Lyases, as biocatalysts

Purkarthofer, T., Skranc, W., Schuster, C. and Griengl, H. (2007) Potential and capabilities of hydroxynitrile lyases as biocatalysts in the chemical industry. Applied Microbiology and Biotechnology, 76, 309—320. 

This section will focus on the development of (R)- and (S)-hydroxynitrile lyases as biocatalysts and their large-scale application. 

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... 

Fechter, M.H. and Griengl, H., Hydroxynitriie lyases biological sources and application as biocatalysts. Food Technol. BiotechnoL, 2004, 42, 287. 

A biocatalytic enantioselective addition of ammonia to a C=C bond of an a,)9-unsaturated compound, namely fumaric acid, makes the manufacture of L-aspartic acid possible on an industrial scale. This process, which is applied by, e. g., Kyowa Hakko Kogyo and Tanabe Seiyaku, is based on the use of an aspartate ammonia lyase as a biocatalyst [119]. Another comparable reaction is the asymmetric biocatalytic addition of ammonia to trans-cinnamic acid, which represents a technically feasible process for the production of L-phenyl-alanine [120]. 

Combined use of microbial enzymes as biocatalysts with chemical synthesis has its origin in the steroid transformation developed in the USA in the early 1950s. Arima and his group [11] invented a unique microbial conversion process, in which the aliphatic side-chain of cholesterol was cleaved to produce a steroid core as a starting material for chemical synthesis of steroid hormones. Yamada et al. discovered the reverse reaction of the pyridoxal-containing L-amino acid lyases and applied them to synthesize L-tryptophan and l-DOPA [12] from pyruvate, ammonia and corresponding aromatic compounds. Since these early achievements, a variety of unique processes with newly screened microbial enzymes as biocatalysts have been invented. 

Yeast contains a variety of enzymes, and in some cases use of a single purified enzyme is preferable. These arc divided into oxidorcductases, transferases, hydrolases, lyases, isomerases, and lipases. Many of these are commercially available (but expensive). Purified reductases usually require expensive cofactors. In addition individual microbes can be used as biocatalysts. A general review of microbial asymmetric reductions is available.5 These reductions can be the opposite of those of yeast. 

Turner, N.J. (2011) Ammonia lyases and aminomutases as biocatalysts for the synthesis of a-amino and P-amino acids. Curr. Opin. Chem. Biol, 15 (2), 234-240. 

This chapter covers some general aspects of the use of enzymes in aqueous and organic media. Although lipases are the most common biocatalysts in these processes [4], other hydrolytic enzymes such as esterases and nitrilases have also shown their utility in the manufacture of pharmaceuticals. In addition, some representative examples using oxidoreductases and lyases will be also discussed. 

A single enzyme, L-aspartate ammonia lyase obtained from E. coli is used acting on ammonium fumarate substrate. Little cell activity was lost upon immobilisation. Initially polyacrylamide was used as the immobilisation medium, and later cross-linked K-carrageenan was used, as higher operational life-times for the biocatalyst were obtained. The immobilized cell activity is very stable with a half-life of 120 days, while achieving 95% conversion of substrate into product. 

Figure 10-20) these are changed into cis unsaturated aldehydes by hydroperoxide lyase. The cis unsaturated aldehydes are transformed by isomerase into the corresponding trans isomers. These same substances in another matrix would be experienced as off-flavors. The use of lipoxygenase as a versatile biocatalyst has been described by Gardner (1996). 

The aldol reaction is of central importance to synthetic organic chemistry in carbon skeletal elaboration. Furthermore it generates at least one and often two new stereogenic centers. Since an abundance of natural aldolases have now been identified and characterized, interest in the application of aldolases as catalysts in synthetic organic chemistry continues to increase. However, despite the potential synthetic utility of aldolase chemistry, the lyase class of enzymes is still underutilized. In contrast, the oxido-reductase and hydrolase class of enzymes have demonstrated substantial synthetic utility and are the two most utilized class of biocatalysts. 

L-Phenylalanine can be s)mdiesised from fnms-dnnamic add (Hgure AS. 12) catalysed by a L-phenylalanine ammonia-lyase horn Rhodococcus glutinis. The commercialisation of the process was limited by the low conversion (70%), low stability of die biocatalyst and the severe inhibition exerted by fnms-dnnamic add. These problems were laig overcome by researchers at Cenex. The process, commercialised for a short period 1 Cenex, involves a cell-hee preparation of phenylalanine-ammonia-lyase activity from Rhodotorula rubra. 

The enzymatic production of diverse amino acids has been described by quite a lot of companies over the last 20-30 years and many processes are based on the use of lyases [138-141]. With regard to the use of immobilized biocatalysts only two new processes shall be introduced here the production of L-as-partic acid (169) using an immobilized cell catalyst by Nippon Shokubai Ka-gaku Kogyo, Japan [142, 143] and the use of an immobilized enzyme by BioCatalytics [7] (both Scheme 55). 

The application of the biocatalysts in this overview is limited to these stable enzymes, which do not need cofactors, such as the various hydrolytic enzymes, some lyases, transferases and isomerases. In addition to these groups, oxidoreductases, which demand NAD or NADP as cofactors, some pyridoxyl-phosphate dependent lyases with simple systems for cofactor regeration and finally, various aldolases in combination with L-glycerol- phosphate oxidase and catalase are useable to some extent in cell-free form. 

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