14 Chapter 2 oxygenases Similarly, many oxygenation reactions (Table 2.1), which also require cofactors, are usually performed using whole micro-organisms. Collectively, oxidoreductases and oxygenases account for around 30% of all reported biotransformations. Enzymes such as lyases, transferases and isomerases(Table 2. 1) account for most of the of industrially applied biotransformations. 2. 3. 1 Enzyme catalysed processes Enzymes isolated from micro-organisms have many desirable properties as catalysts for he synthesis of industrial chemicals, but there are associated problems associated the provision of enzyme cofactors can be expensi most enzyme reactions are carried out in water and the enzymes must be separated from the product stream; the product stream is often very dilute, presenting problems of product preparation of a crude or purified cell-free enzyme preparation is necessary Advances in genetic and chemical enzyme modifications, enzyme immobilisation and immobilisation enzymatic reactions in organic solvents, have increased the actual use and potential of enzymes in the production of industrial chemicals. Enzyme immobilisation, in particular, has proved to be a valuable approach to the use of enzymes in chemical synthesis. The term denotes enzymes that are physically confined or localised in a defined region in with retention of their catalytic activities. a detailed consideration of immobilisation techniques is beyond the scope of this chapter; the subject is covered adequately in the biotol text entitled'Technological Applications of Biocatalyst enzyme Enzyme immobilisation allows the construction of enzyme reactors in which the bioreactor enzyme can be reused. Furthermore, the process operates continuously and can be sign readily controlled Enzyme reactors currently in use include those illustrated in Figure
14 Chapter 2 oxygenases associated problems enzyme immobilisation enzyme boreactor design Similarly, many oxygenation reactions (Table Z.l), which also require cofactors, are usually performed using whole micro-organisms. Collectively, oxidoreductases and oxygenases account for around 30% of all reported biotransformations. Enzymes such as lyases, transferases and isomerases (Table 2.1) account for most of the remainder of industrially applied biotransformations. 2.3.1 Enzyme catalysed processes Enzymes isolated from micro-organisms have many desirable properties as catalysts for the synthesis of industrial chemicals, but there are associated problems: their protein structure may not be stable under non physiological conditions which may be detrimental to their long term use, especially at elevated temperatures; the provision of enzyme cofactors can be expensive; 0 most enzyme reactions are carried out in water and the enzymes must be separated from the product stream; the product stream is often very dilute, presenting problems of product concentration and recovery; 0 preparation of a crude or purified cell-free enzyme preparation is necessary. Advances in genetic and chemical enzyme modifications, enzyme immobilisation and enzymatic reactions in organic solvents, have increased the actual use and potential of enzymes in the production of industrial chemicals. Enzyme immobilisation, in particular, has proved to be a valuable approach to the use of enzymes in chemical synthesis. The term denotes enzymes that are physically confined or localised in a defined region in space with retention of their catalytic activities. A detailed consideration of immobilisation techniques is beyond the scope of this chapter; the subject is covered adequately in the BIOTOL text entitled 'Technological Applications of Biocatalysts'. Enzyme immobilisation allows the construction of enzyme reactors in which the enzyme can be reused. Furthermore, the process operates continuously and can be readily controlled. Enzyme reactors currently in use include those illustrated in Figure 2.1
Biocatalysts in organic chemical synthesis b .. Hproduct immobilised immobilised substrat product column substrate substrate immobilised substrat ( fluidised) ultrafiltra enzyme Figure 2. 1 Examples of enzyme bioreactor design Bioreactors: a)batch stirred tank; b )continuous stirred tank; c)continuous packed-bed i) downward flow, i)upward flow and ii recycle; d) continuous fluidised-bed; e)continuous ultrafiltration. Redrawn from Katchalski-Katzir E(1993)Trends in Biotechnology 1,471-477. advant Some of the potential advantages of enzyme immobilisation are: · high enzyme loads enzyme can often be regenerated by suitable ment and used again, the ability to recycle products · high flow rates; reduction in cost(energy and labour), energy and waste products easy to scale up to large systems; high yields of pure materials higher substrate concentrations can be used
Biocatalysts in organic chemical synthesis 15 Figure 2.1 Examples of enzyme bioreactor design. Bioreactors: a) batch stirred tank; b) continuous stirred tank; c) continuous packed-bed i) downward flow, ii) upward flow and iii) recycle; d) continuous fluidised-bed; e) continuous ultrafittration. Redrawn from Katchalski - Katzir E. (1993) Trends in Biotechnology I/. 471 -477. Some of the potential advantages of enzyme immobilisation are: 0 high enzyme loads; 0 prolonged enzyme activity; 0 enzyme can often be regenerated by suitable treatment and used again; the ability to recycle products; 0 high flow rates; reduction in cost (energy and labour), energy and waste products; easy to scale up to large systems; high yields of pure materials; higher substrate concentrations can be used. advantages of enzyme irnmobilisation
16 Chapter 2 2.3.2 Whole cell process e can also use microbial cells(fermentation) containing the desired catalytic activity without isolating the enzymes responsibl Advantages of whole cell processes include: cell disruption not necessary enzyme isolation not necessary; cofactor regeneration not a problem reduced catalyst preparation costs Among the disadvantages of whole cell processes are system not fully understood(black box situation) product contamination by cellular enzymes or other end products of metabolism reduced catalytic specific activity cell structures acting as diffusion barriers; contamination by other micro-organisms may be a problem. Synthesis of industrial chemicals by microbial cells may be by fermentation (free, living ells), immobilised growing cells, immobilised resting cells or immobilised dead cells immobilised Immobilised cells have all the advantages of immobilised enzymes. cell immobilisation cell is preferred for reactions catalysed by intracellular enzymes because it avoids tedious and expensive extraction and purification procedures, which often result in preparations of low yield and stability SAQ 2.1 Identify which of the following statements are true for immobilised biocatalysts hen compared to free enzyme or free cell system 1)Conversions carried out by immobilised cells give higher yields than those carried out by growing and dividing cells 2)Downstream processing can be much easier 3)Smaller reactor volumes achieve similar rates of product formation. 4) The volume of effluent can be reduced SAQ 2.2 Benzene dioxygenase is a complex enzyme consisting of three protein components, that catalyse the conversion of benzene to benzene cis-dihydrodio Give two reasons why this biotransformation should be carried out using whole cells as opposed to using enzyme preparations
16 Chapter 2 2.3.2 Whole cell processes We can also use microbial cells (fermentation) containing the desired catalytic activity without isolating the enzymes responsible. Advantages of whole cell processes include: cell disruption not necessary; enzyme isolation not necessary; advantages 0 more suited to multiple step processes; 0 cofactor regeneration not a problem; 0 increased enzyme stability; reduced catalyst preparation costs. Among the disadvantages of whole cell processes are: 0 system not fully understood (black box situation); 0 product contamination by cellular enzymes or other end products of metabolism; 0 cell structures acting as diffusion barriers; contamination by other micro-organisms may be a problem. Synthesis of industrial chemicals by microbial cells may be by fermentation (free, living cells), immobilised growing cells, immobilised resting cells or immobilised dead cells. Immobilised cells have all the advantages of immobilised enzymes. Cell immobilisation is preferred for reactions catalysed by intracellular enzymes because it avoids tedious and expensive extraction and purification procedures, which often result in preparations of low yield and stability. disadvantages 0 reduced catalytic specific activity; immobilii cells Idenbfy which of the following statements are true for immobilised biocatalysts, when compared to free enzyme or free cell systems. 1) Conversions carried out by immobilised cells give higher yields than those carried out by growing and dividing cells. 2) Downstream processing can be much easier. 3) Smaller reactor volumes achieve similar rates of product formation. 4) The volume of effluent can be reduced. Benzene dioxygenase is a complex enzyme consisting of three protein components, that catalyse the conversion of benzene to benzene cis-dihydrodiol. Give two reasons why this biotransformation should be canied out using whole cells as opposed to using enzyme preparations
Biocatalysts in organic chemical synthesis 2.4 Scale of production The decision as to which approach -free enzyme, immobilised enzyme, fermentation, immobilised cells-is mainly dictated by economIcs ommercial aspects of bioprocess development are considered in Section 2. 6. The analysis of the various factors involved is a critical part of the decision-making process and involves inputs from entists engineers and marketing personnel. For products derived from micro-organisms, process design can be based mainly on biochemical engineering considerations or on microbial physiology considerations When compared to the improvements achieved bulk chemicals manufacture and in petroleum refining, the application of communication biochemical engineering principles to microbial proo has not been as successful over the past thirty years. It is now accepted that individual factors affecting overall optimisation of microbial processes are best handled by individual specialists microbial biotechnologists and chemical engineers. The biotechnologist, in addition to having an in-depth knowledge in a particular field, must also have the appropriate skills and knowledge to communicate and interact effectively with chemical engineers Such interaction is thought to be essential for technological innovation and commercial success of microbial processes of the future. The necessity for interaction between biotechnologists and chemical engineers increases with the scale of production. In chemical manufacture three categories of product can be defined according to the scale of production(Table 2.2) production range examples of blotechnology sIngle plant products ne chemicals 100 kg/annum- 100 vitamins. vaccines, nucleotides onnes/annum amino acids) usually batch reactors antibiotics ntermediate volume 0,000 tonnes/annum h or continuous reactors citric acid food industry bionics fo enzymes industr ermented foods and bulk chemicals > 20.000 tonnes/annum single cell protein, ethanol for usually continuous flow industry biopolymers for enhanced oil recovery), biogas (methane), sewage and wastewater treatment plants Table 2.2 Product categories in chemical manufacture The design of production plants for the manufacture of the three categories of product a lly produced in batch olume and also be used for the production of a variety of similar products. Fine chemicals usually bulk chemicals have demanding product quality specifications and uently, a significant fraction of the production costs are involved in product purification and testing. Intermediate volume chemicals have less rigorous quality specifications than fine chemicals and usually manufactured in product-specific-plants, either as batch or continuous flow processes. Bulk chemical production plants usually operate continuous flow processes
Biocatalysts in organic chemical synthesis 17 k.4 Scale of production The decision as to which approach - free enzyme, immobilised enzyme, fermentation, P obilised cells - is mainly dictated by economics. Commercial aspects of bioprocess evelopment are considered in Section 2.6. The analysis of the various factors involved is a critical part of the decision-making process and involves inputs from scientists, engineers and marketing personnel. For products derived from micrmrganisms, process design can be based mainly on biochemical engineering considerations or on microbial physiology considerations. When compared to the improvements achieved in bulk chemicals manufacture and in petroleum refining, the application of biochemical engineering principles to microbial processes has not been as successful over the past thirty years. It is now accepted that individual factors affecting overall optimisation of microbial processes are best handled by individual specialists - microbial biotechnologists and chemical engineers. The biotechnologist, in addition to having an in-depth knowledge in a particular field, must also have the appropriate skills and knowledge to communicate and interact effectively with chemical engineers. Such interaction is thought to be essential for technological innovation and commercial success of microbial processes of the future. The necessity for interaction between biotechnologists and chemical engineers increases with the scale of production. In chemical manufacture three categories of product can be defined according to the scale of production (Table 2.2). -ww needfor communication production range examples of biotechnology single piant products fine chemicals 100 kg/annum-100 vitamins, vaccines, nucletides, tonneslannum amino acids) usually batch reactors antibiotics ) some enzymes ) intermediate volume 100-20,000 tonnes/annum glutamic acid) chemicals batch or continuous reactors citric acid ) food industry lactic acid ) antibiotics for agriculture, enzymes for industry, many fermented foods and beverages industry, biopolymers (for enhanced oil recovery), biogas wastewater treatment plants bulk chemicals > 20,000 tonnes/annum single cell protein, ethanol for usually continuous flow I (methane), sewage and I Table 2.2 Product categories in chemical manufacture. The design of production plants for the manufacture of the three categories of product varies considerably. Fine chemicals are usually produced in batch reactors, which may also be used for the production of a variety of similar products. Fine chemicals usually have demanding product quality specifications and, consequently, a significant fraction of the production costs are involved in product purification and testing. Intermediate volume chemicals have less rigorous quality specifications than fine chemicals and are usually manufactured in product-spe4c-plants, either as batch or continuous flow processes. Bulk chemical production plants usually operate continuous flow processes fine, volume and bulk demimls