Fermentation technology

GENETIC IMPROVEMENT OF FERMENTATION PROCESSES

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The genome of the organism ultimately controls its metabolism. Although improved fermenter engineering design and optimal cultural conditions can quantitatively enhance the microbial products, this will only be up to a limit. Genetic improvement of the organism is fundamental to the success of fermentation technology. Mutation and recombination are the two ways to meet this end.

A. MUTATION

A certain amount of mutational change in the genome occurs as a natural process, though the probability is small. Exposing a culture of a micro-organism to UV light, ionising radiation or certain chemicals, enhances the rate of occurrence of mutations. But it is a tremendous task for the industrial geneticist to screen the very large number of randomly produced mutants and to select the ones with the desired qualities.

The synthesis of a number of products of cell metabolism is controlled by a 'feed-back inhibition'. When a compound reaches a particular level of accumulation, its synthesis is stopped. Synthesis starts again when the level of the compound falls below the specific level. If a mutant is produced, in which the feedback signalling is suppressed, the product is synthesised continuously. By such a manipulation, a high producing strain of Corynebacterium glutamacium was developed to recover very high quantities of lysine. Such strains that do not produce controlling end products are called auxotrophs.

B. RECOMBINATION

Recombination is defined as any process that brings together genes from different sources.

A strain of Brevibacterium flavum is a high producer of lysine, but is limited by its poor capacity to absorb glucose. Another strain of the bacterium, which is an efficient absorber of glucose but which does not produce lysine, was used to develop a recombinant strain, through protoplast fusion. The new strain utilises high levels of glucose and yields higher levels of lysine.

A gene for the synthesis of phenylalanine was transferred to a chosen strain of Escherichia coli, which was a non-producer, but a good experimental and production tool.

Transformation of a high cephalosporin producing strain of Cephalosporium acremonium with a plasmid containing the gene REXH has significantly increased the titre.

A number of human proteins, such as insulin, human growth hormone, bone growth factor, alpha, beta and gamma interferons, interleukin-2, tumour necrosis factor, tissue plasminogen activator, blood clotting factor VIII, epidermal growth factor, granulocyte colony stimulating factor, erythropoietin, etc., are being produced through recombinant micro-organisms.

C. DNA MANIPULATION

In vitro DNA technology was used to increase the number of copies of a critical pathway gene (operon), as for example the production of threonine in Escherichia coli, at rates 40 to 50 times higher than usual.

TYPES OF CULTURE SYSTEMS

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A. BATCH PROCESSING OR CULTURE

At about the onset of the stationary phase, the culture is disbanded for the recovery of its biomass (cells, organism) or the compounds that accumulated in the medium (alcohol, amino acids), and a new batch is set up. This is batch processing or batch culture.

The best advantage of batch processing is the optimum levels of product recovery. The disadvantages are the wastage of unused nutrients, the peaked input of labour and the time lost between batches.

B. CONTINUOUS PROCESSING OR CULTURE

The culture medium may be designed such that growth is limited by the availability of one or two components of the medium. When the initial quantity of this component is exhausted, growth ceases and a steady state is reached, but growth is renewed by the addition of the limiting component. A certain amount of the whole culture medium (aliquot) can also be added periodically, at the time when steady state sets in. The addition of nutrients will increase the volume of the medium in the fermentation vessel. It is so arranged that the increased volume will drain off as an overflow, which is collected and used for recovery of products. At each step of addition of the medium, the medium becomes dilute both in terms of the concentration of the biomass and the products. New growth, stimulated by the added medium, will increase the biomass and the products, till another steady state sets in; and another aliquot of medium will reverse the process.

This is continuous culture or processing. Since the growth of the organism is controlled by the availability of growth limiting chemical component of the medium, this system is called a chemostat. The rate at which aliquots are added is the dilution rate that is in effect the factor that dictates the rate of growth.

The events in a continuous culture are:

a) the growth rate of cells will be less than the dilution rate and they will be washed out of the vessel at a rate greater than they are being produced, resulting in a decrease of biomass concentration both within the vessel and in the overflow;

b) the substrate concentration in the vessel will rise because fewer cells are left in the vessel to consume it;

c) the increased substrate concentration in the vessel will result in the cells growing at a rate greater than the dilution rate and biomass concentration will increase; and

d) the steady state will be re-established.

Hence, a chemostat is a nutrient limited self-balancing culture system, which may be maintained in a steady state over a wide range of sub-maximum specific growth rates.

The continuous processing offers the most control over the growth of cells.

Commercial adaptation of continuous processing is confined to biomass production, and to a limited extent to the production of potable and industrial alcohol.

The steady state of continuous processing is advantageous as the system is far easier to control. During batch processing, heat output, acid or alkali production, and oxygen consumption will range from very low rates at the start to very high rates during the late exponential phase. The control of the environmental factors of the system becomes difficult. In the continuous processing, the rates of consumption of nutrients and those of the output chemicals are maintainable at optimal levels. Besides, the labour demand is also more uniform.

Continuous processing may suffer from contamination, both from within and outside. The fermenter design, along with strict operational control, should actually take care of this problem.

The production of growth associated products like ethanol is more efficient in continuous processing, particularly for industrial use.

Continuous culturing is highly selective and favours the propagation of the best-adapted organism in culture.

A commercial organism is highly mutated such that it will produce very high amounts of the desired product. But physiologically such strains are inefficient and give way in culture to inferior producers--a kind of contamination from within.

C. FED-BATCH CULTURE OR PROCESSING

In the fed-batch system, a fresh aliquot of the medium is continuously or periodically added, without the removal of the culture fluid. The fermenter is designed to accommodate the increasing volumes. The system is always at a quasi-steady state.

Fed-batch achieved some appreciable degree of process and product control.

A low but constantly replenished medium has the following advantages:

a) maintaining conditions in the culture within the aeration capacity of the fermenter;

b) removing the repressive effects of medium components such as rapidly used carbon and nitrogen sources and phosphate;

c) avoiding the toxic effects of a medium component; and

d) providing limiting level of a required nutrient for an auxotrophic strain.

Production of baker's yeast is mostly by fed-batch culture, where biomass is the desired product. Diluting the culture with a batch of fresh medium prevents the production of ethanol, at the expense of biomass; the moment traces of ethanol were detected in the exhaust gas.

The production of penicillin, a secondary metabolite, is also by fed-batch method. Penicillin process has two stages: an initial growth phase followed by the production phase called the 'idiophase'. The culture is maintained at low levels of biomass and phenyl acetic acid, the precursor of penicillin, is fed into the fermenter continuously, but at a low rate, as the precursor is toxic to the organism at higher concentrations.

DESIGN OF INDUSTRIAL FERMENTATION PROCESS

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The fermentation process requires the following:

a) a pure culture of the chosen organism, in sufficient quantity and in the correct physiological state;

b) sterilised, carefully composed medium for growth of the organism;

c) a seed fermenter, a mini-model of production fermenter to develop an inoculum to initiate the process in the main fermenter;

d) a production fermenter, the functional large model; and

e) equipment for i) drawing the culture medium in steady state, ii) cell separation, iii) collection of cell free supernatant, iv) product purification, and v) effluent treatment.

Items a) to c) above constitute the upstream and e) constitutes the downstream, of the fermentation process,

Fermenters/bioreactors are equipped with an aerator to supply oxygen in aerobic processes, a stirrer to keep the concentration of the medium uniform, and a thermostat to regulate temperature, a pH detector and similar control devices.

PHASES OF MICROBIAL GROWTH

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When a particular organism is introduced into a selected growth medium, the medium is inoculated with the particular organism. Growth of the inoculum does not occur immediately, but takes a little while. This is the period of adaptation, called the lag phase.

Following the lag phase, the rate of growth of the organism steadily increases, for a certain period--this period is the log or exponential phase.

After a certain time of exponential phase, the rate of growth slows down, due to the continuously falling concentrations of nutrients and/or a continuously increasing (accumulating) concentrations of toxic substances. This phase, where the increase of the rate of growth is checked, is the deceleration phase.

After the deceleration phase, growth ceases and the culture enters a stationary phase or a steady state. The biomass remains constant, except when certain accumulated chemicals in the culture lyse the cells (chemolysis). Unless other micro-organisms contaminate the culture, the chemical constitution remains unchanged. Mutation of the organism in the culture can also be a source of contamination, called internal contamination

Microbial growth kinetics while fermentation

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I.1. Lag phase

II.2. Acceleration phase

III.3. Exponential (logrithmic) phase

IV.4. Deceleration phase

V.5. Stationary phase

VI.6. Accelerated death phase

VII.7. Exponential death phase

VIII.8. Survival phase

From: EL-Mansi and Bryce (1999) ,Fermentation Microbiology and Biotechnology.

Typical Microbial Production Process

•Create Production Host

–2-4 weeks

•Fermentation Process

–3-20 days

•Recovery Process/Formulation

–2-10 days

•

The Fermenter -Design and Engineering aspects

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Different types of Fermenters

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FERMENTERS AND BIOREACTORS DESIGN

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FERMENTERS AND BIOREACTORS DESIGN

There are many requirements that need to be met in the design of a large production scale fermentation facility. Aspects of design to be considered include design yield basis, operating schedule, media sterilization, fermenter and ancillary vessel design, piping systems, CIP/SIP and CGMP compliance. To be successful, a well thought-out and well-designed sanitary/sterile envelope is therefore crucial to the fermentation/biotech facility.

A fermenter is the set up to carry out the process of fermentation. The fermenters vary from laboratory experimental models of one or two litres capacity, to industrial models of several hundred litres capacity, which refers to the volume of the main fermenting vessel.

A bioreactor differs from a fermenter in that the former is used for the mass culture of plant or animal cells, instead of micro-organisms. The chemical compounds synthesised by these cultured cells, such as therapeutic agents, can be extracted easily from the cell biomass.

The design engineering and operational parameters of both fermenters and bioreactors are identical. With the involvement of micro-organisms as elicitors in some situations, the distinction between the two concepts is being gradually obliterated.

Functions/Requirements of Fermenter

1) Provide operation free from contamination;

2) Maintain a specific temperature;

3) Provide adequate mixing and aeration;

4) Control the pH of the culture;

5) Allow monitoring and/or control of dissolved oxygen;

6) Allow feeding of nutrient solutions and reagents;

7) Provide access points for inoculation and sampling;

8) Minimize liquid loss from the vessel;

9) Facilitate the growth of a wide range of organisms.

(Allman A.R., 1999: Fermentation Microbiology and Biotechnology)

Main parts of a Benchtop fermentor

1) Mechanical: drive motor, heater, pump, gas control, etc;

2) Vessel and accessories;

3) Peripheral equipment such as reagent bottles;

4) Instrumentation and sensors.

Types of Fermenter

1. Activated sludge Fermenter
2.Air Lift Fermenter

3.Bubble cap Fermenter

4. Loop Fermenter

5.Mist Fermenter

6. Packed Bed Fermenter

7.Rotating Drug Fermenter

8. Tower Fermenter

9.Trickling Film Fermenter

Fermentation Introduction

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Introduction

Pharmaceutical proteins produced via fermentation in transgenic microbes or mammalian cell culture systems provide economical systems for production of therapeutic proteins. These include antibodies, vaccines, blood proteins, etc.

Biopharmaceuticals are medical drugs (see pharmacology) produced using biotechnology. They are proteins (including antibodies), nucleic acids (DNA, RNA or antisense oligonucleotides) used for therapeutic or in vivo diagnostic purposes, and are produced by means other than direct extraction from a native (non-engineered) biological source

Dozens of new pharmaceuticals produced via fermentation in transgenic microbes have been approved for therapeutic use in the USA. Hundreds of additional biotech drug candidates are in various stages of research or clinical trials. Fermentation systems can be scaled up to produce quantities of pharmaceuticals that are difficult or impossible to produce via traditional methods. Pharmaceutical quality may also be improved. For example, pharmaceuticals produced from blood must be carefully purified to ensure no transmission of viruses as accidental contaminants in the pharmaceutical product. Microbial systems that do not allow human viruses to replicate enable pharmaceutical production with little or no risk of virus contamination.

The total biotech exports in 2005-06 were at Rs 3,357.17 crore, while the domestic business reported Rs 3,163.83 crore in sales. The exports accounted for 51.48 percent share of the total industry. BioPharma exports accounted for 74.33 percent of the total exports of Rs 3,357.17 crore, clocking Rs 2,495.24 crore in revenues. BioAgri's share of exports was the lowest at 1.07 percent and exports from this segment stood at Rs 35.88 crore. BioServices overseas revenues were Rs 684 crore. BioIndustrial and Bioinformatics sector accounted for 1.23 percent and 3 percent share of the total exports respectively.

Classification of biopharmaceuticals

Blood factors (Factor VIII and Factor IX)
Thrombolytic agents (tissue plasminogen activator)
Hormones (Insulin, growth hormone, gonadotrophins)
Haematopoietic growth factors (Erythropoietin, colony stimulating factors)
Interferons (Interferons-α, -β, -γ)
Interleukin-based products (Interleukin-2)
Vaccines (Hepatitis B surface antigen)
Monoclonal antibodies (Various)
Additional products (tumour necrosis factor, therapeutic enzymes)

Definition of Fermentation:

Fermentation technology is the oldest of all biotechnological processes. The term is derived from the Latin verb fevere, to boil--the appearance of fruit extracts or malted grain acted upon by yeast, during the production of alcohol.

Fermentation is a process of chemical change caused by organisms or their products, usually producing effervescence and heat.

Microbiologists consider fermentation as 'any process for the production of a product by means of mass culture of micro-organisms'.

Biochemists consider fermentation as 'an energy-generating process in which organic compounds act both as electron donors and acceptors'; hence fermentation is ‘an anaerobic process where energy is produced without the participation of oxygen or other inorganic electron acceptors’.

One process by which carbon-containing compounds are broken down in an energy yielding process. Fermentation occurs during times of low oxygen supply and is therefore known as a type of anaerobic respiration.

The anaerobic enzymatic conversion of organic compounds, especially carbohydrates, to simpler compounds , especially to ethyl alcohol, resulting in energy in the form of adenosine Triphosphate (ATP).

The process is used in the production of alcohol, bread, vinegar and other food or industrial products. It differs from respiration in that organic substances rather than molecular oxygen are used as electron acceptors

Fermentation occurs widely in bacteria and yeasts, the process usually being identified by the product formed, for example, acetic, alcoholic, butyric and lactic fermentation are those that result in the formation of acetic acid, alcohol, butyric acid and lactic acid, respectively.

Commercially important Fermentation

Microbial cells or Biomass as the product: Eg. Bakers Yeast, Lactic acid bacillus, Bacillus sp.
Microbial Enzymes: Catalase, Amylase, Protease, Pectinase, Glucose isomerase, Cellulase, Hemicellulase, Lipase, Lactase, Streptokinase etc.
Microbial metabolites :
1. Primary metabolites – Ethanol, Citric acid, Glutamic acid, Lysine, Vitamins, Polysaccharides etc.
2. Secondary metabolites: All antibiotic fermentation
Recombinant products : Insulin, HBV, Interferon, GCSF, Streptokinase
Biotransformations: Eg. Phenyl acetyl carbinol,Steroid Biotransformation

Nutrient sources for industrial fermentation

Medium for Industrial Fermentations

Any Microbe requires Water, Oxygen, Energy source, Carbon source, Nitrogen source and Micronutrients for the growth.

Carbon & Energy source + Nitrogen source + O₂ + other requirements → Biomass + Product + byproducts + CO₂ + H₂O + heat

Nutrient	Raw material
Carbon
Glucose	Corn sugar, Starch, Cellulose
Sucrose	Sugarcane, Sugar beet molasses
Lactose	Milk whey
Fats	Vegetable oils
Hydrocarbons	Petroleum fractions
Nitrogen
Protein	Soybean meal, Cornsteep liquor, Distillers' solubles
Ammonia	Pure ammonia or ammonium salts Urea
Nitrate	Nitrate salts
Phosphorus source	Phosphate salts

Nutrient sources for industrial fermentation

Nutrient Raw material

Carbon source

Glucose Corn sugar, Starch, Cellulose

Sucrose Sugarcane, Sugar beet molasses

Lactose Milk whey

Fats Vegetable oils

Hydrocarbons Petroleum fractions

Nitrogen source

Protein Soybean meal, Cornsteep liquor, Distillers' solubles

Ammonia Pure ammonia or ammonium salts

Nitrate Nitrate salts

Nitrogen Air

Phosphorous source Phosphate salts

Trace elements : Fe, Zn, Cu, Mn, Mo, Co

Antifoaming agents : Esters, Fatty acids, Silicones, Sulphonates, Polypropylene

Buffers: Calcium carbonate, Phosphates

Growth factors: Some microorganisms cannot synthesize the required cell components themselves and need to be supplemented: E.g. Thiamine, Biotin, Calcium pentothenate

Precursors: Directly incorporated into the desired product: Phenyl ethylamine into Benzyl penicillin, Phenyl acetic acid into Penicillin G

Inhibitors: To get the specific products: e.g. Sodium barbital for Rifamycin

Inducers: Majority of the enzymes are inducible and are synthesized in response of inducers: e.g. Starch for Amylases, Maltose for Pollulanase, Pectin for Pectinase

Chelators: Chelaters are the chemicals used to avoid the precipitation of metal ions. Chelaters like EDTA, Citric acid, Polyphosphates are used in low concentrations.

For more details on industrial fermentation read

1. Biochemical Engineering Fundamentals by J.E. Bailey and P.F. Ollis, McGraw Hill Publication 2. . Principles of fermentation technology by Stansbury, P.F., A. Whitaker and S.J. Hall, 1997

Biotechnology Companies Developing Pharmaceuticals via Fermentation in Transgenic Microbes or Mammalian Cell Cultures

PRODUCTS OF FERMENTATION PROCESSES

The growth of micro-organisms or other cells results in a wide range of products. Each culture operation has one or few set objectives. The process has to be monitored carefully and continuously, to maintain the precise conditions needed and recover optimum levels of products. Accordingly, fermentation processes aim at one or more of the following:

a) production of cells (biomass) such as yeasts;

b) extraction of metabolic products such amino acids, proteins (including enzymes), vitamins, alcohol, etc., for human and/or animal consumption or industrial use such as fertiliser production;

c) modification of compounds (through the mediation of elicitors or through biotransformation); and

d) production of recombinant products.

A. MICROBIAL BIOMASS

Microbial biomass is produced commercially as single cell protein (SCP) using such unicellular algae as species of Chlorella or Spirulina for human or animal consumption, or viable yeast cells needed for the baking industry, which was also used as human feed at one time. Bacterial biomass is used as animal feed. The biomass of Fusarium graminearum is also produced, for a similar use.

B. MICROBIAL METABOLITES

i) Primary metabolites:

During the log or exponential phase organisms produce a variety of substances that are essential for their growth, such as nucleotides, nucleic acids, amino acids, proteins, carbohydrates, lipids, etc., or by- products of energy yielding metabolism such as ethanol, acetone, butanol, etc. This phase is described as the tropophase, and the products are usually called primary metabolites. Commercial examples of such products are given in Table 2.

TABLE 2

Examples of commercially produced primary metabolites

Primary Organism Significance

Metabolite

Ethanol Saccharomyces cerevisiae alcoholic beverages

Kluyveromyces fragilis

Citric acid Aspergillus niger food industry

Acetone and Clostridium

butanol acetobutyricum solvents

Lysine Corynebacterium nutritional additive

Glutamic acid glutamacium flavour enhancer

Riboflavin Ashbya gossipii nutritional

Eremothecium ashbyi

Vitamin B12 Pseudomonas denitrificans nutritional

Propionibacterium shermanii

Dextran Leuconostoc mesenteroides industrial

Xanthan gum Xanthomonas campestris industrial

ii) Secondary metabolites:

Organisms produce a number of products, other than the primary metabolites. The phase, during which products that have no obvious role in metabolism of the culture organisms are produced, is called the idiophase, and the products are called secondary metabolites.

In reality, the distinction between the primary and secondary metabolites is not a straightjacket situation. Many secondary metabolites are produced from intermediates and end products of secondary metabolism. Some like those of the Enterobacteriaceae do not undergo secondary metabolism. Examples of secondary metabolites are given in Table 3.

TABLE 3

Examples of commercially produced secondary metabolites

Metabolite Species Significance

Penicillin Penicillium chrysogenum antibiotic

Erythromycin Streptomyces erythreus antibiotic

Streptomycin Streptomyces griseus antibiotic

Cephalosporin Cephalosporium acrimonium antibiotic

Griseofulvin Penicillium griseofulvin antifungal antibiotic

Cyclosporin A Tolypocladium inflatum immunosuppressant

Gibberellin Gibberella fujikuroi plant growth regulator

Secondary metabolism may be repressed in certain cases. Glucose represses the production of actinomycin, penicillin, neomycin and streptomycin; phosphate represses streptomycin and tetracyclin production. Hence, the culture medium for secondary metabolite production should be carefully chosen.

C. PRODUCTION ENZYMES

Industrial production of enzymes is needed for the commercial production of food and beverages. Enzymes are also used in clinical or industrial analysis and now they are even added to washing powders (cellulase, protease, lipase). Enzymes may be produced by microbial, plant or animal cultures. Even plant and animal enzymes can be produced by microbial fermentation. While most enzymes are produced in the tropophase, some like the amylases (by Bacillus stearothermophilus) are produced in the idiophase, and hence are secondary metabolites. Examples of enzymes produced through fermentation processes are given in Table 4.

TABLE 4

Examples of commercially produced enzymes

Organism Enzyme

Aspergillus oryzae Amylases

Aspergillus niger Glucamylase

Trichoderma reesii Cellulase

Saccharomyces cerevisiea Invertase

Kluyveromyces fragilis Lactase

Saccharomycopsis lipolytica Lipase

Aspergillus species Pectinases and proteases

Bacillus species Proteases

Mucor pusillus Microbial rennet

Mucor meihei Microbial rennet

D. FOOD INDUSTRY PRODUCTS

A very wide range of innumerable products of the food industry, such as sour cream, yoghurt, cheeses, fermented meats, bread and other bakery products, alcoholic beverages, vinegar, fermented vegetables and pickles, etc., are produced through microbial fermentation processes. The efficiency of the strains of the organisms used, and the processes are being continuously improved to market quality products at more reasonable costs.

E. RECOMBINANT PRODUCTS

Recombinant DNA technology has made it possible to introduce genes from any organism into micro-organisms and vice versa, resulting in transgenic organisms and the latter are made to produce the gene product. Genetically manipulated Escherichia coli, Saccharomyces cerevisiae, other yeasts and even filamentous fungi are now being used to produce interferon, insulin, human serum albumin, and several other products.

F. BIOTRANSFORMATION

Production of a structurally similar compound from a particular one, during the fermentation process is transformation, or biotransformation, or bioconversion. The oldest instance of this process is the production of acetic acid from ethanol.

Immobilised plant cells may be used for biotransformation. Using alginate as the immobilising polymer, digitoxin from Digitalis lanata was converted into digoxin, which is a therapeutic agent in great demand. Similarly, codeinone was converted into codeine and tyrosine from Mucuna pruriens was converted into DOPA.

G. ELICITORS

It is possible to induce production or enhance production of a compound in cultures by using elicitors, which may be micro-organisms. For example, Saccharomyces cerevisiae was an efficient elicitor in the production of glyceollin (Glycine max) and berberine (Thalictrum rugosum). Rhizopus arrhizus trebled diosgenin production by Dioscorea deltoidea. The production of morphine and codeine by Papaver somniferum was increased 18 times by Verticillium dahliae.

EXAMPLES :

Vitamins

Figure 11.13, Vitamin B₁₂
Used as supplements for human food and animal feeds
Nearly $1B/year
Synthesized chemically, but some by biocatalysis
Selected high-yield strains for B₁₂ produce up to 60 mg/L
For riboflavin, up to 7 g/L

Amino Acids in the Food Industry (a short list)

L-Glutamate (MSG) flavor enhancer
Aspartame (phe + asp) sweetener
L-Lysine nutritive additive
DL- Methionine nutritive additive

Industrial Production of Lysine

Figure 11.14a, Biochemical pathway of aspartate to lysine
Figure 11.14g, Structures of lysine and S-aminoethylcysteine
Overproducing strain: Brevibacterium flavum can produce over 60 g lysine/liter

Bioconversion

Supplement to organic synthesis
Growth in a large fermentor
Chemical to be converted added at appropriate time
Incubation
Conversion
Extraction
Purification of product
Figure 11.15, Cortisone production using a microorganism

Enzymes and their Markets

Pharmaceuticals
- Chiral resolution ($25B)
Agricultural
- Genetically engineered crops, animal feed ($5.9B)
Chemical
- Fine chemicals ($45B), specialty chemicals, and polymers
Industrial
- Consumer products, oil well breakers

Enzymes and Industrial Uses

Amylases = used in bread, glucose manufacture, in detergents
Proteases = used in bread, stain removal, meat tenderizing, contact cleaning solns
Cellulases = used in fabric softening, “stone-washed” jeans, detergents
Lipases = used in detergents, break-down of fats
DNA polymerases = used in biotechnology (PCR)

“Extremo”zymes: Enzymes that function under harsh industrial and environmental conditions

There is a need for enyzmes that catalyze desired (and often very specific) chemical conversions under typically harsh industrial and environmental conditions:
- Low- and High pH
- Cold to very hot temperatures
- Activity in non-aqueous solutions

Thermostable Enzymes

Figure 11.16b, Thermostability of pullulanase from Pyrococcus woesei
Examples of “Extremo”zymes
Thermus aquaticus
- DNA polymerase @ 75 – 95 C
Thermus thermophilus
- Pyrophosphatase @ 75 C
Thermococcus litoralis
- Pullulanase @ 118 C
Pyrococcus furiosus
- Ferredoxin @ 140 C
Psychrophiles – cold active enzymes
Acidophiles/Alkaliphiles – low-/high pH
Halophiles – High salt concentrations