Biocatalysts in organic chemical synthesis 2.1 Introduction 2.2 Micro-organisms as catalysts of organic synthesis 2.3 Enzyme preparations versus whole cell processes 2.4 Scale of production 2.5 Modes of operation of bioprocesses 2.6 Biotechnological processes verses chemical synthetic processes 2.7 Bioprocess development Summary and obiectives
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10 Chapter 2 Biocatalysts in organic chemical synthesis 1 Introduction traditonal and Biotechnology is often divided into two categories called'traditional biotechnology new and'new biotechnology The major products of the traditional biotechnology industry toachnoogy are industrial alcohol, food and flavour ingredients, antibiotics and citric acid. The new biotechnology involves the newer techniques of genetic engineering and cell fusion to produce organisms capable of making useful products. Products of the new biotechnology are extremely diverse and include steroid derivatives, antibiotics and special proteins for therapeutic use (eg human growth hormone, interferons and The market for products of traditional biotechnology is currently worth around 250 times more than those of the new biotechnology, although it is predicted that the new biotechnology will account for an increasingly larger fraction of the total biotechnology industry The purpose of this chapter is to compare and contrast various production strategies of the biotechnology industry and to consider some of the major decisions that have to be made during bioprocess development. Many of the areas touched upon will be developed in greater detail in other chapters of this book. The book is limited to the use of micro-organisms and enzymes as bioprocess catalysts and does not consider catalysis by plant and animal cells. As you will see later in this chapter, industrial microbiology is the major foundation of biotechnology and there are many reasons why micro-organisms dominate as production organisms in both traditional and new biotechnological processes. 2.2 Micro-organisms as catalysts of organic synthesis Microbial cells are very attractive as a source of catalysts for the production of organic chemicals because of their broad range of enzymes capable of a wide variety of chemical reactions, some of which are illustrated in Table 2.1
10 Chapter 2 Biocatalysts in organic chemical synthesis 2.1 Introduction tradHionaiand new bboe*mb!JY Biotechnology is often divided into two categories called 'traditional biotechnology' and 'new biotechnology'. The major products of the traditional biotechnology industry are industrial alcohol, food and flavour ingredients, antibiotics and citric acid. The new biotechnology involves the newer techniques of genetic engineering and cell fusion to produce organisms capable of making useful products. Products of the new biotechnology are extremely diverse and include steroid derivatives, antibiotics and special proteins for therapeutic use (eg human growth hormone, interferons and interleukins). The market for products of traditional biotechnology is cmntly worth around w) times more than those of the new biotechnology, although it is predicted that the new biotechnology will account for an increasingly larger fraction of the total biotechnology industry. The purpose of this chapter is to compare and contrast various production strategies of the biotechnology industry and to consider some of the mapr decisions that have to be made during bioprocess development. Many of the areas touched upon will be developed in greater detail in other chapters of this book. The book is limited to the use of micro-organisms and enzymes as bioprocess catalysts and does not consider catalysis by plant and animal cells. As you will see later in this chapter, industrial microbiology is the major foundation of biotechnology and there are many reasons why micro-organisms dominate as production organisms in both traditional and new biotechnological processes. 2.2 Micro-organisms as catalysts of organic synthesis Microbial cells are very attractive as a source of catalysts for the production of organic chemicals because of their broad range of enzymes capable of a wide variety of chemical reactions, some of which are illustrated in Table 2.1
Biocatalysts in organic chemical synthesis Esterase enzymes- cleavage of various ester bonds to yield an acid and an alcohol, ie RI COoR2+H20 <- →>R1COOH+R2OH LIpase enzymes(a subgroup of esterase enzymes)-hydrolyse fats into glycerol and fatty acids, eg H2C-, H,COH R1 OOH 2+929 HCOH R2OOH H2C- OR H2CO fatty acids Proteolytic enzymes-hydrolyse proteins selectively, either on terminal groups (exopeptidases)or intemal linkages(endopeptidases),eg A Oxldoreductases-catalyse oxidation-reduction reactions, eg dehydrogenase H.C. CH,OH →H2C.CHo NAD+ NADH +H+ Oxygenases- add one(monooxygenases)or both(dioxygenases)atoms of molecular oxygen to molecules, eg NADH NAD+ cis-dihydrodiol Table 2.1 Some reactions catalysed by microbial enzymes. In principle each enzyme catalyses the reverse process as well
Biocatalysts in organic chemical synthesis 11 Table 2.1 Some reactions catalysed by microbial enzymes. In principle each enzyme catalyses the reverse process as well
Chapter 2 Lyases-catalyse the breakage of c-c bonds, eg socitrate HC- COOH ase CH,COOH H Hoc一cooH CH2COOH + C-COOH Isocitric acid Succinic acid Glyoxylic acid Transferases-catalyse the transfer of functional groups, eg CHOl CH2 OPO3H2 Glucokinase D-Glucose D-Glucose-6-phosphoric acid es-catalyse the inter-conversion of isomers, eg CH2OH Triosephosphate C=O somerase HCOH CH2OPO3H2 CH2oPO3H2 Dihydroxyacetone phosotnyde-3 Tabe2.1… Continued rather than plant and animal cells, are generally preferred for the production of organic chemicals. There are several reasons for ∏ Make a list of at least five commonly found features of micro-organisms that would benefit their use as catalysts for organic synthesis Your list could have included the following commonly found features high growth rates which allows the generation of large amounts of catalyst advantages of substrates for growth are often cheap and include waste materials from other ing microbial industrial processes they are generally more robust and less fastideous than plant and animal cell cultures
12 Chapter 2 Table 2.1 ...... Continued Microbial cells, rather than plant and animal cells, are generally preferred for the production of organic chemicals. There are several reasons for this. n would benefit their use as catalysts for organic synthesis. Your list could have included the following coINl[lonly found features: high growth rates which allows the generation of large amounts of catalyst (microorganism); substrates for growth are often cheap and include waste materials from other 0 they are generally more robust and less fastideous than plant and animal cell cultures; Make a list of at least five commonly found features of mi<3.o-organisms that advantagesof -9 mia*u industrial processes; Cells
Biocatalysts in organic chemical synthesis are many different types of microbes, each with unique nutritional and ological features, which may be desirable for process development; collectively, micro-organisms have a broad complement of enzymes capable of a of chemical production plants involving micro-organisms are generally independent of climatic conditions and require little space( compared to crop plant production) for some microbes, such as Escherichia coli, the genome is well known and relatively easy to manipulate genetically many microbes are single celled organisms that grow well in stirred tank bioreactors ]though it is possible to obtain cells from whole animals or plants and to cultivate them in suitable nutrient solutions, in general they are not as easy to handle as microbes Nevertheless, plant and animal cells are a valuable genetic resource for biotechnology and many newly developed bioprocesses rely on transfer of their genes to micro-organisms Microbial enzymes can be applied as catalysts for chemical synthesis in biosynthetic processes or in biotransformations(bioconversions). In a biosynthetic process the tion product is formed de novo by the microbial cell from substrates, such as monosaccharides, molasses, soybean and corn steep liquor. In a biotransformation, however, a precursor that is usually chemically synthesised is converted in one or several enzyme catalysed steps into the desired chemical. This chemical may be the end product or may serve as a precursor for further chemical modification 2. 3 Enzyme preparations versus whole cell processes In designing a process we have the choice of using the whole organism or specific enzymes isolated from it. As always both options have pros and cons. Broadly speaking we could say that biosynthetic processes mostly rely on whole cells, whereas biotransformations can be catalysed by whole cells and by enzyme preparations ydrolytic Hydrolytic enzymes such as proteases, esterases and lipases(Table 2.1)account for nzymes more than half of all reported biotransformations. These enzymes are particularly easy to use because: they are available in large amounts from industrial sources they are stable in non-aqueous solvent they do not have cofactor requirements Why do you think many processes based on redox reactions involving dehydrogenase enzymes are still carried out using whole cells? dehydrogenases Dehydrogenase enzymes generally require NADH or NADPH, and although methods for recycling these cofactors are now available on a laboratory scale, little progress has been made in the scale-up to industrial level
Biocatalysts in organic chemical synthesis 13 there are many different types of microbes, each with unique nutritional and physiological features, which may be desirable for process development; 0 collectively, micro-organisms have a broad complement of enzymes capable of a wide variety of chemical reactions; production plants involving micro-organisms are generally independent of climatic conditions and require little space (compared to crop plant produdion); 0 for some microbes, such as Esc/~m-ichia coli, the genome is well known and relatively easy to manipulate genetically; 0 many microbes are single celled organisms that grow well in stirred tank bioreactors (fermentors). Although it is possible to obtain cells from whole animals or plants and to cultivate them in suitable nutrient solutions, in general they are not as easy to handle as microbes. Nevertheless, plant and animal cells are a valuable genetic resource for biotechnology and many newly developed bioprocesses rely on transfer of their genes to micro-organisms. Microbial enzymes can be applied as catalysts for chemical synthesis in biosynthetic processes or in biotransformations (bioconversions). In a biosynthetic process the product is formed de novo by the microbial cell from substrates, such as monosaccharides, molasses, soybean and corn steep liquor. In a biotransformation, however, a precursor that is usually chemically synthesised is converted in one or several enzyme catalysed steps into the desired chemical. This chemical may be the end product or may serve as a pmrsor for further chemical modification. biosynthetic Pr-wsand bornsforination 2.3 Enzyme preparations versus whole cell processes In designing a process we have the choice of using the whole organism or specific enzymes isolated from it. As always both options have pro's and cons. Broadly speaking we could say that biosynthetic processes mostly rely on whole cells, whereas biotransformations can be catalysed by whole cells and by enzyme preparations. Hydrolytic enzymes such as proteases, esterases and lipases (Table 2.1) account for more than half of all reported biotransformations. These enzymes are particularly easy to use because: hydrolytic 0 they are available in large amounts from industrial sources; they are stable in non-aqueous solvent; they do not have cofactor requirements. Why do you think many processes based on redox reactions involving n dehydrogenase enzymes are still carried out using whole cells? Dehydrogenase enzymes generally require NADH or NADPH, and although methods for recycling these cofactors are now available on a laboratory scale, little progress has been made in the scale-up to industrial level. dehydrogenases