Enzymes hydroxynitrile lyase

The highest reported heterologous production of a protein in yeast comes from the work by Hasslacher et al. (1997). The enzyme hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis was reported to produce levels of 22g/L intracellularly in P. pastoris. In the same study, S. cerevisiae and E. coli were tested in parallel experiments but were not competitive. Levels of proteins produced by yeasts have more typically been in the range of 1-15 g/L (Schmidt, 2004). A list of reported yields for expression of proteins in P. pastoris is provided by Cregg at http / faculty.kgi.edu/cregg/index.htm... 

Zuegg, J., Gruber, K., Gugganig, M. et al. (1999) Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis. Protein Science A Publication of the Protein Society, 8, 1990-2000. 

Lauble, H., Miehlich, B., Forster, S. et al. (2002) Crystal structure of hydroxynitrile lyase from Sorghum bicolor in complex with the inhibitor benzoic acid a novel cyanogenic enzyme. Biochemistry, 41, 12043-12050. 

Bauer, M., Griengl, H. and Steiner, W. (1999) Kinetic studies on the enzyme (S)-hydroxynitrile lyase from Hevea hyusilh iisis using initial rale methods and progress curve analysis. Biotechnology and Bioengineering, 62,20-29. 

Willeman, W.F., Straathof, A.J.J. and Heijnen, J.J. (2002) Reaction temperature optimization procedure for the synthesis of (/ )-mandelonitrile by Primus amygdalus hydroxynitrile lyase using a process model approach. Enzyme and Microbial Technology, 30, 200-208. 

Persson, M., Costes, D., Wehtje, E. and Adlercreutz, P. (2002) Effects of solvent, water activity and temperature on lipase and hydroxynitrile lyase enantioselectivity. Enzyme and Microbial Technology, 30, 916-923. 

Hickel, A., Radke, C.J. and Blanch, H.W. (1999) Hydroxynitrile lyase at the diisopropyl ether/water interface evidence for interfacial enzyme activity. Biotechnology and Bioengineering, 65, 425—136. 

Rhodococcus erythropolis NCIMB 11540 has been employed as biocatalyst for the conversion of (R)- or (.S )-cyanohydrins to the corresponding (R)- or (S)-a-hydroxycarboxylic acids with an optical purity of up to >99% enatiomeric excess (ee) [27-29] the chiral cyanohydrins can separately be produced using hydroxynitrile lyase from Hevea braziliensis or from Prunus anygdalis [30]. Using the combined NHase-amidase enzyme system of the Rhodococcus erythropolis NCIMB 11 540, the chiral cyanohydrins were first hydrolyzed to the... 

The production of optically active cyanohydrins, with nitrile and alcohol functional groups that can each be readily derivatized, is an increasingly significant organic synthesis method. Hydroxynitrile lyase (HNL) enzymes have been shown to be very effective biocatalysts for the formation of these compounds from a variety of aldehyde and aliphatic ketone starting materials.Recent work has also expanded the application of HNLs to the asymmetric production of cyanohydrins from aromatic ketones. In particular, commercially available preparations of these enzymes have been utilized for high ee (5)-cyanohydrin synthesis from phenylacetones with a variety of different aromatic substitutions (Figure 8.1). 

Sharma, M., Sharma, N.N. and Bhalla, T.C., Hydroxynitrile lyases at the interface of biology and chemistry. Enzyme Microb. Technol., 2005, 37, 279. 

A new hydroxynitrile lyase (HNL) was isolated from the seed of Japanese apricot Prunus mume). It accepts benzaldehyde and a large number of unnatural substrates for the addition of HCN to produce the corresponding (7 )-cyanohydrins in excellent optical and chemical yields. A new high-performance liquid chromatography (HPLC)-based enantioselective assay technique was developed for the enzyme, which promotes the addition of KCN to benzaldehyde in a buffered solution (pH 4.0). Asymmetric synthesis of (7 )-cyanohydrins by a new HNL is described (Figure 8.4). ... 

We have found a new (/ )-hydroxynitrile lyase from Japanese apricot (P. mume). The new enzyme accepts a broad array of substrates, ranging from aromatic, heteroaromatic, bicyclic to aliphatic carbonyl compounds, and yields the corresponding cyanohydrins with excellent enantioselection. 

This enzyme [EC 4.1.2.11], also known as hydroxyman-delonitrile lyase or hydroxynitrile lyase, catalyzes the conversion of (5 )-4-hydroxymandelonitrile to cyanide and 4-hydroxybenzaldehyde. Aliphatic hydroxynitrhes do not serve as substrates for this enzyme, unlike that of (5 )-hydroxynitrilase [EC 4.1.2.39]. 

This enzyme [EC 4.1.2.10], also known as hydroxynitrile lyase and (i )-oxynitrilase, catalyzes the conversion of mandelonitrile to cyanide and benzaldehyde. 

Enzymes for Carboligation - 2-Ketoacid Decarboxylases and Hydroxynitrile Lyases 327... 

The detailed characterization of hydroxynitrile lyases from Sorghum hicolor (E.C. 4.1.2.11) and Linum usitassimum (E.C. 4.1.2.37) has been hampered for a long time due to the lack of a recombinant expression system. Therefore our studies were focused on cloning of the coding genes, recombinant expression, and characterization of these enzymes. 

HNLs comprise a heterogenous enzyme family, since hydroxynitrile lyase activity has evolved in different structural frames by convergent evolution [17, 18]. Thus, (S) -specific HNLs based on an a/P-hydrolase fold framework from Manihot esculmta (cassava) [19-21], Hevea hrasilensis (rubber tree) [22-26], and Sorghum hicolor (millet) [27-33] have been described. (R)-specific HNLs based on the structural framework of oxidoreductases were isolated from Linum usitatissimum (flax) [30, 34-37] and Rosaceae (e.g., bitter almonds) [31, 38]. Despite their potential in biocatalysis only few HNLs (from cassava and rubber tree) are available by recombinant gene expression, which is a prerequisite for their technical application [20, 24]. Thus, cloning, recombinant expression, and... 

M. Hasslacher, C. Kratky, H. Griengl, H. Schwab, S. D. Kohlwein, Hydroxynitrile lyase from Hevea hrasiliensis, molecular characterization and mechanism of enzyme catalysis. Proteins 1997, 27, 438-449. 

Although many biochemical reactions take place in the bulk aqueous phase, there are several, catalyzed by hydroxynitrile lyases, where only the enzyme molecules close to the interface are involved in the reaction, unlike those enzyme molecules that remain idly suspended in the bulk aqueous phase [6, 50, 51]. This mechanism has no relation to the interfacial activation mechanism typical of lipases and phospholipases. Promoting biocatalysis in the interface may prove fruitful, particularly if substrates are dissolved in both aqueous phases, provided that interfacial stress is minimized. This approach was put into practice recently for the enzymatic epoxidation of styrene [52]. By binding the enzyme to the interface through conjugation of chloroperoxidase with polystyrene, a platform that protected the enzyme from interfacial stress and minimized product hydrolysis was obtained. It also allowed a significant increase in productivity, as compared to the use of free enzyme, and simultaneously allowed continuous feeding, which further enhanced productivity. 

The application of crude enzyme preparations also constitutes a convenient method to find new sources for hydroxynitrile lyases [56-58]. 

Aliphatic aldehydes have been converted to their (R)-cyanohydrins using a bipha-sic system to accommodate hydroxynitrile lyase enzyme (from the Japanese apricot, Prunus mume) as the enantioselective catalyst.251... 

The hydroxynitrile lyase (HNL) class of enzymes, also referred to as oxynitrilases, consists of enzymes that catalyze the formation of chiral cyanohydrins by the stereospecific addition of hydrogen cyanide (HCN) to aldehydes and ketones (Scheme 19.36).275 279 These chiral cyanohydrins are versatile synthons, which can be further modified to prepare chiral a-hydroxy acids, a-hydroxy aldehydes and ketones, acyloins, vicinal diols, ethanolamines, and a- and P-amino acids, to name a few.280 Both (R)- and (.S )-selective HNLs have been isolated, usually from plant sources, where their natural substrates play a role in defense mechanisms of the plant through the release of HCN. In addition to there being HNLs with different stereo-preferences, two different classifications have been defined, based on whether the HNL contains a flavin adenine dinucleotide (FAD) co-factor. 

In a second approach hydrocyanic acid was added to hydroxypivaldehyde by use of (R)-selective hydroxynitrile lyase from almonds (PaHNL) [11]. (R)-Cyanohydrin was obtained in 84% yield and 89% ee, and was directly cydized to give crude D-pantolactone by acid-catalyzed hydrolysis. Unfortunately, in contrast with O-protected hydroxy and halogenated pivalaldehydes, the technically available starting compound hydroxypivaldehyde requires use of purified enzyme (and high enzyme loading). 

For the synthesis of cyanohydrins nature provides the chemist with R- and S-selective enzymes, the hydroxynitrile lyases (HNL) [4-7]. These HNLs are also known as oxynitrilases and their natural function is to catalyze the release of HCN from natural cyanohydrins like mandelonitrile and acetone cyanohydrin. This is a defense reaction of many plants. It occurs if a predator injures the plant cell. The reaction also takes place when we eat almonds. Ironically the benzaldhyde released together with the HCN from the almonds is actually the flavor that attracts us to eat them. 

In conclusion, the bienzymatic transformation of aldehydes and HCN into the enantiomerically pure 2-hydroxycarboxylic acids is feasible. The stereochemistry can be steered either by the hydroxynitrile lyase or by both enzymes in combination and the hydrocyanation equilibrium is no longer an issue because it can be shifted to complete conversion. The formation of large amounts of amide, in particular (S)-4a, somewhat reduces the immediate practical value of our procedure. Ways to obviate this unwanted side-reaction will be discussed later. 

Oxynitrilases or hydroxynitrile lyases (HNL) constitute a group of enzymes that catalyze the reversible addition of HCN to ketones and aldehydes. The natural role of these enzymes is a defence mechanism of higher plants against herbivores, whereby HCN is liberated from cyanoglucosides such as prunasin (almond, cherry, apple) by the action of a glycosidase and a hydroxynitrile lyase. 

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