Abstract:

Hydroxynitrile lyase is a key enzyme for the process of cyanogenesis. Cyanogenesis is defense mechanism in which HCN is released form damaged tissue. The released HCN act as a defense mechanism against herbivoral and fungal attack. This process is widely distributed in several families of plants. Its presence is also discovered in bacteria, fungus, and insect. Hydroxynitrile catalyzes the reversible cleavage of cyanohydrins.  Optically pure cyanohydrins are used as an intermediate compound for the synthesis of various other compounds of industrial importance. It is also used in pharmaceuticals. In chemical industries, hydroxynitrile lyases are used as an important industrial biocatalyst for the synthesis of chiral cyanohydrins by catalyzing the reversible enzymatic reaction.  Hydroxynitrile lyase is divided into two main classes on the basis of presence or absence of cofactor FAD (Flavin adenine dinucleotide). This enzyme is immobilized by using different materials. By using X-ray crystallography and other techniques, its three dimensional structure is analyzed. Substrate specificity for this enzyme is checked by altering the active site amino acid by side directed mutagenesis. Hydroxynitrile lyase is cloned and expressed in E.coli, Pichia pectoris and Saccharomyces cerevisiae, for its large scale production.

Introduction:

 

Hydroxynitrile lyases (EC 4. 1. 2. 39) are the group of enzymes which catalyze the reaction involving enantioselective cleavage and synthesis of cyanohydrins. Cyanohydrins are alcohol containing a cyano group i.e. having a cyano and a hydroxyl group attached to the same carbon atom. In this mechanism of cyanogenesis, HCN is released .The release of HCN is used for defense against herbivores and fungal attack is widely distributed in higher plants, in important food crops such as cassava and sorghum, in several species of fern, bacteria, fungi and insects. Hydroxynitrile lyase is a key enzyme for the process.  (Gruber, K. et al., 2004)

 

During cyanogenesis cyanohydrin-O-glycosides are decomposed to a sugar molecule, Hydrogen cyanide and carbonyl compound in two step reactions. In the First step, β-galactocidase do hydrolysis of glycoside molecule into corresponding cyanohydrin. In second reaction, these cyanohydrins are hydrolyzed into HCN and aldehyde or ketone respectively. The second step occurs either spontaneously or by the action of enzyme α-hydroxynitrile lyase (HNL). Hydroxynitrile lyase not only cleaves the cyanohydrins but also catalyze the formation of cyanohydrins from carbonyl compounds and HCN. (Effenberger, 1991) The process of cyanogenesis is observed in more than 3000 plant species. (Wagner et al., 1996)

 

Cyanogenesis has been reported in more than 3000 species of vascular plant taxa comprising 105 families of flowering plants, pteridophytes (ferns) and gymnosperms. Prominent plants are belonging to families like Linaceae, Euphorbiaceae, Clusiaceae, Olacaceae, Rosaceae, Gramineae (monocotyledons) and Filitaceae (ferns). Cyanogenesis is also observed in diverse group of bacteria (Chromobacterium violaceum and few species of Pseudomonas), fungi, lichen, millipedes (Apheloria corrugata), arthropods and insects (Zygaena trifollii, a moth) (Monica et al., 2005).

 

Hydroxynitrile lyase isolated and purified from different plant species and divided into two main classes; HNL I and HNL II. This classification is based on the presence and absence of flavin adenine dinucleotide (FAD). Removal of this cofactor can cause the inactivation of enzyme, it also provides structural stability to enzyme or it can be present as an evolutionary remnant. Flavin adenine dinucleotide binds covalently with hydrophobic region near the catalytic site of enzyme. (Wagner et al., 1996)

 

FAD dependent Hnl’s are highly glycosylated, single-chain proteins with molecular masses of between 50–80 kDa; they accept (R) (+) mandelonitrile as their natural substrate, and show sequence homology with FAD-dependent oxidoreductases. These enzymes have been isolated from plants belonging to Prunoideae and Maloideae subfamilies of the Rosaceae as major seed storage proteins which play an active role in cyanogenesis. (Wagner et al., 1996)

 

FAD independent Hnl’s found in Sorghum bicolor, Manihot esculenta, Phlebodium aureum and Linum usitatissimum is less prevalent protein and has diverse physicochemical properties like substrate specificity, mass, glycosylation, isoelectric point, structure and amino acid sequence. This type of Hnl exhibit molecular masses of between 20–42 kDa, and accept a variety of (R) or (S) configured cyanohydrins as substrates. (Wagner et al., 1996)

The primary role of Hydroxynitrile lyase is in cyanogenesis for plant defense against herbivoral and fungal attack. A second physiological role of cyanogenic glycosides has also been suggested in the biosynthesis of L-asparagine. Moreover, Hydroxynitrile lyase can be used for detoxification of cyanogenic food crops, which cause a potential health hazards to consumers mainly in the third world. The most important of these crops is cassava (Manihot esculenta), which forms the basic nutriment for several hundred million people and has an estimated annual production of 150 million metric tons.  (Monica et al., 2005)

 

Besides their biological interest, HNL’s have gained attention as important biocatalysts for the synthesis of optically pure cyanohydrins. Chiral cyanohydrins are important synthetic compounds for the production of a large number of pharmaceuticals and agrochemicals. In chemical industries, hydroxynitrile lyases are used as an important industrial biocatalyst for the synthesis of chiral cyanohydrins by catalyzing the reversible enzymatic reaction. Cyanohydrins are biologically active compounds used in synthesis of alpha amino alcohols, alpha hydroxy ketones and alpha hydroxy acids, which have importance as chemicals, pharmaceuticals and agrochemicals.  (Monica et al., 2005) In 2000, the insecticidal activities of several cyanohydrins, cyanohydrin esters were evaluated. (Peterson, 2000)

 

 

 

Figure: The Biological Role of Hydroxynitrile Lyases in Plant Cyanogenesis

 

 

 

 

Historical perspective:

 

A century ago, in 1908, Rosenthaler prepared the mandelonitrile from benzaldehyde and HCN in the presence of a crude enzyme preparation extracted from almonds (named as emulsin). This was the first biocatalytic application of an HNL. (Effenburger et al., 2000)

 

This reaction later occurred in the presence of various plant extracts. But it was not confirmed that which enzyme is involved in this process. Enzymatic activity was also demonstrated in extract of almonds. After partial purification and characterization, it was proved that Hydroxynitrile lyase was involved in this process. (Effenburger et al., 2000)

In 1960, (R) - Hydroxynitrile lyases purified from almonds (Prunus amygdalus), were used in catalysis. In 1960, these enzymes gained importance for the preparation of cyanohydrins. Then in 1987, the synthetic application of PaHNL was rediscovered for the preparation of cyanohydrins with high enantiomeric purity. Another (R)-selective oxynitrilase (LuHNL) has been recognized and isolated from flax (Linum usitatissimum).  (S)- SbHNL isolated from plant Sorghum bicolor was the first (S)-HNL isolated and used in organic solvents for the synthesis of (S)-cyanohydrins. The substrate range was limited to aromatic and heteroarmatic aldehyde. (Effenburger et al., 2000)

Then in 1990, bulk quantities of this enzyme was prepared and used in the synthesis of various products. This enzyme was isolated, cloned and over-expressed, after the recognition of their important use as biocatalysts for the preparation of cyanohydrins. Further study of this enzyme was done in recent years.

 

 

 

The investigation of 3-Dimensional Structure of enzyme substrate complex of HNL from Hevea brasiliensis:

Zuegg and his coworkers determined the 3D structure of complexes between Hydroxynitrile lyase from Hevea brasiliensis with various substrates and inhibitor molecules (trichloracetaldehyde, hexafluoracetone, acetone, and rhodanide) by using x-ray crystallography. The complex between enzyme and trichloracetaldehyde, showed a covalent linkage which had apparent by the nucleophilic attack of the catalytic Ser80-Ogamma. Other complexes showed Hydrogen bonding between protein and substrate or inhibitor molecule. When native structure of enzyme was determined at cryo-temperature and at room temperature, it lead to the observation of two conserved water molecules. One molecule was found to be conserved in all complex structure and appears to have structural importance. The second water molecule is found to be conserved in all complex structure except in complex with rhodanide; but it is hydrogen bonded to the imidazole of catalytic His235 and appears to affect the catalyzed reactions of Hb-HN. From 3D structural data, it was observed that the incoming substrate is activated by H-bonding with carbonyl oxygen to Ser80 and The11 hydroxyl groups. Water molecule is replaced by the hydrogen cyanide and deprotonated by the His235 base. This deprotonation is facilitated by the positive charge of residue Lysine236. (Zuegg et al., 1991)

Reaction Parameters influencing activity and enantiospecificity of Hydroxynitrile lyase:

The effects of the different reaction parameters on the enzymatic reaction rate and product’s enantiomeric excess (e.e.) were studied in 1991.  It was observed that reaction rate increase with solvent hyrophobicity.  But highly hydrophobic solvent cannot be used for high HCN concentrations because they can cause loss of activity and product enantiomeric excess.  The reaction catalyzed by (S)- hydroxynitrile lyases isolated from H. brasiliensis for the synthesis of 3-phenylpropionaldehyde, an enantiomeric excess value of 88 ± 1% was produced  under optimized conditions of reaction.  But enantiomeric value decreased in the presence of high concentration of hydrogen cyanide, High solvent log Ρ and low enzyme loading because they deactivate the enzyme. The e.e value increased for all four enzymes at low temperatures (low from -5°C). Cyanohydrins with different eanntiopurities were obtained from enzymes purified from different sources under same optimized conditions. (Costesa et al., 1991) In

 

2002, the effect of reaction parameters was further studied. It was observed that in all HNL catalyzed reaction, by decreasing the temperature, enantioselectivity increased. By increasing water content enantioselectivity increased in HNL reactions. Log Ρ did not influence the enantioselectivity of HNL catalyzed reactions during this study. (Mattias et al., 2002)

Stability and Stabilization of Hydroxynitrile Lyase in Organic Solvents:

In 2001 stability of Hydroxynitrile lyase purified from Hevea brasiliensis was studied in different organic solvents.  It was determined that, the enzyme had half-lives in the range 1400-2500 hours, in dry solvents. The enzyme half life became low in medium which was water saturated. It was found that substrates like hydrogen cyanide and aldehyde can cause deactivation of enzyme. And this deactivation increased by increasing concentration of substrate, but it was reduced in hydrophilic solvents. When substrate concentration increased (2M) in tertiary-butyl methyl ether, the enzyme half life was 1.7 hours in the presence of hydrogen cyanide. Enzyme half life was 1.0 hours when 3-phenylpropionaldehyde was used.  It was also observed that the addition of polyethylenamine (125mg per g of enzyme), was increased the half life of enzyme up to 110 hour when incubated in the presence of hydrogen cyanide, and with 3-phenylpropanaldehyde in tertiary-butyl methyl ether, enzyme half life was 3.2 hour.  Same stabilization effects were observed for albumin and poly ethylene glycol. (David et al., 2001)

Role of Tryptophan 128 of hydroxynitrile lyase from Manihot esculenta in substrate specificity:

Hanspeter and his team studied the role of Tryptophan 128 of hydroxynitrile lyase of Manihot esculenta (MeHNL). This residue was replaced the mutant MeHNL-W128A by alanine to study its importance for the substrate specificity of the enzyme. Wild-type MeHNL and MeHNL-W128A showed comparable activity on the natural substrate acetone cyanohydrins. However, the specific activities of MeHNL-W128A for the unnatural substrates mandelonitrile and 4-hydroxymandelonitrile are increased 9-fold and ∼450-fold respectively. The crystal structure of the MeHNL-W128A in substrate-free form at 2.1 Å resolution indicates that the W128A substitution has enlarged the active-site channel entrance. The MeHNL-W128A–4-hydroxybenzaldehyde complex structure at 2.1 Å resolution shows the presence of two hydroxy benzaldehyde molecules in a sandwich type arrangement in the

 

active site. To find out change in substrate specificity of the mutant enzyme, they determined the X-ray crystal structure of MeHNL-W128A in complex with 4-hydroxybenzaldehyde at 2.1 Å resolution. The values of specific activities and km were compared for wild type and mutant form. The K_m value of MeHNL-W128A for acetone cyanohydrin is slightly higher than that for the wild-type enzyme, whereas the K_m values for the substrate mandelonitrile is seven times lower .For 4-hydroxymandelonitrile, a K_m value of wild-type MeHNL could not be determined. Due to the slow conversion rate of the substrate, the enzyme activity was below the detection limit at low substrate concentrations. Because the specific activity and the K_m value, for 4-hydroxymandelonitrile were clearly improved in comparison with mandelonitrile, the defined arrangement of a second molecule 4-hydroxybenzaldehyde in the active site of MeHNL-W128A showed an additional influence on the catalytic mechanism. (Hanspeter et al., 2002)

 

Table: Relative Specific activities and Km values for wild type MeHNL and MeHNL- W128A

 

	Relative specific activity	Km Value (mM)
Substrate	Wild Type MeHNL	MeHNL- W128A	Wild-type MeHNL	MeHNL- W128A
Acetone cyanohydrin	1	0.7	67	150
Mandelonitrile	1	9	30	4.5
4-Hydroxymandelonitrile	1	445	__	0.625

 

 

 

 

 Screening for hydroxynitrile lyase activity in crude preparation of edible plants:

Liliana and his coworkers, screened hydroxynitrile lyase activity in crude preparations of various edible plants by using the following criteria (i) readily available plants. (ii) In plants where cyanogenic glycoside is well established. (iii) Plants which are taxonomically related to other known sources of Hydroxynitrile lyase. They used crude enzyme preparations from available fresh fruits and leaves from garden tree. They selected the synthesis of mandelonitrile from benzaldehyde for the screening of the hydroxynitrile lyase activity of each crude preparation and then results were observed.  Hydroxynitrile activity was observed in seeds of melon, quince, anona and cherimoya. In leaves of cherry, plum, peach and mamey, HNL activity was also present. Crude preparations of enzyme from these sources, enantioselectively catalyzed the transformation of benzaldehyde into mandelonitrile. This methodology represents an HNL enzyme as attractive alternative for the preparation of quiral non-racemic cyanohydrins. (Liliana et al., 2004) The defatted meal of guanabana (Annona muricata) seeds catalyzed the (S)-selective addition of HCN to heteroaromatic, aromatic and α/β-unsaturated aldehydes, but did not catalyze the aliphatic aldehydes (Martin and Herfried, 2004). Different plant sources were examined for (R)- selective hydroxynitrile lyase. Apple, apricot, cherry and plum meals were prepared from the seeds or kernels of mature garden fruits. (Martin and Herfried, 2004)

In 2005, various other plant species were examined to check HNL activity. Yasuhisa and his team used HPLC method, and 163 species of plants form 74 families were examined for (R) – and (S) hydroxynitrile lyase activity. They discovered that leaves of Baliospermum montanum have (S)-HNL activity and leaves of Passiflora edulus, Prunus mume, prunus persica and Sorbus eucoparia exhibit (R)-HNL activity.  (Yasuhisa et al., 2005) Samik and his coworkers, isolated new hydroxynitrile lyase form seeds of Prunus mume. It is FAD containing enzyme and accepts a wide range of unnatural substrates to produce cyanohydrins in excellent optical and chemical yields. (Samik et al., 2005)

 

 

 

 

Table # 2: Isolated and characterized (R)-selective hydroxynitrile lyases:

(Martin and Herfried, 2004)

 

Species	E.C. No.	Enzyme source	Natural substrate
Prunus amygdalus	4.1.2.10	Almond bran, over expression in Pichia pastoris	(R)-mandelonitrile
Prunus serotina	4.1.2.10	black cherry kernels	(R)-mandelonitrile
Prunus domestica	4.1.2.10	plum kernel and leaves	(R)-mandelonitrile
Prunus avium	4.1.2.10	cherry kernel and leaves	(R)-mandelonitrile
Prunus persica	4.1.2.10	peach – kernel and leaves	(R)-mandelonitrile
Malus pumila	4.1.2.10	apple seed meal	(R)-mandelonitrile
Eriobotrya japonica	4.1.2.10	loquat seed meal	(R)-mandelonitrile
Linum usitatissimum	4.1.2.37	young flax plants over expression in E.coli &P.  pastoris	(R)-2-butanone-cyanhydrin

 

 

 

 

Table#3: Isolated and characterized (S)-selective hydroxynitrile lyases

(Martin and Herfried, 2004)

 

Species	E.C. No.	Enzyme source	Natural substrate
Manihot esculenta	4.1.2.38	manioc leaves over-expression in E. coli	acetone-cyanhydrin
Hevea brasiliensis	4.1.2.38	Rubber tree leaves overexpression in P. pastoris	acetone-cyanhydrin
Sorghum bicolor	4.1.2.11	millet seedlings	(S)-4-hydroxy-mandelonitrile
Ximenia americana	_	guanabana seed meal	(S)-mandelonitrile
Annonia muricata	_	cherimoya seed meal	_
Annonia cherimolia	_	anona seed meal	_

 

 

 

 

 

Production of Recombinant Hydroxynitrile Lyases:

Hydroxynitrile lyase of some plant species have been cloned in microbial systems for large-scale production.  In E.coli, HbHNL gene is over expressed with the help of strong inducible tac promoter, inclusion bodies are formed in the form of products which are insoluble and inactive. Although the most heterologous expression of enzyme HbHNL was achieved in Saccharomyces cerevisiae and methanol inducible Pichia pastoris expression system in soluble and highly active form comprising about 60% of total protein of the cell. MeHNL was also cloned and expressed in E.coli whereas LuHNL has been cloned and expressed in E. coli as well as in P. pastoris. But SbHNL recombinant expression was failed because it requires complex post-transcriptional modification of native enzyme.  A number of recombinant HNLs have been expressed in E. coli, Saccharomyces cerevisiae, and Pichia pastoris, leading to their wild application in industry (Monica et al., 2005). Hydroxynitrile lyase purified from Manihot esculenta (MeHNL) was cloned and expressed in multi-auxotrophic mutant of Saccharomyces cerevisiae cells. During this study, about 29 kg of (S)-mandelonitrile was produced from 23.3 kg of benzaldehyde, giving 98 mol% yield and a mean enantio excess of 98.9% ee. They concluded that this methodology can be applied in (S)-mandelonitrile production on a commercial ton scale. A number of recombinant HNLs have been expressed in E. coli, Saccharomyces cerevisiae, and Pichia pastoris, leading to their wild application in industry. (Hisashi et al., 2008)

Characterization of Two Bacterial Hydroxynitrile Lyases with High Similarity to Cupin Superfamily Proteins:

In 2012, bacterial proteins with HNL activity were reported. Usually plants are colonized by a range of different bacterial species.  These endophytes promote plant growth and health through various mechanisms, including the production of various substances. In pseudomonas and some other bacterial species, HCN production is reported.  In an experiment, gene libraries of some entophytic bacteria were isolated from potato. These were constructed in pZero-2 vector by standard procedures and then screened for HNL activity for (R/S)-mandelonitrile by using colony based colorimetric assay (67 mM (R/S)-mandelonitrile in 30 mM citrate-phosphate buffer (pH 3.5) at room temperature). In the gene library of a strain related to Pseudomonas mephitica, HNL activity was detected. Then this gene library was sub cloned and rescreened, the transformant which exhibited the strongest activity for mandelonitrile was selected for characterization. In characterization, sequence revealed that plasmid had 1,659-bp insert carrying two open reading frames (ORFs). Sequence of ORF 1 showed high similarity for protein of cupin superfamily, while second ORF sequence had similarity for drug resistant protein. Both ORF sequences showed no similarity with known HNL sequences. Then further study identified the two possible start codons for ORF 1. Upstream of both start codons of ORF1 motifs resembled to shine-Dalgarno sequences and in front of the second possible start codon of ORF1-short, a putative promoter sequence was identified. ORF1-long, ORF1-short, and ORF2 were cloned into pEamTA, and the clones carrying these genes were screened to check their performance on (R/S)-mandelonitrile. The drug resistance protein exhibited no activity, the cupin-like protein displayed HNL activity. Thus, the protein encoded by ORF1 was named PsmHNL. A protein isolated from Burkholderia phytofirmans strain PsJN was examined to verify its HNL functionality. As a result, significant enzymatic activity could be observed in colony based filter assay, and that protein was named as BpHNL. This result showed that HNL activity could be present in protein of cupin family. (Zahid et al., 2012)

Immobilization of Hydroxynitrile lyase:

HNLs isolated from different plant families such as Prunus amygdalus (PaHNL), Manihot esculenta (MeHNL), and Hevea brasiliensis (HbHNL) were immobilized in sol-gels. The cross-linked aggregate of HbHNL was prepared. Immobilization of HNLs on supports such as celite, cellulose, nitrocellulose, polyvinyl alcohol hydro gels, and aqua-gels has been reported to improve the enzyme robustness in biphasic media. These immobilized enzymes have been used for enantioselective synthesis of cyanohydrins. When enzyme activities of these immobilized enzymes and free enzymes were compared, it was concluded that immobilized enzymes perform better than free enzymes. (Hanefeld et al.,2008) Hydroxynitrile lyases are diverse group of enzymes that are used as biocatalyst in laboratory and industries. Due to their importance they have been immobilized using different methodologies.  (Hanefeld, 2013)

 

 

 

 

 

 

Conclusion:

Hydroxynitrile lyase is an important enzyme for the production of large number of synthetic compounds. Although, this enzyme was discovered before 1960 but it gained importance for the production of cyanohydrins, in 1960.  It can be isolated from various plant sources which are easily available.  Thus, we can prepare enantioselectively, wide range of compounds for industries by using this enzyme. These compounds are also used as in pharmaceutical and agrochemical industries. In addition, Hydroxynitrile lyase activity can be increased by using optimum conditions for reaction, and it can be used in the synthesis of various useful compounds. Its production is also increased by cloning techniques. Researchers have done a lot of work to discover the various aspects of its usefulness. Recently, the modeling calculations provided possible explanations for the substrate affinity for HNL, its reaction mechanism and enantioselectivity of this enzyme that provides the basis for future protein engineering efforts to modify the structure and function of such enzymes for the preparation  of cyanohydrins of high optical and enantiomeric value.

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