (%) were 100?99, 100?21, 100?99, 83?91, 100?99, 100?72, and 100?14%, respectively, for (R)‐mandelonitrile, (R)‐2‐chloromandelonitrile, (R)‐3,4‐dihydroxymandelonitrile, (R)‐2‐hydroxy‐4‐phenyl butyronitrile, (R)‐4‐bromomandelonitrile, (R)‐4‐fluoromandelonitrile, and (R)‐4‐nitromandelonitrile. The results show that PdHNL‐CLEA offers a promising potential for the preparation of enantiopure (R)‐mandelonitrile, (R)‐3,4‐dihydroxymandelonitrile, (R)‐2‐hydroxy‐4‐phenyl butyronitrile, and (R)‐4‐bromomandelonitrile with a high yield and enantiopurity. © 2014 American Institute of Chemical Engineers Biotechnol. Prog, 30:818–827, 2014     

Seasonal variation in antifungal,antibacterial and acetylcholinesterase activity in seven South African seaweeds

Seven seaweeds were collected from the intertidal zone at Rocky Bay on the east coast of South Africa. The species were Caulerpa racemosa var. laetevirens, Codium capitatum, Halimeda cuneata, Ulva fasciata, Amphiroa bowerbankii, Amphiroa ephedraea and Dictyota humifusa. Six bimonthly collections were made within a few days of the new moon to correspond with spring tide. Methanol extracts were tested for antifungal, antibacterial and acetylcholinesterase (AChE) inhibitory activity. No seasonal variation was observed in antifungal activity, with D. humifusa extracts being the most active. The seaweed extracts inhibited the growth of the Gram-positive bacteria, with Bacillus subtilis being more susceptible than Staphylococcus aureus. Dictyota humifusa was the only seaweed able to inhibit the Gram-negative Escherichia coli. Seasonal variation in antibacterial activity was observed, with the extracts generally having no activity in summer and having antibacterial activity in late winter (July collection) and early spring (September and November collections). Dictyota humifusa was the most effective seaweed species, having antibacterial activity throughout the year. All the extracts tested had AChE inhibitory activity, with no seasonal variation in the levels of activity. Dictyota humifusa extracts were the most effective at inhibiting AChE activity.     

Biologically Active Polypeptides and Hydrolytic Enzymes in Sea Anemones of Northern Temperate Waters

The content of biologically active polypeptides in aqueous and ethanol extracts of seven sea anemone species collected near Sakhalin Island (Sea of Okhotsk) and in Posyet Bay (Sea of Japan) was analyzed. Water extracts of the sea anemone Cribrinopsis similis showed the highest hemolytic activity, while ethanol extracts proved to have toxic properties. The levels of toxic and hemolytic activity of extracts of sea anemones inhabiting northern temperate waters were 2 to 3 orders of magnitude lower, compared to tropic species. The reason for this is likely to be the differences in the habitat conditions and biological traits of these animals. The water extracts of all species possessed proteolytic, phospholipase A2, and low DNAase activities, except Actinostola sp., whose aqueous extract contained a high activity alkaline DNAase. The species studied contained a wide range of proteinase inhibitors, O-glycosyl hydrolases (glycosidases and polysaccharide hydrolases). Water extracts of C. similis and Stomphia coccinea possessed the highest laminarinase activity. High activity of N-galactopyranosidase was found in water extracts of S. coccinea and Oulactis orientalis.     

<Emphasis Type="Italic">In vitro</Emphasis> antifungal activity of propolis samples of Czech and Slovak origin

Propolis has been used in traditional folk medicine for ages owing to a number of biological effects. Four propolis samples of Czech and one of Slovak origin were extracted using Soxhlet apparatus and analysed by thin-layer chromatography. Raw propolis samples and their extracts were tested by microdilution broth method to determine minimal inhibitory concentration (MIC) in eight strains of human pathogenic fungi. Raw propolis samples showed a lower in vitro antifungal activity than their extracts. In general, the petroleum ether extracts exhibited the highest in vitro antifungal activity (MIC range of 16–64 μg/ml). The content of flavonoids in the samples varied according to region. The highest amount of flavonoids was found in sample A that originated from Broumov (4%). The most susceptible to the propolis extracts were Trichophyton mentagrophytes and Candida albicans. The propolis samples of Czech and Slovak origin and their extracts showed a considerable in vitro antifungal effect which was associated especially with nonpolar petroleum ether and toluene extracts. There was only a partial correlation between flavonoids content and in vitro antifungal activity.