22.1 INTRODUCTION
Amino acids are building blocks of proteins. In mammals, and especially in man, a number of amino acids cannot be formed by generally known biosynthetic mechanism. This is basically because man cannot synthesize the α-keto acids needed for the synthesis of corresponding amino acids. Such amino acids are called essential amino acids (it is more correct to call them indispensable amino acids), and they must be supplied externally, for instance, through diet.
22.2 PRODUCTION ASPECT
Amino acid can be synthesized quite economically by chemical means. Chemically synthesized amino acids are usually racemic mixtures of D- and isomers. It is to be noted, only the L-isomer is of value for flavor application or for food/feed supplement. The D-L-isomers are biologically inactive. To date, chemical resolution of DL-racemic mixtures has been relatively expensive, although a Japanese process is apparently in commercial use.
Microbiological production of amino acids on the other hand does not involve operational difficulties of high temperature and pressure often encountered in chemical catalytic processes.
The microbial process can be carried out under ambient conditions. Besides, the end products produced by them can be obtained in a pure form because the enzyme systems in an organism are known for high selectivity.
22.3 PRODUCTION OF L-GLUTAMIC ACID
According to an estimate of 1995, the annual production of glutamic acid was 370,000 MT. The main producers of glutamic acid are Japanese companies: Ajinomoto Co., and Kyowa Hakko Kogyo Co.
Glutamic acid is a negatively charged dicarboxylic acid having the structure:
HOOCCH2CH2CHCOOH NH2
α
It is not an indispensable amino acid.
22.3.1 USES
Monosodium salt of glutamic acid (MSG) is used as flavor enhancer. It enhances the flavor of meat and meat products. Glutamic acid is also the starting material for a variety of specialty chemicals. N-acyl glutamate is used in cosmetics, soaps and shampoos. Oxypyrrolidone carboxylic acid is used as a natural moisturizer. Amides of glutamates can be used as gelatinizing agents: it can gelatinize mineral oil spilled in the ocean. In particular, this property can be gainfully utilized for marine antipollution purposes.
22.3.2 MICROORGANISMS
Most glutamic acid producing bacteria are Gram-positive, non-spore forming, non-motile, and biotin dependent. Examples of some of the more important glutamic acid bacteria are given in
229 table 22.1. Overproduction of glutamic acid is possible through the use of organisms dependent on biotin, oleic acid, or glycerol (they are auxotrophic mutants).
Table 22. 1: Examples of commercially employed glutamic acid bacteria
Genus Representative organism
Brevibacterium B. divericatum, B. flavum Corynebacterium C. glutamicum, C. lilium Microbacterium M. flavum var glutamicum Arthrobacter A. globiformis
22.3.3 BIOSYNTHESIS OF GLUTAMIC ACID
Glutamic acid is synthesized through (i) glyoxylate cycle as an oxaloacetate generating system (without CO2 fixation), and (ii) through Phosphoenol pyruvate (PEP) to form oxaloacetate with CO2 fixation. The bacteria use both EMP and HMP pathways. Compounds from these pathways are fed into TCA cycle. In all, the bacteria use about 16 enzymatic steps. The final product is formed by reductive amination of α-ketoglutarate. See figure 22.1.
Two enzymes play very important role in the biosynthesis of glutamic acid. They are (i) PEP carboxylase, and (ii) α-ketoglutarate dehydrogenase. The efficiency of CO2- fixation depends on PEP carboxylase activity. ketoglutarate dehydrogenase can transform α-ketoglutarate to glutamic acid as well as CO2 + water via succinyl-ScoA. Bacterial α-ketoglutarate dehydrogenase is such that it carries out the preferential synthesis of glutamic acid at a rate several times faster than that for the oxidation. See figure 22.1 for the outline of biosynthesis of glutamic acid.
22.3.4 GLUTAMIC ACID EXCRETION AND CELL WALL PERMEABILITY
The overproduction of glutamic acid is in fact a function of cell wall permeability of the bacterium. Under normal condition, the cell wall is impervious enough to block the flow of glutamic acid that has been synthesized inside the cell. Accumulation of glutamic acid inside the cell soon exerts product inhibition and the organism, in response to this, stops the synthetic reaction. This is an undesirable aspect when it comes to overproduction of glutamic acid. The elucidation of biochemistry of glutamic acid biosynthesis has made it possible to overcome this effect. The basic strategy used in this case is to weaken the cell wall of the bacterium. This can be achieved by adding, during the growth phase, agents capable of inhibiting cell wall synthesis, e.g., penicillin, cephalosporin, detergents, etc. An equivalent effect can be achieved by limiting biotin content in the medium or by supplying saturated C16 and C18 fatty acids. The explanation for the last sentence runs as follows: biotin is a cofactor of acetyl-ScoA carboxylase, an enzyme responsible for the conversion of acetyl-ScoA to malonyl-ScoA, the starting compound for the synthesis of fatty acid, viz., oleic acid. Good supply of biotin in the medium leads to normal production of oleic acid. Oleic acid in turn combines with inositol, mannose, etc., to form phospholipids, which is the component of cell membrane. Limiting biotin in the medium leads to synthesis of weak cell membrane thus producing leaky cells. Leaky cells cannot withhold the glutamic acid synthesized inside the cell. The addition of saturated fatty acids also has similar function. They repress the synthesis of oleic acid.
The elucidation of biochemistry of glutamic acid biosynthesis has been a turning point. It has made possible to use molasses for glutamic acid production. The initial failure was, of course, due to high biotin content in the medium. See figure 22.2 also.
230
Glucose
Glucose-6-P
Triose-P Pentose-P
Acetyl-SCoA
Citrate
Isocitrate Glyoxylate
Malate Oxaloacetate
Succinate Fumarate
α-ketoglutarate NH3, NADH+H+
CO2 CO2
CO2
CO2 CO2 CO2
CO2
L-glutamic acid H2O, NAD+
Figure 22. 1: Synthesis of glutamic acid
Cell wall (inhibition) Cell membrane Penicillin
Cephalosporin Glucose Glutamate
Acetyl-ScoA Oleic acid Glycerol
Mannose Inositol Excretion
Biotin (promotion)
C16 and C18 (repression)
Phospholipids
Figure 22. 2: Relation between cell wall and excretion in glutamic acid bacteria
22.3.5 PRODUCTION METHODS
Glutamic acid can be produced by four methods:
1. By the hydrolysis of wheat gluten, soybean cake, or other proteinaceous materials 2. By cleavage of pyrrolidone carboxylic acid (5-oxoproline).
3. By one-stage fermentation (involving one organism)
4. By two-stage fermentation process (one organism produces α-ketoglutarate and another organism produces glutamic acid from α-ketoglutarate)
22.3.5.1 ONE STAGE FERMENTATION PROCESS
The most widely used carbon and energy source is the carbohydrate, such as molasses and starch hydrolysates. Certain strains can also utilize non-carbohydrate materials such as acetic acid. When molasses is used, most of the growth requirements are met. The nitrogen requirement is met normally
N COOH O H
MICROBIAL PRODUCTION OF AMINO ACIDS by supplying gaseous ammonia. Gaseous ammonia fulfils several objectives: (i) supplies nitrogen, (ii) maintains pH by neutralizing the accumulated acid, and (ii) avoids unwanted dilution of the medium.
The fermentation is a highly aerobic one. Air pressure in the fermentation vessel is critically maintained slightly above the actual requirement for cellular respiration. Inadequate air supply leads to accumulation of lactic and succinic acid while excess air supply promotes α-ketoglutarate accumulation.
Fermentation is carried out in a continuous or batch mode. The medium is sterilized in either continuous or batch mode. The sugar concentration is maintained at 10% glucose equivalent. pH is maintained near neutrality with ammonia gas. The biotin content in the medium should be suboptimal: it has been worked out that biotin should be 1-5 µg/L of medium. Penicillin and similar other agents are added during the growth phase. The temperature for the fermentation is 30-35°C. The duration of fermentation is around 3 days. Glutamic acid excretion starts after the intracellular concentration of glutamic acid has reached 50 mg/g of dry cell.
22.3.5.1.1 YIELD
After the growth phase, an ideal fermentation should proceed as:
C6H12O6 + NH3 + 1.5 O2 → C5H9O4N + 3 H2O
This represents a 100% molar conversion, or 81.7% weight conversion of glucose anhydrous to glutamic acid. Under practical condition, the molar conversion is 50-75% by resting cells.
22.3.5.2 COMMERCIAL FED-BATCH FERMENTATION PROCESS
At the beginning of the fermentation, 0.65ml oleic acid/liter of medium is added. The pH is set at 8.5 with ammonia and automatically maintained at 7.8 during the course of fermentation.
After about 14hrs, the temperature is increased from an initial 32-33°C to 38°C due to growth.
After the metabolism of glucose down to the level of 0.5-2% (from an initial of about 12%
glucose equivalent in the molasses medium) glucose feeding is done until the fermentation is complete. On an average, 160g/liter of broth is added. Glutamic acid content is analyzed hourly.
Aeration is controlled in such a way that CO2 of the exhaust gas does not exceed 4.5% by volume. Fermentation is stopped after 30-35 hrs after the glutamic acid production reaches about 100g/liter.
22.4 MICROBIAL PRODUCTION OF L-TRYPTOPHAN
L-tryptophan is an aromatic, indispensable amino acid. Its chemical name is α-amino-β-indole propionic acid. The condensed structure is:
N H NH2
CH2CHCOOH
This amino acid is produced by Japanese companies, viz., Ajinomoto Co. and Tanabe Seiyaku.
The annual world production in 1995 was 400MT.
22.4.1 USES
Therapeutically, it is used as a component solution for transfusion. Since it is an indispensable amino acid, it can also be used for the fortification of food commodities like corn (maize) that contain limiting amounts of tryptophan.
INDUSTRIAL MICROBIOLOGY 22.4.2 MICROORGANISMS USED
Microorganisms used for the production of tryptophan are highly improved strains of bacteria.
Some of the industrially exploited or tested microorganisms are: Corynebacterium glutamicum, Brevibacterium flavum, Bacillus subtilis, Candida fumicola, Achromobacter liquidium, Pseudomonas putida, etc.
22.4.3 BIOSYNTHETIC PATHWAY
The microorganism uses chorismic acid pathway for the synthesis of tryptophan. Since the synthesis of phenylalanine and tyrosine also share the same pathway, it is obvious that tryptophan-producing strains are auxotrophic mutants. The outline of the biosynthetic pathway followed by Corynebacterium glutamicum strain is shown in figure 22.3.
Erythrose-4-phosphate
Phosphoenol pyruvate
3-Deoxy-2-Keto-D-Arabino-Heptulosonic acid
-7-phosphate (DAHP)
Shikimic acid
Prephenic acid
Phenylalanine Tyrosine
L-tryptophan Anthranilic acid
NH2
COOH COOH
OH HO OH
Chorismic acid
Repression Metabolic block
due to mutation Feedback
inhibition
OCCOOH COOH
OH O
Figure 22. 3: Biosynthetic pathway of tryptophan in C. glutamicum
22.4.4 PRODUCTION METHODS
There are three main methods for the production of tryptophan:
1. Production by fermentation 2. Production by microbial conversion 3. Production by enzymatic method
Detailed information on any of the above methods is not available. Literatures are therefore based only on classical researches and patents filed for the method.
22.4.4.1 PRODUCTION BY FERMENTATION
In the overproduction of tryptophan by fermentation, the basic strategy is to obtain auxotrophs and/or analog resistant strains by mutation. Mutants carrying multiple markers are more suitable as they are more stable. The classical work carried out by Nakayama (1976) is used here as an example.
He used Corynebacterium glutamicum KY 9456, a double auxotroph of phenylalanine and tyrosine for further mutation. A stepwise mutation finally produced a strain called Px-115-97 that produced significantly higher amounts of tryptophan. The parent strain was mutated in a stepwise manner to develop resistance to 5-methyl tryptophan (5MTr), tryptophan hydroxamate (TrpHxr), 6-fluoro tryptophan (6FTr), 4-methyl tryptophan (4MTr), parafluoro phenylalanine (PFPr), paraamino phenylalanine (PAPr), tyrosine hydroxamate (TyrHxr), and phenylalanine hydroxamate (PheHxr).
233 The auxotrophy produced metabolic block while the analog resistance released the bacterium from repression by tryptophan. The yield gradually increased from a mere 0.15 g/L to final of 12 g/L in a nutritionally balanced cane molasses medium of following composition: Cane molasses (10%
glucose equivalent), MgSO4.7H2O (0.025%), KH2PO4 (0.05%), K2HPO4 (0.05%), (NH4)2SO4 (2%), CaCO3 (2%), and Cornsteep liquor (2%). The pH was kept at 7.2. The organism was still sensitive to phenylalanine and tyrosine, which implied that there was further scope for the development by building multiple analog resistances. The genealogy of the bacterium used in the study appears in table 22.2
Table 22. 2: Genealogy of C. glutamicum mutated for tryptophan production
K Y 9456 Phe-, Tyr
-
4M T-11
PFP-2-32
PAP-126-50 Tx-49
Px-115-97
5M Tr, TrpH xr, 6FTr, 4M Tr PFPr
PAPr TyrH xr Ph eH xr G enealogy/m utation
0.15
4.9
5.7
7.1 10
12 Production (g/L)
22.4.4.2 PRODUCTION BY MICROBIAL CONVERSION
Various microorganisms including auxotrophic- and regulatory mutants were selected by different workers. Candida fumicola, Corynebacterium glutamicum, Bacillus subtilis, and E. coli were studied. In this method, precursor of tryptophan such as anthranilic acid or indole is used in the medium. These precursors are chemically synthesized.
22.4.4.3 PRODUCTION BY ENZYMATIC METHOD
This method utilizes the enzyme tryptophanase, which catalyzes reversible synthesis of tryptophan.
The enzyme is produced by bacteria such as Achromobacter liquidium, Pseudomonas putida, etc.
Depending on the organism used, the major substrate can be indole or indole derivatives.
Interesting as the above two methods appear, they have not gained commercial importance because the costs of precursors such as indole and anthranilate are prohibitively high.
22.4.5 RECOVERY
The culture broth is subjected to strongly acidic cation exchange resin. The adsorbed tryptophan is then eluted from the resin with 0.5N aqueous ammonia, and crystallized to obtain crude crystals. The latter is dissolved in a small amount of hot, 50% aqueous ethanol, decolorized with activated carbon, and recrystallized to obtain pure tryptophan crystals.
22.5 MICROBIAL PRODUCTION OF L-LYSINE
L-lysine or α, ε-diaminocaproic acid is indispensable to man. It is a limiting amino acid in cereal grains. Its condensed formula is:
234 CH2CH2CH2CH2CHCOOH
NH2 NH2
Commercially, lysine is available as lysine monohydrochloride. The amino acid can be produced by chemical as well as microbial method. The annual world production in 1995 was 70,000 MT. Microbial methods of production can be classified as:
1. Production by homoserine auxotrophs 2. Production by multiply improved strains 3. Enzymatic method
22.5.1 BIOSYNTHESIS OF L-LYSINE
Lysine biosynthesis can occur by two different pathways, viz.,
Diaminopimelate pathway, and (ii) Aminoadipate pathway. The former pathway is found in bacteria, certain lower fungi, algae, and higher plants while the latter is found in classes of lower fungi, higher fungi, and Euglena (flagellated protozoa).
In the diaminopimelate pathway, the carbon chain is synthesized from pyruvate and aspartate (and thus categorized as member of aspartate family). Other members that share diaminopimelate pathway are threonine, isoleucine, and methionine. The important steps of diaminopimelate pathway are given in figure 22.4.
Aspartic β-semialdehyde
Isofunctional enzymes
Aspartate Aspartyl phosphate Homoserine Threonine
Isoleucine Methionine
Dihydrodipicolinate
Diaminopimelate
L-lysine Pyruvate
Feedback inhibition
Feedback inhibition
Repression Metabolic block in mutants
Feedback inhibition
Figure 22. 4: Diaminopimelate pathway for lysine synthesis
22.5.2 PRODUCTION BY HOMOSERINE AUXOTROPHS
The microorganisms used here are auxotrophic mutants of C. glutamicum. Double auxotrophs, which require in addition to homoserine at least one of amino acids, viz., thre, met or ile for growth have been found to be highly stabilized. It may be stated that the overproduction is due to the release of aspartokinase from concerted feedback inhibition by branch end products due to the metabolic block.
22.5.2.1 CULTURAL CONDITIONS
The carbon sources for the production can be molasses, starch hydrolysates, and in some cases, acetic acid and ethanol. Cane molasses is the most important, though. The fermentation occurs at neutrality. NH3 can be added to control the pH and meet nitrogen requirement. Biotin is very
MICROBIAL PRODUCTION OF AMINO ACIDS important for growth and production: it must be greater than 30 µg/L. The requirement of biotin is variable and so is the explanation behind it. In biotin-dependent strains, the excretion results from the leaky cell wall. On the other hand, biotin is a coenzyme needed for the decarboxylation-conversion diaminopimelate to lysine.
The seed culture is prepared in stages. The final seed culture necessarily contains cane molasses for acclimatizing the organism to future environment. The production medium contains 20% glucose (from cane molasses) and 1.8% soybean meal hydrolysate. The amounts of growth factors (homoserine or threonine and methionine) are added in suboptimal levels. Fermentation is carried out at about 28°C. Aeration is a crucial aspect of lysine fermentation. It is kept at greater than the actual requirement for respiratory growth. Oxygen deficiency may lead to lactic acid production at the cost of lysine, although not as significant as in the case of glutamate production. The duration of fermentation is about 3 days. The yield is about 30-40% based on sugar consumed. The composition of seed culture medium is given in table 22.3. Since the most important intermediate is aspartic acid, its inclusion in the medium increases the yield of lysine.
Table 22. 3: Composition of the seed culture medium for lysine production Seed culture 1 Seed culture 2 Main culture
Glucose 2% Cane molasses 5% Molasses (20% glucose equivalent) Peptone 1% (NH4)2SO4 2%
Meat extract 0.5% Cornsteep liquor 5%
NaCl 0.25% CaCO3 1%
Soybean meal hydrolysate
1.8%
22.5.3 PRODUCTION BY ENZYMATIC METHOD
This method is used by Toray Company, Japan. The method in principle utilizes two enzymes, viz., racemase and hydrolase to transform DL-α-aminocaprolactam (DL-ACL) to lysine. In the industrial process, DL-ACL is produced synthetically using chemicals such as NOCl, cyclohexane, NH3, HCl, etc., in a series of reaction steps. Racemization and hydrolysis are the final reactions for producing L-lysine. The reactions may be outlined as:
D L-ACL Racemization L-am inocaprolactam L-lysine H ydro lysis
Aminocaprolactam racemase is produced by Achromobacter obae using DL-ACL as an inducer.
Cryptococcus laurentii, another organism, produces hydrolase inductively in a medium containing L-ACL, glucose and other components. A similar optimum pH values for both the enzymes allows efficient conversion, which appears to be a single step.
Industrially, resting cells of the above two organisms are used for the production of enzymes. There is no detailed information about the use of enzyme but literatures on its study are available. It has been reported that incubation of 100 ml of 10% DL-ACL (pH adjusted to 8 with HCl) with 0.1 g of acetone-dried cells of C. laurentii and A. obae nov. sp. at 40°C for 24 hours resulted in 99.8%
conversion of DL-ACL to L-lysine.
The amino acid produced by enzymatic means is relatively free from debris. It is therefore much easier to purify the amino acid. A very high-grade lysine can be obtained by carbon treatment and crystallization. See figure 22.5
INDUSTRIAL MICROBIOLOGY
NOCl Cyclohexane
NH3
α-amino cyclohexane oxime DL--amino caprolactam Beckmann rearrangement
Hydrolase Racemase
L-lysine NH2
NOH
α NH2
O α HN
Figure 22. 5: Outline of enzymatic synthesis of L-lysine
CHAPTER 23