Biochemical processes (Section 2)

What is biochemical Process? Biochemistry is the study of a chemical process in living things. A process that uses living cells or biomolecules to carry out a chemical transformation leading to the production and ultimate recovery of valuable products. Biochemists study microorganisms like proteins and carbohydrates. Fine chemicals The category fine chemicals within the context of biotechnology broadly interpreted to bioproduction of high value molecules and bioconversion of high value starting materials to yet more valuable products. Some products of biotechnology: 1. Enzyme 2. Antibiotics 3. Recombinant DNA Technology

1. ENZYME
Enzymes are usually proteins of high molecular weight that act as catalysts. Enzymes are specific, versatile and very effective biological catalysts, resulting in much higher reaction rates as compared to chemically catalyzed reactions under ambient conditions. More than 2000 enzymes are known. How enzyme works Enzymes lower the activation energy of the reaction catalyzed by binding the substrate and forming an enzyme substrate complex. Enzymes do not affect the free energy change or the equilibrium constant. The interaction between the enzyme and its substrate is usually by weak forces. In most cases, van der waals forces and hydrogen bonding are responsible for the formation of ES complexes. Methods of Enzyme Production Isolation of Microorganisms, Strain Development and Preparation of Inoculum Medium Formulation and Preparation Sterilization and Inoculation of Medium, Maintenance of Culture and Fluid Filtration Purification of Enzyme Large-scale production of enzymes Among various enzymes produced at large scale are proteases, isomerases and oxidases. These enzymes are produced using overproducing strains of certain organisms. Separation and purification of an enzyme from an organism require disruption of cell, removal of cell debris and nucleic acid, precipitation of proteins, ultrafiltration of the desired enzyme, chromatographic separations (optional), crystallization and drying. STEPS: The first step in the large scale production of enzyme is to cultivate the organisms producing the desired enzyme. Proteases are produced by using overproducing strains of Bacillus aspergillus , Rhizopus and Mucor; Pectinaeses are produced by Aspergillus niger; Lactases are produced by yeast and aspergillus Lipases are produced by certain strains of yeast and fungi; Glucose isomerase is produced by Flavobacterium arborescens or Bacillus coagulans

 After the cultivation step, cells are separated from the media usually by filtration or sometimes by centrifugation. Depending of intracellular or extracellular nature of the enzyme either the cells or the fermentation broth is further processed to separate and purify the enzyme. Medical and industrial utilization of enzymes Proteases are used in: food processing such as cheese making (rennet), baking, meat tenderization (papain, trypsin), and brewing (trypsin, pepsin) in detergents for the hydrolysis of protein stains (subtilisin carlsberg) tanning and the medical treatment of wounds Enzymes are commonly used in medicine for diagnosis, therapy, and treatment purposes. Trypsin and Streptokinase can be used as an antiinflammatory agent. Lysozyme which hydrolyses cell wall of gram-positive bacteria, is used as an antibacterial agent Urokinase is used in dissolving and preventing blood clots Advantages of using enzyme They are specific in their action and are therefore less likely to produce unwanted byproducts. They are biodegradable and therefore cause less environmental pollution. They work in mild conditions, i.e. low temperatures, neutral pH and normal atmospheric pressure, and therefore are energy saving Disadvantages of using enzyme They are highly sensitive to changes in physical and chemical conditions surrounding them. They are easily denatured by even a small increase in temperature and are highly susceptible to poisons and changes in pH. Therefore the conditions in which they work must be tightly controlled. The enzyme substrate mixture must be uncontaminated with other substances that might affect the reaction.

Environmental Factors Enzymes are used in other many other industries including textile, paper and leather manufacturers. Experts state that harsh chemical treatments used in these industries' production can present potential hazards to staffers. The U.S. Department of Agriculture reports that using commercial enzymes to produce grain ethanol can reduce greenhouse gas emissions associated with the manufacture of ethanol.

 There are significant municipal concerns relating to the use of surfactants and enzymes in the process of wastewater treatment. Many municipalities across the country are banning the use of surfactants and enzymes entering wastewater treatment because of the harmful effects these products have on the sewer lines and the damage is costing cities much money in repair.

2. ANTIBIOTICS
The Greek word anti means "against", and the Greek word bios means "life" (bacteria are life forms). An antibiotic is a drug that kills or slows the growth of bacteria. These are used to treat infections caused by bacteria. How do antibiotics work? Although there are a number of different types of antibiotic they all work in one of two ways: A bactericidal antibiotic kills the bacteria. A bactericidal usually either interferes with the formation of the bacterium's cell wall or its cell contents. o Penicillin is a bactericidal. A bacteriostatic stops bacteria from multiplying. What are antibiotics for? An antibiotic is given for the treatment of an infection caused by bacteria. Antibiotics target microorganisms such as bacteria, fungi and parasites. However, they are not effective against viruses. If you have an infection it is important to know whether it is caused by bacteria or a virus. INDUSTRIAL PRODUCTION OF ANTIBIOTIC- PENICILLIN The industrial production of penicillin was broadly classified in to two processes namely: Upstream processing Downstream processing 1. UPSTREAM PROCESSING: Upstream processing encompasses any technology that leads to the synthesis of a product. Upstream includes the exploration, development and production. SELECTION & IMPROVEMENT OF STRAIN INOCULUM PREPARATION 2. DOWNSTREAM PROCESSING: The extraction and purification of a biotechnological product from fermentation is referred to as downstream processing. RAW MATERIALS: CARBON SOURCES: Lactose acts as a very satisfactory carbon compound, provided that is used in a concentration of 6%. Others such as glucose & sucrose may be used. NITROGEN SOURCES: Ammonium sulphate and ammonium acetate can be used as nitrogenous sources. MINERAL SOURCES: Elements namely potassium, phosphorus, magnesium, sulphur, zinc and copper are essential for penicillin production. Some of these are applied by corn steep liquor. FERMENTATION PROCESS: The fermentation process is usually carried out in a large fermenter. The fermentation medium is formulated and fed in to the fermenter. The inoculum is also maintained properly.

DOWNSTREAM PROCESSING SOLID LIQUID SEPARATION: The first step in product recovery is the separation of whole cells and other insoluble ingredients from the culture broth. Several methods such as floatation, flocculation etc, are used to serve this purpose. CONCENTRATION: This is a step followed to concentrate the desired product. The methods used to concentrate includes adsorption, precipitation etc. PURIFICATION: Chromatographic techniques are generally used to purify the product. As concerned with the production of antibiotics ion exchange chromatography seems to be the better option EXTRACTION AND PURIFICATION OF PENICILLIN: There are some special techniques used for the extraction of penicillin from the fermentation medium. They are as follow: REMOVAL OF MYCELIUM COUNTERCURRENT SOLVENT EXTRACTION OF PENICILLIN TREATMENT OF CRUDE EXTRACT: The solvent extract recovered in the previous stage is carefully extracted back with aqueous sodium hydroxide. This is followed by charcoal treatment to eliminate pyrogens and by sterilization

Innovations in the production of Antibiotics Researchers describe an approach that is more efficient--and environmentally friendly--in developing new antibiotics, those needed to kill the increasing number of infections resistant to multiple drugs. One way to increase the number of antibiotics for fighting infections is to start where nature stopped and engineer the enzymes to produce new molecules, and thus new antibiotics The interdisciplinary team of scientists, including research professors David H. Sherman and Janet L. Smith from the University of Michigan's Life Sciences Institute and Fecik of University of Minnesota College of Pharmacy, is the first to crystallize an enzyme in the process of closing the antibiotic ring, which illustrates exactly how the ring is formed. Researchers describe a method that can be used to get the bacteria itself to produce new compounds that turn into the ring structure and may be useful as new drugs. Typical drug development involves chemical manipulations that result in chemical waste, which can be difficult to dispose of and is hazardous to the environment.

This research implies it is realistic to develop a more environmentally friendly way to discover more potential drug compounds with less chemical manipulation, and thus less chemical waste. BENEFITS OF ANTIBIOTICS Benefit to Animal Welfare or severely restricting the use of antimicrobials in animals may negatively impact a veterinarians ability to protect animal health and prevent suffering from disease, which can lead to poor animal welfare. Benefit to Food Safety Food safety begins with healthy animals, continues in the harvesting process with good hygiene and application of hazard analysis/critical control point regulations, and extends to the packing and handling of the food product in both the market and the home. All of these steps work in tandem, and skipping any one of them increases the risk of foodborne illness. Benefits of Antibiotics Use to Agriculture Antibiotics are used in animal feeds to increase animal weight, increase efficiency of feed utilization, increase reproductive efficiency and decrease morbidity and mortality. These benefits to animals and animal producers are reflected in decreases in food costs to humans. There are also benefits to human health from use of antibiotics in food animals. By reducing the incidence of animal health problems, use of antibiotics in food animals reduce the transference of animal infections to humans. Disadvantages of using antibiotics Heavy indiscriminate use of antibiotics in poultry (meat, dairy products) production may result in the transfer of residual antibiotics to human food and the transfer of resistant bacteria to humans. The " resistant gene " of mutant may be transferred to another pathogenic species thus spreading a health hazard. Impact to the environment Most compounds or at least most groups of compounds acting via the same mechanisms are found in hospital effluent and in some cases even in municipal sewage in concentrations that are high enough to warrant further risk assessment and risk management. The emission of antibiotics into the environment should be reduced as an important part of the risk management. For this reason, unused therapeutic drugs should not be flushed down the drain and physicians must be made aware that antibiotics are not completely metabolized by patients. The environmental significance of therapeutic drugs, disinfectaants and diagnostics should be included in the undergraduate curricula of medical students and pharmacists. Antibiotics act as an ecological factor in the environment that could potentially affect microbial communities. The effects include phylogenetic structure alteration, resistance expansion, and ecological function disturbance in the micro-ecosystem. Numerous studies have detected changes of microbial community structure upon addition of antibiotics in soil and water environment. Effects of antibiotics on ecological functions have also been discovered, including nitrogen transformation, methanogenesis, and sulfate reduction.

3. RECOMBINANT DNA TECHNOLOGY Also known as in vitro recombination, is a technique involved in creating and purifying desired genes. The process of in vitro recombination makes it possible to cut different strands of DNA with a restriction enzyme and join the DNA molecules together via complementary base pairing. Emerged as a response to the need for specific DNA segments in amounts sufficient for biochemical analysis. Recombinant DNA and biotechnology can be used to form proteins not normally produced in a cell. In addition, bacteria that carry recombinant DNA can be released into the environment to increase the fertility of the soil, serve as an insecticide, or relieve pollution. The technology of recombinant DNA has been made possible in part by extensive research on microorganisms during the last century. One important microorganism in recombinant DNA research is Escherichia coli (E. coli). The biochemistry and genetics of E. coli are well known, and its DNA has been isolated and made to accept new genes. The DNA can then be forced into fresh cells of E. coli, and the bacteria will begin to produce the proteins specified by the foreign genes. Such altered bacteria are said to have been transformed. Discovery of rdna technology Arthur Kornberg Replication of viral DNA in a breakthrough that served as a proof-of-concept for cloning. Werner Arber Discovery of restriction enzymes in a bacteria that degrade foreign viral DNA molecules while sparing their own DNA. Ligase could be used to glue DNA molecules. Herbert Boyer and Stanley Cohen founded the first company, Genetech, to work with rDNA in its drug development labs Insertion of rDNA into bacteria in such a way that the foreign DNA would replicate naturally Techniques Some of the molecular biology techniques utilized during recombinant DNA include: The study and/or modification of gene expression patterns Gene cloning DNA sequencing Creation of transgenic plants and animals

Processes A summary of the process of making recombinant DNA: Treat the DNA taken from both sources with the same restriction enzyme, which cuts both molecules at the same site. The overhanging single-stranded ends of the cut are called sticky ends, which pair with its complementary sticky end, when mixed. DNA ligase is used to covalently link the two strands into a molecule of recombinant DNA. The DNA is replicated many times.

Methods of introducing foreign dna Transformation Host cells are bacteria Non-bacterial transformation Host cells are not bacteria Phage Introduction Host cells are phage

Advantages of rdna technology Development of cure for genetic and acquired diseases Development of DNA vaccines Development of cure for infertility Development of genetically modified plants Has the potential to provide more food to the world Improved medicines Improved livestock Lowering the cost of medicines Typical problems The sharp bands give the wrong impression that the molecules are perfectly separated. The sticky ends can degrade. It is possible that the plasmids werent cut completely A small portion of the plasmid was not even cut at all.

Disadvantages of rdna technology Safety concerns Environmental concerns Ethical dilemmas Potential for experimental abuse Germline treatment going from treating diseases to a method for picking the traits you want in a child.

Concerns Economic and Access Concerns Environmental and Monitoring Concerns Allergenicity Antibiotic Resistance Transfer Concerns with Naturally Occurring Toxicants

Applications Recombinant vaccines Prevention and cure of sickle cell anemia Prevention and cure of cystic fibrosis Production of clotting factors Production of insulin Production of recombinant pharmaceuticals Plants Germ line and somatic cell gene therapy Application of rdna in some industries Agriculture Genetically modified crops, pesticide resistant crops Pharmacology Artificial insulin production, drug delivery to target sites Medicine Gene therapy, antiviral therapy, vaccination, synthesizing clotting factors Others: Fluorescent fishes, glowing plants, etc. Potential risks to humans, plants and animals Pathogenicity The potential ability of living organisms and viruses to cause disease in man, animals and plants. Safe handling of Bulk Quantities of Micro-organisms Safety of Biologically Active Products Environmental concerns Altered plants pollinating nearby plants. It could change some characteristics of said plants Products Insulin Human growth hormone Tissue plasminogen activator Interferon Vaccines