Natural antifreeze proteins for increased cold resistance

The unpalatable ice crystals that give ice cream and other frozen foods a coarse texture or a rock-hard consistency may one day be freezer pests of the past thanks to naturally occurring antifreeze.

Researchers at the Protein Engineering Network of Centres of Excellence (PENCE) are studying and enhancing antifreeze proteins (AFPs), the tiny cellular constituents that have been found to inhibit the growth of ice crystals in various organisms.

These remarkable proteins can mean the difference between life and death for many species. They were first discovered approximately 30 years ago as an explanation for the ability of cold water fish like flounders and cod to survive the mere 1°C difference in freezing temperature between their blood and the seawater. Since then, researchers have also found AFPs in insects and plants that encounter sub-zero temperatures, such as moths, beetles, and certain varieties of grass.

Dr. Peter Davies, whose research is funded in part by the Medical Research Council (MRC), is the head of the PENCE antifreeze project and a professor of biochemistry at Queen's University. He explains that the AFPs from all of these different organisms share a common ability: they bind to the surface of ice and make it harder for water to join to the crystal, thus preventing its growth.

Because ice is thermally unstable, no one has actually observed this surface interaction. Researchers suggest that rather than completely covering the surface, the bound AFPs resemble buttons on a mattress. In their absence, the crystal would expand rapidly.

One consequence of AFP-binding is "thermal hysteresis," or the lowering of the freezing point of an organism. While fish AFPs can produce between 1^° and 1.5^°C of thermal hysteresis, antifreeze from moths and beetles is considerably more potent, allowing these insects to resist freezing at temperatures 5^°C below the normal freezing point.

But even organisms that produce high concentrations of AFPs can only resist freezing down to certain temperatures. Fortunately, AFPs also work in the frozen state to keep ice crystals small, thus limiting freezing injury and enabling organisms to tolerate colder conditions. This feature holds great promise for the food industry.

Dr. Davies explains that while periodic rises in temperature are useful to prevent the formation of frost in frost-free freezers, they also lead to the undesirable creation of ice crystals in frozen foods.

"When you freeze beans or ice cream or waffles you're not actually looking at one big ice crystal but at a lot of little ice crystals," he says. "As you get closer to the melting temperature, you get a little bit of fluid water even within the frozen product. This redistribution of water is actually what helps the small crystals grow into big ones. The bigger crystals can do more damage to the tissue; they can lead to quite a lot of spoilage of the food products or to larger ice crystals that are less palatable, in ice cream for example."

Ice cream and frozen yogurt are in fact two of the lead products for the incorporation of AFPs. As an additive for improved shelf-life, lower storage temperatures and better textural qualities, AFPs could generate sales in the multi-million dollar range.

AFPs could also increase frost and freeze tolerance for crops. In Canada, losses due to cold, frost and freezing represent an average of 10% to 15% of total crop insurance indemnities, or a sum of approximately $800 million to $1,200 million since 1960. The potential benefits from increased crop protection and the extension of the growing season are particularly attractive features of natural antifreeze.

Several companies have already expressed an interest in the properties of AFPs. The Canadian/US biotechnology company A/F Protein Inc. has joined forces with PENCE in a $190,000 collaboration that will accelerate both the development and the eventual marketing of antifreeze proteins.

Dr. Michael Erisman, Vice President of Business Development at A/F Protein Inc., estimates that his company has sold over a million dollars worth of the proteins.

"We have done that essentially at cost," he says. "We are trying to make AFPs available to the corporate and academic research communities to better understand the dynamics of how these proteins operate, so that we can attempt to develop commercial applications."

In one project, the company is investigating the use of AFPs in topically applied creams that would protect the skin from cold weather damage like frostbite.

Dr. Erisman estimates that the food and cosmetic applications of AFPs could be available within three years. But he adds that development is a relatively long-term and unpredictable process. Any attempts to undertake a successful commercialization of the AFP technology must be preceded by several considerations.

So far, AFPs have been obtained almost exclusively from fish. As Dr. Davies points out, not only is that a messy and expensive task, but it is dependent on a limited supply of fish as well. These are some of the factors behind the growing interest in both the optimization of naturally occurring AFPs, and the production of synthetic ones. PENCE researchers have progressed rapidly in both of these areas since the project's inauguration in 1995.

"We're looking for ways to make a cheaper and in some cases a better antifreeze protein," says Dr. Davies. "If you can learn from a better natural antifreeze what it is that makes it better, then you can hopefully redesign the ones you're working with to incorporate some of those features."

The key tool in this endeavour is protein engineering, the science of modifying existing proteins or custom-designing new ones for a wide range of objectives. PENCE researchers have succeeded in creating synthetic AFPs, and the aim is now to enhance them for eventual biotechnological and biomedical applications.

For example, because they limit ice crystal damage to tissues, AFPs could be used in the cryopreservation (preservation by freezing) of organs, blood, sperm, and even embryos. According to Dr. Erisman, this type of medical application of AFPs would require between six and eight years of extensive clinical and safety evaluation before becoming commercially available. Similarly, AFPs could also be engineered to confer freeze tolerance to unprotected organisms, like the Atlantic salmon.

Given the skepticism surrounding the genetic manipulation of animals and food products, the thought of applying protein engineering to living organisms is certain to raise many a quizzical eyebrow. However, protein engineering does not tamper with the genetic constitution of living beings.

"If you engineer a protein, that product by itself doesn't have a life of its own," explains Dr. Davies. "I think where people start to get nervous is when you change the genetic make-up of an organism. If that organism has some kind of advantage over an actual one, then the danger is that it might completely supplant it. Those are legitimate concerns that have to be addressed."

"People need to know that these things are being done extremely cautiously," he adds.

As one of 11 federal Networks of Centres of Excellence (NCE), PENCE links 37 industrial affiliates with over 50 of Canada's best researchers at 14 universities, hospitals and government organizations. The NCE program is jointly funded by the Natural Sciences and Engineering Research Council (NSERC), the Medical Research Council (MRC), and the Social Sciences and Humanities Research Council (SSHRC) in partnership with Industry Canada. In 1997-98, the NCE funds going to PENCE represented an investment of $3.3 million in Canadian research and development.

For more information please visit the PENCE Web site.

by Jasmine Solomonescu