Biocatalysis

Biocatalysis refers to catalysis by enzymes. The enzyme may be introduced into the reaction in a purified isolated form or as a whole-cell micro-organism. Enzymes are highly complex proteins, typically made up of 100 to 400 amino acid units. The catalytic properties of an enzyme depend on the actual sequence of amino acids, which also determines its three-dimensional structure. In this respect the location of cysteine groups is particularly important since these form stable disulfide linkages, which hold the structure in place. This three-dimensional structure, whilst not directly involved in the catalysis, plays an important role by holding the active site or sites on the enzyme in the correct orientation to act as a catalyst. Some important aspects of enzyme catalysis, relevant to green chemistry, are summarized in Table 4.3. 

Since enzymes are composed of amino acids they may be assumed to act as either acid or base catalysts through groups such as -COOH, -NH2 and -CONH2. The scope of activity, however, is enhanced considerably through coordination with metallic ions found in the body such as Mg, Fe, Fe , Ca and Zn +. Enzymes have been classified into six functional types according to the reactions they catalyse  

High degree of substrate specificity due to limited flexibility of active site 

Natural operation under conditions found in body 

Possibility for tandem reactions when using whole organisms 

The time is ripe for the widespread application of biocatalysis in industrial organic synthesis and according to a recent estimate [113] more than 130 processes have been commercialised. Advances in recombinant DNA techniques have made it, in principle, possible to produce virtually any enzyme for a commercially acceptable price. Advances in protein engineering have made it possible, using techniques such as site directed mutagenesis and in vitro evolution, to manipulate enzymes such that they exhibit the desired substrate specificity, activity, stability, pH profile, etc. [114]. Furthermore, the development of effective immobilisation techniques has paved the way for optimising the performance and recovery and recycling of enzymes. 

In contrast, enzymatic cleavage of penicillin G (Fig. 1.37) is performed in water at 37 °C and the only reagent used is NH3 (0.9 kg per kg of 6-APA), to adjust the pH. The enzymatic process currently accounts for the majority of the several thousand tons of 6-APA produced annually on a world-wide basis. 

Another advantage of biocatalysis is the high degree of chemo-, regio- and stereoselectivities which are difficult or impossible to achieve by chemical means. A pertinent example is the production of the artificial sweetener, aspartame. The enzymatic process, operated by the Holland Sweetener Company (a joint venture of DSM and Tosoh) is completely regio- and enantiospecific (Fig. 1.38) [117]. 

The above-mentioned processes employ isolated enzymes - penicillin G acy-lase and thermolysin - and the key to their success was an efficient production of the enzyme. As with chemical catalysts, another key to success in biocatalytic processes is an effective method for immobilisation, providing for efficient recovery and re-use. 

DuPont has developed a process for the manufacture of glyoxylic acid, a large volume fine chemical, by aerobic oxidation of glycolic acid, mediated by resting 

Enzymes can operate under relatively mild conditions and usually exhibit a very high degree of substrate-, chemo-, regio-, and enantioselectivity. In this chapter we cannot cover the whole area of biocatalysis. We will give preference to biocatalysts used in industrial production processes. 

Subsequently, selected apphcations of biocatalysts will be examined, used as either isolated enzymes or enzymes that operate in immobilized or permeabihzed cells. Synthesis routes in which one or all of the steps are biocatalytic have advanced dramatically in recent years. Increasingly, biocatalysts are combined with chemical catalysts or utilized in a network of reactions in a whole cell. It can be pointed out, that biocatalysts do not operate by different scientific principles from usual catalysts. All enzyme actions can be explained by rational chemical and physical principles. However, enzymes can create imusual and superior reaction conditions such as extremely low p/fa values or a high positive potential for a redox metal ion. 

Industrial Catalysis A Practical Approach, Second Edition. Jens Hagen Copyright 2006 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim ISBN 3-527-31144-0 

Enzymes show some advantages and disadvantages with other kinds of catalysts (Table 4-1). Whereas enzymes often exhibit great advantages in terms of selectivity, their stability is often insufficient. Furthermore, long development times of new biocatalysts remain a problem and a challenge. 

Very high enantioselectivity often low specific activity 

One of the areas to benefit from the speed and efficiency of reaction optimization afforded by continuous flow processes is that ofbiochemical transformations involving enzymes, whole cells, or lysates [78]. Biocatalysis is an important area of synthetic chemistry that has been extensively studied for application within industry for the synthesis of amino adds, lipids, sugars, pharmaceuticals, and fine chemicals however, the long-term instability of biocatalysts predudes application within industry. 

An early example by Reetz and co-workers [79] demonstrated the evaluation of a series of biocatalysts for the hydrolytic kinetic resolution of chiral glycidyl phenyl ethers. Employing a fused-silica reactor, the authors developed an integrated reaction system capable of performing biocatalytic hydrolysis, along with separation and detection of the reaction products. Using the enantioselective hydrolysis of 2-phenoxymethyloxirane (136) to 3-phenoxypropane-l,2-diol (137) as a model reaction (Table 6.14), the authors evaluated the biocatalytic activity of a series of epoxide 

A more recent example of enzyme-catalyzed synthesis performed in micro reactors was reported by Rutjes and co-workers [81], who demonstrated the use of crude enzyme lysates, containing hydroxynitrile lyase, for the enantioselective synthesis of cyanohydrins. Employing a wet-etched borosilicate glass micro reaction channel, containing pillars to promote biphasic laminar flow, the authors evaluated the 

In addition to concerns associated with the cost of enzymes and their efficient re-use with respect to preparative-scale synthesis, the difficulties associated with the recovery and re-use of enzyme cofactors, which are frequently more expensive than 

Biocatalysts are enzymes used from naturally occurring organisms such as yeasts, bacteria, and plants. Biocatalysts can be applied to nearly any field involving chemical reactions. Many chemical companies are interested in this new market such as Dow Chemical, BASF, DuPont, and Cargill, just to name a few. Biocatalysis uses 

Chirality plays a major role in the development of drugs. A chiral molecule is defined as nonsuperimposable on its mirror image. Two chiral molecules commonly called enantiomers are often compared to the right and left hands. The same type and number of atoms are in the two enantiomers but the spatial arrangement of atoms is different Usually one enantiomer is preferred over the other. In the pharmaceutical industry chiral molecules constitute a large portion of pharmaceutical sales. More than half the drugs approved worldwide are chiral. Examples include Lipitor and Zocor , as well as ibuprofen sold under the common brand names Motrin and Advil . 

The key chiral building block is hydroxynitrile (HN) or ethyl (7 )-4-cyano-3-hydroxybutyrate whose demand is estimated to be about 200 metric tons annually. Traditional commercial processes are based on  

The three-step green process designed by Codexis is centered around the activity, selectivity, and stability of three enzymes created using cutting-edge genetic methods. This new process involves  

neutral conditions with fewer steps. 

The examples of bioorganic chemistry in the previous paragraph are all concerned with the known biocatalytic assays, in which the effects of miniaturization on the efficiency of the analytical method were investigated. Recently, a new development has started in which the biocatalytic process itself has become the center of attention. Biocatalysis in microreactors, as described in here, deal with the investigation of the use of enzymes for the production of molecules. Two different approaches can be identified. In one line of investigation, the miniaturized reaction environment is used to screen the efficiency of an enzyme. In this case, only small amounts of 

Biological species such as enzymes, whole cells, antibodies and even bacteria can all be successfully entrapped in silica sol-gel matrices, often with enhancement of activity with respect to the free biologicals. In these cases, the process is adapted to eliminate toxic alcohols which are typically released in conventional sol-gel processes based on the hydrolysis of silicon alkoxides. Two such methods are the use of silicon alkoxide 

Enzymes as nature s catalysts are able to perform an outstanding array of regio- and stereoselective reactions. Therefore, as water is nature s solvent, it is not surprising that many biocatalytic reactions have been performed in the aqueous phase.However, in typical reactions, the substrates are limited to hydrophilic compounds because of a desire for reaction homogeneity. It should also be noted that, in most cases, the aqueous medium is a buffer solution of an ideal pH for the enzyme to function effectively. 

Reaction studies include cyanations using hydroxy nitrile lyases, hydrolysis of amides using acylases, amidases or lipases, and even dehydration reactions of aldoximes to nitriles using aldoxime dehydratase. This reaction is quite 

Enzymes may also catalyze transformations of a highly selective and nniqne natnre snch as in transformations of small molecnles. Examples are the fragmentation of 1-amino-cyclopropane-1-carboxylate to the frnit ripening hormone ethylene, the cycloreversion of thymine dimers in DNA repair, the synthesis of isopenicUhn and the rednction of molecn-lai nitrogen (N2) to ammonia 

Enzymes demonstrate both high specificities and significant reaction rate accelerations. The relative values of enzymic over non-enzymic reactions may be from 10 ° to 10 (orotidine decarboxylase) and the turnover numbers range from one catalytic event per minute to 10 per second (hydration of CO2 to HC03 by carbonic anhydrase). The molecular entities of enzymes cover proteins, ribozymes and catalytic antibodies. 

Tlie sequential reactions in elongating acyl transfers in the synthesis of polyketide natural products and non-ribosomal peptide antibiotics such as erythromycin, rapamycin, epotliilone, lovastatin, penicillins, cyclosporin and vancomycin resemble molecular solid-state assembly lines. Such multimodular enzymes may be utilized in combinatorial biosynthesis by way of reprogramming for the manufacture of unnaUiral analogs of natural products. 

IPNS belongs to a family of iron-containing enzymes which use Fe to activate O2 and simultaneously the specific co-substrate for redox reactions whereby both atoms of O2 aie reduced to water and the tripeptide ACV undergoes oxidation with C-S and C-C bond formation to generate P-lactam molecules. 

A related enzyme, the expandase enzyme, is deployed by cephalosporin-producing organisms to expand the five-membered penicillin ring to the six-membered cephalosporin ring. 

Enzymes have many potential advantages when used as catalysts for chemical synthesis. The unique properties offered by these biocatalysts are, first of all, their often outstanding chemo-, regio-, and, in particular, stereoselectivity. Furthermore, enzymes are highly efEcient catalysts working under very mild conditions. However, enzymes do also have some drawbacks that may limit their potential use, such as ability to accept a limited substrate pool only, and a moderate operational stability. Ways of overcoming most of these potential limitations exist and they pose in most cases more of a perceived than a real problem. Well over 100 different biocatalytic processes have been implemented on an industrial scale [1]. A few processes are 

A new tool has recently been added to the area of biocatalysis as a variety of enzymes within the last few years have been shown to exhibit both stability and activity in ionic liquids (ILs). This sechon will give a short review of these findings and discuss what potential impact they can have on the area of biocatalysis in general. 

Ionic Liquids and Enzymes Solvent Properties of Ionic Liquids 

By nature ILs are polar compounds, a property that is easily studied with solvatochromic compounds such as Reichardt s dye [4]. On the normalized polarity scale ( ) from 0.0 (tetramethylsilane, TMS) to 1.0 (water), most ILs can be foimd around 0.6-0.7. By comparison, for ethanol is 0.654 [4]. In general, the polarity is largely controlled by the nature of the cahon, whereas the ability of the ILs to parhcipate in hydrogen bonding seems to depend on the anion. On the other hand, the miscibility of ILs with water seems unpredictable. A well-known example is [BMIMjjBFJ which is water-miscible, while [BMIM][PFj] is not. Regarding biocatalysis, it has been pointed out that even trace amoimts of ionic impurihes can significantly affect the properties of the IL as well as the activity of added enzyme. 

Kazlauskas and co-workers found a dramatically improved enzymatic performance in some tetrafluoroborate ILs after treatment with Na2C03 [5], The authors speculate that the effect could be due to precipitation of Ag2C03 or neutralization of traces of acid, but can potentially also be explained by a more well-defined water content [6]. Traces of halide ions can also be a possible cause of poor enzymatic performance [7]. 

Licia M. Pera Mario D. Baigori Ashok Pandey, Guillermo R. Castro  

Industrial Bior neries and White Biotechnology http //dx.doi.Org/10.1016/B978-0-444-63453-5.00012-4 

There are a number of strategies for screening the biocatalysts. These include the use and development of synthetic substrates, fermentation techniques, metagenomic methods, and genetic databases. 

Recently, a universal enzyme-coupled fluorescence assay for glycosyl transferases was developed. This method is extremely cost-effective and is based on the quantification of nucleotides produced in the glycosyl transfer reaction. The guanosine diphosphate (GDP), uridine diphosphate (UDP), and cytidine monophosphate (CMP) are phos-phorylated with nucleotide kinase in the presence of excess of ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase,glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for the conversion of resazurin to resorufin, which is then quantified by fluorescence measurement. 

There are studies on the use of chromogenic substrates such as p-nitrophenyl deri-vates of caprate, laurate, palmitate, and/or stearate for a selectivity-based analysis of lipase preparations toward transesterification or hydrolysis in organic medium as well as to 

Values of 50 g VS 1 biofilm have been repoiTed [90], where VS (volatile sohds) is a general measure for biomass as used in environmental engineering. 

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J. S. Dordick, ed.. Biocatalysis Torlndustry, Plenum Publishing Corp., New York, 1991. 

Stuckey, D. C., Caridis, K. A., Leak, D. J., Kinetics of Mycobacterium M156 for chiral biotransformations. Biotechnology 94, Indlnt. Symp. on Applied Biocatalysis, Brighton, pp.37-39, 1994. 

Biocatalysis Chemical reactions mediated by biological systems (microbial communities, whole organisms or cells, cell-free extracts, or purified enzymes aka catalytic proteins). 

The term biotransformation or biocatalysis is used for processes in which a starting material (precursor) is converted into the desired product in just one step. This can be done by use either of whole cells or of (partially) purified enzymes. Product examples range from bulk chemicals (such as acrylamide) to fine chemicals and chiral synthons (chiral amines or alcohols, for example). There are several books and reviews dealing with the use of bio transformations either at laboratory or at industrial scales [1, 10-13]. 

In order to broaden the field of biocatalysis in ionic liquids, other enzyme classes have also been screened. Of special interest are oxidoreductases for the enan-tioselective reduction of prochiral ketones [40]. Formate dehydrogenase from Candida boidinii was found to be stable and active in mixtures of [MMIM][MeS04] with buffer (Entry 12) [41]. So far, however, we have not been able to find an alcohol dehydrogenase that is active in the presence of ionic liquids in order to make use of another advantage of ionic liquids that they increase the solubility of hydrophobic compounds in aqueous systems. On addition of 40 % v/v of [MMIM][MeS04] to water, for example, the solubility of acetophenone is increased from 20 mmol to 200 mmol L ... 

There is still a long way to go before ionic liquids can become commonly used in biocatalysis. This will require ... 

T. Hartmann, E. Schwabe, T. Scheper, Enzyme catalysis in supercritical fluids in R. Patel, Stereoselective Biocatalysis, Marcel Dekker, 2000, 799. 

C. Veeger, in Biocatalysis in organic media (C. Laane, J. Tramper, M. D. Lilly eds.), Elsevier, Amsterdam, 1987. P. Bonhote, A. P. Dias, K. Papageor-eiou, M. Gratzel, Inorg. Chem. 1996,... 

Before discussing the medium engineering phenomenon and its synthetic relevance in details, it is useful to offer a brief overview of the fundamentals of biocatalysis in organic media. 

The aspects of medium engineering summarized so far were a hot topic in biocatalysis research during the 1980s and 1990s [5]. Nowadays, all of them constitute a well-established methodology that is successfully employed by chemists in synthetic applications, both in academia and industry. In turn, the main research interests of medium engineering have moved toward the use of ionic liquids as reaction media and the employment of additives. 

In recent years ionic liquids have also been employed as media for reactions catalyzed both by isolated enzymes and by whole cells, and excellent reviews on this topic are already available [47]. Biocatalysis has been mainly conducted in those room-temperature ionic liquids that are composed of a 1,3-dialkylimidazolium or N-alkylpyridinium cation and a noncoordinating anion [47aj. 

Nowadays biocatalysis is a well-assessed methodology that has moved from the original status of academic curiosity to become a widely exploited technique for preparative-scale reactions, up to the point that the so-called industrial biotechnology (to which biocatalysis contributes to the most extent) is one of the three pillars of the modern sustainable chemistry. 

Directed Enzyme Evolution Screening and Selection Methods, Humana Press, Totowa. Vol. 230. (b) Brakmann, S. and Johnsson, K. (eds)(2002) Directed Molecular Evolution of Proteins (or How to Improve Enzymes for Biocatalysis), Wdey-VCH Verlag GmbH, Weinheim. (c) Brakmann, S. and Schwienhorst, A. (eds)... 

In the enzyme design approach, as discussed in the first part of this chapter, one attempts to utilize the mechanistic understanding of chemical reactions and enzyme structure to create a new catalyst. This approach represents a largely academic research field aiming at fundamental understanding of biocatalysis. Indeed, the invention of functional artificial enzymes can be considered to be the ultimate test for any theory on enzyme mechanisms. Most artificial enzymes, to date, do not fulfill the conditions of catalytic efficiency and price per unit necessary for industrial applications. 

Biocatalysis has emerged as an important tool for the enantioselective synthesis of chiral pharmaceutical intermediates and several review articles have been published in recent years [133-137]. For example, quinuclidinol is a common pharmacophore of neuromodulators acting on muscarinic receptors (Figure 6.50). (JJ)-Quinudidin-3-ol was prepared via Aspergillus melleus protease-mediated enantioselective hydrolysis of the racemic butyrate [54,138]. Calcium hydroxide served as a scavenger of butyric acid to prevent enzyme inhibition and the unwanted (R) enantiomer was racemized over Raney Co under hydrogen for recycling. 

In the last decade, biocatalysis in nonaqueous media, using hydrolases, has been widely used for organic chemists. The possibilities that these biocatalysts offer for the preparation of different types of organic compounds, depending upon the nucleophile... 

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