9.3.4 Substrate Inhibition

In a number of cases, the substrate itself can act as an inhibitor. In the case of uncompetitive inhibition, the inactive molecule (S · E · S) is formed by the reaction

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Consequently we see that by replacing (I) by (S) in Equation (9-40), the rate law for –rS is

9-44

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We see that at low substrate concentrations

9-45

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then

9-46

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and the rate increases linearly with increasing substrate concentration.

At high substrate concentrations ((S)2 / KI) >>(KM + (S)), then

9-47

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Substrate inhibition

and we see that the rate decreases as the substrate concentration increases. Consequently, the rate of reaction goes through a maximum in the substrate concentration, as shown in Figure 9-16. We also see that there is an optimum substrate concentration at which to operate. This maximum is found by setting the derivative of –rS in Equation (9-44) wrt S equal to 0, to obtain

9-48

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Figure 9-16. Substrate reaction rate as a function of substrate concentration for substrate inhibition.

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When substrate inhibition is possible, a semibatch reactor called a fed batch is often used as a CSTR to maximize the reaction rate and conversion.

Our discussion of enzymes is continued in the Professional Reference Shelf on the DVD-ROM and on the Web where we describe multiple enzyme and substrate systems, enzyme regeneration, and enzyme co-factors (see R9.6).

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9.4 Bioreactors and Biosynthesis

A bioreactor is a reactor that sustains and supports life for cells and tissue cultures. Virtually all cellular reactions necessary to maintain life are mediated by enzymes as they catalyze various aspects of cell metabolism such as the transformation of chemical energy and the construction, breakdown, and digestion of cellular components. Because enzymatic reactions are involved in the growth of microorganisms (biomass), we now proceed to study microbial growth and bioreactors. Not surprisingly, the Monod equation, which describes the growth law for a number of bacteria, is similar to the Michaelis–Menten equation. Consequently, even though bioreactors are not truly homogeneous because of the presence of living cells, we include them in this chapter as a logical progression from enzymatic reactions.

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The use of living cells to produce marketable chemical products is becoming increasingly important. The number of chemicals, agricultural products, and food products produced by biosynthesis has risen dramatically. In 2007, companies in this sector raised over $24 billion of new financing.13 Both microorganisms and mammalian cells are being used to produce a variety of products, such as insulin, most antibiotics, and polymers. It is expected that in the future a number of organic chemicals currently derived from petroleum will be produced by living cells. The advantages of bioconversions are mild reaction conditions; high yields (e.g., 100% conversion of glucose to gluconic acid with Aspergillus niger); and the fact that organisms contain several enzymes that can catalyze successive steps in a reaction and, most importantly, act as stereospecific catalysts. A common example of specificity in bioconversion production of a single desired isomer that when produced chemically yields a mixture of isomers is the conversion of cis-proenylphosphonic acid to the antibiotic (–) cis-1,2-epoxypropyl-phosphonic acid. Bacteria can also be modified and turned into living chemical factories. For example, using recombinant DNA, Biotechnic International engineered a bacteria to produce fertilizer by turning nitrogen into nitrates.14

The growth of biotechnology

$24 billion

More recently, the synthesis of biomass (i.e., cell/organisms) has become an important alternative energy source. In 2009, ExxonMobil invested over 600 million dollars to develop algae growth and harvest in waste ponds. It is estimated that one acre of algae can provide 2,000 gallons of gasoline per year.

In biosynthesis, the cells, also referred to as the biomass, consume nutrients to grow and produce more cells and important products. Internally, a cell uses its nutrients to produce energy and more cells. This transformation of nutrients to energy and bioproducts is accomplished through a cell’s use of a number of different enzymes in a series of reactions to produce metabolic products. These products can either remain in the cell (intracellular) or be secreted from the cells (extracellular). In the former case, the cells must be lysed (ruptured) and the product filtered and purified from the whole broth (reaction mixture). A schematic of a cell is shown in Figure 9-17.

Figure 9-17. (a) Schematic of cell; (b) photo of E. coli cell dividing.

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Courtesy of kentoh/Shutterstock.

The cell consists of a cell wall and an outer membrane that encloses the cytoplasm containing a nuclear region and ribosomes. The cell wall protects the cell from external influences. The cell membrane provides for selective transport of materials into and out of the cell. Other substances can attach to the cell membrane to carry out important cell functions. The cytoplasm contains the ribosomes that contain ribonucleic acid (RNA), which are important in the synthesis of proteins. The nuclear region contains deoxyribonucleic acid (DNA), which provides the genetic information for the production of proteins and other cellular substances and structures.15

The reactions in the cell all take place simultaneously and are classified as either class (I) nutrient degradation (fueling reactions), class (II) synthesis of small molecules (amino acids), or class (III) synthesis of large molecules (polymerization, e.g., RNA, DNA). A rough overview with only a fraction of the reactions and metabolic pathways is shown in Figure 9-18. A more detailed model is given in Figures 5-1 and 6-14 of Shuler and Kargi.16 In the Class I reactions, adenosine triphosphate (ATP) participates in the degradation of nutrients to form products to be used in the biosynthesis reactions (Class II) of small molecules (e.g., amino acids), which are then polymerized to form RNA and DNA (Class III). ATP also transfers the energy it releases when it loses a phosphonate group to form adenosine diphosphate (ADP).

ATP + H2O → ADP + P + H2O + Energy

Figure 9-18. Examples of reactions occurring in the cell.

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