FERMENTATION (ENZYME IN ORGANIC SYNTHESIS)Introduction of Fermentation:-Fermentation is the process that produces alcoholic beverages or acidic dairy products. For a cell, fermentation is a way of getting energy without using oxygen. In general, fermentation involves the breaking down of complex organic substances into simpler ones. The microbial or animal cell obtains energy through glycolysis, splitting a sugar molecule and removing electrons from the molecule. The electrons are then passed to an organic molecule such as pyruvic acid. This results in the formation of a waste product that is excreted from the cell. Waste products formed in this way include ethyl alcohol, butyl alcohol, lactic acid, and acetone--the substances vital to our utilization of fermentation.PRODUCTION OF DRUG INTERMEDIATES BY FERMENTATION:-Shikimic acid is a key intermediate for the synthesis of the antiviral drug oseltamivir (Tamiflu). Shikimic acid can be produced via chemical synthesis, microbial fermentation and extraction from certain plants. An alternative production route is via biotransformation of the more readily available quinic acid. Much of the current supply of shikimic acid is sourced from the seeds of Chinese star anise (Illicium verum). Supply from star anise seeds has experienced difficulties and is susceptible to vagaries of weather. Star anise tree takes around six-years from planting to bear fruit, but remains productive for long. Extraction and purification from seeds are expensive. Production via fermentation is increasing. Other production methods are too expensive, or insufficiently developed. In the future, production in recombinant microorganisms via fermentation may become established as the preferred route.Production of chiral hydroxyl acids:- More than 5 million tons of starches are produced per year in the European Economic Community from agricultural products such as maize, wheat, barley and potatoes. Some of this is used as fermentation feed stock. D-Glucose, the basic building unit of starch can be formed in situ by the action of amalyse enzymes. The glucose can then be converted to a variety of commercially interesting hydroxyl acids which are primary products of carbohydrate metabolism. The commercially most important is citric acid, which is achiral, but a variety of chiral hydroxyl acids are also produced by carbohydrate metabolism (fig 1).

Fig. 1:- chiral hydroxyl acids available from industrial fermentation.Both enantiomer of lactic acid are produced commercially, the more important one being the (S) = L isomer. About 30,000 tons per annum are produced by fermentation of D-glucose with lactobacillus delbruckii or lactose (whey from cheese manufacture) with Lactbacillus bulgaricus. The (R) = D enantiomer is produced on about a 1000 ton scale as an intermediate for optically active herbicides. L Tartaric acid can be produced by fermentation but the most economical sources are still the cream of tartar by product of wine making. Similarly, L malic acid is available by fermentation of glucose but is more economically produced by the enzymatic hydration of fumaric acid (reaction 1). The latter can also be carried out as fermentation with the fumarase containing Brevibacterium ammoniagenes.

About 45,000 tons per amount of D-gluconic acid are produced worldwide using a variety of microbes (e.g. Aspergilius Niger). 2-keto-D-gluconic acid is produced by fermentation of glucose with acetobacter suboxydans and is an intermediate in the production of isoascorbic acid (isovitamin C) (reaction 2).

About 30,000 tons per amount of ascorbic acid (vitamin C) are produced by the Reichstein-Grussner process which dates from 1934. The key step in this process is the microbial oxidation of D-Sorbital to L-Sorbose mediated by Acetobacter Suboxydans (fig 2).

Fig.2:- L-Sorbose production by fermentation of D-Sorbitol.

Fig.3:- Direct fermentation of glucose to 2-keto-L-gluconic acid by a recombinant Strain of Erwinia herbicola. r-DNA technology has been used to engineer a microorganism capable of producing 2-keto-L-gluconic acid, the precursor of ascorbic acid, by direct fermentation of glucose. Thus, several species of Acetobacter, gluconobacter, Erwinia are able to efficiently oxidize glucose to 2, 5-diketogluconic acid (fig3). The conversion of the latter to 2-keto-L-gluconic acid is mediated by corynebacterium sp. In order to produce a recombinant strain for the one step conversion of glucose, the 2, 5-diketogluconic acid reductase gene was isolated from coryne-bacterium sp. DNA and inserted into a plasmid, Erwinia herbicola. However, this elegant process can not as yet complete with the more circuit tons Reichstein Grussner process due to the low productivities obtained.Production of amino acids:- All 19 of the proteinogenic amino acids are available as primary metabolites in fermentation processes. However, most wild strains isolated from nature are not able to produce industrially significant amount because over-production of amino acids is prevented by metabolic regulation mechanisms (e.g., feedback inhibition). Moreover, the cell membrane permeability barrier prevents the life-sustaining amino acids from escaping into the environment. In 1957, two groups in japan discovered wild bacteria capable of producing high yields of glutamic acid. Monosodium glutamate had been used in japan as a flavour enhancer from the beginning of this century and the discovery of a commercially viable microbial synthesis laid the foundation for an amino acid fermentation industry that is still dominated by Japanese companies. Subsequently many strains of Brevibacterium and Corynebacterium were found to overproduce glutamic acid. Although glucose and molasses are the major carbon sources, strains have also been developed that utilize ethanol, acetic acid, or hydrocarbons. Further research led to the development of mutant strains with enhanced cell membrane permeability. One strategy adopted for this purpose is to grow c. glutamicum in a medium containing less than the optimum amount of the vitamin biotin; the cell membrane then becomes deficient in phospholipids and develops leaks, which allows more glutamic acid to be excreted. Following the success achieved with glutamic acid fermentation, methods were developed for the microbial synthesis of other amino acids (e.g., lysine, threomine, proline, phenylalanine, and tryptophan; (table 1). The two most important examples, glutamic acid and lysine are produced on scales of 3, 50,000 and 70,000 tons, respectively which rank with bulk chemicals. The extensive application of r-DNA technology to amino acid fermentations has resulted in further strain improvements. The only limiting factor appears to be the volume of product necessary to warrant the large development costs. L-phenylalanine is a good example; once a large volume application emerged, in the form of the artificial sweetener aspartame, the fermentation process was significantly improved. Similarly demand for L-proline as an intermediate for ACE- Inhibitors stimulated the further development of the fermentation method for its production and a substantial reduction in its price. Fermentation is not most economical production method for all the natural amino acids. For e.g., Aspartic acid, the raw material for aspartame is more economically made by enzymatic acid (reaction 3, analogous to the formation of L-malic acid by enzymatic hydration of the same substance (reaction 1). Table (1) shows fermentation process of interest for industrial amino acid production. Amino acid organism Concentration (g/l)

L ArginineBrevibacterium flavum 35

L Glutamic acidCorynebacterium sp. 100

L GlutamineCorynebacterium glutamicum 37

L HistidineCorynebacterium glutamicum 15

L IsoleucineBrevibacterium flavum 30

L LeucineBrevibacterium lactofermntum 30

L LysineBrevibacterium lactofermntum 70

L Phenyl alanineBrevibacterium lactofermntum 25

L ProlineBrevibacterium flavum 35

L ThreonineSerratia narcescenes 25

L Tryptophan Brevibacterium flavum 20

L ValineCorynebacterium glutamicum 30

Commercial production of aspartic acid, by tarabe, involves the use of a packed column of immobilized cells of E.Coli in a continuous process.Production of Beta Lactam Antibiotics:- An enormous number of antibiotics are produced by fermentation. The most imp classic the Beta-lactum antibiotics, comprising the penicillins and cephalosporins. Their commercial importance is underscored by the fact that five of the ten pharmaceuticals are beta-lactum antibiotics. The fermentation process for the manufacture of penicillin has along and colorful history and has earned the accolade queen of fermentations. The worldwide production of Penicillin G and Penicillin V (mostly the former) is about 25,000 tons. The current commercial Penicillin producing strains of Penicillin chrysogenum are the result of many rounds of strain improvement programs. The fermentation is carried out on a very large scale, with fermentators having volumes upto 200 cubic meters. Productivities have also been significantly improved by developments in process engineering. (E.g. initroduction of fed batch process). Penicillin G concentrations of more than 20 g/l can be obtained, which represents a 1,000 fold increase over the original yields obtained by Florey and chain. The biosynthetic pathway for penicillin production involves the fermentation of a tripeptide from valine, cysteine and L--aminoadipic acid. The tripeptide is converted to isopenicillin N by isopenicillin N synthase. Penicillin G and Penicillin V are formed by enzymatic transcylation with phenylacetic or phenoxy acetic acid, respectively, that are added to the fermentation broth (fig 4). Penicillic G is converted to a wide range of Semisynthetic Penicililins and cephalosporins via the intermediacy of 6-aminopenicillanic acid (6-APA) and 7-aminodeacetoxy cephalosporanic acid (7-ADCA). Originally 7-ADCA was produced from cephalosporin C, the latter being obtained by fermentation of Cephalosporium acremonium. However, due to the low productivity of this fermentation, production of 7-ADCA by chemical conversion of 6-APA proved more economical. An enzymatic process has been developed by Gist Brocades for the deacylation of Pen G to 6-APA and the analogous synthesis of 7-ADCA from its phenylacetyl derivative (fig 5). The enzymatic process for 6-APA uses an amidase (penicillin acylase) that was originally developed in the 1960S but could not compete with the chemical method. However, dramatic improvements in the enzyme production using engineering techniques and more efficient utilization via immobilization have led to Substantial cost reduction in the enzymatic process. This, coupled with increasing environmental pressure or the chemical process (e.g., the use of chlorinated hydrocarbon solvents, etc.), appears to have tipped the balance in flavor of the enzymatic process which is more environmently friendly. A second group of Semisynthetic cephalosporins is derived from 7-amino-cephalosporanic acid (7-ACA). The latter is produced by chemical deacylation of cephalosporin C, the product of fermentation (fig 6). In the penicillins, only modifications at position 6 have led to therapeutically useful products whereas useful cephalosporins arise from modifications at both the 7 and 3 positions.

Fig 4:- Biosynthetic pathway for penicillin.

Fig 5:- Conversion of Pen-G to Semisyntheric Penicillins and Cephalosporins.

Fig 6:- Production of 7-ACA from cephalosporin C. The discovery and development of new beta-lactam antibiotics still continues unbated and has led to the introduction of new classes such as the carbapenems (e.g., thienamycin) and the monobactam (e.g., azthreonam)

Interestingly, although they are available by fermentation, thienamycin and azthreonam are both made by chemical synthesis, presumably due to very low productivities in fermentation.Vitamins:- Vitamins are organic substances that are needed generally in trace quantities, for the functioning of most life forms, mostly as coenzymes in biosynthetic pathways. Most of the 13 mammalian vitamins (table 2) are produced by chemical synthesis.Table 2:- Vitamins; function and production method. VitaminFunctionMethod of production

Lipid soluble

A 11-Cis-RetinalVisionChemical

*D Ergosterol (Provitamin D)Calcium regulationBy product of yeast fermentation

*E -TocopherolIntracellular AntioxidantChemical

*K Phylloquinone Prothombin biosynthesisChemical/resolution

Water soluble

B ThiamineCoenzymeChemical

*B RiboflavinCoenzymeChemical/resolution

B Pyridoxal phosphateCoenzymeChemical

*B Coenzyme BCoenzymefermentation

*B Pantothenic acid (coenzyme A) CoenzymeChemical

*BC Tetrahydrofolic acidCoenzymeChemical

*C Ascorbic acidCosubstrate of monooxygenaseChemical

*H BiotinCoenzymeChemical

*PP Niacin(nicotin acid)CoenzymeChemical

Note:-*chiral compounds: - Note, however, that vitamin E and K are produced synthetically as mixture of optical isomers.+ Tetrahydrofollic acid is manufactured from L-glutamic acid which is available from fermentation.But another exception is the complex molecule vitamin B. It is produced by fermentation with Pseudomonas denitrificans. Strain improvement programs have resulted in productivity increases from 0.6 mg/l to about 60 mg/l at reaction times of 90 hrs (about 0.001 g/l/h!). Some vitamins are produced by a combination of fermentation with chemical steps (e.g., Vitamin C). Similarly, riboflavin is available by denovo fermentation but is more economically produced by high yield fermentation (70 g/l) of glucose to D-ribose, using mutant strains of B. subtiles or B. punilus, followed by chemical conversion.Synthesis of chemicals via microbial transformation (precursor fermentation):- The products discussed in predicting sections were mainly primary or secondary metabolites produced by denovo fermentation from glucose or a glucose equivalent. A second imp category of fermentation processes is the so called precursor fermentations or microbial transformations, whereby the conversion of a foreign substrate is mediated by a growing microbe. It usually involves a single step and cofactor-dependent enzyme. Carrying out the

Fig 7:- Production of riboflavin (vitamin B) by fermentation chemical synthesis.Reaction as a fermentation obviates the need for expensive cofactor regeneration precursor fermentations generally involve redox processes or condensation reactions.Microbial oxidation:- The commercially most imp type of precursor fermentation is undoubtedly microbial oxidation. The archetype of such process is the regioselective microbial oxidation of D- Sorbitol to L-sorbose mentioned earlier. The most imp examples of microbial oxidation are probably the regio- and stereoselective hydroxylations of the steroid nucleus which constitute key steps in the manufacture of steroid drugs with a value of more than a billion dollars. The selective conversion of progesterone to 11-Progesterone has already have been mentioned. By a suitable choice of microorganism (fungi, antinomycetes and bacteria are used). The same substrate can be converted to other imp derivatives (fig 8). Similarly, microbial oxidation can also be utilized for regioselective hydroxylation of aromatic compounds. A commercially relevant example is the Lonza process for the regioselective hydroxylation of nicotinic acid (nician) to

Fig-8:- Microbial oxidation of Progesterone.Afford 6-hydroxynicotinic acid, an insecticide intermediate (reaction 4). The commercial process takes advantage of the much lower water solubility of the magnesium salt of the product compared to the starting material. The magnesium salt of 6-hydroxynicotinic acid precipitates during the fermentation and is collected in a settler.

R R Optical purity

H CH 95% ee

H CHCH 93% ee

H CHCHCH 96% ee

H CHCHCHCH 95% ee

CH H 97% ee

CHCH H 99% ee

Fig 9:- Selective microbial hydroxylation of carboxylic acids. Another industrially useful microbial transformation is the regio- and enantioselective beta-hydroxylation of aliphatic carboxylic acids mediated by yeasts such as candida rugosa and C. parapsitosis. For e.g., mutant strains of C.rugosa gave concentrations of the (R)-hydroxy carboxylic acid of the order of 5-10 g/l in 24 hours (fig 9). The reaction proceeds via initial enzymatic dehydrogenation to , -unsaturated acid followed by enantioselective enzymatic hydration, as illustrated in (fig 10) for isobutyric acid hydroxylation. Kanegafuchi has commercialized processes for the production of (R)--hydroxyisobutyric acid and (R)--hydroxy-n-butyric acid using this technology. The former is a chiral synthon in the manufacture of the ACE-inhibitor, captopril and the latter in a route to carbapenems intermediates. An alternative route to (R)--hydroxybutyric acid (esters) involves acid-catalyzed depolymerization of poly-(R)-3-hydroxybutyrate. The latter is produced commercially by ICI by fermentation of glucose with Alca ligenes eutrophus bacteria. In practice, the fermentation can be controlled to give a copolymer containing varying proportions of (R)-3-hydroxybutyrate and (R)-3-hydroxyvalerate, with yields up to 80% of the dry weight of the biomass. Depolymerization thus provides acess to both optically pure acids and their esters. Analogous to the above microbial hydroxylations, Lonza has commercialized a process for the production of (R)-Carnitine by fermentation of butyrobetaine or crotonebetaine (fig 11). A soil microorganism (taxonomically situated between Agrobacterium and Rhizobium) was isolated that grows on crotonbetaine or butyrobetaine as the sole source of carbon, nitrogen and energy under aerobic conditions. Amutant strain of this microbe produced (R)-Carnitine in concentrations up to 100 g/l and productivities of about 5g/l/h.

Fig 10:- Mechanism of candida rugosa mediated hydroxylation of isobutyric acid. Microbial oxidation can also be used for the kinetic resolution of racemates, the classical example being Pasteurs resolution of tartaric acid with penicillium glaucum an example is the conversion of racemic 3-chloro-1, 2-propanediol to the (R)-isomer by fermentation with serratia marescenes. The (R)-isomer is versatile chiral C - Synthon that can, in principle, be converted to a variety of commercially interesting products (fig 12). Unfortunately, this method involves sacrificing half of the starting material; however, the methodology has been refined to provide an elegant method for complete conversion of racemic diols to the (S)-enantiomer by microbial stereoinversion (fig 13). For example, fermentation of racemic 1, 2-pentanediol with candida parapsilosis produced 28 g/l of the (S)-enantiomer in 95% yield and 100% ee. Stereoinversion involves the coupling of NAD-dependent, (R) - Specific alcohol dehydrogenase with a NADPH dependent (S)-Specific keto-reductase (fig 13).

Fig 11:- Pathway for the formation of (R)-Carnitine from butyrobetaine, (R)-Carnitine dehydrogenase is blocked in the mutant strain.

Fig 12:- (R)-3-Chloro-1, 2-propanediol by microbial kinetic resolution. Another Potentially useful precursor fermentation is the enantioselective microbial epoxidation of prochiral olefins. For e.g. Nocardia Corallina converts a variety of olefins to the (R)-epoxides (e.g., reaction 5) with ees in the range of 60-94%. However, productivities are generally low due to severe product inhibition, which raises doubts about the commercial potential of the method.

Fig 13:- Mechanism of candida parvapsilosis-mediated stereoinversion of 1, 2-diols.Microbial Reductions:- An Enantioselective reduction with fermentating bakers yeast (Saccharomyces cerevisiae) is a widely used technique in organic synthesis. The observation, by Mamol and Vercellone that fermenting yeast reduces 17-keto steroids to 17-hydroxy steroids eventually led to commercial processes for the manufacture of steroid hormones. (Reaction 6)

Reduction of -keto esters to the corresponding (S) - alcohols is probably the most studied reaction. In common with most yeast reductions, high ees are obtained only when substrate concentrations are kept at 1g/l or less, as typical reaction times are 24-28 hrs this translate into meager productivities (0.02-0.04 g/l/h).Table 3:- A comparison of yeast reduction with catalytic asymmetric hydrogenation.

Bakers yeast reduction

Asymmetric hydrogenation

Methyl esters, 40 gm water, 2600ml Yeast, 200 gm Sucrose, 500 gm. Room temperature, 80 h Filtration + extraction + distillation, 59-76% yield; S in 85% ee* productivity: = 0.01g/l/h (< 0.01 for >97% ee)

Ethyl ester, 40gm methanol, 40ml Ru BINAP, 0.14 gm H, 20-100 barRoom temperature, 40 h isolation by distillation, 96% yield; R or S in >99% eeProductivity:=12g/l/h.

Figure 14:- Variety of transformations by yeast reduction.

Figure 15:- Enantioselective reduction of potassium ketopantoate to D-pantoic acid.Interestingly, seebach has compared yeast reduction with catalytic asymmetric hydrogenation of -keto ester (Table 3). Obviously, the asymmetric hydrogenation wins hands down on productivity and ease of work-up. Indeed, this is a serious handicap for yeast reductions on an industrial scale and in most cases alternative methods (e.g., catalytic asymmetric hydrogenation or enzymatic kinetic resolution) will be more economically viable. Nevertheless, yeast reductions are interesting for a variety of transformation as shown in the following examples (fig 14) and perhaps the productivities are amenable to future improvement by genetic engineering. Microbial reductions are not limited to bakers yeast, as is illustrated by the enantioselective reduction of potassium to D-pantoic acid (fig 15), the precursor of panthothetic acid (vitamin B), By species of Agrobacterium. In order to find a suitable microbe, 188 strains of bacteria, 84 antinomycetes, 231 yeasts, 203 molds, and 353 microorganism isolated from soil samples were screened! Optimum activities were found with Agrobacterium spp. That yielded D-pantoic acid in >98% ee. Under optimized conditions, concentrations of about 120 g/l were obtained at 90% conversion in 5days (i.e., a productivity of 1g/l/h). This compares favourably with the reduction of ketopantoyl lactone by candida cells (81 g/l and 80% ee at substrate concentrations maintained below 3%) and is a perfect example of perseverance pays.Condensation reactions:- An example of microbial condensation reaction of commercial significance is the bakers yeast-mediated acyloins condensation of aromatic and , -unsaturated aldehydes with acetaldehyde, the latter being formed in situ by glucose fermentation (Reactions 7 and 8). When benzaldehyde is the substrate, this constitutes the key step in the knoll process for the manufacture (591) of ephedrine and pseudoephedrine (fig 16). Furthermore, Fuganti and coworkers have used the general reactions 4-7 and 4-8 as a key step in the synthesis of a variety of natural products.

Fig 16:- Manufacture of ephedrine and pseudoephedrine.Introduction of L-ephedrine:-Ephedrine is asympathomimetic amine commonly used as astimulant,appetite suppressant, concentration aid,decongestant, and to treathypotensionassociated with anaesthesia.Ephedrine is similar in molecular structure to the well-known drugsphenylpropanolamineandmethamphetamine, as well as to the important neurotransmitterepinephrine(adrenalin). Chemically, it is analkaloidwith aphenethylamineskeleton found in various plants in the genusEphedra (familyEphedraceae). It works mainly by increasing the activity ofnorepinephrine(noradrenalin) onreceptors. It is most usually marketed as thehydrochlorideorsulfatesalt.The herb used intraditional Chinese medicine, contains ephedrine andpseudoephedrineas its principal active constituents. The same may be true of other herbal products containing extracts from other ephedra species.Synthesis of L-ephedrine:-Ephedrine can be synthesized frombenzaldehydein a few different ways. According to the first, benzaldehyde is condensed withnitro ethane, giving 2-methyl-2-nitro-1-phenylethanol, which is reduced to 2-methyl-2-amino-1-phenylethanol.The necessary L-isomer is isolated from the mixture of isomers by crystallization.Methylationof this gives ephedrine.

The second method consists of the fermentation of glucose yeast carboligase in the presence of benzaldehyde, which during the process turns into phenylacetylcarbinol. This is reduced by hydrogen in the presence of methylamine to give desired ephedrine.

Development of Practical Synthesis of Chemicals via Transformations Using Isolated Enzymes in Immobilized (Solid-Supported) Form:-

The option of recycling a catalyst has, in general, often been realized by means of Immobilization of the catalyst on a solid support, which enables simple separation of the heterogeneous catalyst from the reaction mixture and its synthetic re-use. However, it also should be mentioned that many other techniques for catalyst Immobilization has been developed. As for enzymes, one example of a very successful application of this concept of Heterogeneous enzyme catalysis is the established biocatalytic synthesis of 6-amino penicillanic acid (6-APA), which is applied with an annual production volume exceeding 10,000 tons per year. The catalytic concept is shown in Scheme 1. Compared with the alternative chemical route, the use of an immobilized Pen G acylase enables cleavage of the unwanted side-chain without the need for significant amounts of a range of hazardous chemicals. As a solid support, Eupergit beads turned out to be highly efficient for the Pen G acylase catalyst. Notably, the immobilized enzyme catalyst can be re-used more than 850 times, thus delivering a highly efficient production process and very low overall enzyme loading of the heterogenized enzyme catalyst per kg of 6-aminopenicillanic acid.

Scheme 1. Immobilized penicillin G acylase (Pen G acylase) in the production of 6-APA. Furthermore, heterogeneous enzyme catalysis has also been carried out very successfully in organic reaction media. For example, when using immobilized lipase, direct ester formation starting from an acid and an alcohol enables efficient formation of the ester in a solvent-free medium. Such a process technology has been industrially established for fatty acid ester manufacture, for example, at Unichema Chemie and Degussa AG (now: Evonik Degussa GmbH). In the field of racemic resolution, the BASF process for the production of chiral amines is based on an enantioselective acylation when starting from a racemic amine in the presence of an immobilized lipase. The production volume of this process technology is in the >1000 tons per year range.

Development of Practical Synthesis of Chemicals via Transformations Using Isolated Enzymes in Free Form:- The use of solid-supported catalysts, however, also means that there is a switch from a catalytic reaction in a homogeneous reaction medium (as in case of .free. enzymes) for example, by means of a so-called enzyme-membrane reactor (Figure 1).In an EMR, the enzyme reacts as a free enzyme but is prevented from leaving theReactor by a membrane. This membrane has a specific molecular-weight cut-off, that is, only molecules that have a molecular weight below the cut-off can cross the membrane. These membrane-permeable molecules are typically substrate and product. The structure and concept of such an enzyme-membrane reactor is shown in Figure 1, exemplified for the synthesis of L-methionine by means of anAminoacylase. Notably, the EMR runs also very successfully on an industrial scale,Producing L-methionine on a multi-hundred tons scale annually. ThisIndustrial process has been developed and established jointly by the Wandrey group and researchers at Degussa AG (now: Evonik Degussa GmbH).

Fig 1:- Production of L-methionine in an enzyme-membrane reaction.

A further elegant example of the combination of organic chemistry with aBiocatalyst used in the form of an isolated enzyme is the synthesis of the artificialsweetener aspartame in homogeneous reaction medium (Scheme 2). Thisprocess also represents an efficient and well-established industrial process running at about the 2500 tons scale annually. The condensation reaction of the protected aspartate and phenylalanine methyl ester is catalyzed by thermolysin from a Bacillus strain in aqueous homogenous medium. In the downstream processing a filtration step is included that ensures enzyme separation and recovery for re-use. The reaction, which proceeds with excellent selectivity (>99.9%), originally had much advantage as an efficient resolution of racemic phenylalanine, until the supply of L-phenylalanine was started by the fermentative method.

Fig 2:- Enzymatic synthesis of L-aspartame with thermolysin.

A further process option, which is of particular of interest when producing hydrophobic molecules, is to run the reaction in a two-phase reaction medium consisting of an aqueous phase and a water-immiscible organic solvent. This concept is, of course, not only suitable for enzymes but also for whole-cell catalysts. Whereas the enzymatic reaction proceeds in the aqueous phase, product accumulation takes place in the organic phase. After completion of the reaction, simple phase separation enables recovery of the enzyme (dissolved in the aqueous phase and ready for use in the next reaction run) and isolation of the product (dissolved in the organic phase). A prerequisite for such a process with enzyme recycling is a high stability of the enzyme towards the reaction medium and substrates/products, leading to sufficient remaining enzyme activity after the biotransformation and phase-separation steps. Today many enzymes fulfill this prerequisite. Notably, among them are not only hydrolases but also enzymes from other enzyme classes.

Fig: - Hydrocyanation process with free, non immobilized enzyme.

A selected example of such a process concept with non-immobilized enzymes is the hydrocyanation of m-phenoxybenzaldehyde in the presence of a recombinant oxynitrilase from Hevea brasiliensis (Scheme 3). This biotransformation, leading to an asymmetric C-C bond formation, which has been developed by the Griengl group, has found an industrial application at DSM, running at a hundred tons scale per year. Notably, an impressive spacetime yield of 1 kg /l /day has been achieved. The enantioselectivity is also excellent, leading to the desired (S)-cyanohydrin with 98.5% e.e. Thus, today, various options are readily available for the development of efficient biotransformations with isolated enzymes. These methodologies consist of the use of enzymes in immobilized, solid-supported form or as free enzymes. In the latter case, enzyme separation from the reaction mixture and re-use for the next synthetic cycle can be realized by means of, for example, enzyme-membrane reactor technology or the use of two-phase systems and a phase-separation after the reaction is completed.

Reference:-

Chirotechnology:- Industrial synthesis of optically active Compounds by Roger A. Sheldon, 1993. Enzyme catalysis in organic synthesis, third edition. Edited by Karlheinz Drauz, Harold Groger, and Oliver May, Wiley-VCH Verlag GmbH & Co KgaA, 2012. www. Wikipadia.com.