Biocatalysis lyases

The present chapter reviews applications in biocatalysis of the ONIOM method. The focus is on studies performed in our research group, in most cases using the two-layer ONIOM(QM MM) approach as implemented in Gaussian [23], The studied systems include methane monooxygenase (MMO), ribonucleotide reductase (RNR) [24, 25], isopenicillin N synthase (IPNS) [26], mammalian Glutathione peroxidase (GPx) [27,28], Bi2-dependent methylmalonyl-CoA mutase [29] and PLP-dependent P-lyase [30], These systems will be described in more detail in the following sections. ONIOM applications to enzymatic systems performed by other research groups will be only briefly described. 

Effenherger, F., Hydroxynitriie lyases in stereoselective synthesis. In Stereoselective Biocatalysis, Patel, R.N. (ed.). Dekker New York, 2000 pp. 321-342. 

Enzymes that are suited for application in biocatalysis are mostly hydrolases, bnt also oxidorednctases, lyases and, to a lesser extent, transferases are useful. Obviously, the focus of bulk enzyme producers is different from the main interests of those who want to apply enzymes in biocatalytic applications. Fortunately, a growing number of companies has become active in the field of enzyme prodnction for biotransformations and by now a large nnmber of enzymes suited for biotransformations has become commercially available (Table 5.1). 

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... 

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. 

Effenberger F (2000) Hydroxynitiile lyases in stereoselective synthesis. In Patel RN (ed) Stereoselective biocatalysis. Marcel Dekker, New York/Basel, pp 321—342... 

Pohl M, Liese A. Industrial processes using lyases for C-C, C-N, and C-C bond formation. In Biocatalysis in the Pharmaceutical and Biotechnology Industries, Ed. Patel RN. CRC Press, Boca Raton, FL, 2007, p. 661. 

In Abramowicz DA (ed). Biocatalysis. Van Nostrand Reinhold, New York, pp 277-318 Nasser W, Chalet F, Robert-Baudouy J (1990) Purification and characterization of extracellular pectate lyase from Bacillus subtilis. Biochimie 72(9) 689-695 Neidleman SL (1991) Historical perspective on the industrial uses of biocattilysts. In Dordick JS (ed). Biocatalysts for industry. Plenum Press, New York, pp 21-33 Neuhaus W, Novalin S, Klimacek, M et al. (2006) Optimization of an innovative hoUow-fiber process to produce lactose-reduced skim milk. Appl Biochem Biotechnol 134(1) 1-14 Nield BS, Willows RD, Torda AE et al. (2002) New enzymes from environmental cassette arrays functional attributes of a phosphotransferase and an RNA-methyltransferase. Protein Sci 13 1651-1659... 

Kumagai H (1999b) Tyrosine phenol-lyase. In Flicinger MC, Drew SW (eds) Encyclopedia of bioprocess, technology fermentetion, biocatalysis and bioseparation. Wiley, New York, pp 2605—2609 Mori H, Shibasaki T, Uozald Y, Ochiai K, Ozaki A (1996) Detection of novel proline 3-hydroxylase activities in Streptomyces and Bacillus spp. by regio-and stereospecific hydroxylation of L-prohne. Appl Environ Microbiol 62 1903-1907... 

Enzymes from plants have been used since andent times, even though their nature remained obscure until the nineteenth century. Malting of cereals and the hydrolysis of complex polysaccharides are two of the oldest uses of enzymes in human history. Like their animal counterparts, most commerdally available plant-derived enzymes are hydrolases, particularly lipases and proteases. Papain, a cysteine protease from papaya, is the best-known plant-derived hydrolytic enzyme. Bromelain from pineapple and ficain from fig latex are similar cysteine proteases that have also found applications in biocatalysis. Horseradish peroxidase is a versatile oxidative enzyme obtained from its namesake that oxidizes many organic compounds, especially phenols. Hydroxynitrile lyase (often called oxynitrilase) from bitter almond is one of the most important plant-derived... 

Traditional techniques such as physical adsorption and covalent linkage onto solid supports, entrapment in polymer matrices, and microencapsulation have long been used for immobilizing such enzymes as lipases, proteases, hydantoinases, acylases, amidases, oxidases, isomerases, lyases, and transferases [12-18]. Encapsulation and adsorption have also proved their utility in the immobilization of bacterial, fungal, animal, and plant cells [12-21]. However, as biocatalysis applications have grown, so the drawbacks and limitations of traditional approaches have become increasingly evident. The forefront issues now facing bioimmobilization are indicated in Table 1. 

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