Chap 14 Amino Acids. I. Amino acids: Introduction

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1 Chap 14 Amino Acids Introduction Biotechnology has numerous applications: Medicine: the advent of genetic engineering has advanced the drug design to molecular levels. Molecular biology methods and genomics-based discovery have also contributed to the drug discovery. Food and agriculture: transgenic animals and plants, genetically modified food, genetically modified pesticides. Others (many chemicals ) I. Amino acids: monosodium glutamate (MSG 味精 ) is used as a flavor enhancer (as early as in 1908 by Dr. Ikeda). Early commercialization was based on isolation from vegetable proteins, but yield was low. Chemical synthesis of D-glutamate 1 was partially successful but D-Glu was tasteless and of little use. In 1957, Corynebacterium glutamicum (a Gram + bacterium isolated from soil) was found to secrete L-glutamate (by Drs. Udaka and Kinoshiba). The commercialization of this product also boosted the fermentation industry and the need for fermentation devices. Nucleotide production for use as flavor enhancers was also developed in 1970s. Commercial use of amino acids Glycine is used as a sweetener in juices. L-aspartate and L-phenylalanine are required for the synthesis of the sweetener: aspartame. 1 Many molecules have chiral symmetry and the amino acids can be D or L form depending on the mirror symmetry. All a.a. derived from proteins have the L-stereochemical configuration. 1

2 Chemical industry requires a.a. as building blocks for a diversity of compounds (e.g. polymers, cosmetics). Also a large market is in the animal feed additive because typical feedstuffs, such as soybean meal for pigs, are poor in some essential methionine. Other common additives include L-lysine and L-threonine. Pharmaceutical industry needs a.a. The market for a.a is growing 5 to 10% annually (has grown 20-fold over the past two decades). The prices of the a.a are inversely proportional to the product supply. The price, in turn, dictates the location of the production factories (cost of carbon source and the local market). For example: the main production factories of L-glutamate are located in the Far East. About one third of L-lysin (worldwide) is produced in North America because 1/3 of market is there and access to maize (the feedstock material for fermentation) is convenient. Production methods and tools 1. Chemical synthesis: 2. Acid hydrolysis of proteins: for low market volume products such as L-Cys. 3. Enzyme catalysis: for L-Asp production 2

3 4. Fermentation with bacteria: Classical strain development: Bacteria do not normally secrete a.a. in significant amounts, therefore, mutants that oversynthesize the respective a.a. must be generated by mutagenesis and screening programs. Procedures: Examples: 5-bromouracil (BU): analogue of thymine, incorporated into DNA chain in place of T (to pair with A), but during further DNA synthesis, BU has a higher tendency to pair with G original AT pair is replaced by CG. 2-aminopurine (AP): analogue of A, incorporated into DNA in place of A, but pairs with C in further synthesis. UV: causes two neighboring pyrimidines (e.g. two T or two C) to become covalently bonded the dimer distorts the shape of DNA and prevents replication past the dimer. Applications of new techniques Analyze mechanisms of flux control and identify the key enzyme that limits the production yield. Rapidly develop new producer strains based on the classically obtained strain, by increasing the key enzyme activities (e.g. by site-directed mutagenesis) Tools: Reliable quantification of the carbon fluxes in the cell, For instance, RNA analysis using gene chips can detect whether a specific gene is upregulated or down-regulated under certain conditions and discover the role of this gene and the possible effect of the mutations. 3

4 II. L-glutamate C. glutamicum is still the strain to use. Glu is mainly derived from -ketoglutarate in the TCA cycle, so high capability to replenish TCA cycle is necessary for high production of Glu. C. glutamicum has pyruvate dehydrogenase (PyrDH) shuffling Acetyl-CoA into TCA cycle, but has two enzymes supplying oxaloacetate: pyruvate carboxylase (Pyrc) and phosphoenolpyruvate carboxylase (PEPC). Since C. glutamicum possesses both enzymes, it has enormous flexibility for replenishing TCA cycle intermediates upon their withdrawal. Glu can be present in the cell at a concentration as high as 150 mm (other a.a. are usually <10 mm). This high concentration ensures the supply of Glu for cell synthesis and for the supply of amino groups for various cellular reactions. As much as 70% of the amino groups in cell materials stems from L-Glu. Production process Key factors: Ammonium concentration: Large amount is necessary for sugar conversion to L- Glu, but high concentration is inhibitory to growth O2: poor oxygenation in excess of O2 4

5 The production fermenter can be as large as 500 m 3, and precultivation is needed. L-Glu is synthesized intracellularly and needs treatment to induce excretion. Thus the onset of L- Glu excretion is controlled by surfactants, e.g. Tween 40. Yields of 60-70% L-Glu, based on glucose used, have been reported. At the end, the fermentation broth contains the ammonium salt. The broth is subjected to. From the eluates, MSG may be crystallized directly followed by further conditioning steps (e.g. decolorization and sieving) to yield a food-grade quality. III. L-Lysine (CH2)4NH2 Also produced using C. glutamicum. Common C sources: molasses, sucrose or starch hydrolysates. Molasses (similar to syrup) is the by-product of processing of sugar cane, grapes or sugar beets into sugar, so molasses is cheap. But utilization of molasses produces wastes and the seasonal 5

6 availability of molasses causes ageing effect in its quality during storage. Therefore, hydrolysed starches are often used instead. Growth factors are provided by plant protein hydrolysates, liquor or by the addition of defined compounds. After initial consumption of sugar, the substrates are added continuously and L-Lysine accumulates up to 170 g/l. Ammonium sulfate is added to neutralize the accumulating basic lysine, so lysine is present in the broth as its sulfate form. Downstream processing (3 different processes in use): Biomass separation Biomass separation Biomass separation IV. L-Aspartate Demand increases due to the introduction of aspartame as an artificial sweetener. Aspartame: Dipeptide consisting of L-aspartate and L-phenylalanine. 200-fold sweeter than sugar and is low-calorie. Now produced exclusively using The enzyme is immobilized in Carrageenan (a cross-linked polymer of glutaraldehyde and hexamethylene diamine, Table 14.3), which can be reused and catalyze the production of By immobilization, the enzyme half-life is increased from. Production process: 6

7 Heat treatment of E. coli which contains aspartase. The heat treatment inactivates the fumarase (which partially converts fumarate to malate as in TCA cycle). Immobilize the cells to multistage packed-bed columns for 2 reasons: The continuous process enables full automation and control to achieve optimal output with the highest product quality. 7