Chapter 8 SAQ 8. 1 1)Name three sulphur containing amino acids 2)Name five of the eight essential amino acids. 3)Name two amino acids that contain a heterocyclic ring. 4)Name the amino acid with the simplest structur 5) Name the amino acid considered to be a dimer 6)Name an amino acid that is produced industrially only by enzymatic 7) Name an amino acid that is produced industrially only be chemical 8.3 Stereochemistry of amino acids The French physicist Biot discovered during the early nineteen th century, that a number of naturally occurring organic compounds rotate the plane of polarisation of an incident beam of polarised light. In the latter part of the nineteenth century it was found that many pairs of compounds seemed to have an identical structure and identical physical properties, such as melting point and solubility. Compounds in each pair were differentiated by the fact that even in solution they rotated polarised light in equal amounts but in opposite direction. Such compounds are called optical isomers and are symmetric arrangement of groups around a tetrahedral carbon atom. The gomes? described as being optically active. Optical activity requires, and is explained by an tetrahedral properties of a tetrahedron are such that if there are four different substituents attached to a carbon atom, the molecule does not contain a plane of symmetry, and there are two kinds of geometrical arrangements which the molecule can have. These two arrangements(configurations)are different in that it is not possible to simultaneously superimpose all the atoms of one figure on the like atoms of the other The two configurations are, in fact non-superimposable mirror images An illustrative example is given in Figure 8.1
236 Chapter 8 1) Name thee sulphur containing amino acids. 2) Name five of the eight essential amino acids. 3) Name two amino acids that contain a heterocyclic ring. 4) Name the amino acid with the simplest structure. 5) Name the amino acid considered to be a dimer. 6) Name an amino acid that is produced industrially only by enzymatic 7) Name an amino acid that is produced industrially only be chemical catalysis. synthesis. 8.3 Stereochemistry of amino acids The French physicist Biot discovered during the early nineteenth century, that a number of naturally occumng organic compounds rotate the plane of polarisation of an incident beam of polarised light. In the latter part of the nineteenth century, it was found that many pairs of compounds seemed to have an identical structure and identical physical properties, such as melting point and solubility. Compounds in each pair were differentiated by the fact that even in solution they rotated polarised light in equal amounts but in opposite direction. Such compounds are called optical isomers and are described as being optically active. Optical activity requires, and is explained by an asymmetric arrangement of groups around a tetrahedral carbon atom. The geometric properties of a tetrahedron are such that if there are four different substituents attached to a carbon atom, the molecule does not contain a plane of symmetry, and there are two kinds of geometrical arrangements which the molecule can have. These two arrangements (configurations) are different in that it is not possible to simultaneously superimpose all the atoms of one figure on the like atoms of the other. The two configurations are, in fact non-superimposable mirror images. An illustrative example is given in Figure 8.1. Optical isomers mra~~edra~ cabon a~m
Industrial production of amino acids by fermentation and chemo-enzymatic methods HOC YAY CO2H HO2C Figure 8. 1 Non-superimposable mirror images metric Such molecules result when the four groups attached to the carbon atom are all diffe arbon and a molecule of this kind is said to be asymmetric, or to contain an asymmetric car Molecules that are not superimposable on their mirror images are chiral. If two enantiomers compounds are related as non-superimposable mirror images, they are called enantiomers I cany you explain why the amino acid alanine is optically active, whereas glycine We can see from Table 8. 2 that the a-carbon of alanine is asymmetric (four different groups attached), whereas that of glycine is not. Optical activity requires an asymmetric carbon atom racemic If two enantiomers are mixed together in equal amounts the result is a racemic mixture mxtures We meet a number of enantiomeric items in daily life. The left hand, for example, is the mirror image of the right hand and they are not superimposable(see Figure 8.1). This becomes obvious if we try to put a right glove on a left hand. Similarly, a pair of shoes is an enantiomeric relationship while the stock in a shoe store constitutes a racemic mixture Representation of and the nomenclature for stereo-isomers are given in Appendix 8. 3. 1 Importance of chirality If we consider natural synthetic processes, enzymes are seen to exert complete control over the enantiomeric purity of biomolecules(see Figure 8.2). They are able to achieve this because they are made of single enantiomers of amino acids. The resulting enantiomer of the enzymes functions as a template for the synthesis of only one enantiomer of the product. Moreover, the interaction of an enzyme with the two enantiomers of a given substrate molecule will be different. Biologically important molecules often show effective activity as one enantiomer the other is at best ineffective or at worst detrimental
Industrial production of amino acids by fermentation and chemo-enzymatic methods 237 I I Figure 8.1 Non-superimposable mirror images. Such molecules result when the four groups attached to the carbon atom are all different and a molecule of this kind is said to be asymmetric, or to contain an asymmetric carbon. Molecules that are not superimposable on their mirror images are chiral. If two compounds are related as non-superimposable mirror images, they are called Can you explain why the amino acid alanine is optically active, whereas glycine n is not (refer to Table 8.2)? We can see from Table 8.2 that the a-carbon of alanine is asymmetric (four different groups attached), whereas that of glycine is not. Optical activity requires an asymmetric carbon atom. If two enantiomers are mixed together in equal amounts the result is a racemic mixture. We meet a number of enantiomeric items in daily life. The left hand, for example, is the mirror image of the right hand and they are not superimposable (see Figure 8.1). This becomes obvious if we try to put a right glove on a left hand. Similarly, a pair of shoes is an enantiomeric relationship while the stock in a shoe store constitutes a racemic mixture. Representation of and the nomenclature for stereo-isomers are given in Appendix 1. 8.3.1 Importance of chirality If we consider natural synthetic processes, enzymes are seen to exert complete control over the enantiomeric purity of biomolecules (see Figure 8.2). They are able to achieve this because they are made of single enantiomers of amino acids. The resulting enantiomer of the enzymes functions as a template for the synthesis of only one enantiomer of the product. Moreover, the interaction of an enzyme with the two enantiomers of a given substrate molecule will be different. Biologically important molecules often show effective activity as one enantiomer, the other is at best ineffective or at worst detrimental. asymmetric carbon enantiomem enantiomers. racemic mixtures enantiomeric PJw
238 enantiomers n enzyme receptor Figure 8.2 Enzyme interaction with two enantiomers of a given substrate molecule In some cases the unwanted enantiomer can perturb other biological processes and cause catastrophic side effects. The use of enantiomerically pure compounds thus action permits more specific drug action and the reduction in the amount of drug involved in its metabolism before secretion can be avoided Numerous examples of the different biological effects of enantiomers are available. One of the enantiomers of limonene smells of lemons, the other of oranges; one of carvone smells of caraway, the other of spearmint. These differences obviously have important
230 Chapter 8 Figure 8.2 Enzyme interaction with two enantiomers of a given substrate molecule. In some cases the unwanted enantiomer can perturb other biological processes and cause catastrophic side effects. The use of enantiomerically pure compounds thus permits more specific drug action and the reduction in the amount of drug administered. Even in the cases where the other enantiomer is inactive, the work involved in its metabolism before secretion can be avoided. Numerous examples of the different biological effects of enantiomers are available. One of the enantiomers of limonene smells of lemons, the other of oranges; one of carvone smells of caraway, the other of spearmint. These differences obviously have important sped:iz
Industrial production of amino acids by fermentation and chemo-enzymatic methods consequences for the perfume and flavour industries. Both enantiomers of sucrose are equally sweet, but only the naturally occurring D-enantiomer is metabolised, making the synthetic L-enantiomer a potential dietary sweetener In the protection of crops from insects, one enantiomer of a compound may be a repellant while the other is an attractant, and the racemic mixture is ineffective. One enantiomer of penicillamine(D-)exhibits antiarthritic properties but the other is highly toxic(Figure 8.3). The teratogenic effects of thalidomide were induced by one enantiomer, the other exhibited the beneficial effects against moming sickness. Different optical enantiomers of amino acids also have different properties L-asparagine, for example, tastes bitter while D-asparagine tastes sweet(see Figure 8.3) demonstrate the importance of the use of homochiral compounde ( figure a 3).When achiral l-phenylalanine is a constituent of the artificial sweetener aspartame (Figure 8.3).When compounds one uses D-phenylalanine the same compound tastes bitter. These examples clearly 1)penicillamine H3C CH3 HOC NH2 extreme toxicity 2)asparagine CH2-CH-C-NH-CH sweet Figure 8.3 Examples of diferent biological effects of enantiomers. s and R refer to a particular system of nomenclature used to describe chiral carbon. (see Appendix A8. 1) SAQ 8.2 List possible advantages of using enantiomerically pure compounds as drugs, as opposed to using racemic mixtures
Industrial production of amino acids by fermentation and chemo-enzymatic methods 239 consequences for the perfume and flavour industries. Both enantiomers of sucrose are equally sweet, but only the naturally occurring D-enantiomer is metabolid, making the synthetic L-enantiomer a potential dietary sweetener. In the protection of mps from insects, one enantiomer of a compound may be a repellant while the other is an attractant, and the racemic mixture is ineffective. One enantiomer of penicillamine (D-) exhibits antiarthritic properties but the other is highly toxic (Figure 8.3). The teratogenic effects of thalidomide were induced by one enantiomer, the other exhibited the beneficial effects against morning sickness. Different optical enantiomers of amino acids also have different properties. L-asparagine, for example, tastes bitter while D-asparagine tastes sweet (see Figure 8.3). L-Phenylalanine is a constituent of the artificial sweetener aspartame (Figure 8.3). When one uses D-phenylalanine the same compound tastes bitter. These examples clearly demonstrate the importance of the use of homochiral compounds. hvnce Of hanochlral compo,,,,^ ~ ~~~ ~~ Figure 8.3 Examples of different biological effects of enantiomers. S and R refer to a particular system of nomenclature used to describe chiral carbon. (see Appendix A8.1) List possible advantages of using enantiomerically pure compounds as drugs, as opposed to using racemic mixtures
240 Chapter 8 8. 4 Amino acid fermentation wild strains Many micro-organisms accumulate amino acids in culture media. Indeed wild strains have proved to be effective producers of amino acids like alanine, glutamic acid and valine Since amino acids are used as essential components of the microbial cells and their biosynthesis is regulated to maintain an optimal level, they are normally synthesised in limited amounts and are subject to negative feedback control. The main problem using wild strains is, therefore, the production of minor amounts of amino acids at an early tage in the fermentation, giving rise to feedback control To achieve overproduction of amino acids the follo improvement of the uptake of the raw material(starting materiaL); hindrance of the side reactions stimulation of the enzymes that are involved in the synthesis inhibition of the degradation of the desired amino acid; stimulation of excretion of the amino acid that is produce ategies for The most successful way to achieve overproduction is to make use of mutants. Another rproducton way to overcome feedback regulation is to make use of a kind of semi-fermentation process called precursor addition fermentation; this will be considered later in this chapte Amino acids produced by fermentation on an industrial scale are listed in table 8.3 Ino acids tonnes/year ppllcatlons ca8,000 westener) synthesis of alan glutamic acid ca.270,00 flavours, pharmaceuticals ca.90,000 ca.8,000 aspartame(sweetener) ca.500 dietary ca.100 pharmaceuticals, dietary Table 8.3 Amino acids industrially produced by fermentation ∏ Examine the list of procedures to achieve overproduction(shown above)and identify which ones could be achieved by mutation of a wild strain. Since all the procedures listed involve enzymes they all could be achieved by mutation This emphasises the potential of using mutation for amino acid production
240 Chapter 8 8.4 Amino acid fermentation wild strains Many micro-organisrns accumulate amino acids in culture media. Indeed, wild strains have proved to be effective producers of amino acids like alanine, glutamic acid and valine. Since amino acids are used as essential components of the microbial cells and their biosynthesis is regulated to maintain an optimal level, they are normally synthesised in limited amounts and are subject to negative feedback control. The main problem using wild strains is, therefore, the production of minor amounts of amino acids at an early stage in the fermentation, giving rise to feedback control. To achieve overproduction of amino acids the following procedures can be used: 0 improvement of the uptake of the raw material (starting material); 0 hindrance of the side reactions; tzgz mw 0 stimulation of the enzymes that are involved in the synthesis; 0 inhibition of the degradation of the desired amino acid; 0 stimulation of excretion of the amino acid that is produced. The most successful way to achieve overproduction is to make use of mutants. Another way to overcome feedback rrgulation is to make use of a kind of semi-fermentation process called precursor addition fermentation; this will be considered later in this chapter. Amino acids produced by fermentation on an industrial scale are listed in Table 8.3. smegies br overpcoddm amino aclds tonneelyear aspartic acid ca. 8,000 glutamic acid lysine phenylalanine threonine tryptophan ca. 270,000 ca. 90,000 ca. 8,000 ca. 500 ca. 100 applications aspartame (sweetener) enzymatic synthesis of alanine and phenylanine flavours, pharmaceuticals dietary aspartame (sweetener) dietary pharmaceuticals, dietary Table 8.3 Amino adds industrially produced by fermentation. Examine the list of p'ocedures to achieve overproduction (shown above) and n idenbfy which ones could be achieved by mutation of a wild strain. Since all the procedures listed involve enzymes they all could be achieved by mutation. This emphasises the potential of using mutation for amino acid production