Industrial production of amino acids by fermentation and chemo-enzymatic methods 8. 1 Introduction 8.2 Essential and nonessential amino acid 3 Stereochemistry of amino acids 8. 4 Amino acid fermentation 8.5 Recovery of the amino acid from the fermentation broth 248 8.6 Case study: fermentative production of L-phenylalanine from glucose 8.7 Case study: The production of L-phenylalanine by enzymatic Summary and objectives Appendix 8. 1: Representation of and the nomenclature for stereo-isomers Appendix 8.2 Examples of industrial production of amino adds by enzymatic method A8.2.1 Enzymatic resolution of racemates A8. 2.2 Enzymatic asymmetric synthesis A8. 2.3 Oxidation-Reduction reactions
231 Industrial production of amino acids by fermentation and chemo-enzymatic methods 8.1 Introduction 8.2 Essential and nonessential amino acids 8.3 Stereochemistry of amino acids 8.4 Amino acid fermentation 8.5 Recovery of the amino acid from the fermentation broth 8.6 Case study: fermentative production of L-phenylalanine from glucose 8.7 Case study: The production of L-phenylalanine by enzymatic methods Summary and objectives Appendix 8.1: Representation of and the nomenclature for stereeisomers Appendix 8.2 Examples of industrial production of amino acids by A8.2.1 Enzymatic resolution of racemates enzymatic methods A8.2.2 Enzymatic asymmetric synthesis A8.2.3 Oxidation - Reduction reactions 232 234 236 240 248 253 262 272 273 277 277 286 289
232 Chapter 8 Industrial production of amino acids by fermentation and chemo-enzymatic methods 8.1 Introduction Amino acids have always played animportant role in the biology of life, in biochemistry and in (industrial) chemistry. There are several reasons why they are of commercial amino acids are the building blocks of proteins and they play an essential role in the regulation of the metabolism of living organisms. Large-scale essential chemical and microbial producton processes have been commercialised for a number no aads of essential amino acids The use of glutamic acid lysine and methionine as food and feed additives is well established nowadays. Secondly, current interest in developing on-proteino. peptide-derived chemotherapeutics has heightened the importance of rare and genic amino non-proteinogenic pure amino acids. For example, D-phenylglycine and acds D-p-hydroxy-phenylglycine are building blocks for the broad spectrum B-lactam antibiotics ampicillin and amoxycillin, respectively. The natural amino acid L-valine is used as feedstock in the fermentative production of the cyclic peptide cyclosporin A, which has immuno-suppressive activity and is used in human transplant surgery Thirdly, amino acids are versatile chiral (optically active)building blocks for a whole range of fine chemicals. In the last two decades, there has been a growing public precursors for awareness and concern with regard to the exposure of man and his environment to an fine chemical ever increasing number of chemicals. The benefits, however, arising from the use synthesis therapeutic agents, pesticides, food and feed additives, etc are enormous. hence there is still an ever increasing demand for more selective drugs and pesticides which are targeted in their mode of action, exhibit less toxic side-effects and are more environmentally acceptable. To this end a central role will be played by chiral compounds, as nature at the molecular level is intrinsically chiral. Consequently, this provides an important stimulus for companies to market chiral products as pure optical isomers. This in turn results in an increasing need for efficient methods for the industrial synthesis of optically active compounds Amino acids are, therefore, important as nutrients (food and feed ), as seasoning flavourings and starting material for pharmaceuticals, cosmetics and other chemicals They can be produced in a variety of ways(see Table 8.1) · chemical synthesis; isolation from natural materials(extraction) amino acid fermentations(using micro-organisms); chemo-enzymatic methods In this chapter we consider amino acid production by fermentation and by chemo-enzymatic methods. we first consider the stereochemistry of amino acids and the importance of chirality in chemical synthesis. General approaches to amino acid fermentation and recovery of amino acids from fermentation broths are then dealt with followed by a detailed consideration of the production of L-phenylalanine by direct fermentation. Later in this chapter, chemo-enzymatic methods of amino acid
232 Chapter 8 essential ammo adds non-pcoteinogenic amino acids precursors for me chemical synthesis Industrial production of amino acids by fermentation and cherno-enzymatic methods 8.1 Introduction Amino acids have always played an important role in the biology of life, in biochemistry and in (industrial) chemistry. There are several reasons why they are of commercial interest. Firstly, amino acids are the building blocks of proteins and they play an essential role in the reguiation of the metabolism of living organisms. Largescale chemical and microbial production processes have been commercialised for a number of essential amino acids. The use of glutamic acid, lysine and methionine as food and feed additives is well established nowadays. Secondly, current interest in developing peptide-derived chemotherapeutics has heightened the importance of rare and non-proteinogenic pure amino acids. For example, D-phenylglycine and D-phydroxy-phenylglycine are building blocks for the broad spectrum f3-ladam antibiotics ampicillin and amoxycillin, respectively. The natural amino acid L-valine is used as feedstock in the fermentative production of the cyclic peptide cyclosporin A, which has immuno-suppressive activity and is used in human transplant surgery. Thirdly, amino acids are versatile chiral (optically active) building blocks for a whole range of fine chemicals. In the last two decades, there has been a growing public awareness and concern with regard to the exposure of man and his environment to an ever increasing number of chemicals. The benefits, however, arising from the use of therapeutic agents, pesticides, food and feed additives, etc are enormous. Hence there is still an ever increasing demand for more selective drugs and pesticides which are targeted in their mode of action, exhibit less toxic side-effects and are more environmentally acceptable. To this end a central role will be played by chiral compounds, as nature at the molecular level is intrinsically chiral. Consequently, this provides an important stimulus for companies to market chiral products as pure optical isomers. This in turn results in an increasing need for efficient methods for the industrial synthesis of optically active compounds. Amino acids are, therefore, important as nutrients (food and feed), as seasoning, flavourings and starting material for pharmaceuticals, cosmetics and other chemicals. They can be produced in a variety of ways (see Table 8.1): 0 chemical synthesis; 0 isolation from natural materials (extraction); amino acid fermentations (using micro-organisms); 0 chemo-enzymatic methods. In this chapter we consider amino acid production by fermentation and by chemoenzymatic methods. We first consider the stereochemistry of amino acids and the importance of chirality in chemical synthesis. General approaches to amino acid fermentation and recovery of amino acids from fermentation broths are then dealt with, followed by a detailed consideration of the production of L-phenylalanine by direct fermentation. Later in this chapter, chemo-enzymatic methods of amino acid
Industrial production of amino acids by fermentation and chemo-enzymatic methods fermentation are examined. We first consider general aspects of the approach, followed by more detailed case studies. We have again selected L-phenylalanine for detailed consideration, since this important amino acid can be produced by direct fermentation and by a variety of chemo-enzymatic methods. This allows comparisons between the two different approaches to be made, including a consideration of economic aspects of large scale production of the amino acid Two appendices are included at the end of this chapter The first is intended to serve as a reminder, for those of you who might need it, of the nomenclature and representation of stereoisomers. The second appendix contains descriptions of various chemo-enzymatic methods of amino acid production. This appendix has been constructed largely from the recent primary literature and includes many new advances in the field. It is not necessary for you to consult the appendix to satisfy the learning objectives of the chapter, rather the information is provided to illustrate the extensive range of methodology associated with chemo-enzymatic approaches to amino acid production. It is therefore available for those of you who may wish to extend your knowledge in this area. Where available, data derived from the literature are used to illustrate methods and to discuss economic aspects of large-scale production amino acld chemical extraction fermentation enzymatIc synthesls LLL -histidine(HCI L-phenylalanine (+) L-threonine L-tyrosine L-valine (+) Table 8. 1 Production methods of proteinogenic amino acids
Industrial production of amino acids by fermentation and chemo-enzymatic methods 233 fennentation are examined. We first consider general aspects of the approach, followed by more detailed case studies. We have again selected L-phenylalanine for detailed consideration, since this important amino acid can be produced by direct fermentation and by a variety of chemo-enzymatic methods. This allows comparisons between the two different approaches to be made, including a consideration of economic aspects of large scale production of the amino acid. Two appendices are included at the end of this chapter. The first is intended to serve as a reminder, for those of you who might need it, of the nomenclature and representation of stereoisomers. The second appendix contains descriptions of various chemo-enzymatic methods of amino acid production. This appendix has been constructed largely from the recent primary literature and includes many new advances in the field. It is not necessary for you to consult the appendix to satisfy the learning objectives of the chapter, rather the information is provided to illustrate the extensive range of methodology associated with chemo-enzymatic approaches to amino acid production. It is therefore available for those of you who may wish to extend your knowledge in this area. Where available, data derived from the literature are used to illustrate methods and to discuss economic aspects of large-scale production. + amino ackl chemical extradlon fermentation enzymatic synthesls CatalySlS L-alanine + + L-arginine + + L-aspartic acid + + L-cystine + L-glutamic acid (Na) (+I + L-histidine (.HCI) + + L-isoleucine + + L-leucine + L-lysine (.HCI) + + L-methionine + L-phenylalanine (+) (+) + L-proline + (+I L-serine + + L-threonine + + L-tryptophan + + L-tyrosine + L-valine + (+) + L-cysteine Table 8.1 Production methods of proteinogenic amino acids
Chapter 8 8.2 Essential and nonessential amino acids An amino acid is defined as a compound that possesses both amino and carboxyl roups. Some amino acids are aminocarboxylic acids such as proline while others are sulphur containing amino acids, such as cysteine and methionine table 8.2). Over 10 amino acids have been isolated and identified from natural sources to date. The great majority of these naturally occurring amino acids have the amino group attached to the a-carbon carbon a to the carboxylic acid. With very few exceptions, the a-carbon also be ydrogen atom. The fourth bond of the a-carbon is joined to a group which has 100 variations. Thus, most of the naturally occurring amino acids differ only structure of the organic residue attached to the a-carbon An interesting and important fact is that almost all amino acids isolated from proteins L-configuration have the L-configuration at the a-carbon, although some amino acids isolated from microbiological sources are the mirror image isomers, ie in the D-configuration. We shall consider amino acid stereochemistry in more detail in section 8.3 Of the amino acids isolated from living material only about 20 are naturally occurring components of proteins. Some of these are shown in Table 8. 2. The remainder, non-proteinogenic amino acids, are found as intermediates or end products of One of the amino adids commonly found in protein hydrolysates is called cystine; it has the following structure HOOC- CH- CH2 -S-S- CH2- CH-COOH NH H2 of cysteine, where the thiol of two monomers spaced intervals in the polypeptide are j a disulphide bridge asic amino acid is cysteine and consequently the is not included here
234 Chapter 8 8.2 Essential and nonessential amino acids a-carbon Lconfiguration dimer An amino acid is defined as a compound that possesses both amino and carboxyl groups. Some amino acids are iminocarboxylic acids, such as proline while others are sulphur containing amino acids, such as cysteine and methionine (Table 8.2). Over 100 amino acids have been isolated and identified from natural sources to date. The great majority of these naturally occumng amino acids have the amino group attached to the carbon a to the carboxylic acid. With very few exceptions, the a-carbon also bears a hydrogen atom. The fourth bond of the a-carbon is pined to a group which has over 100 variations. Thus, most of the naturally occurring amino acids differ only in the structure of the organic residue attached to the a-carbon. An interesting and important fact is that almost all amino acids isolated from proteins have the L-configuration at the a-carbon, although some amino acids isolated from microbiological sources are the mirror image isomers, ie in the Dconfiguration. We shall consider amino acid steremhemistry in more detail in section 8.3. Of the amino acids isolated from living material, only about 20 are naturally Occurring components of proteins. Some of these are shown in Table 8.2. The remainder, non-proteinogenic amino acids, are found as intermediates or end products of metabolism. One of the amino acids commonly found in protein hydrolysates is called cystine; it has the following structure: HOOC- CH-CH,-S-S-CH,-CH-COOH (CYSCYS) I I NH2 NH2 cystine It is clearly a dimer of cysteine, where the thiol groups have been oxidised to form a disulphide linkage. The dimer actually results because of two monomers at widely spaced intervals in the polypeptide are joined together by a disulphide bridge. Thus the basic amino acid is cysteine and consequently, the dimer is not included here
Industrial production of amino acids by fermentation and chemo-enzymatic methods 2H COH L-alanine CO2H rtic acid tophan HsCS CO2H NH2 L-methionine L-glutamine L-phenylalanine (L-phe Table 8. 2 Structure of some proteinogenic a-amino acids All living species are able to synthesise amino acids. Many species, however, are deficient in their ability to synthesise within their own metabolic system all the amino esential acids necessary for life. The eight amino acids with this special significance for the human species are called essential amino acids, these are ·L- valine L-leucine L-threonine · L-methionine; L-phenylalanine; L-tryptopha They are essential not because they are the only amino acids required for human functioning, but because they are essential in the diet of the human species
Industrial production of amino acids by fermentation and chemo-enzymatic methods 235 Table 8.2 structure of some proteinogenic a-amino acids. All living species are able to synthesise amino acids. Many species, however, are deficient in their ability to synthesise within their own metabolic system all the amino acids necessary for life. The eight amino acids with this special significance for the human species are called essential amino acids, these are: L-valine; 0 L-leucine; L-isoleucine; 0 L-threonine; 0 L-methionine; L-phenylalanine; 0 L-tryptophan; L-lysine. They are essential not because they are the only amino acids requred for human functioning, but because they are essential in the diet of the human species. essential amino acids