Manihot

Until 1987, the (R)-PaHNL from almonds was the only HNL used as catalyst in the enantioselective preparation of cyanohydrins. Therefore, it was of great interest to get access to HNLs which catalyze the formation of (5 )-cyanohydrins. (5 )-SbHNL [EC 4.1.2.11], isolated from Sorghum bicolor, was the first HNL used for the preparation of (5 )-cyanohydrins. Since the substrate range of SbHNL is limited to aromatic and heteroaromatic aldehydes as substrates, other enzymes with (5 )-cyanoglycosides have been investigated as catalysts for the synthesis of (5 )-cyanohydrins. The (5 )-HNLs from cassava (Manihot esculenta, MeHNL) and from Hevea brasiliensis (HbHNL) proved to be highly promising candidates for the preparation of (5 )-cyanohydrins. Both MeHNL and HbHNL have been overexpressed successfully in Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris. 

Figure 8.12 Conversion of benzaldehyde into enantiomerically pure (S)-mandelic acid by the sequential addition of HCN catalyzed by the (.S )-selective oxynitrilase from Manihot esculenta (MeHnL), and subsequent hydrolysis of the resultant (5)-mandelonitrile by the nitrilase from Pseudomonas fluorescens ECB 191 (PfNLase)...

Cassava, Manihot esculenta, roots, total cyanide (mg/kg FW) ... 

Dufour, D.L. 1988. Cyanide content of cassava (Manihot esculenta, Euphorbiaceae) cultivars used by Tukanoan indians in northwest Amazonia. Econ. Botany 42 255-266. 

ANDERSEN, M.D., M0LLER, B.L., Cytochromes P450 from Cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin cloning, functional expression in Pichia pastoris and substrate specificity of the isolated recombinant enzymes, J. Biol. Chem., 2000,275, 1966-1975. 

Jackson LC. 1988. Behavioral effects of chronic sublethal dietary cyanide in an animal model Implications for humans consuming cassava (Manihot esculenta). Hum Biol 60 597-614. 

Kamalu BP. 1993. Pathological changes in growing dogs fed on a balanced cassava (Manihot esculenta Crantz) diet. BR J Nutr 69(3) 921-934. 

Starch is one of the most abimdant plant polysaccharides and is a major source of carbohydrates and energy in the human diet (Zobel and Stephen, 1995). Starch is the most widely used hydrocolloid in the food industry (Wanous, 2004), and is also a widely used industrial substrate polymer. Total annual world production of starch is approximately 60 million MT and it is predicted to increase by additional approximately 10 million MT by 2010 (FAO, 2006b LMC International, 2002 S. K. Patil and Associates, 2007). Com/maize Zea mays L.), cassava (also known as tapioca—Manihot escu-lenta Crantn.), sweet potato Ipomoea batatas L.), wheat Triticum aestivum L.), and potato Solanum tuberosum L.) are the major sources of starch, while rice Oryza sativa L.), barley Hordeum vulgare L.), sago Cycas spp.), arrowroot Tacca leontopetaloides (L.) Kimtze), buckwheat Fagopyrum esculentum Moench), etc. contribute in lesser amounts to total global production. 

Chiral cyanohydrins are versatile intermediates in the synthesis of a-hydroxy acids, /3-amino alcohols, amino nitriles, a-hydroxy ketones and aziridines. For the synthesis of enantiopure cyanohydrins, the use of hydroxynitrile lyases is currently the most effective approach.Application of an organic-solvent-free system allows thermodynamically hindered substrates to be converted with moderate to excellent yields. With the use of the highly selective hydroxynitrile lyase from Manihot esculenta, the syntheses of several acetophenone cyanohydrins with excellent enantioselectivities were developed (Figure 8.2). (5)-Acetophenone cyanohydrin was synthesized on a preparative scale. ... 

Manihot esculenta (manioc) leaves NO 92-124 28.5-30 acetone cyanohydrin HO CN 33, 34... 

More successfully, the (S)-Hnl from Manihot esculenta has also been overexpressed in E. coli [41] and the lysate of the transformed cells showed an enzyme activity of 0.5 units per ml of the culture. A culture of 801 volume of the recombinant MeHnl followed by a short purification procedure [41] yielded 40,000 U. To obtain the equivalent amount of enzyme from the parent plant material would require the processing of 100 -200 kg of dried cassava leaves and thus this recombinant method for the production of MeHnl is a significant practical development. Hence, this recombinant MeHnl has allowed a study of (S)-cyano-hydrin production to be performed [41]. 

Scheme 3. The proposed mechanism for cyanohydrin cleavage by the Hnl from Manihot...

Manihot esculenta cytochrome P450 Conversion of vahne and isoleucine to aliphatic aldoximes 103... 

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... 

J. Hughes, Z. Keresztessy, K. Brown, S. Suhandono, M. A. Hughes, Genomic organization and structure of % hydroxynitrile lyase in cassava (Manihot esculenta Grantz). Arch. Biochem. Biophys. 1998, 356,107-115. 

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