Enzymes: New, Fresh and Better Than Ever : Protein Focus of Major Research
After nearly a decade of disuse, enzymes that once removed food and grass stains from clothing are making a comeback in laundry detergents.
These naturally occurring proteins, which carry out specific chemical reactions, were largely phased out in the early 1970s because they caused skin irritation among workers at detergent factories.
But scientists never quite gave up on enzymes. They eventually figured out a way to encapsulate these enzymes so that they could be added to powdered detergents without coming into direct contact with workers. Meanwhile, though, liquid detergents have become vastly popular, presenting scientists with a new problem: When mixed with liquids, the enzymes break down and become useless.
To overcome that problem, scientists now are tinkering with the structure of the enzymes, trying to produce new molecules that will be more resistant to the effects of heat, acidity and long-term storage.
The impact of such protein engineering extends far beyond the detergent industry. Protein engineers also are attempting to redesign enzymes to carry out chemical reactions that nature never intended.
One company, for example, is trying to alter an enzyme so that it would destroy nerve gases stockpiled for chemical warfare. Another, Amgen Inc. of Thousand Oaks, has made custom-designed forms of interferon and interleukin that seem to have greater effectiveness against certain types of cancer than their natural counterparts.
No Longer a Stepchild
Protein engineering, in which biologists use the techniques of genetic engineering to change the characteristics of specific molecules within the organisms, long has been considered something of a stepchild of genetic engineering. But no more.
In the last two to three years, the field has begun to emerge as a powerful force in its own right. And scientists today are speaking of custom-made “designer proteins” in the same wide-eyed manner that they recently talked about designer genes. In genetic engineering, scientists manipulate DNA to alter the characteristics of organisms or to give them new capabilities.
Designer enzymes “promise to be of great use in producing chemicals, foods, drugs and fuels,” according to biochemist Alexander M. Klibanov of the Massachusetts Institute of Technology. “They can be used to break down industrial waste and analyze chemicals. Indeed, virtually any imaginable chemical process can be (carried out) by some enzyme. . . .”
Many biotechnology companies that once devoted themselves exclusively to genetic engineering, such as Genentech, Genex and Amgen, have beefed up their protein engineering departments or created new subsidiaries to study the technology.
Research Groups Created
Industrial giants such as DuPont have also created new research groups to study protein structure and modification. “Every major drug or chemical company involved in biotechnology is now at least putting a big toe in the water,” said biochemist Kevin M. Ulmer, director of the new Center for Advanced Research in Biotechnology in Shady Grove, Md., which is devoted primarily to protein engineering.
Interest in protein engineering is not restricted to the United States. In 1986, Japan’s Ministry of International Trade and Industry, for example, initiated an eight-year, 30-billion-yen ($189 million) research program in the area.
“My impression is that the field as a whole is where genetic engineering was in 1976,” Ulmer said. “We have numerous proofs that the principle is sound and that the technology works, but we haven’t had the first big commercial breakthrough to really get things moving.”
The still-young biotechnology industry has learned a lot from its past experiences, Ulmer said, and when the big breakthrough occurs, “I think protein engineering will move forward much faster than genetic engineering did.”
DNA, or deoxyribonucleic acid, is often called the blueprint of life. By that analogy, proteins are the brick and mortar from which organisms are constructed--as well as the masons and carpenters who put the organisms together and the maintenance crew that keeps them running.
Proteins are composed of tens to hundreds of individual amino acids strung together like beads on a necklace and then folded like origami into intricate balls. They are a major structural component of body tissues.
Others of the roughly 1 million proteins thought to occur in nature are enzymes, which carry out all the chemical reactions necessary for manufacturing everything in the body as well as for converting food into energy.
Antibodies, which play a major role in fighting off infections and disease, also are proteins.
Potential Target
Each type of protein represents a potential target for protein engineers, but the greatest interest at the moment revolves around enzymes. Enzymes are catalysts that carry out specific chemical reactions without themselves being consumed in the process. A very small amount of an enzyme in yeast, for instance, converts sugar in dough into the large volume of carbon dioxide that gives bread and rolls their characteristic texture.
Industrial chemists eye enzymes longingly because of two traits: They typically carry out only one reaction and produce only one product, and they operate under very mild conditions of temperature and acidity, essentially the same conditions found in the body.
Chemical Byproducts
Virtually all conventional industrial chemical reactions, in contrast, produce many unwanted byproducts in addition to the desired product, so that valuable starting materials are wasted and expensive separation techniques must be employed.
Amino acids, for example, are widely used as food additives, in animal feed and in medicine. Each amino acid exists in two forms, called D and L, that have identical chemical compositions, but that bear the same relationship to each other that a left-handed glove bears to a right-handed glove.
The L-form has nutritive value and is useful in medicine. The D-form, in contrast, has no nutritive value and is useless. Conventional chemical reactions produce identical amounts of the D- and L-forms, so that expensive separation procedures must be used to isolate the L-form and half the starting material is wasted.
Enzymes produce only the L-form.
Problems Could Be Avoided
Many industrial chemical reactions are also carried out at high temperatures and pressures, necessitating the use of expensive containment vessels and significant amounts of energy.
Those problems could be avoided and costs reduced by using enzymes.
Though thousands of enzymes have been identified in nature, only 16 are isolated from microorganisms in significant quantities, while another 200 are isolated in limited quantities, according to Klibanov. If the specific reaction that a chemist is interested in is not carried out by one of these 216 enzymes, he is out of luck because the other enzymes are going to be much more expensive to obtain.
Or, as is the case with detergent enzymes, the enzymes may not function properly if they are used under adverse conditions.
Altering the Structure
What protein engineers would like to do in the short term is change the structure of an enzyme slightly so that it can better stand up to adverse conditions. In the long run, they would like to take one of these readily available, inexpensive enzymes--or some other cheap protein--and change its structure even more so that it carries out a completely different reaction than the one nature designed it for.
The most common way to engineer proteins is to change the identity of one or more amino acids in the molecule. This can readily be done by making small changes in the DNA that codes the protein.
Biochemist David A. Estell and his colleagues at Genencor, a South San Francisco-based subsidiary of Genentech and Corning Glass Works, have developed a technique to replace any amino acid in an enzyme with each of the 19 other common amino acids, producing 19 new proteins. This process takes about three weeks, but determining the characteristics of each new variant takes longer.
They have used this technique to make and study more than 80 different variants of the bacterial enzyme subtilisin, a well-studied enzyme that breaks down proteins and is very similar to the enzymes used in detergents. In the process, they have found several variants that are more resistant to heat than the natural enzyme, can be stored longer, or can function under more alkaline conditions.
Estell has subsequently produced equivalent versions of the commercial enzyme and these may find their way into detergents.
More important, however, is what the researchers have learned.
Already, Estell noted, they are applying the knowledge they have gained about subtilisin to engineer another enzyme--which he will not name--so that it will convert an inexpensive fat such as palm oil into higher-priced fats such as those in cocoa butter, which is used in candy and other foods. Such an enzyme, Estell said, would have great value in manufacturing such foods because it would reduce the cost of the raw materials.
An Intuitive Process
Deciding which amino acids in a protein to alter is still very much of an intuitive process. “It’s an imperfect science,” added biochemist Phil Whitcome of Amgen. “At best, it is like looking through a fog bank and trying to see what is inside.”
To eliminate some of the uncertainties, chemist Arieh Warshel of USC is developing a computer program to predict the effect of changes more precisely. But the experimental approach is time consuming, requiring about 10 hours on a Cray super computer, and very expensive. But Warshel is confident that once his approach has been proved, “people will build computers (designed specifically) for this type of calculation and they will be much cheaper than the Cray.”
A new and completely different way to make designer proteins has been used by teams at University of California, Berkeley, and at Scripps Clinic in La Jolla. Scientists reported in December that they have made antibodies that catalyze chemical reactions just like enzymes.
And chemists at Genex Corp. in Gaithersburg, Md., have used protein-engineering techniques to construct synthetic antibodies that are much smaller and more stable than naturally occurring antibodies. Such artificial antibodies might be more useful in fighting diseases such as cancer than the highly touted monoclonal antibodies now made by the biotechnology industry because the synthetic antibodies are smaller and, hence, less likely to set off an immune reaction.
