Okafor N. Modern Industrial Microbiology and Biotechnology

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!& Modern Industrial Microbiology and Biotechnology

Amino acids have the general formula R. CH—COOH and are the main components of

of which proteins are made. The amino acids found in proteins number 20. Of these eight

are essential for animals and must be supplied in their food, since animals cannot

synthesize them.

Each of the 20 amino acids found in proteins can be distinguished by the R-group

substitution on the carbon atom and can be divided into the following groups on that

basis: amino acids with aliphatic R groups, non-aromatic amino acids with hydroxyl R

groups, amino acids with sulfur containing R groups, amino acids with acidic R groups,

amino acids with basic R groups, amino acids with aromatic R groups and amino acids

with imino acids as the R groups. The nature of the R group influences the activity of the

amino acid. Thus, the hydrophilic amino acids which have –OH groups in their R

substituent (e.g. serine) tend to interact with the aqueous environment, are often involved

in the formation of H-bonds and are predominantly found on the exterior surfaces

proteins or in the reactive centers of enzymes. On the other hand, the hydrophobic amino

acids (without –OH groups in the R substituent, for example methionine) tend to repel the

aqueous environment and, therefore, reside predominantly in the interior of proteins.

This class of amino acids does not ionize nor participate in the formation of H-bonds

(Table 21.1).

All the amino acids, except glycine have two optically active isomers, the D – or the L-

form. Natural proteins are usually made up of L- (or the so-called natural amino acids.)

Outside the 20 amino acids found in protein, many other rare amino acids have been

reported in various metabolites such as some antibiotics, other microbiological products

and in non-proteinaceous materials in plants and animals.

21.1 USES OF AMINO ACIDS

Amino acids find use in a large number of activities, including human and animal

nutrition, medicine, cosmetics, and in the synthesis of chemicals.

Production of Amino Acids

by Fermentation

+0)26-4

Production of Amino Acids by Fermentation !&

Table 21.1 Amino acids found in proteins

Amino Acid Symbol Structure

Amino Acids with Aliphatic R-Groups

Glycine Gly - G

H–CH–COOH

Alanine Ala - A

CH –CH –COOH

* Valine Val - V

CH–CH–COOH

* Leucine Leu - L

CH–CH –CH–COOH

* Isoleucine Ile - I

CH–CH–COOH

Non-Aromatic Amino Acids with Hydroxyl R-Groups

Serine Ser - S

HO–CH –CH–COOH

* Threonine Thr - T

CH–CH–COOH

Amino Acids with Sulfur-Containing R-Groups

Cysteine Cys - C

HS–CH –CH–COOH

* Methionine Met-M

HC–S–(CH ) –CH–COOH

322

Acidic Amino Acids and their Amides

Aspartic Acid Asp - D

HOOC–CH –CH–COOH

Asparagine Asn - N

HN–C–CH –CH–COOH

Glutamic Acid Glu - E

HOOC–CH –CH –CH–COOH

Glutamine Gln - Q

HN–C–CH –CH –CH–COOH

222

Basic Amino Acids

Arginine Arg - R

HN–CH –CH –CH –CH–COOH

222

C=NH

* Lysine Lys - K

HN–(CH –CH–COOH

224

)

Histidine His - H

HN N:

—CH –CH–COOH

Contd

!& Modern Industrial Microbiology and Biotechnology

Amino Acids with Aromatic Rings

* Phenylalanine Phe - F

CH –CH–COOH

Tyrosine Tyr - Y

CH –CH–COOH

* Tryptophan Trp-W

CH –CH–COOH

Imino Acids

Proline Pro - P

COOH

*Essential

Table 21.1 Contd.

Amino Acid Symbol Structure

(i) Use in human and animal nutritional supplementation: Proteins are metabolized

constantly in the body and most of the amino acids absorbed are used to replace body

proteins. The remaining are metabolized into various body components including

hormones, and nucleic acid bases. Of the amino acids in protein eight are essential and

the diet must contain them. Foods such as plant proteins lacking in some essential amino

acids are fortified with their addition. Most cereals are particularly low in lysine, the

addition of which greatly improves the quality of the food as determined by the PER

(protein equivalency ration). The PER is a means of comparing the amino acid content of

protein with that of hen’s egg or human milk. It is usually best determined by feeding tests

to rats and mice.

Animal feeds made from inexpensive plant proteins can be greatly improved with

only a small quantity of the limiting amino acids, resulting in higher growth rates in the

animals. L – lysine and DL methionine are widely used as feed improvers.

(ii) Flavor and taste enhancement in foods: Amino acids are important in deciding the

taste of meats and such foods. Mono-sodium glutamate well-known as a flavoring agent,

will be discussed later.

Amino acids influence the taste of foods. Some are very sweet; for example glycine is as

sweet as sugar and is sometimes used in soft drinks and soups. The amino acid is present

in large amounts in shrimps to which it confers its sweet taste. The peptide, L- aspartyl –

L – phenylalanine methyl ester is particularly sweet. Well-known sweetners such as

Aspartame, Sweet and Low, and Splenda contain a dipetide formed from aspartic acid

and phenylalanine Other amino acids e.g. valine are bitter. It is interesting that while the

L- isomers of leucine, phenylalanine, tyrosine, and tryptophane are bitter, the D-isomers

are sweet. The combination of various amino acids influence the taste and flavor of foods.

Thus cheese flavor derives from the combined effect of glutamic acid as well as that of

bitter amino acids such as valine, leucine and methionine.

Production of Amino Acids by Fermentation !&!

(iii) Medical uses: The greatest application of amino acids in medicine is in transfusion;

which is administered when the oral consumption of proteinaceous food is not possible

such as after an operation. In the past only essential amino acids were used in

transfusion; nowadays non-essential ones are added.

Various amino acids are used for ammonia detoxification in blood in liver diseases, in

the treatment of heart failure, in cases of peptic ulcer and male sterility, etc. Table 21.2

summarizes some of these uses.

In addition derivatives of amino acids are widely used in medicine as discussed

below. Methyldopa (L-methy1-3, 4 dihydroxy-phenylalanin) is widely used as an anti-

hypertensive with relatively few side effects. Dopa is used in treating Parkinson’s disease

(Fig. 21.1):

OH CH

CCOOH

OH CH

CHCOOH

Methyl dopa Dopa

Fig. 21.1 Methyl Dopa and Dopa

A derivative of serine, cycloserine is an antibiotic produced by a streptomycete; it is

used for the treatment of tuberculosis.

(iv) Use as an industrial synthetic raw materials: Although numerous studies have been

concluded on the use of amino acids as raw materials in the chemical industry, very few

of these have been put into actual practice. Thus, the manufacture of artificial silk was

considered and dropped. However amino acids are used in the following:

(a) Surface-active agents: A surface-active agent has a water solube (or hydrophilic end)

as well as a water-repellent) or hydrophobic end. The hydrophilic end is dissolved

in the water and as a consequence the surface tension of the water is lowered.

Surface action agents can be prepared from amino acids by introducing long-chain

lipophilic groups to one of the two hydrophilic groups (- COOH, or – NH

) of

amino acids. The resulting surface-active agents is either cationic (if it has a

positive charge) or anionic (negative charge). As they lower the surface tension like

soap, they foam just like soap. Some are even more effective than soap as cleansing

agents. Many of them also have strong bacteriostatic action. Thus sodium lauryl

sarcosinate is used in toothpaste and shampoo because it has a bacteriocidal as

well as foaming action. These derivatives are also used as fungicides and

pesticides.

(b) Production of polymers from amino acids: Polymers derived from amino acids are

used in making synthetic leather, fire-resistant fabrics and anti-static materials.

skin function by regulating pH and a protective action against bacteria. Detergents

(surface action agents) derived from amino acids are less irritating than soaps

!&" Modern Industrial Microbiology and Biotechnology

because the pH of 5.5-6.0 is closer to that of the skin, whereas soap is slightly

alkaline. The addition of different amino acids to shampoo is practiced to achieve

different ends: anti-dandruff shampoos contain cysteine; thioglycolic acid is

employed as a reducing agent for the cold waving of hair.

21.2 METHODS FOR THE MANUFACTURE OF

AMINO ACIDS

The beginning of the development of the amino acid industry can be put at 1908 when

Kikunae Ikeda identified and isolated monosodium glutamate (MSG), the sodium salt of

glutamic acid, as the flavoring agent in ‘kombu’, a traditional seasoning agent used in

Japan, and derived from some marine algae. The Ajinomoto company the following year

started producing MSG by extraction from the acid hydrolysate of wheat gluten or

defatted soy. Glutamic acid, lysine and methionine are the most produced amino acids

globally (Fig. 21.2). Today amino acids are produced by a number of methods.

Table 21.2 Therapeutic uses of some amino acids

Amino Acid Use

Ornithine Used for treating cases of hyperammonemia (excessive

ammonia in blood) because it increases urease activity in the

liver, thus enhancing urease production.

Arginine More common than ornithine.

1. Use for ammonia detoxication described above.

2. Arginine is the main component of spermatic proteins and

hence administered in cases of male sterility due to low

number or weak spermatozoa (Sterilitas virilis).

Aspartic acid 1. Used for ammonia detoxification combined with ornithine.

2. Used as carrier for K

and Mg

in form of potassium or

magnesium aspartate in cases of heart failure, fatigue, etc.

Cysteine and cystine 1. Protect SH-enzymes in the liver from enzyme inhibitors;

used for dealing with poisons generally including cyanide

poisoning.

2. L- cysteine is used in bronchitis and nasal catarrh.

Gluatamic acid Important in brain metabolism hence various analogues of

glutamic acid are used in treating various neuropathic diseases.

Glycine Rarely used as a drug, but only as a sweetener in medicines.

Histidine An amino acid essential for infants but not for adults, it is used in

adults for gastric and duodenal ulcers and is administered in cases

of anaemia because it helps in haemoglobin regeneration.

Methionine A sulfur-containing amino acid, it is important in the metabolism

of various sulfur-containing compounds in the body. It is also

used for detoxification in poisoning by arsenic, chloroform, and

benzene derivaties. A derivative of methionine, Vitamin U, is

used as an anti-ulcer drug, because it neutralizes histamine which

is known to induce ulcer formation.

Tryptophan Used as an anti-depressant.

Production of Amino Acids by Fermentation !&#

(i) Protein hydrolysis: Protein hydrolysis was the original method of amino manufacture.

Hair, keratin, blood meal and feathers are hydrolyzed using acid and the amino acid

extracted. It is not very popular because it depends on the availability of hair, feathers

and the other raw materials. However, cysteine and cystine are still produced by

isolating them from chemically hydrolyzed keratin protein in hair and feathers while

proline and hydroxyproline are precipitated from gelatin hydrolysates.

(ii) Chemical synthesis: Glycine, L-alanine, and DL- methionine are produced by

chemical synthesis. Chemical synthesis can only produce the D,L- (recemic) forms of

amino acids and an additional step involving the use of an immobilized enzyme,

aminoacylase, produced by Aspergillus niger is necessary to obtain the biologically active

L-form. (Fig. 21. 3). This step is expensive and on account of this, few amino acids are

prepared by chemical synthesis. Amino acids produced by chemical synthesis are

glycine and methionine; methionine is said to have the same effect as an animal feed

additive whether in the L- or in the D, L- form.

(iii) Microbiological methods: Microbiological methods are of three types:

1. Semi-fermentation;

2. Use of microbial enzymes or immobilized cells;

3. Direct fermentation.

Fig. 21. 2 World Production of Amino Acids, 1996 (from Mueller and Huebner, 2003)

Fig. 21.3 Action of Aminoacylase on Racemic Mixtures of Amino Acids

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21.2.1 Semi-fermentation

In this process, the metabolic intermediate in the amino acid biosynthesis or its precursor

is added to the medium, which contains carbon and nitrogen sources, and other

nutrients required for growth and production; the metabolite is converted to the amino

acid during fermentation. Sometimes the intermediate could be another amino acid.

Examples of the commercial production of amino acids by semi-fermentation are L-serine

production from glycine and methanol using the methane-utilizing bacterium

Hyphomicrobium sp. or Pseudomonas sp. Other examples are the production of L-

tryptophan from anthranillic acid or indole using E. coli and B. subtilis L-isoleucine

production from DL-=-aminobutyric acid and ethanol by Brevibacterium sp. has been

done commercially by this process (Table 21. 3).

21.2.2 Enzymatic Process

Chemically synthesized substrates can be converted to the corresponding amino acids by

the catalytic action of an enzyme or microbial cells as an enzyme source. Often the

enzymes or the cells may be immobilized. L-alanine production from L-aspartic acid, L-

aspartic acid production from fumaric acid, L-cysteine production from DL-2-

aminothiazoline-4-carboxylic acid (Fig. 21. 5). Others are D-phenylglycine (and D-p-

hydroxyphenylglycine) production from DL-phenylhydantoin (and DL-p-

hydroxyphenylhydantoin), and L-tryptophan production from indole and DL-serine

have been in operation as commercial processes.

Fig. 21.4 Examples of Enzyme Conversions for Producing Amino Acids

Production of Amino Acids by Fermentation !&%

Table 21.3 Amino acid production by semi-fermentation process (from Araki, 2003)

Amino acid produced Precursor added to the medium Amount, mg/mL

D-alanine DL-alanine 48.8

L-isoleucine D-threonine 15

DL-=-aminobutyric acid 15.7

DL-=-hydroxybutyric acid

DL-=-bromobutyric acid

L-methionine L-hydroxy-4-methylthiobutyric acid 10.9

DL-5-(2-methylthioethyl)hydantoin 34

D-threonine

L-phenylalanine acetoamidocinnamic acid 75.9

L-proline L-glutamic acid 108.3

L-serine glycine 16

54.5

L-threonine L-homoserine 16

L-tryptophan anthranilic acid 40

indole 16.7

Fig. 21.5 Metabolic Pathways Involved in the Biosynthesis of Amino Acids from Glucose

!&& Modern Industrial Microbiology and Biotechnology

21.2.3 Production of Amino Acids by the Direct Fermentation

Although other microbiological methods for the commercial production of amino acids

exist, such as the biosynthesis of amino acids using intermediates, and the use of

enzymes, by far the most important method for producing amino acids microbiologically

is by direct fermentation. What method is used in any particular situation depends on

factors such as process economics, the available raw materials, market size, the

environmental regulation operating in the place of production, etc. Nevertheless, the

fermentation method appears to be the dominant one and will be discussed.

The production of amino acids by fermentation was stimulated by the discovery of an

efficient L- glutamic acid producer Corynebacterium glutamincum. Many microorganisms

have been reported to produce amino acids. They are mainly bacteria, but they also

include some molds and yeasts. The four most widely reported bacteria belong to the

following four genera, the typical species of which are given in parenthesis.

Corynebacterium spp. (C. glutamicum; C. lilum)

Brevibacterium spp. (B. divericartum: B. alanicum)

Microbacterium spp. (M. flavum var. glutamicum)

Arthrobacter spp. (A. globiformis; A. aminofaciens)

Auxotrophic and regulatory mutants of glutamic acid producing bacteria are used for

the commercial production of all amino acids outside L- glutamic acid and L-glutamine,

which are produced by the wild type of these organisms (Table 21.4).

Table 21.4 Amino acids produced from wild type and mutant strains of bacteria

Wild-type Auxotrophic Mutants Regulatory Mutants

L- glutamic acid 1 L- citruline 1 L- arginine 1,2,3

L-valine, 1 L- leucine 1 L- histidine 1,3,5

L- lysine 1 L- isoleucine 1,3,5

L- ornithine 1 L- leucine 5,6

L- proline 3 L- lysine 3

L- threonine 4 L- methionine 1

L- tyrosine 1 L- phenylalanine 1,3

L- thereonine 1,3

L- tryptophane 1,3

L- tyrosine 1,3

L- valine 6

1= Corynebacterium glutamicum 2= Bacillus subtilis

3= Brevibacterium flavum 4= Escherichia coli

5= Serratia marcescens 6= Brevibacterium lactofermentum

21.3 PRODUCTION OF GLUTAMIC ACID BY

WILD TYPE BACTERIA

(i) Organisms: Wild type strains of the organisms of the four genera mentioned above are

now used for the production of glutamic acid. The preferred organism is however

Corynebacterium glutamicum. The properties common to the glutamic acid bacteria are: (a)

Production of Amino Acids by Fermentation !&'

they are all Gram-positive and non-motile; (b) they require biotin to grow; (c) they lack or

have very low amounts of the enzyme =-ketoglutarate, which is formed by removal of CO

from isocitrate formed in TCA cycle (citric acid cylce). Since =-ketoglutarate is not

dehydrogenated it is available to form glutarate by reacting with ammonia (Fig. 21.1).

(ii) Conditions of the fermentation: The composition of a medium which has been used for

the production of glutamic acid is as follows (%): glucose, 10; corn steep liquor 0.25;

enzymatic casein hydrolysate 0.25; K

HPO

0.1, Mg. SO

, 7H

O, 0.25; urea, 0.5. It should

be noted that besides glucose, hydrocarbons have served as carbon sources for glutamic

acid production. The optimal temperature is 30° to 35° and a high degree of aeration is

necessary.

(iii) Biochemical basis for glutamic acid production: Studies by several workers have clarified

the basis for glutamic acid production as summarized below.

(a) Glutamic acid production is greatest when biotin is limiting; that is, when it is sub-

optimal. When biotin is optimal, growth is luxuriant and lactic acid, not glutamic

acid, is excreted. The optimal level of biotin is 0.5 mg per gm of dry cells; with

higher amounts glutamic acid production falls.

(b) The isocitrate-succinate part of the TCA cycle (Fig. 21.5) is needed for growth. It is

only after the growth phase that glutamic acid production becomes optimal.

diffusion of glutamic acid, essential for high glutamic acid productivity. This

increased permeability to the acid can be achieved in the following ways: (i)

ensuring biotin deficiency in the medium (ii) treatment with fatty acid derivatives,

(iii) ensuring oleic acid deficiency in mutants requiring oleic acid (C

- C

). (iv)

addition of penicillin during growth of glutamic acid bacteria, Cells treated in one

of the first three ways above have cell membranes in which the saturated to

unsaturated fatty acid ratio is abnormal, therefore the permeability barrier is

destroyed and glutamic acid accumulates in the medium. The major factor in

glutamic acid production by wild type organism is thus altered permeability.

Treatment with penicillin prevents cell-wall formation. Cell wall inhibiting

antibiotics such as penicillin and cephalosporin have enabled the use of molasses

which are rich in biotin for glutamic acid production.

21.4 PRODUCTION OF AMINO ACIDS BY MUTANTS

After wild type strains of C. glutamicum and of other bacteria were found to accumulate

glutamic acid, efforts to find in nature bacteria able to yield high amounts of other amino

acids failed. The reason for this is that microorganisms avoid over-production of amino

acids, producing only the quantity they require. To induce the organism to over produce,

regulatory mechanisms must be disorganized as discussed in Chapter 6. Two major

means of regulating amino acid synthesis are feedback inhibition and repression.

Auxotrotrophic mutants and regulatory mutants are two means by which the organisms’

tendency not to overproduce can be disorganized. In order to over produce an amino acid

which is an intermediate in a synthetic pathway, a mutant auxotroph is produced whose

pathway in the synthesis is blocked. When this mutant is cultivated, limiting nutrient

feedback and/or repression would have been removed and an overproduction of the