Okafor N. Modern Industrial Microbiology and Biotechnology

Подождите немного. Документ загружается.

470 Modern Industrial Microbiology and Biotechnology

A major transformation in which interest has grown sharply in recent times is the

cleavage of the C

side chain of sterols. An important source of steroids for the synthesis

and production of pharmacologically active steroids used in contraceptives,

corticosteroids, geriatic drugs etc. is diosgenin (Fig. 26.1) from Dioscorea spp. Due to the

shortage of diosgenin, interest has shifted to more abundant sterols from phytosterols (i.e.

sterols from plants) and cholesterols from animals. The phytosterols include soy bean

sterols mainly >-sitosterol and stigmasterol and tall oil sterols mainly sitosterol and

campesterol. For these to be used as starting materials for the production of progesterone

and other drugs, the C

side chain must be cleaved hence the interest. The microbial

removal of the side chain offers more promise than chemical means. Unfortunately micro-

organisms which cleave off the side-chain will also attack the D ring to which the chain

is attached. Three methods have therefore been evolved to solve the problem of inhibiting

ring degradation, while cleaving the chain.

(i) The substrate may be modified structurally by chemical means so that the ring is

stable while the side-chain is cleaved. Thus while cholesterol rings are degraded

when the side-chain is cleaved by Nocardia sp. 3-Acetoxy-9- hydroxy-5-cholestene

is not. This later compound can be prepared from cholesterol by three chemical

steps. The cleavage of the side-chain of cholesterol yields esterone which can then

be used for further transformations.

(ii) The enzymes which open the D nucleus may be selectively inhibited. The key stage

in the opening of the ring is at the ninth position and since the enzyme for this

hydroxylation contains metals, the enzyme and its process may be inhibiting by

using chelating agents which remove metals from them.

(iii) Finally, mutants have been developed which will degrade only the side chain. One

of the best known is a mutant of Mycobacterium sp.

26.3.2 Fermentation Conditions Used in Steroid Transformation

The media used are highly variable, but in the main are not very complex. They are

basically mineral salts media containing some carbon source such as glucose, dextrin or

glycerol. Nitrogen sources may be ammonium salts, corn steep liquor, soybean, or a

protein digest. In some cases yeast extract is added.

Steroid and sterols are lipids; they are not water soluble and therefore must be

dissolved in a water-miscible lipid-solvent. Acetone, ethanol, propylene glycol, and

methanol are suitable because they dissolve a reasonable amount of the steroid while

being relatively non-inhibitory to the enzymes; dimethyl formamide dissolves a

reasonable amount of the steroids but has only a minimum of toxicity. Sometimes the

steroid is added in small amounts at a time. In this way, any toxic effect of the solvent is

minimized.

The level of steroid added is variable and depends both on the transforming ability of

the organisms as well as its susceptibility to the toxic effects of the steroid. Normally 200-

800 mg/litre are added but much higher amounts are sometimes used. To solve the

problem of the insolubility of steroids in water, non-ionic surface-acting agents which

reduce surface tension e.g. Tween 80 are often added to the medium. Some poly-

saccharides in the medium e.g. yeast cell wall mannan, bind to the steroids and cause

them to be more available to the organism.

Microbial Transformation of Steroids and Sterols 471

A wide range of microorganisms, mainly fungi and bacteria, are used in the

transformation of steroids. Some of these include the fungi Rhizopus nigricans, Curvularia

lunata, Fusarium spp. Cylindrocarpon radicicola as well as the bacteria Mycobacterium spp.,

Corynedbacterium simplex, and Streptomyces spp. As has been mentioned, there are

organisms to perform just about any conceivable transformation of the steroid molecule.

The transformation may occur at different stages of the growth and the steroid may be

added to the growing cultures either simultaneously with the inoculation of the culture

or the resting or stationary stage of the organism. Fungal spores may sometimes be

inoculated as the steroid is introduced into the medium. In recent times immobilized cells

have been employed in the transformations of steroids.

Steroid transformations require vigorous aeration and a temperature of about 28°C is

usually employed. The fermentation is usually complete in four to five days.

26.4 SCREENING FOR MICROORGANISMS

The screening for microorganisms capable of transforming steroids to yield products of

useful pharmacological properties is a continuing one. The processes which are followed

in the screening are as follows:

(i) The microorganism is isolated from soil or some suitable source and grown in a

suitable medium for 24-28 hours.

(ii) The steroid in a suitable carrier is added to the fermentation and the growth

continues for a further period which could be as long as one week.

(iii) The transformation products are extracted with solvents such as methyl acetate

and purified by chromatography etc.

(iv) The product is tested for pharmacological properties.

(v) Finally, the structure is elucidated by classical methods of organic chemistry.

SUGGESTED READINGS

Flickinger, Michael C., Drew and Stephen W. 1999. Encyclopedia of Bioprocess Technology -

Fermentation, Biocatalysis, and Bioseparation Wiley. Electronic ISBN: 1-59124-457-9.

Martin, C.K.A. 1984. Sterols. In: Biotechnology. Kiesich (ed) Vol 6A Biotransformations Verlag

Chemie. Weinheim: Germany. pp. 79–96.

Morgan, B.P., Moynihan, M.S. 1997. Steroids. Kirk-Othmer Encyclopedia of Chemical

Technology, 2, 71-113.

Smith, L.L. 1984. Steroids. In: Biotechnology. Kiesich (ed) Vol 6A Biotransformations Verlag

Chemie. Weinheim Germany: pp. 31-78.

"% Modern Industrial Microbiology and Biotechnology

27.1 NATURE AND IMPORTANCE OF VACCINES

Vaccines are materials which when introduced into the human body help protect the

vaccinated person against specified communicable diseases. Communicable diseases

are diseases caused by microorganisms, including viruses. Vaccines are preparations of

dead or weakened pathogens, or their products, that when introduced into the body,

stimulate the production of protective antibodies or T cells without causing the disease.

Vaccination is also called active immunization because the immune system of the

body is stimulated to actively develop its own immunity against the pathogen. Passive

immunity, in contrast, results from the injection of antibodies formed by another animal

(e.g., horse, human) which provide immediate, but temporary, protection for the recipient.

The name ‘vaccine’ comes from the Latin vacca (for cow). This is because the earliest

vaccination was done using the cow pox virus (which causes the disease in cow) as a

vaccine against small pox in humans. The English physician, Edward Jenner carried out

the above vaccination in the late 18th century and published his paper in 1798.

Over the past 200 or so years vaccination has contributed greatly to reducing

morbidity and mortality from communicable diseases. The greatest triumph of

vaccination is the eradication of smallpox from the earth; no naturally-occurring cases

has been reported since 1977. A program to try to eliminate another virus disease,

poliomyelitis (polio for short), from the world has been on for some time and the

indications are that the number of cases has drastically dropped. Except for the few cases

caused by oral polio vaccine (OPV) (see below), in which the live virus reverts, the disease

has now been eliminated from the Western hemisphere. Outbreaks of polio still occur in

Africa, the Indian subcontinent, and parts of the Near East. Due to the success of

vaccination near 100% reduction has been obtained in the cases of many diseases which

were previously sources of great mortality and morbidity. These include diphtheria,

measles, mumps, pertusis, rubella and tetanus. Table 27.1 gives a list of the most

commonly used vaccines today.

27.2 BODY DEFENSES AGAINST

COMMUNICABLE DISEASES

In order to better understand the nature of vaccines and their design and production, it is

important that the defenses of the human body against communicable diseases be

Vaccines

+0)26-4

Vaccines "%!

Table 27.1 Vaccines most commonly used in the world

Disease Preparation Notes

Diphtheria Toxoid Often given to children in a

single preparation (DTP; the

Tetanus Toxoid ‘triple vaccine’) or the now-

preferred DTaP using acellular

pertussis

Pertussis Killed bacteria (‘P’) or their

purified components

(acellular pertussis = ‘aP’)

Inactivated virus previously Inactivated polio vaccine: IPV

grown on monkey or (Salk)

human diploid cells

Polio Attenuated virus Oral polio vaccine; OPV (Sabin)

inactivated virus Both vaccines trivalent (types 1,

previously grown on 2, and 3)

monkey or human

diploid cells

Hepatitis B Protein (HBsAg) from Made by genetic engineering

the surface of the virus

Diphtheria, tetanus, uses acellular pertussis and Pediarix®; combination

pertussis, polio, and IPV (Salk) vaccine given in 3 doses to

hepatitis B infants

Measles Attenuated virus

Mumps 1 Attenuated virus Often given as a mixture

2 Vaccine: Live in duck cells (MMR) Does not increase the

risk of autism. (Nor do any

vaccines containing thimerosal

as a preservative.)

Rubella Attenuated virus

Pig, chick embryo or

canine tissue-culture grown

Chickenpox Attenuated virus Caused by the varicella-zoster

(Varicella) virus (VZV)

Influenza Egg-grown virus, formalin Contains hemagglutinins

inactivated, highly purified from the type A and type B

by zonal ultracentrifugation viruses recently in circulation

Hemagglutinins

Pneumococcal Capsular polysaccharides A mixture of the capsular

infections polysaccharides of 23 common

types. Works poorly in infants.

7 capsular polysaccharides Mobilizes helper T cells; works

conjugated to protein well in infants.

Staphylococcal 2 capsular polysaccharides To prevent infection by Staph.

infections conjugated to protein aureus in patients hospitalized

and/or receiving dialysis

Contd.

"%" Modern Industrial Microbiology and Biotechnology

discussed briefly. Ordinarily the human body is surrounded by microorganisms: in the

air it breathes, the water it drinks, in the soil around it and on the clothes he wears. Most

of these are not normally pathogenic. But even the pathogenic ones do not always cause

disease when they come in contact with the human body because the body has evolved

ways of dealing with microorganisms and preventing them from causing disease,

collectively known as the immune system. The immune system is a complex network of

cells and organs which work together to protect the body from communicable diseases. It

has two components: the innate or non-specific immunity and the acquired or specific

methods. While the innate immunity eliminates the organism no matter the type,

acquired or specific immunity specifically recognizes and selectively eliminates the

microorganism or foreign molecule.

Meningococcal disease Polysaccharides Used chiefly to prevent

outbreaks among the military

Hemophilus influenzae, Capsular polysaccharide Prevents ear infections in

type b (Hib) conjugated to protein children

Hepatitis A Inactivated virus Available in single shot with

HBsAg (Twinrix®)

Rabies Inactivated virus Vaccine prepared from human

Active: diploid cell cultures (HDCV)

(1) b-propiolactone- has replaced the duck vaccine

inactivated virus grown in (DEV)

embryonated duck eggs

(2) phenol-inactivated virus

grown in rabbit brain

Passive: equine hyper-

immune serum

Smallpox Attenuated live virus: Despite the global eradication

attenuated by passing of smallpox, is used to protect

through calves against a possible bioterrorist

attack

Anthrax Extract of attenuated bacteria Primarily for veterinarians and

military personnel

Typhoid Three available:

1. killed bacteria

2. live, attenuated

bacteria (oral)

3. polysaccharide

conjugated to protein

Yellow fever Live attenuated virus

Prepared in chick embryo:

Dakar strain or 17D strain

Tuberculosis Live attenuated mycobacte- Rarely used in the US

rium BCG (Bacille calmette

Guerin) strain (BCG)

Table 27.1 Contd.

Disease Preparation Notes

Vaccines "%#

27.2.1 Innate or Non-specific Immunity

The innate or non-specific defense mechanisms are the first line defense against invading

microorganisms. They will act irrespective of the type of microorganism. Briefly they

consist of the following:

(a) Anatomic barriers: these include mechanical barriers such as skin, which physically

keeps out microorganisms and mucous membranes of the alimentary canal

respiratory and urinogenital tracts which entrap microorganisms. In addition the

mucous membranes harbor a normal set or flora of microorganisms which keep out

foreign organisms.

(b) Physiologic barriers: The physiology of the human body keeps out some pathogens.

Thus the high temperature of the human body, including the fever response keeps

out some microorganisms as does the acidic nature of the stomach. Chemical

mediators such as lysozyme found in tears breakdown bacterial cell walls.

organisms, while specialized cells engulf and breakdown foreign particles.

(d) Inflammatory responses: Tissue damage and infection induce leakage of vascular

fluid serum protein with antibacterial fluid and influx of white blood cells leading

to pus formation.

27.2.1.1 Acquired or Specific immunity

Acquired or specific immunity has three important properties among others, which are

crucial in understanding vaccines and how they function.

(i) Antigenic specificity: An antigen is a material usually a protein which binds

specifically to an antibody or to T-cell receptor (see below). Great specifity occurs in

the anatigen-antibody or antigen-T-cell receptor relations. Often a small difference

of a single amino acid can decide whether or not binding to antibody or T-cell will

take place.

(ii) Immunologic memory: Once the acquired immune system has recognized and

responded to an antigen, it exhibits immunologic memory: a second encounter

with the same antigen induces an increased response.

(iii) Self/non-self recognition: Specific immunity recognizes foreign bodies in

contrast to those of the body and seeks to destroy the intruders. In rare cases the

system of recognition breaks down and the system fails to recognize body cells and

proceeds to destroy them, giving rise to auto-immune diseases.

Specific immunity has two components, humoral and cell-mediated immunity which

are mediated by white blood cells known as lymphocytes, and as will be seen below both

sectors are linked. Humoral immunity is mediated by B Lymphocytes, while cell-

mediated immunity is brought about by T Lymphocytes. White blood cells including

lymphocytes and like red blood cells are produced from stem cells in the bone marrow.

The B lymphocytes, remain in the bone marrow to mature, while T lymphocytes mature in

the thymus, a small organ located above the heart.

27.2.2.2 Specific immunity: humoral immunity

Humoral immunity is also known as antibody immunity. Antibodies are soluble proteins

in the blood which bind to foreign agents and mark them for destruction, or neutralize

"%$ Modern Industrial Microbiology and Biotechnology

toxins produced by microorganisms. Also known as immunoglobulins, antibodies are

glycoproteins by nature (i.e. proteins to which carbohydrates are conjugated). (see Fig.

27.1)

Fig. 27.1 General Structure of an Antibody Molecule

-S-S-, Disulphide bonds; CHO, carbohydrate molecule attached to the constant region of the heavy chain;

2, C

3, Constant regions of the heavy chain in the biological activity end of the antigen molecule; C

Constant region of the light chain; V

, Variable region of the light chain; C

1, Constant region of the heavy

chain in the constant part antigen binding end; V

, Variable region of the light chain (see text)

When B lymphocytes mature each has one unique antigen-binding molecule, an

antibody attached to its membrane; up to 10 different antibody molecules may be carried

on the B lymphocytes. When such a mature B lymphocyte which has not encountered any

antigen, known as a naïve lymphocyte, encounters an antigen for which its membrane

bound antibody is specific, it begins to divide rapidly and differentiates into two types of

cells: memory cells and plasma cells. The memory cells have a longer span of life and

continue to express membrane-bound antibody just like the original parent cell. The

plasma cells, on the other hand live for four to five days and do not have cell-membrane

bound antibodies; instead they produce antibody in a form in which it can be secreted,

often in huge amounts, sometimes reaching 2,000 molecules per second. The memory

cells are the source of the long-term protection which vaccines confer.

Antibodies (immunoglobulins) are Y- shaped and consist of two identical light chains

and two identical heavy chains (Fig. 27.1). The upper end of the Y of the light and heavy

chains of the antibody molecule is the variable region. The amino acids in this region vary

greatly among different antibodies and this variability confers on antibodies the vast

specificity for which they are known. The lower ends of the light and heavy chains are the

‘constant’ regions and do not show the variability found at the tips of the Y. The

variability in protein composition in the ‘constant’ region of the heavy chains (Fig. 27. 1)

Vaccines "%%

leads to antibodies being divided into five major classes, each with a different and

distinct property: IgG, IgA, IgD, IgM, and IgE. IgG is the most abundant (80%) of all Igs,

and it is the only one able to cross the placenta, helping to confer maternal immunity on

the newborn.

An antibody recognizes an antigen in a specific manner and the immune system

acquires memory towards it. The first encounter with an antigen is known as the primary

response. Re-encounter with the same antigen causes a secondary response that is more

rapid and powerful. This is the basis on which vaccines function; they induce the

memory lymphocytes to proliferate and the resulting plasma cells to produce soluble

antibodies (Fig. 27.2).

Antibodies are proteins known as immumoglogulins (Ig). The are five different kinds of immunoglobulins

IgA, IgD, IgE, IgG, and IgM. The animal body produces antibodies when challemged with materials to which

the body can react by producing antibodies (known as antigens). When the animal body is challenged with

the same antigen a second time the production of antibodies is not only produced in a shorter time, but the

antibody production is more pronounced as shown in the figure above. In the figure above IgM is produced

in the first challenge and IgM in the second. (see text).

Fig. 27.2 Antibody Response of the Animal Body to a Second Challenge of an Antigen

27.2.2.3 Specific immunity: cell-mediated immunity

While B lymphocytes mediate antibody or humoral immunity, T lymphocytes are

responsible for cell-mediated immunity. T lymphocytes do not have cell membrane

bound antibodies, nor do they secrete antibodies. Instead they have T-cell receptors

(TCRs). Unlike antibodies which can recognize antigens directly, T-cell receptors can

recognize an antigen only if the antigen is associated with cell membrane proteins

known as major histocompatibility compatibility (MHC) molecules, of which two classes

exist: MHC I and MHC II.

When a naïve T cell encounters an antigen associated with an MHC on a cell, the T cell

proliferates and differentiates into memory T cells, T helper cells (TH), and T cytotoxic

cells (TC). T

and T

cellscarry on their membranes different glycoproteins. T

cells carry

glycoprotein CD4, while T

cells carry CD8.

"%& Modern Industrial Microbiology and Biotechnology

When a T

cells interacts with an antigen linked to an MHC II compound, it is

activated to produce cytokins which also activate B cells to produce antibodies. Cytokins

also activate T

cells when they interact with an antigen linked to an MHC I compound,

to differentiate into cytoxic T lymphocytes (CTLs). CTLs do not secrete cytokins; instead

they monitor the body cells and eliminate any cells which are foreign or contain foreign

bodies such as cancer cells or cells containing viruses. To ensure that self cells are not

attacked by CTLs, they attack only cells displaying foreign foreigns complexed to an

MHC molecule on the surface of cells called antigen presenting cells (APCs). Antigen

presenting cells adsorb foreign antigens such as viruses, digest them and display the

peptides from them on their surfaces. CTLs identify such cells and destroy them. APCS

are specialized white blood cells. The relationships between B lymphocytes, T

lymphocytes, cytokins, Tc cells and CTLs are depicted in Fig. 27.3. The cell-mediated

immune response is important in cases where the pathogen is intracellular as in viruses

Fig. 27.3 Scheme Showing Immune System in Man

27.2.2.3 Antigens and Epitopes

Antigens are macromolecules that elicit an immune response in the body. Antigens can

be proteins, polysaccharides or conjugates of lipids with proteins (lipoproteins) and

polysaccharides (glycolipids). Antigens are generally very large and complex and the

lymphocytes may not recognize all the sites of a particular antigen. Rather both B and T

lymphocytes recognize discreet sites on an antigen known as epitopes or antigenic

determinants. The aim in vaccine production is to ensure that epitopes exist on the vaccine

which will elicit humoral or cell-mediated response.

Vaccines "%'

27.3 TRADITIONAL AND MODERN METHODS OF

VACCINE PRODUCTION

Traditionally three types of vaccines have been used: attenuated live vaccines, killed

vaccines and bacterial toxoids. Recent advances in molecular biology and genomic

science have spilled over into vaccine production. This chapter discusses the traditional

vaccines, but will also discuss the newer approaches which have been influenced by

advances in molecular biology and genomic science.

27.3.1 Traditional Vaccines

27.3.1.1 Live attenuated organisms

In live attenuated vaccines, the organism has been cultured so as to reduce its pathogenicity,

but still retains some of the antigens of the virulent form. They consist of the living patho-

gens whose virulence has been reduced (attenuated) by passaging them through hosts

different from the usual. Alternatively, non-virulent strains of the pathogen may be used.

Live agents may be used for one or more of the following reasons:

(i) When the protection-inducing substance is produced as a diffusible product of

metabolizing organisms e.g. Bacillus anthracis.

(ii) When it is not feasible to produce sufficient amounts of nonviable agents and a

small concentration of the living agent can propagate within the vaccinated

subject to overcome the deficiency.

(iii) When immunity is induced by the modification of parasitized cells.

Live vaccines in use include those against polio (Sabin oral polio vaccine - OPV), foot

and mouth disease of farm animals, mumps, measles, rubella (German measles),

tuberculosis, rabies and yellow fever. For tuberculosis the vaccine is derived the Bacillus

Calmette-Guérin (BCG) strain of Mycobacterium tuberculosis, a weakened version of the

bacterium that causes tuberculosis in cows. BCG is used as a vaccine against

tuberculosis in many European countries; it is however not commonly used in the U. S.

The OPV has advantages and disadvantages when compared with the (inactivated)

Salk polio vaccine (IPV). OPV can be given by mouth rather than by injection, and it can

spread to the other members of the vaccinee’s family thus immunizing them as well. Its

disadvantage is that on rare occasions, the virus regains full virulence and cause the

disease. On account of this, the Salk vaccine has gained prominence over the Sabin

vaccine in some countries.

27.3.1.2 Killed vaccines

These consist of suspensions of fully virulent organisms (bacteria or viruses) killed as

mildly as possible in order not to destroy the antigenic determinants on the organism.

Killing can be achieved by heat, (usually about 60°C for 1 hour) chemicals (phenol,

alcohol, formalin, b-propiolactone) or ultraviolet irradiation. Killed vaccines do not

provide as prolonged antigenic stimuli as living vaccines and two, three or more sub-

cutaneous injections are required to give adequate protection. Examples of killed

vaccines include TAB vaccine against typhoid fever and which consists of heat-killed

phenol-preserved suspension of Salmonella typhii and Salm. Paratyphii A & B, whooping

cough, cholera, and the Salk IPV.