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Monday, August 29, 2011

Artificial Blood and Controversy

Pharmaceutical companies developed a few varieties of artificial blood in the 1980s and 1990s, but many abandoned their research after heart attacks, strokes and deaths in human trials. Some early formulas also caused capillaries to collapse and blood pressure to skyrocket. However, additional research has led to several specific blood substitutes in two classes -- hemoglobin-based oxygen carriers (HBOCs) and perflourocarbons (PFCs). Some of these substitutes are nearing the end of their testing phase and may be available to hospitals soon. Others are already in use. For example, an HBOC called Hemopure is currently used in hospitals in South Africa, where the spread of HIV has threatened the blood supply. A PFC-based oxygen carrier called Oxygent is in the late stages of human trials in Europe and North America.

artificial blood

The two types have dramatically different chemical structures, but they both work primarily through passive diffusion. Passive diffusion takes advantage of gasses' tendency to move from areas of greater concentration to areas lesser concentration until it reaches a state of equilibrium. In the human body, oxygen moves from the lungs (high concentration) to the blood (low concentration). Then, once the blood reaches the capillaries, the oxygen moves from the blood (high concentration) to the tissues (low concentration).

Artificial Blood Controversy

At first glance, artificial blood seems like a good thing. It has a longer shelf life than human blood. Since the manufacturing process can include sterilization, it doesn't carry the risk for disease transmission. Doctors can administer it to patients of any blood type. In addition, many people who cannot accept blood transfusions for religious reasons can accept artificial blood, particularly PFCs, which are not derived from blood.
However, artificial blood has been at the center of several controversies. Doctors abandoned the use of HemAssist, the first HBOC tested on humans in the United States, after patients who received the HBOC died more often than those who received donated blood. Sometimes, pharmaceutical companies have had trouble proving that their oxygen carriers are effective. Part of this is because artificial blood is different from real blood, so it can be difficult to develop accurate methods for comparison. In other cases, such as when artificial blood is used to deliver oxygen through swollen brain tissue, the results can be hard to quantify.
Another source of controversy has involved artificial blood studies. From 2004 to 2006, Northfield Laboratories began testing an HBOC called PolyHeme on trauma patients. The study took place at more than 20 hospitals around the United States. Since many trauma patients are unconscious and can't give consent for medical procedures, the Food and Drug Administration (FDA) approved the test as a no-consent study. In other words, doctors could give patients PolyHeme instead of real blood without asking first.
Complete details on Artificial Blood and how it works can be found in the below link

Tuesday, August 23, 2011

Enzyme used in Fruit Juice Industry

One of the major problems in the preparation of fruit juices and wine is cloudiness due primarily to the presence of pectins. These consist primarily of a-1,4-anhydrogalacturonic acid polymers, with varying degrees of methyl esterification. They are associated with other plant polymers and, after homogenisation, with the cell debris. The cloudiness that they cause is difficult to remove except by enzymic hydrolysis. Such treatment also has the additional benefits of reducing the solution viscosity, increasing the volume of juice produced (e.g. the yield of juice from white grapes can be raised by 15%), subtle but generally beneficial changes in the flavour and, in the case of wine-making, shorter fermentation times. Insoluble plant material is easily removed by filtration, or settling and decantation, once the stabilising effect of the pectins on the colloidal haze has been removed.
Commercial pectolytic enzyme preparations are produced from Aspergillus niger and consist of a synergistic mixture of enzymes:
  1. polygalacturonase (EC, responsible for the random hydrolysis of 1,4-a-D-galactosiduronic linkages;
  2. pectinesterase (EC, which releases methanol from the pectyl methyl esters, a necessary stage before the polygalacturonase can act fully (the increase in the methanol content of such treated juice is generally less than the natural concentrations and poses no health risk);
  3. pectin lyase (EC, which cleaves the pectin, by an elimination reaction releasing oligosaccharides with non-reducing terminal 4-deoxymethyl-a-D-galact-4-enuronosyl residues, without the necessity of pectin methyl esterase action; and
  4. hemicellulase (a mixture of hydrolytic enzymes including: xylan endo-1,3-b-xylosidase, EC; xylan 1,4-b-xylosidase, EC; and a-L-arabinofuranosidase, EC, strictly not a pectinase but its adventitious presence is encouraged in order to reduce hemicellulose levels.
The optimal activity of these enzymes is at a pH between 4 and 5 and generally below 50 degree Celcius. They are suitable for direct addition to the fruit pulps at levels around 20 U l-1 (net activity). Enzymes with improved characteristics of greater heat stability and lower pH optimum are currently being sought.
In brewing, barley malt supplies the major proportion of the enzyme needed for saccharification prior to fermentation. Often other starch containing material (adjuncts) are used to increase the fermentable sugar and reduce the relative costs of the fermentation. Although malt enzyme may also be used to hydrolyse these adjuncts, for maximum economic return extra enzymes are added to achieve their rapid saccharification. It not necessary nor desirable to saccharify the starch totally, as non-fermentable dextrins are needed to give the drink 'body' and stabilise its foam 'head'. For this reason the saccharification process is stopped, by boiling the 'wort', after about 75% of the starch has been converted into fermentable sugar.
The enzymes used in brewing are needed for saccharification of starch (bacterial and fungal a-amylases), breakdown of barley b-1,4- and b-1,3- linked glucan (b-glucanase) and hydrolysis of protein (neutral protease) to increase the (later) fermentation rate, particularly in the production of high-gravity beer, where extra protein is added. Cellulases are also occasionally used, particularly where wheat is used as adjunct but also to help breakdown the barley b-glucans. Due to the extreme heat stability of the B. amyloliquefaciens a-amylase, where this is used the wort must be boiled for a much longer period (e.g. 30 min) to inactivate it prior to fermentation. Papain is used in the later post-fermentation stages of beer-making to prevent the occurrence of protein- and tannin-containing 'chill-haze' otherwise formed on cooling the beer.
Recently, 'light' beers, of lower calorific content, have become more popular. These require a higher degree of saccharification at lower starch concentrations to reduce the alcohol and total solids contents of the beer. This may be achieved by the use of glucoamylase and/or fungal a-amylase during the fermentation.
A great variety of carbohydrate sources are used world wide to produce distilled alcoholic drinks. Many of these contain sufficient quantities of fermentable sugar (e.g. rum from molasses and brandy from grapes), others contain mainly starch and must be saccharified before use (e.g. whiskey from barley malt, corn or rye). In the distilling industry, saccharification continues throughout the fermentation period. In some cases (e.g. Scotch malt whisky manufacture uses barley malt exclusively) the enzymes are naturally present but in others (e.g. grain spirits production) the more heat-stable bacterial a-amylases may be used in the saccharification.


Gateway Cloning

Gateway® Cloning is a universal cloning technique developed by Invitrogen life technologies. Gateway® Cloning Technique allows transfer of DNA fragments between different cloning vectors while maintaining the reading frame. It has effectively replaced the use of restriction endonucleases and ligases. Using Gateway®, one can clone/sub clone DNA segments for functional analysis.

Gateway® Cloning Mechanism

(a) The BP Reaction (PCR fragment + Donor vector = Entry Clone)

The BP Reaction is a recombination reaction which is explained in the following lines. For the reaction to take place, the gene of interest is amplified with the help of an attB tagged primer pair. The donor vector includes attP sites. The PCR product that includes the attB sites combines with the donor vector that includes the attP sites resulting in the formation of an entry clone. This integration reaction between the attB and the attP sites forms the basis of this reaction. The resulting entry clone contains the gene of interest flanked by attL sites.

(b)The LR reaction (Entry Clone + Destination Vector = Expression Clone)

The LR Reaction, again is a recombination reaction between attL and attR sites. The reaction generates an expression clone and is catalyzed by recombinant proteins. The entry clone generated from the BP reaction includes the attL sites. The Destination vector is designed to include the attR sites. The LR reaction is carried out to transfer the sequence of interest to one or more destination vectors in simultaneous reactions, making the technology high throughput. The entry clone is mixed with the appropriate Gateway® vector and Gateway® Clonase enzyme. Recombination between these sites generates two molecules. One molecule contains the DNA segment of interest, the other molecule is a by-product.

  1. Allows subcloning from one vector backbone to another.
  2. Every subcloning reaction maintains the appropriate reading frame.
  3. Subcloning process is fast and facilitates reaction automation.
  4. Supports site specific recombination.
  5. Multiple genes can be transferred to one or more vectors in one experiment.


Friday, August 19, 2011

Cell Immobilization with Calcium Alginate


To investigate the conversion of glucose to ethanol by entrapped yeast cells in a continuous reactor.


This experiment is all about an immobilized cell fermentor. Yeast cells will be entrapped in calcium alginate gels by using the similar techniques as in enzyme immobilization. Other cell entrapment media that have been previously attempted include polyacrylamide, gelatin, chitosan, and k-carrageenan gels.
Due to the constraint in the available equipment to carry out the immobilization procedure aseptically, the experiment will be conducted without autoclaving. The immobilized cell reactor will be employed to convert glucose into ethanol anaerobically. The reasons for choosing this system of microorganism and product are many folds. First, the anaerobic condition will eliminate the need for aeration, which causes many technical problems. Secondly, the lack of oxygen will prevent the uncontrolled growth of aerobic contaminants in an unsterilized fermentor. The presence of high levels of ethanol should also discourage most microorganisms from taking over the fermentor. To reduce further the chance of contamination by bacteria, the pH of the fermentor will be kept low; a value of 4.0 should drastically slow down the growth of most bacteria but only slightly affect the yeast's ethanol producing capacity.
The production of ethanol in an immobilized bioreactor is a relatively well studied process. As high as 95% of the theoretical yield of alcohol based on glucose (8.5 % ethanol from 14% glucose) has been reported. A high space velocity, defined as the volume of nutrient feed per hour per gel volume, of 0.4-0.5 hr-1 is commonly used to maximize the ethanol productivity. An ethanol productivity of 20 g/l-hr can be achieved.
Both the steady state response and the transient approach to the steady state will be studied in this experiment .

List of Reagents and Instruments

A. Equipment

  • Beakers
  • Graduated cylinder
  • Balance
  • Pipets
  • Magnetic stirrer
  • Syringe & needle
  • Spectrophotometer with flow through cell
  • pH probe and controller
  • Microcomputer with data acquisition capabilities

B. Reagents

  • Growth medium, see the recipe in Experiment No. 9.
  • Alginic acid, sodium salt
  • CaCl2
  • Yeast culture
  • NH4OH
  • Reagents for glucose analysis
  • Reagents for ethanol analysis (or a GC)


  1. Immobilized Cell Preparation:
    • Dissolve 9 g of sodium alginate in 300 ml of growth medium, following the same procedure adopted in enzyme immobilization to avoid clump formation. Stir until all sodium alginate is completely dissolved. The final solution contains 3% alginate by weight. See Note 1.
    • Thoroughly suspend about 250 g of wet cells in the alginate solution prepared in the previous step. Let air bubbles escape. See Note 2.
    • Drip the yeast-alginate mixture from a height of 20 cm into 1000 ml of crosslinking solution. (The crosslinking solution is prepared by adding an additional 0.05M of CaCl2 to the growth media. The calcium crosslinking solution is agitated on a magnetic stirrer. Gel formation can be achieved at room temperature as soon as the sodium alginate drops come in direct contact with the calcium solution. Relatively small alginate beads are preferred to minimize the mass transfer resistance. A diameter of 0.5-2 mm can be readily achieved with a syringe and a needle. The beads should fully harden in 1-2 hours. Note that the concentration of the CaCl2 is about one fourth of the strength used for enzyme immobilization.
    • Wash the beads with a fresh calcium crosslinking solution.
  2. Immobilized Cell Reactor Construction:
    • Construct an immobilized cell reactor with a 500ml Erlenmeyer flask. Place the hardened beads in the flask and seal it with a rubber stopper with appropriate hose connections.
    • Make all necessary connections. Start the experiment by filling the flask with the growth media (100g/l glucose) to the working volume of 350ml.
  3. Immobilized Cell Reactor:
      Then following sequence of events will be monitored both on-line and off-line. The responsibilities of on-line data acquisition and off-line sample collection and analysis will be shared by the entire class; the exact assignment will be determined in class. A microcomputer will be programmed to take data on the glucose concentration and the rate of NH4OH addition needed to maintain the pH at 4.0. The off-line samples will be analyzed for the optical density (for free cell concentration), glucose concentration, and ethanol concentration. Furthermore, the liquid and gas flow rate will be measured with a graduated cylinder as indicated in Figure 2.
    • The reactor will be operated in a batch manner until no more glucose is utilized. This can be detected with the leveling off in the glucose concentration.
    • Substrate feeding will then commence at the rate of 0.4/hr. Record the substrate flow rate. The approach to the first steady-state during the start-up will be followed.
    • Various parameters (nitrogen consumption rate, carbon dioxide evolution rate, glucose concentration, ethanol concentration, and free cell level) at the high steady state are recorded.
    • Decrease the substrate feeding rate to 0.2 /hr Measure the substrate flow rate and follow the transient approach to the new low steady state.
    • Repeat part 2c) for the new steady state.
  4. If time permits, continue shifting the flow rate and obtain more information on steady states. Continue operating the bioreactor until noticeable deterioration in the performance is detected due to gel swelling, cell death, or severe contamination.


  1. To avoid the premature gel formation, the phosphate concentration in the medium must be adjusted to less than 100┬ÁM.
  2. Because cell growth can break the bead and is generally considered undesirable beyond what is needed to compensate for the endogenous decay, the cells used for immobilization ideally should have just entered the stationary phase. An equivalent amount of dried cell culture may also be used in lieu of wet cell paste. The actual cell loading may be varied according to the substrate concentration in the feed and the desired product levels. The ratio of wet weight to dry weight is approximately 4 for most cells.


Basically, immobilization of live cells is very similar to the enzyme counterpart. In the past, various cells have been immobilized: bacteria, yeasts, fungi, plant tissues, mammalian tissues, and insect tissues. However, true successes are limited to only a few cases. One of the problems is the mass transfer resistance imposed by the fact that the substrate has to diffuse to the reaction site and inhibitory or toxic products must be removed to the environment. Oxygen transfer is often the rate limiting step in a suspended cell culture, and it is more so in an immobilized cell culture. Oxygenation in an immobilized cell culture is one of the major technical problems that remain to be solved. In light of the oxygenation problems, immobilization techniques have been mainly confined to anaerobic processes in which either obligate (strict) anaerobes are employed or only the anaerobic components of the facultative metabolic mechanisms are selectively utilized.
The lower microorganisms (bacteria, yeasts, and fungi) can be rather easily immobilized with a number of methods: entrapment, ion exchange adsorption, porous ceramics, and even covalent bonding. In terms of dollar values, chemicals of plant origin account for the lion's share of the market. Some examples of plant extracts are drugs, flavors, and perfumes. Despite the recent surge in research activities in animal cell culture throughout the country, few applications actually exist beyond the production of monoclonal antibodies. Immobilized insect tissues have been used in pesticide research and has a potentially quite large commercial market in agriculture.
Most of the principles involved in enzyme immobilization are directly applicable to cell immobilization. Covalent bonding, affinity bonding, physical adsorption, and entrapment in synthetic and natural polymer matrices. The most popular and practical immobilization technique deals with cell recycle with an ultrafiltration membrane or a hollow fiber cartridge. Although this process is not ordinarily viewed as cell immobilization at all, it is functionally equivalent, the cell recycle devices effectively retaining the catalysts in a bioreactor and accomplishing the same objective as cell immobilization.
An immobilized cell bioreactor is well suited for those cells whose growth phases and product formation phases are uncoupled. Cell biomass and primary metabolites are growth associated products, but secondary metabolites such as antibiotics and various enzymes are produced during the stationary phase. The uncoupling of the phases means that productive cells cannot compete with the nonproductive cells in a continuously operated suspension fermentor because the productive cells spend the nutritional and energy resources producing chemicals in quantities far above the amount necessary for their survival, instead of reproducing themselves to propagate further. On the contrary, cell growth in an immobilized cell reactor must be severely limited if gel swelling or breakage is to be avoided. However, once the cells are immobilized, the cell viability must be concomitantly sustained over a long period of time. Thus, immobilization is advantageous for sustaining slowly growing cells, especially plant tissues. In summary, one wishes to keep the immobilized cells alive without multiplying.


  1. Mattiasson, Bo Immobilized Cells and Organelles, Volume I and II, CRC Press, 1983.
  2. Venkatsubramanian, K., Immobilized Microbial Cells, in ACS Symposium Series, 106, American Chemical Society, Washington, D.C., 1979.
  3. Nagashima, M., Azuma, M., and Noguchi, S., Technology developments in biomass alcohol production in Japan: continuous alcohol production with immobilized microbial cells, Ann. N.Y. Acad. Sci., 413, 457, 1983.

Enzyme Purification by Isoelectric Precipitation


To recover proteins/enzymes from a solution by changing the pH of the solution.


The solubility of protein depends on, among other things, the pH of the solution. Similar to the amino acids that comprise protein, protein itself can be either positively or negatively charged overall due to the terminal amine -NH2 and carboxyl (-COOH) groups and the groups on the side chain. It is positively charged at low pH and negatively charged at high pH. The intermediate pH at which a protein molecule has a net charge of zero is called the isoelectric point of that protein. In general, the net charge on the protein, either positive or negative, can interact with water molecules, meaning that it is more likely for a protein molecule to dissociate itself from other protein molecules, thus, more soluble. As a result, protein is the least soluble when the pH of the solution is at its isoelectric point.

                                        The isoelectric point (pI) is the pH of a solution at which the net primary charge of a protein becomes zero. At a solution pH that is above the pI the surface of the protein is predominantly negatively charged and therefore like-charged molecules will exhibit repulsive forces. Likewise, at a solution pH that is below the pI, the surface of the protein is predominantly positively charged and repulsion between proteins occurs. However, at the pI the negative and positive charges cancel, repulsive electrostatic forces are reduced and the attraction forces predominate. The attraction forces will cause aggregation and precipitation. The pI of most proteins is in the pH range of 4-6. Mineral acids, such as hydrochloric and sulfuric acid are used as precipitants. The greatest disadvantage to isoelectric point precipitation is the irreversible denaturation caused by the mineral acids. For this reason isoelectric point precipitation is most often used to precipitate contaminant proteins, rather than the target protein. The precipitation of casein during cheesemaking, or during production of sodium caseinate, is an isoelectric precipitation.
When microorganisms grow in milk, they often produce acids and lowers the pH of the milk. The phenomenon of precipitation or coagulation of milk protein (casein) at low pH as milk becomes spoiled is one of the common examples of protein isolation due to changes in the pH.

List of Reagents and Instruments

A. Equipment

  • Test tubes
  • Graduated cylinder
  • Pipets
  • Balance
  • Centrifuge
  • Filtration devices

B. Reagents

  • Protein solution, 5.0 g/l (albumin, gelatine, casein)
  • Enzymes solution, 10 g/l (alpha-amylase, protease)
  • NaOH or KOH solution, 1N
  • Acetic Acid solution, 0.1N


  1. Precipitation of Protein in Acidified Solution:
    • Add 5.0 g of casein to 200 ml of 1N NaOH solution.
    • Pipet 4 ml of the protein solution into a test tube.
    • While stirring, add the acid solution drop-wise to the alkaline protein solution from a graduated pipet or a buret until precipitates start to form. Stir thoroughly to avoid the localization of low pH spots in the solution. Note the volume of the acid solution added at the incipient of precipitation. Since precipitation is not an instantaneous process, let the test tube stand undisturbed for 30 minutes.
    • Repeat the same process for a series of test tubes, each containing 4 ml of the alkaline protein solution. To each test tube, add slightly less acid solution than the previous one so that a series of pH values can be established. Let each test tube stand for 30 minutes. Measure the pH of each solution and note the pH region around which the amount of precipitate is the maximum