chapter 5 microbial biotechnology

BTEC3301

MICROORGANISMS AS TOOLS

INDUSTRIAL USE OF ENZYMES

Several thermostable enzymes, like the Taq polymerase have been identified and widely used in PCR and other reactions.

Cellulase is obtained from E.coli and degrades cellulose, a polysaccharide in plant cells.

Microbial Microbial BiotechnologyBiotechnology

The denim jean is treated with cellulase, from fungi Trichoderma reesei and Aspergillus niger, to give the faded look and texture.

The protease subtilisin, from Bacillus subtilis, forms component of Laundry detergent to remove and degrade protein stains.

Microbial BiotechnologyMicrobial Biotechnology

MICROORGANISMS AS MICROORGANISMS AS TOOLS TOOLS INDUSTRIAL USE OF INDUSTRIAL USE OF

ENZYMESENZYMES

Enzymes can rightly be called the catalytic 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.



ENZYMESENZYMES

Similar microbial enzyme reactions of acid 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 .



ENZYMESENZYMES


Enzyme Production by Microbial Fermentation

Extracellular enzymes are secreted outside the cell makes the recovery and purification process much simpler compared to production of intracellular enzymes .


Intracellular enzymes must be purified from thousands of different cell proteins and other components.

The organism producing the enzymes should 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 .


MICROORGANISMS AS TOOLS MICROORGANISMS AS TOOLS Enzyme Production by Microbial Enzyme Production by Microbial

FermentationFermentation

The organism should be able to produce high amount of the desired enzyme in a reasonable time frame.

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 .


MICROORGANISMS AS TOOLS MICROORGANISMS AS TOOLS Enzyme Production by Microbial FermentationEnzyme Production by Microbial Fermentation

Most of the industrially used microorganisms have been genetically modified to overproduce the desired activity and not to produce undesired side activities.


MICROORGANISMS AS TOOLS MICROORGANISMS AS TOOLS Enzyme Production by Microbial FermentationEnzyme Production by Microbial Fermentation


Protein engineering

Often enzymes do not have the desired properties for an industrial application. E.g extreme thermo stability or overproduction of the enzyme.


Protein engineering is used to improve commercially available enzyme to be a better industrial catalyst.

Several enzymes have already been engineered to function better in industrial processes. These include proteinases, lipases, cellulases and few amylases


MICROORGANISMS AS TOOLSMICROORGANISMS AS TOOLS Protein EngineeringProtein Engineering

Xylanase from fungus Trichoderma sps. is a good example of an industrial enzyme, used in pulp and paper industry and needs to be stable in high 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 pH-optimum shifted towards alkaline region by one pH-unit.


MICROORGANISMS AS TOOLSMICROORGANISMS AS TOOLS Protein EngineeringProtein Engineering


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 .


Example liquefaction of starch with amylases, bleaching of cellulose pulp with xylanases or use of enzymes in animal feed.

An alternative way to use enzymes is to immobilize them so that they can be reused.

One method of immobilization is to use ultrafiltration 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 .


MICROORGANISMS AS TOOLSMICROORGANISMS AS TOOLS Enzyme TechnologyEnzyme Technology

Large scale enzyme applicationsDetergents

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 several different chemicals present.

Reading Assignment for Reading Assignment for Quiz:Detergent, Food & Quiz:Detergent, Food & BeveragesAnimal BeveragesAnimal feed,personal care etc slide feed,personal care etc slide 15-4015-40

Lipid degrading enzymes, lipase, were used in powder and liquid detergents to decompose fats.

Lipase is produced in large scale by Aspergillus oryzae host after cloning the Humicola gene into this organism.

Amylases are used in detergents to remove starch based stains.


MICROORGANISMS AS TOOLSMICROORGANISMS AS TOOLS Large scale enzyme Large scale enzyme

applicationsapplicationsDetergentsDetergents

Cellulases have been part of detergents since early 90s. Cellulase is actually an enzyme complex capable of degrading crystalline cellulose to glucose.

In textile cellulases remove cellulose microfibrils, which are formed during washing.

Alkaline cellulases are produced by Bacillus strains and neutral and acidic cellulases by Trichoderma and Humicola fungi.


MICROORGANISMS AS TOOLSMICROORGANISMS AS TOOLS Large scale enzyme Large scale enzyme

applicationsapplicationsDetergentsDetergents

Large scale enzyme applicationsFoods/Beverages produced by Microbial Activity

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 BiotechnologyMicrobial BiotechnologyMICROORGANISMS AS TOOLSMICROORGANISMS AS TOOLS

The industrial microbiologist/ biotechnologist 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.


MICROORGANISMS AS TOOLS MICROORGANISMS AS TOOLS Large scale enzyme applications Large scale enzyme applications Foods/Beverages produced by Foods/Beverages produced by

Microbial ActivityMicrobial Activity

Enzymes have many applications in drink industry. The use of chymosin in cheese making to coagulate milk protein.

Another enzyme used in milk industry is beta-galactosidase or lactase, which splits milk-sugar lactose into glucose and galactose. This process is used for milk products that are consumed by lactose intolerant consumers.




Enzymes are used also in fruit juice 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.




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.




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.




Large scale enzyme applications

Foods/Beverages cured or improved by microbial activity

Production of coffee, tea, cocoa, summer sausage, vanilla, cheese, olives and tobacco all require

microbial activity.



Food flavoring agents and preservatives

Organic acids such as citric, malic and ascorbic acids and monosodium glutamate are microbial products

commonly used in foods.



Foods Mushrooms, truffles and some red and

green algae are consumed directly. Yeasts are used as food supplements

for humans and animals.



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 enhanced by some bacteria (microbial leaching).


Large scale enzyme applications Waste and Wastewater

Management

Isolating or developing microbial strains capable of degrading and detoxifying hydrocarbon and halogenated hydrocarbon waste (for example organophosphates, acetylcholinesterase inhibitors) of industrial, agricultural or military origin is essential in waste management.



Textiles

The use of enzymes in textile industry is one of the most rapidly growing fields in industrial enzymology.

Starch has for a long time been used as a protective glue of fibers in weaving of fabrics. This is called sizing.


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 to degrade lignin the aromatic polymer found in all plant materials .

MICROORGANISMS AS TOOLSMICROORGANISMS AS TOOLSLarge scale enzyme Large scale enzyme

applicationsapplicationsTextilesTextiles


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 wheat-based 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.

MICROORGANISMS AS TOOLSMICROORGANISMS AS TOOLSLarge scale enzyme Large scale enzyme

applicationsapplicationsAnimal FeedAnimal Feed


Baking

Alpha-amylases have been most widely studied in connection with improved bread quality and increased shelf life.

Both fungal and bacterial amylases are used in bread making and excess may lead to sticky dough.



Pulp and Paper

The major application is the use of xylanases in pulp bleaching for paper.



Leather

Leather industry uses proteolytic and lipolytic enzymes in leather processing.

Enzymes are used to remove animal skin, hair, and any unwanted parts.



Leather

The used enzymes are typically alkaline bacterial proteases.

Lipases are used in this phase or in bating phase to specifically remove grease.



Enzymes in Personal Care products

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.


Large scale enzyme applicationsEnzymes in Personal Care

products One application is contact lens cleaning.

Proteinase and lipase containing enzyme solutions are used for this purpose. Hydrogen peroxide is used in disinfections of contact lenses. The residual hydrogen peroxide after disinfections can be removed by a heme containing catalase enzyme, which degrades hydrogen peroxide.


Large scale enzyme applicationsEnzymes in Personal Care products

Some toothpaste contains glucoamylase and glucose oxidase.

Dentures can be cleaned with protein degrading enzyme solutions.

Enzymes are also used for applications in

skin and hair care products.


Large scale enzyme applicationsEnzymes in DNA-technology

DNA-technology has revolutionized both traditional biotechnology and opened totally new fields for scientific study.

Recombinant DNA-technology allows one to produce new enzymes in traditional overproducing and safe organisms .



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.



Enzymes in DNA-technology

Genetic engineering means reading and modifying this language. Enzymes are crucial tools in this process. E.g.:



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.

Microbial Microbial BiotechnologyBiotechnologyMICROORGANISMS AS MICROORGANISMS AS TOOLSTOOLS

Therapeutic Proteins by Gene Transfer

Recombinant DNA technology led to the rapid development and production of Therapeutic protein.

There are many proteins essential to good health that some people cannot produce because of genetic defects.



These proteins include various blood-clotting 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.



If the gene inserted into the plasmid of bacteria is 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.


Courtesy © John J. Cardamone, Courtesy © John J. Cardamone, Jr.Jr.

Insertion of a DNA section into a plasmid


cDNA:

Human genes composed of coding and non- coding 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.


Transfer of the Insulin gene into a plasmid vector (schematic)


Cloning the Insulin gene (Mechanism):

Insulin was first synthesized in 1979 in E. coli cells through the use of recombinant DNA techniques.

Insulin is produced by beta cells in pancreas in humans.


Human insulin has two polypeptides subunits called the A (21 amino acids) and the B (30 amino acids) which are bonded by disulphide bond to create the active insulin.

When a human gene for insulin is cloned, gene for each of the subunit is inserted into plasmid vector separately (Fig 5.9).

Microbial BiotechnologyMicrobial BiotechnologyMICROORGANISMS AS TOOLSMICROORGANISMS AS TOOLSTherapeutic Proteins by Gene TransferTherapeutic Proteins by Gene Transfer Cloning the Insulin gene (Mechanism):Cloning the Insulin gene (Mechanism):

The vector has the Lac z gene encoding for the enzyme β-galactosidase (β-gal).

The genes that code for the two insulin chains in 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 .


With the recombinant DNA molecule successfully 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 β-gal attached to human insulin protein and this is called a fusion protein .


The fusion protein here is called β-gal-insulin fusion protein.

When the bacteria divide, the plasmids are 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.


After the chains are synthesized, the bonds that hold the insulin molecule to the beta-galactosidase are cleaved with cyanogen bromide.

Affinity column is used to separate the two proteins.

The two chains are then purified to give native insulin.

This form of insulin is an exact match to that which is made in the body.


Fig 5.9: Using bacteria to produce Human insulin

Type 1 or insulin-dependent diabetes mellitus & study steps

in Fig.5.9.

NEW & VIEWS


Microbes against microbes

Antibiotics are antimicrobial drugs used against microbes.

An antibiotic is a substance, usually produced by a microorganism which, in very small quantities, inhibits or kills other microorganisms .


Both natural and chemically enhanced microbial products can be used to control human, animal and plant diseases.

Using traditional genetics or recombinant DNA techniques, the microorganism can be modified to improve the yield or action of antibiotics and other antimicrobial agents.

Microbial BiotechnologyMicrobial BiotechnologyMICROORGANISMS AS TOOLSMICROORGANISMS AS TOOLSTherapeutic Proteins by Gene TransferTherapeutic Proteins by Gene Transfer Microbes against microbesMicrobes against microbes

New research directions are aimed at discovering microbial metabolites with pharmacological activities useful in the treatment of hypertension, obesity, coronary heart disease, cancer and inflammation.

Microbial BiotechnologyMicrobial BiotechnologyMICROORGANISMS AS TOOLSMICROORGANISMS AS TOOLSTherapeutic Proteins by Gene TransferTherapeutic Proteins by Gene Transfer Microbes against microbesMicrobes against microbes

MICROORGANISMS AS TOOLSVaccinesIntroduction

A number of diseases are caused by micro-organisms

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).


For many bacterial diseases, and some fungal diseases, there are antibiotics, produced by other micro-organisms.

There are very few means of fighting viral 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.


Vaccines:Vaccines: IntroductionIntroduction

Biotechnology has made it now possible to produce immunological agents to afford protection from diseases to large numbers of people.

This area is immunotechnology, an arm of biotechnology.


Vaccines:Vaccines: IntroductionIntroduction

MICROORGANISMS AS TOOLSVaccines

What are Vaccines A vaccine is an agent, sourced from

the pathogen, and is deliberately introduced into the 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.


Vaccines contain antigens (that elicit the 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 antigen-antibody complex that neutralises the harmful effects of the antigens or the organisms that produce them.


Vaccines:Vaccines: What are VaccinesWhat are Vaccines


VaccinesVaccination/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.



Vaccines

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.



Vaccines

Types of vaccines:

Inactivated vaccines: The pathogen is killed using heat

or formalin, as for example, typhoid vaccines .


Attenuated 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 .

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.


Vaccines:Vaccines: Types of vaccines:Types of vaccines:

Toxoids: The toxin from the pathogen is used

as an antigen to produce the vaccine .

Acellular vaccines: Only the antigenic component of the

organism is used instead of the whole organism, as in Haemophilus influenza B vaccine .



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 .



DNA vaccines: 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



Hepatitis B vaccine? Immunisation by DNA

injection?

2-WAY- LEARNING

Bioterrorism

(Chapter 9)

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 Laboratory of Bioprocess Engineering, Helsinki University of Technology,

Finland Hans Schoemaker, DSM Research, MD Geleen, The Netherlands

http://www.accessexcellence.org/RC/VL/GG/transfer_and.html

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

REFERENCES:

chapter 5 microbial biotechnology

Documents

soluble fermentable sugars

personal care products

gene transfer cloning

gene transfer microbes

microbial biotechnology microorganisms

tools therapeutic proteins

gene transfer

animal feed