Prepared by
Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111

Table of Contents


To hydrolyze protein-based stains in fabrics into soluble amino acids.


In today's laundry detergents, enzymes such as proteases and amylases are some of the active ingredients. In the U.S., about 50% of liquid detergents, 25% of powder detergents, and almost all powdered bleach additives now contain enzymes to help break down stains that are otherwise hard to remove with conventional surfactants alone. For example, amylase catalyzes the breakdown of starch-based stains to smaller segments that make up the larger starch molecule. Oligosaccharides and dextrins released from the enzyme's hydrolytic action are soluble; thus, the stain is physically cut off from the surface of the fabric piece by piece, with the enzyme acting as scissors. The action of proteases, as implied by the name itself, is similar to that of amylase, except that a large protein molecule is hydrolyzed. During the process of hydrolysis, the peptide bonds that hold various amino acids together to form a protein molecule are broken down, releasing smaller polypeptides and individual amino acid units. Generally, polymers made of less than approximately one hundred amino acid monomer units are called polypeptides and larger ones are called proteins.

In this experiment, the same milk protein casein that we have experienced in the previous experiment on cheese making is bound with a blue dye. A small piece of fabric soiled by the dyed casein will be provided to each student. The purpose of this experiment is to observe the hydrolytic action of bacterial protease in removing protein-based stains. Because detergents, especially bath soaps, are generally formulated to degrade mainly oil and grease, protein-based stains have traditionally been among the hardest to remove. Proteins can act as strong natural bonding agents that make all sorts of dirt adhere stubbornly to textile fibers. Anyone trying to wash away blood stains can testify to this effect. Other proteinaceous dirt includes perspiration, grass, and slime stains. This exercise demonstrates that it takes protein to get out protein, as some television commercials claim.

List of Reagents and Instruments

A. Equipment

B. Reagents


  1. Dissolve 1 g of household laundry detergent in 200 ml of hot (60ºC) water in a 250 ml flask. Colored detergent obviously interferes with the quantitative determination of the rate of protein degradation by the colorimetric method; thus, it should not be used if quantitative information is desired. Because the commercial detergent is not completely soluble in water, filter the detergent solution before use.
  2. Similarly, mix 0.1 g of powder protease in 200 ml of hot water in a second flask. Because certain protease is not water soluble, removal of the insoluble solids by filtration or centrifugation, leaving behind only supernatant, may retain only very little protease activity.
  3. Place a small piece of the dyed casein or gelatin cloth into each washing liquid.
  4. Seal the top of each flask with paraffin or with a rubber stopper. Swirl both flasks gently to simulate the agitating washing motion. A thermostated flask shaker may be used for this purpose.
  5. Periodically take out a small (5-10 ml) portion of the washing liquid and measure the absorbance with a spectrophotometer until the changes in the color intensity level off. The stain should be removed in approximately 20-30 minutes, but sometimes it takes as long as a few hours for the complete removal of the color. Because of the small particles of protein released during the wash, one may need to filter the sample again with a 25mm syringe filtration unit fitted with a 10 ml plastic syringe. This should be done immediately before measuring the absorbance.
  6. After briefly rinsing the cloth under running tap water, dry it with a clothing iron.
  7. Compare the effectiveness of the protease with your detergent. Also compare the cloth washed with your detergent with the other students' to see how effective your detergent is in cutting the stain.
  8. Simulate a "warm" (40ºC) and a "cold" (20ºC) wash cycle by repeating the above experiment at appropriate temperatures. (Note that all the temperatures can be run simultaneously.)


An enzyme was first used to improve the effectiveness of a laundry detergent in 1913 by a German named Otto RÖohm, the founder of the giant chemical company Rohm and Hass. The proteolytic enzyme he used, derived from milled animal pancreases, was quite crude and contained many impurities which, in turn, sometimes stained the very textile it was supposed to clean. Neither was the process of enzyme extraction economical enough to include it routinely in household detergents. Currently, these enzymes are manufactured commercially in large quantities through fermentation by common soil bacteria Bacillus subtilis or Bacillus licheniformis. This was made possible in the last two decades by the rapid advances in enzymology and fermentation technology. Although numerous other microorganisms produce proteases and amylases, the types secreted by the above strains have the advantage that they work best at the warm alkaline conditions prevailing in washing liquids. They also must not lose their activity in an environment which contains a multitude of potentially inhibitory chemicals routinely formulated into laundry detergents such as surface active agents, magnesium or calcium ions, builders (sodium tripolyphosphate), perfumes, and other additives.


  1. Plot the color intensity as a function of time for both washing liquids. Which one has stronger cleaning power, your detergent or protease? Explain the temperature effect.
  2. From the above curve, comment on the possible rate expression for the enzymatic reaction.
  3. How would you determine the amount of protein removed if no dye was used?
  4. How does the temperature affect the rate of stain removal?
  5. List some of the biological functions of the pancreas. What type of digestive fluid does it secrete? Is it surprising that one may obtain protease from milled dehydrated and defatted animal pancreas?
  6. List ways in which enzymes can be utilized to break down toxic wastes, thus controlling pollution. Are there any commercial processes at the present that utilize the unique selective catalytic capability of an enzyme in waste treatment?
  7. There is a great concern over the phosphate content in a detergent because of the problems of eutrophication, the almost complete depletion of dissolved oxygen in a body of water resulting from the explosive growth of marine microorganisms in the presence of excess nutrient. Do you consider the enzymes contained in the laundry liquid as water pollutants? What about the possibility of the denatured protein as a source of scarce amino acids? Justify your answer.
  8. All enzymes are proteins, and protease is no exception. Does protease attack each other canabalistiaclly? If not, what prevents protease from digesting each other? (Hint: does protease digest all proteins, or can it recognize only some and exerts its action on only specific ones?)
  9. Comment on ways to improve the experiment.


  1. Duffy, J.I., Chemicals by Enzymatic and Microbial Processes, Noyes Data Corp., New Jersey, 1980, p368-373.

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Enzymes in Laundry Detergents
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Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111
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