Chemistry Everyday for Everyone edited by
Products of Chemistry
George B. Kauffman California State University Fresno, CA 93740
Applications of Biocatalysis to Industrial Processes John T. Sime Zylepsis Ltd., 6 Highpoint, Henwood Business Estate, Ashford, Kent TN24 8DH, UK
The industrial world is making use of a wide range of chemical reactions to provide consumers with the products they require. Each year the types of reactions available increase as researchers discover and create new catalysts and devise novel reaction systems. As long as life has existed, however, there has been a remarkable variety of chemistry carried out in biological systems. This chemistry of life comprises many different, very specific, chemical conversions, which are often similar to those used by the conventional chemical industries. A major difference, however, is that in biological systems the reactions are effected under conditions that are usually very mild: no strong acids or bases, but pH levels in the range 4–9; no extremes of temperatures, but often in the range 10–60 °C; often no need for heavy metal catalysts. Biological systems are able to do this elaborate and selective chemistry by the use of enzymes, which are natural catalysts that are responsible for all the chemical transformations necessary to sustain life, both for primary and secondary metabolism. In fact, enzymes can catalyze many reactions that are not possible by conventional chemistry. Most metabolic processes are extremely specific. For example, an enzyme that indiscriminately oxidized everything it contacted would prove lethal, so enzymes have evolved to give the desired reactions and to control the extent of reaction by switching off at certain times. This adds to the usefulness and specificity of enzyme catalysis. Also, since enzymes are true catalysts they can facilitate reactions in both directions (forward and back) under the correct conditions. It is possible to employ these biological catalytic systems for synthetic purposes outside living organisms and even to employ the enzymes contained in organisms to chemistry they would not normally come into contact with in the normal life of the organism. Advantages of this biocatalysis or biotransformation, over conventional chemistry, are that the reactions carried out can be more specific and selective, they use less energy because heating to high temperatures is not required, there is no highly acidic or highly alkaline residue to be cleaned up, and there are no metal catalyst residues to be treated for disposal. Biocatalysis is, therefore, regarded as an environmentally friendly method of doing chemistry and can, in many instances, lead to cost reduction in overall processes. Enzyme-catalyzed processes have been used industrially for centuries and fermentation processes are well known in history, for example in making wine, beer, bread, and cheese. Biotransformation differs from fermentation in that it involves the use of particular, often single, enzymes as applied to a particular reactant to give high levels of conversion to a single product: most often this is by functional group transformation. By contrast, in fermentation, a microorganism is provided with an energy source (such as sugars) and other essential nutrients, and it produces products (e.g., penicillin) as part of its metabolic process, but in relatively low yields. In this way biocatalysis is more like conventional chemistry than fermentation. 1658
Biocatalytic conversions have found uses in industry throughout this century, but there was a dramatic upturn in the number of such applications during the 1960s, coinciding with the application of the enzyme penicillin acylase to hydrolyze the side chain of natural penicillins (made by fermentation) to give aminopenicillanic acid. This is the nucleus of the penicillin structure and to it can be added nonnaturally-occurring side chains to give rise to the multitude of semisynthetic penicillin-based antibiotics we know today (1). In the last 20 years or so the scientific understanding of enzyme catalysis has evolved greatly as new techniques have been developed and this in turn has led to an increase in the application of these systems to industrial (and research) use. Biocatalysis has found applications in the pharmaceutical industry, food and beverage industries, and industries such as cosmetics, fine chemicals, flavors, and fragrances. Examples from many of these areas are given in this brief overview. Breakdown of Amides One of the prime examples of biocatalysis is the use of penicillin acylase (also known as penicillin amidase) mentioned above (2). Reasons why this is such a well-known example may include the facts that the biotransformation has been used widely across the world by many companies and it facilitated the introduction of the (then) new generation of semisynthetic antibiotics that had such an impact on the treatment of disease. In addition, the technology was crossapplied to similar reactions in other industrial areas, and this conversion is probably still the most commercially significant example of a biotransformation in the world, although it is not the biggest volume bioconversion being employed. The reaction can be summarized by eq 1. PhCONH
S
penicillin H2N acylase
N O
S N
CO2H
O
H 2O
6-APA + PhCO2H
CO2H RCO2H
H2O
(1) RCONH
S N
O
CO2H
Penicillins produced by fermentation, such as penicillin G shown, can be converted to 6-aminopenicillanic acid (6-APA) by the penicillin acylase enzyme, which catalyzes the hydrolysis of the amide bond in the side chain of the molecule to give the amine. It is the 6-APA product that is then converted to a wide range of new penicillin derivatives by reaction of the 6-amino function to give different side chains—hence expanding the available range of antimicrobial agents to
Journal of Chemical Education • Vol. 76 No. 12 December 1999 • JChemEd.chem.wisc.edu
Chemistry Everyday for Everyone
allow, for example, extended activity or orally available compounds. The hydrolysis of the amide bond in the presence of the penicillin nucleus with conventional chemistry is not trivial because conditions that promote hydrolysis of the sidechain amide bond also give rise to hydrolysis of the cyclic amide (β -lactam) in the nucleus. Also, 6-APA is itself a relatively unstable molecule. The biotransformation is carried out at close to neutral pH and without the need for elevated temperature; that is, under the mild conditions required to avoid decomposition of the 6-APA nucleus. The water-soluble enzyme can be attached to insoluble supports such as resins, which means that the resulting immobilized enzyme can be easily removed from the reaction mixture and reused many times, a factor that greatly enhances the economics of the process. It is estimated that at least 16,000 tonnes of 6-APA is produced by biocatalysis each year. Similar enzymes capable of hydrolyzing amide bonds (amidases) are used to produce amino acids. Amino acids are the chemical building blocks for proteins, and in nature these occur as only one of the possible enantiomers with defined stereochemistry. The high levels of selectivity of some enzymes are used to differentiate between the isomeric forms of reactants, only one enantiomer being a substrate for the enzyme, in order to produce stereochemically pure products from a racemic substrate. Thus, for example, the acetyl group in Nacetylmethionine will be hydrolyzed by an amidase from the organism Aspergillus oryzae, but only one of the enantiomers is a substrate for the enzyme (eq 2), so that L -methionine is a product of the reaction and the D-enantiomer of the acetylated substrate remains unreacted. In this way, singlestereochemistry amino acids can be produced for use as fine chemicals or as food supplements. O S OH O H
O S
O
+ CH3COOH
NH2
L-methionine
N
amidase
+
S
OH H
+ OH HO
H 2O O
N S
N -acetyl-D,L-methionine
OH HO N
(2)
N -acetyl-D-methionine
Formation of Amides These are examples of the enzyme-catalyzed hydrolysis of amides but, since amidases are catalysts, they can also assist in the reverse reaction: that is, formation of amide bonds from amines and acids. This type of reaction has been commercialized in the biocatalytic production of aspartame, a sweetener used extensively in the food and beverage industries that is 200 times as sweet as sucrose. Aspartame is formed by linking the methyl ester of phenylalanine with aspartic acid. This apparently simple chemical reaction has to circumvent a number of problems, however. The amine group of the aspartic acid has to be protected to prevent its reacting with another molecule of aspartic acid to give unwanted by-products; the correct single enantiomer of each of the reactants must be used to give the required stereochemistry of aspartame; there are two reactive acid groups in aspartic acid (designated α
and γ) and the reaction needs only one of these (α) to take part in the condensation reaction; and the methyl ester will hydrolyze under extreme conditions. The use of biotransformation can help overcome some of these difficulties (eq 3). Ph
HO2C
Ph
HO2C
thermolysin
+ CO2H
PhCH2OCNH
H 2N
CO2Me H2O
N -Cbz-L-aspartic acid
CNH CO2Me
PhCH2OCNH
O
O
(3)
O
D,L-phenylalanine
Cbz-aspartame
methyl ester
Thermolysin, the enzyme used to form the amide bond in this case, is stable to heat and organic solvents and is able to work in the presence of an organic solvent such as ethyl acetate. It is able to “recognize” the difference between the α acid group of aspartic acid and the γ acid group and to promote reaction only at the α functionality, so that none of the other by-product is formed. The enzyme is also able to differentiate between the enantiomers of the methyl ester of phenylalanine, so that a racemic mixture of isomers can be used in the reaction and only the required L-enantiomer takes part in the condensation reaction. This means that the cheaper racemic substrate can be used; the unreacted D-enantiomer left in the reaction mixture forms a salt of the product, thereby aiding recovery of the aspartame precursor. This reaction typically is operated at pH values between 6 and 8 and at 40 °C, and the amine protecting group of the reaction product, Cbz (i.e., benzyloxycarbonyl, PhCH2OCO), is removed to yield aspartame itself. At present the enzymic production of aspartame by this enzyme-catalyzed route is at least 2000 tonnes a year and is probably rising. Reactions of Esters Hydrolysis of esters to acids and alcohols by esterases and lipases is conceptually and mechanistically very similar to the amidase reactions above, and these are also widely employed in commercial operations. Synthesis of the calciumantagonist drug diltiazem makes use of an esterase that is capable of differentiating between enantiomers of substrate esters. The substrate for the biotransformation is the ester shown in eq 4, a molecule that also contains the epoxide function, which is a reactive cyclic group prone to decomposition under harsh conditions. The enzyme selectively hydrolyzes one enantiomer of a racemic mixture of the substrate esters to an acid with single, specific stereochemistry. This acid is then further elaborated (chemically) to the final drug compound (steps not shown), which has two asymmetric stereocenters. The enzyme thus makes use of both the mild reaction conditions under which it will operate and also its stereoselectivity. OMe O
MeO2C
OMe esterase
O
(R,R )
HO2C
racemate OMe
(4) S
(S,S )
Diltiazem
O N H
O
O
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Chemistry Everyday for Everyone
Another biotransformation process, which operates at the scale of hundreds of tonnes a year, also makes use of ester transforming activity but in the synthetic direction, analogous to the enzyme-catalyzed synthesis of amides described earlier. Myristic acid is condensed with isopropyl alcohol (IPA) to give isopropyl myristate (eq 5), an emollient used in skin care products to give a “velvety” feel to the skin. The enzyme used is obtained from a species of the yeast Candida and the reaction, which requires removal of the water produced in the condensation, operates at 60 °C and gives >99% yields. CO2H
HO +
myristic acid IPA
of acrylamide for manufacture of polymers and synthetic fibers. Acrylamide is the monomeric raw material and it is obtained by hydration of the cyanide function of acrylonitrile. The traditional industrial process involves reaction of acrylonitrile with water in the presence of sulfuric acid or a metal catalyst, often copper. A major problem with this method of synthesis is that the reaction must be stopped to prevent the acrylamide itself being further converted to acrylic acid, which is a contaminant that has to be removed at the final stage. Acrylamide without any trace of acid is purer and commands a commercial dividend in the marketplace. In 1985 Nitto Chemical Industry started to use a process in which the acrylonitrile is hydrated by the enzyme nitrile hydratase (eq 8).
lipase
CN
H2O
O
Once again, this enzyme technology is used because there is a real financial driver and advantage to doing the reaction by biotransformation rather than by more traditional chemical ester formation reactions. Addition to Multiple Bonds The reactions described so far have involved hydrolysis and condensations that include water in the reaction scheme. There is another family of enzymes that also use water in the reaction scheme but add the water across a double bond rather than using it to cleave a bond. One of the methods of producing malic acid uses such an enzyme, called fumarase, to add water across the carbon–carbon double bond found in fumaric acid to give only one enantiomer of malic acid (eq 6). HO2C fumarase
HO2C H HO
CO2H H 2O fumaric acid
CO2H
L-malic
H2O
acid
acid is used in the food industry as an acidulant, and the biotransformation route has been used since the 1960s. Upwards from 1800 tonnes a year of L-malic acid are produced by this biocatalytic method. In a similar reaction, ammonia can be added across a double bond with selectivity. Thus, fumaric acid can be converted into L-aspartic acid when enzyme catalysis is used (eq 7). The enzyme is produced in growing cells of Escherichia coli. The cells are immobilized in a gel, which can be used in a continuous reaction, the reactants being passed over the cells. In this format the activity of the enzyme has a half-life of about 3 years. Again, this type of biotransformation has been in industrial use since the 1960s.
CO2
NH3 fumaric acid
acrylonitrile and water
make catalyst
grow cells
hydrate at 100 °C
hydrate at 10 °C
remove copper
remove cells
remove unreacted starting material
decolor
decolor
L-aspartic
(7)
acid
Yet another type of enzyme-catalyzed reaction that adds water across a multiple bond is to be found in the synthesis 1660
Microbial process
H H 2N
CO2H
Copper process
remove copper ions
HO2C aspartase
Conversions with the biotransformation go to greater than 99.9% yields and a gram of cells can produce many kilograms of acrylamide. Because of the selectivity of the enzyme, acrylic acid is not detectable in the product. This process has been developed during the intervening years and the productivity of the cells has been increased many times. Apart from the purity of the product obtained by this method, a comparison of the biotransformation with a conventional synthesis indicates that fewer process steps are involved (Fig. 1). Thousands of tonnes a year of acrylamide are produced by this method.
(6)
L-Malic
HO2C
CONH2
(8)
(5) O
isopropyl myristate
nitrile hydratase
acrylamide
Figure 1. Comparison of the steps in the synthesis of acrylamide by use of copper catalysis and biocatalysis.
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Chemistry Everyday for Everyone
Removal of Halogen An important intermediate for the synthesis of an agrochemical herbicide is the single isomer (S)-2-chloropropanoic acid, which is required with a high level of stereopurity. Once again, biotransformation has provided a method for the synthesis of this intermediate, on this occasion starting from the more easily prepared mixture of isomers. When the (R ,S ) racemic mixture of the 2-chloropropanoic acids is allowed to react with a dehalogenase (4 ) originally found in a strain of Pseudomonas putida, one isomer is converted to lactic acid and the other, (S )-2-chloropropanoic acid, remains unreacted and can be isolated from the reaction mixture (eq 9).
In this process γ-butyrobetaine is directly hydroxylated by the enzymes in species of Rhizobiaceae to give the product (5). Isomerization The largest-scale biotransformation currently being run in the world uses the enzyme glucose isomerase, which can convert glucose to the sweeter fructose (eq 12). HO OH HO
O
OH
glucose isomerase
OH
glucose
HO
O HO
OH
OH
OH
(12)
fructose
Cl
H HO2C
CH3
HCl
H 2O
H
+ H HO2C
HO2C
Cl
Cl
H +
CH3
dehalogenase (S )-2-chloropropanoic acid
CH3
OH
HO2C
CH3
L-lactic
acid
(9)
As is often the case with this type of reaction, the researchers who developed the biotransformation were in a race against nonbiological chemical processes that were also being developed to give the same product. The biotransformation was brought to full-scale manufacture in 1991, and currently 2000 tonnes a year of the intermediate are produced in this manner. Generation of a Hydroxy Product L-Carnitine is a substance found naturally in the body and it is also given as a nutritional supplement. Again, only one enantiomer of the compound is used. Two biocatalytic routes are available for the synthesis of this product. One involves the reduction of a ketone by addition of hydrogen across a carbonyl group, to give the intermediate (R)-γ-chloroβ-hydroxybutanoic acid octyl ester (eq 10). O Cl
O
reductase OC8H17
γ-chloroacetoacetic acid octyl ester
HO Cl
H
O OC8H17
(R )-γ-chloro-β-hydroxybutanoic acid octyl ester
(10) HO Me3N
H
O OH
L-carnitine
Once again, the selectivity of the enzyme-catalyzed reaction gives only one of the two possible enantiomers of the product. This intermediate can then be elaborated to L-carnitine by replacement of the chlorine by trimethylamine and hydrolysis of the octyl ester group to the corresponding acid. The enzyme system for this conversion is found in cells of the yeast Saccharomyces cerevisiae and the industrial process is used to make thousands of tonnes of L-carnitine a year. A more recent process for the synthesis of L-carnitine uses a very different transformation: hydroxylation (eq 11). O Me3N
hydroxylase OH
γ-butyrobetaine
HO Me3N
H
O
L-carnitine
OH
(11)
A number of companies are running this process, which converts a concentrated glucose solution into a syrup mixture that contains more fructose than the starting material, the final fructose content being between 40% and 60%. This product is referred to as high-fructose syrup and is used as a sweetening agent by food and soft drink manufacturers. The reaction runs at pH 6–8 and at temperatures between 50 and 75 °C. About 10 million tonnes of the product are made by the enzyme process every year. Prospects for the Future There are many more examples of industrial processes that employ enzyme chemistry and exemplify different types of reactions not mentioned here; it would be a major undertaking to try to list all of these. It is evident, however, that biotransformations have much to offer industrial chemistry, and the technology competes on a commercial basis with alternative approaches. For some of the industrial sectors that use biotransformations (such as food, beverages, and cosmetics) there are added advantages, in that this method of treating materials is considered to be natural and can therefore, in some cases, offer a route to final products that can legitimately be called natural (6 ). The extensive range of reactions possible using this technology provides great potential for many more large-scale processes to incorporate enzymic conversions. Although biocatalysis does not provide the answer to all synthetic chemistry problems, there is a lack of general awareness about the extent to which it is currently used. With the growing demand for processes to be environmentally neutral with respect to effluent and waste production and also the amount of energy used, it is likely that biotransformation will be found in more processes in the future. Acknowledgments I wish to thank Nigel Banister and Andrew Willetts for comment, discussion, and contribution to this manuscript. Literature Cited 1. Tramper, J. In Applied Biocatalysis; Cabral, J. M. S.; Best, D.; Boross, L.; Tramper, J., Eds.; Harwood Academic Publishing: Langhorne, PA, 1994; Chapter 1. 2. Lowe, D. A. In Developments in Industrial Microbiology; Cooney, J.; Sebek, O., Eds.; Society for Industrial Microbiology: Fairfax, VA, 1989; Vol. 30, Chapter 11. 3. Yamada, H.; Kobayashi, M. Biosci. Biotechnol. Biochem. 1996, 60, 1391–1400. 4. Taylor, S. Chem. Br. 1998, 34(5), 23. 5. Kulla, H. Chimia 1991, 45(3), 81. 6. Sime, J. Chem. Br. 1998, 34(5), 26.
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