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BTEC3301
Microbial Biotechnology
polymerase have been identified and widely used in PCR and other reactions.
Cellulase is obtained from E.coli and degrades
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fungi Trichoderma reesei and Aspergillus niger, to give the faded look and texture.
The protease subtilisin, from Bacillus subtilis,
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MICROORGANISMS be called Enzymes can rightly AS TOOLS the catalytic INDUSTRIAL USE OF ENZYMES machinery of living systems.
Enzymes are responsible for fermentation of sugar to ethanol by yeasts, a reaction that forms the bases of beer and wine manufacturing.
Enzymes oxidize ethanol to acetic acid. This reaction has been used in vinegar production for thousands of years.
Microbial Biotechnology
MICROORGANISMS AS TOOLS Similar microbial enzyme reactions of acid INDUSTRIAL USE OF ENZYMES forming bacteria and yeasts are responsible for aroma forming activities in bread making.
Presently more than 2000 different enzymes have been isolated and characterized. More than 75% of industrial enzymes are hydrolases. 40% of all enzyme sales are Protein-degrading enzymes .
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of intracellular enzymes .
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Enzyme Production by Microbial Fermentation Intracellular enzymes must be purified from
have a GRAS-status, which means that it is Generally Regarded as Safe. This is especially important when the enzyme produced by the organism is used in food processes .
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The industrial strains typically produce over 50-g/l extracellular enzyme proteins.
Most of the industrial enzymes are produced
by a relatively few microbial hosts like Aspergillus and Trichoderma fungi, Streptomyces and Bacillus .
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Most Enzyme Production by Microbial Fermentation of the industrially used microorganisms have been genetically modified to overproduce the desired activity and not to
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Protein engineering
Often enzymes do not have the desired
properties for an industrial application. E.g extreme thermo stability or overproduction of the enzyme.
engineered to function better in industrial processes. These include proteinases, lipases, cellulases and few amylases
temperature.
Xylanases is a good example of engineered enzyme from Trichoderma. Its xylanase has been
purified and crystallized. By designed mutagenesis its thermal stability has been increased about 2000 times at 70oC and its pHoptimum shifted towards alkaline region by one pH-unit.
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Enzyme Technology
Enzyme technology involves how to use
enzymes. The simplest way to use enzymes is to add them into a process stream where they catalyse the desired reaction and are gradually inactivated during the process .
membranes in the reactor system. The large enzyme molecules cannot pass the membrane but the small molecular reaction products can. Therefore enzymes are retained in a reaction system and the products leave the system continuously .
Reading Assignment for Quiz:Detergent, Food & BeveragesAnimal feed,personal care etc slide 15-40
Detergents were the first large scale application for microbial enzymes.
Bacterial proteinases are still the most important detergent enzymes. Some products have been genetically engineered to be more stable in the hostile environment of washing machines with
Microbial Biotechnology MICROORGANISMS AS TOOLS Lipid degrading enzymes, lipase, were used in
oryzae host after cloning the Humicola gene into this organism.
Amylases are used in detergents to remove starch based stains.
Microbial Biotechnology MICROORGANISMS part of Cellulases have beenAS TOOLS detergents since
early 90s. Cellulase is actually an enzyme complex capable of degrading crystalline cellulose to glucose.
In textile cellulases remove cellulose
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Yogurt, cheese, chocolate, butter, pickles, sauerkraut, soy sauce, food supplements (such as vitamins and amino acids), food thickeners
(produced from microbial polysaccharides), alcohol (beer, whiskeys, wines) and silage for animals are all products of microbial activity.
Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity
may be involved in producing concentrated microbial inocula for fermentations or the maintenance of fermentation systems utilized in production facilities.
The use of starch degrading enzymes,
amylase, was the first large-scale application of microbial enzymes in food industry.
Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity
process is used for milk products that are consumed by lactose intolerant consumers.
Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity
manufacturing. Pectins are substances in fruit lamella and cell walls. The cell wall contains also hemicelluloses and cellulose. Pectinase, xylanase and cellulase improve the liberation of the juice from the pulp.
Pectinases and amylases are used in juice clarification.
Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity
process, which increases the enzyme levels in the grain. In the mashing process the enzymes, amylase, are liberated and they hydrolyse (Break down) the starch into soluble fermentable sugars like maltose, which is a glucose disaccharide.
Similarly enzymes are widely used in wine production to obtain a better extraction of the necessary components and thus improving the
yield.
Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity
Brewing is an enzymatic process. Malting is a process, which increases the enzyme levels in the grain. In the mashing process the enzymes, amylase, are liberated and they hydrolyse (Break
down) the starch into soluble fermentable sugars like maltose, which is a glucose disaccharide.
Similarly enzymes are widely used in wine production to obtain a better extraction of the necessary components and thus improving the
yield.
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microbial activity.
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Foods
Mushrooms, truffles and some red and green algae
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Oil recovery/mining
Oil recovery may be facilitated by the development of unique bacteria which produce a surfactant that forces trapped oil out of rocks.
Extraction of minerals from low-grade ores is
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organophosphates, acetylcholinesterase inhibitors) of industrial, agricultural or military origin is essential in waste management.
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Textiles
The use of enzymes in textile industry is one of the
Enzymes are used to remove the starch in a process called desizing. Amylases are used in this process since they do not harm the textile fibers.
Laccase a polyphenol oxidase from fungi is used
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Animal Feed
The net effect of enzyme usage in feed has been increased animal weight.
The first commercial success was addition of beta-
glucanase into barley based feed diets. Barley contains beta-glucan, which causes high viscosity in the chicken gut.
Xylanase, from Trichoderma, are added to wheatbased broiler feed and are nowadays routinely used in feed formulations and animals gain
weight.
Enzymes have become an important aspect of
animal feed industry. In addition to poultry, enzymes are used in pig feeds and turkey feeds.
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Baking
Alpha-amylases have been most widely studied in
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Leather
Leather industry uses proteolytic and
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Leather
The used enzymes are typically alkaline
bacterial proteases.
Lipases are used in this phase or in bating
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Personal care products are a relatively new area for enzymes and the amounts used are small but worth to mention as a future growth area.
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glucose oxidase. Dentures can be cleaned with protein degrading enzyme solutions. Enzymes are also used for applications in skin and
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Enzymes in DNA-technology
DNA-technology has revolutionized both traditional biotechnology and opened totally new fields for scientific study.
Recombinant DNA-technology allows one to
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Protein engineering is used to modify and improve existing enzymes. DNA is basically a long chain of deoxyribose sugars
linked together by phosphodiester bonds. Organic bases, adenine, thymine, guanine and cytosine are linked to the sugars and form the alphabet of genes. The specific order of the organic bases in the chain constitutes the genetic language.
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Genetic engineering means reading and modifying this language. Enzymes are crucial tools in this process.
E.g.:
1. Restriction enzymes recognise specific DNA sequences and cut the chain at these recognition sites.
2. DNA modifying enzymes synthesize nucleic acids,
degrade them, join pieces together and remove parts of the DNA.
3. DNA-polymerases synthesize new DNA-chains. Many of them need a model template, which they copy. 4. Ligases join adjacent nucleotides together.
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factors causing hemophilia, insulin (resulting in diabetes), growth hormone (resulting in lack of proper growth), and other proteins, the administration of which corrects pathological conditions or results in other therapeutic benefits.
Plasmids are used to transfer human genes to bacterial cells.
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the human gene for insulin, for example, the bacteria into which this gene is inserted produces human insulin.
Bacteria as such do not produce insulin, but the recombinant bacterial cells do produce insulin, it was an outstanding example of microbial biotechnology.
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cDNA:
Human genes composed of coding and noncoding sequences. The copy of the coding sequences is called cDNA. The synthesis of the insulin cDNA will allow the production of a functional insulin molecule.
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humans.
Microbial Biotechnology two polypeptides subunits Human insulin has MICROORGANISMS AS TOOLS
Therapeutic Proteins by Gene Transfer Cloning the Insulin gene (Mechanism):
called the A (21 amino acids) and the B (30 amino acids) which are bonded by disulphide bond to create the active insulin.
for each of the subunit is inserted into plasmid vector separately (Fig 5.9).
MicrobialvectorTOOLS the Lac z gene encoding for the The Biotechnology MICROORGANISMS AS has
Therapeutic Proteins by Gene Transfer Cloning the Insulin gene (Mechanism):
human are fused to the E. coli gene (Lac z) encoding for beta-galactosidase.
The plasmid is then transformed into E.coli . Plasmids enter the bacteria in a process called transfection .
Microbial Biotechnology With the AS TOOLS MICROORGANISMS recombinant DNA molecule successfully
Therapeutic Proteins by Gene Transfer Cloning the Insulin gene (Mechanism):
inserted into the bacterial host, another property of plasmids can be exploited - their capacity to replicate.
Once inside a bacterium, the plasmid containing the human cDNA can multiply to yield several dozen copies. Because the insulin genes are connected to the lac z gene, when bacteria synthesizes proteins from these plasmids, they produce a protein containing -gal attached to human insulin protein and this is called a fusion protein .
fusion protein.
divided between the two daughter cells and the plasmids continue to reproduce.
With cells dividing rapidly (every 20 minutes), a
bacterium containing human cDNA) will shortly produce many millions of similar cells (clones) containing the same human gene.
Microbial Biotechnology After the chains are synthesized, the bonds that MICROORGANISMS AS TOOLS
Therapeutic Proteins by Gene Transfer Cloning the Insulin gene (Mechanism):
hold the insulin molecule to the betagalactosidase are cleaved with cyanogen bromide.
insulin. This form of insulin is an exact match to that which is made in the body.
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against microbes.
An antibiotic is a substance, usually produced
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techniques, the microorganism can be modified to improve the yield or action of antibiotics and other antimicrobial agents.
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New research directions are aimed at discovering microbial metabolites with pharmacological activities useful in the
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Vaccines
Introduction
A number of diseases are caused by microorganisms Vaccines are essential to protect humans and animals from microbial diseases. Recombinant DNA technology has allowed the production of
novel vaccines that offer protection without the risk of infection (e.g. hepatitis B vaccine).
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Vaccines: Introduction
diseases. The production of vaccines against the microbial pathogens, and more particularly the pathogenic viruses, in order to immunise the susceptible populations, is a safe and more certain recourse.
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Vaccines: Introduction
produce immunological agents to afford protection from diseases to large numbers of people.
This area is immunotechnology, an arm of
biotechnology.
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Vaccines
mammalian system in order to impart a memory of the pathogen or its pathogenic component The memory is imparted on the first contact of the vaccine with the mammalian immune system.
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production of antibodies), or immunogens (that trigger the cellular component of immune response)
In the event of an encounter with the
corresponding antibodies, only the antigens can bind with the antibodies, and form an antigenantibody complex that neutralises the harmful effects of the antigens or the organisms that produce them.
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Vaccines
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Vaccination/Immunisation
The process of the deliberate introduction of a vaccine into the organism is vaccination, for which the term inoculation is also often
used. Since vaccination immunises the organism, the process is also called immunisation When an organism is vaccinated, the immune system is readied to show an immune response by way producing antibodies against the pathogen.
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Composition of vaccines
Vaccines are suspensions, in saline , of
weakened pathogenic organisms or the proteins they secrete, which have the potential to cause a disease.
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Types of vaccines:
Inactivated vaccines: The pathogen is killed using heat or formalin, as for example, typhoid vaccines .
Attenuated vaccines: Vaccines: Types of vaccines: The pathogen is weakened (attenuated) by aging or altering growth conditions, but is alive, as in the case of measles, mumps and rubella vaccines .
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Avirulent organisms: A non-pathogenic strain of a pathogenic organism is used as a vaccine, as in BCG (Bacillus Calmette Guerin) vaccine against Mycobacterium tuberculosis, the tuberculosis bacterium.
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Toxoids:
Acellular vaccines:
Only the antigenic component of the organism
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Subunit vaccines:
Genetic engineering techniques have now made
it possible to use as a vaccine only a part of an organism that is adequate to stimulate the immune response e.g in Hepatitis B vaccine a segment of genetic material is isolated from the pathogens and introduced into bacteria or yeasts .
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DNA vaccines are an offshoot of gene therapy Selected segments of DNA, when introduced into the patients system synthesise and deliver proteins that are needed to replace the defective enzyme system or tag a cell for destruction. Viruses or lipid vehicles are used to deliver the DNA into the cells. This recent technology is being tried to produce vaccines against HIV, by a direct injection of plasmid borne DNA
2-WAY- LEARNING
Hepatitis B vaccine? Immunisation by DNA injection?
Bioterrorism (Chapter 9)
REFERENCES:
Introduction to Biotechnology by W.J. Thieman and M.A. Palladino. Pearson & Benjamin Cummings 2nd edition. http://en.wikipedia.org Matti Leisola, Jouni Jokela, Ossi Pastinen, Ossi Turunen
PRODUCTION OF THERAPEUTIC PROTEINS BY GENETIC ENGINEERING - IMPACT No. 299 May 1998 Duane T. G
http://www.fbae.org/Channels/biotech_in_medicine/vaccines.htmish