It's time to stop thinking of enzymes as delicate entities that fall apart under the slightest pressure. Richard Corfield introduces us to the amazing world of thermophilic enzymes and extremophiles.

It’s time to stop thinking of enzymes as delicate entities that fall apart under the slightest pressure. Richard Corfield introduces us to the amazing world of thermophilic enzymes and extremophiles.

One of the most extraordinary ships in maritime history will be undergoing sea trials in the Pacific ocean by the time you read this. The Chikyu (’planet Earth’ in Japanese) is currently being readied in the southern Japanese port of Nagasaki. The ship is one of three platforms that the Integrated Ocean Drilling Program, an international marine research programme, will be using to explore the secrets of the Earth under the oceans.  

What makes the Chikyu special is that it will use riser drilling: an outer casing allows drilling fluid to be circulated to maintain pressure in the borehole. This, together with a blowout protector, means it will be able to drill several thousand metres into the Earth. It is the first such drilling ship to be so equipped and it will transform the science of deep sea drilling. One of the things that Chikyu will be searching for is the new holy grail of biotechnology: bacteria that inhabit some of the most extreme environments on Earth - the so-called extremophiles.  


Source: © JAMSTEC / CDEX

Drilling ship Chikyu on a mission to study bacteria that can live in extreme environments

Extremophiles can live and flourish in temperatures up to 100?C (hyperthermophile) and down to the freezing point of water (psychrophiles). They can live in environments that are so salty nothing else can live there (halophiles), or so acid or alkaline that other organisms would burn (acidophiles and alkalinophiles respectively). But what makes these extremophiles so valuable is that they are a rich source of enzymes for the biotechnology industry.  

As recently as 20 years ago it was an immutable fact of biology classes everywhere that enzymes were incredibly delicate. Many readers will remember having it grilled into them in school biology lessons that enzymes would denature - come unravelled - at temperatures above 32?C. Also, any other type of adverse chemical milieu, such as acidity or alkalinity, would have the same effect. Enzymes - nature’s catalysts - were functional only because they were safely isolated from the environment in the tissues and organs of plants and animals. 

How anyone could harbour ideas like this as late as the 1980s seems incredible now. Hindsight makes it obvious that such a narrow view of enzymes ignores what we now know is the greatest branch of the tree of life - archaea and bacteria. These organisms are not locked away from the environment in the safely cradling arms of highly evolved bodies. Far from it, they inhabit some of the Earth’s most extreme environments with nothing but their cell membranes for protection.  

The reason for the shift in the way enzymes are viewed by biology lies with the discovery in the 1980s of the enzyme Taq polymerase. Extracted from the heat-loving bacterium Thermus aquaticus (native to the boiling geysers of Yellowstone National Park in Wyoming, US), Taq polymerase can catalyse biological reactions in temperatures up to 100?C. Such a biological oddity might have excited little more than raised eyebrows among specialists had it not been for the then recent invention of the polymerase chain reaction (PCR).  

Accurate amplification 

In 1984, while driving home from work, biotechnology maverick Kary Mullis came up with a novel idea of accurately amplifying DNA in geometrically larger amounts. It was a discovery that single-handedly started the biotechnology revolution which has given us genetically modified food, the human genome project and the spectacle of 
OJ Simpson on trial.  

However, a drawback to Mullis’ original PCR protocol was that the enzyme DNA polymerase, central to the process, could not stand the heating steps that were also required. It denatured and had to be replaced after every PCR cycle, thereby limiting the speed with which DNA could be amplified.  

The discovery of Taq polymerase, preadapted for the ultimate hot-water lifestyle, meant that PCR could be truly automated. It was the start of the golden age of biotechnology, which has spilled over into popular consciousness with ideas such as designer organs, the cloned dinosaurs of Jurassic Park and the genetically-engineered android warriors of Blade Runner


Source: © iStockphotos

Hot springs can contain the bacterium Thermus aquaticus - a good source of heat-stable DNA polymerase

However, Taq polymerase has its own set of problems; the greatest of which is that it is not as faithful at copying DNA as is DNA polymerase itself. And this brings us back to the deep sea, for another hyperthermophile. Thermococcus litoralis, which lives near the black smoker vents commonly associated with undersea tectonically active areas, provides a similar DNA polymerase to Taq but this vent polymerase is a much more faithful replicator of DNA. Today vent polymerase accounts for an increasing number of sales of thermostable polymerases.  

The discovery of enzymes such as vent polymerase opened the door for the burgeoning industrial exploitation of hyperthermophiles from deep ocean vents and explains the interest of ships such as Chikyu. Vent polymerase is only one of dozens, if not hundreds, of new enzymes that are being harvested from these strange ecologies. 

The oil bug  

At first sight one of the least likely applications for deep sea enzymes is searching for oil and gas. Yields decrease after a field is initially opened and it is common for a hydraulic fracturing fluid - a proppant such as sand carried in a guar gum matrix - to be pumped into the well to fracture the rocks at depth and encourage flow. However, to exploit the increased yield the fracturing fluid must be broken down so it can be pumped away.  

Guar gum is a galactomannan - a polysaccharide with a mannose backbone and galactose side groups extracted from the bean of the guar tree (Cyamopsis tetragonoloba). It is also commonly used in the food industry as a thickening agent (in ice cream), the paper industry (for sizing paper) and the pharmaceutical and explosive industries (as a thickener and waterproofing agent respectively). 

"At first sight one of the least likely applications for deep sea enzymes is in the search for oil and gas"

Chemical oxidisers such as the persulfates are widely used in the oil industry to break the guar gum after injection and the resulting slurry can then be easily pumped away. However, these persulfate breakers often have detrimental environmental effects and tend to react before the fracturing process is complete resulting in reduced well stimulation. Because of these drawbacks breakers based on enzymes are being developed. These are incorporated into the fluid and can catalyse the reaction in situ.  

Hyperthermophilic enzymes are central to these efforts because their optimum temperature range is effectively identical to the temperatures where they must work (because of the geothermal gradient) and also because they will not react at the lower temperature near the surface. 

Driving ambition 

Hyperthermophilic enzymes are also useful in other processes where high temperature catalysis is desirable but using conventional enzymatic catalysts is undesirable. Perhaps the most important of these, as we face up to climate change, is replacing gasoline with ethanol in vehicle fuels. Ethanol is cleaner than gasoline and is also more versatile; in the interim it can be used as an oxygenate for conventional gasoline powered engines while in the longer term it seems likely that it will become an attractive source of protons needed for the fuel cells that will eventually underpin the much vaunted hydrogen economy. 

However, for ethanol to be a viable alternative fuel of the future it must be produced sustainably. Ethanol is made, predominantly, via the fermentation of sugars using various carbohydrate sources (corn, potatoes and potato waste are all popular) which are then hydrolysed using enzymes such as  a -amylase, pullulanase and  a -glucosidase. These enzymes work in tandem to break down the complex carbohydrate molecules.  

However, this hydrolysis process can be improved by using hyperthermophilic enzymes that operate at higher temperatures where the solubility of starch is greater, diffusion rates are higher and the chances of contamination by yeasts is reduced. But perhaps the most exciting innovation in bioethanol production is to exploit the lassitude of hyperthermophilic enzymes at low temperature and genetically introduce them into the plants that provide the carbohydrates in the first place.  

As an example, sweet potatoes grow optimally at 18?C - a temperature at which hyperthermophilic enzymes are inactive so will not interfere with the normal growth of the plant. However, when heated the hyperthermophilic enzymes will activate and the plant will effectively self-ferment, vastly improving the speed and efficiency of yield. 

Such a process sounds almost the stuff of science fiction but there are good reasons to suppose that these transgenic one-pot fuel producers are just around the corner.  

The production of the enzyme 


-amaylase has already been induced in genetically modified potatoes using a fusion gene created from the hyperthermophile bacteria Thermus thermophilus and Bacillus stearothermophilus

Generating food 

The latest, and perhaps most controversial use of transgenic hyperthermophile fusion techniques is in generating foodstuffs. Experiments to produce high fructose corn syrup as a sweetener in drinks is already well under way. But here the pH resistance of the enzymes is as important as their ability to work at elevated temperatures.  

The mixture starts with an initial, rather acidic pH of 4.2. But during the next step, where the starch is liquefied, the solution’s pH has to be stabilised close to equilibrium, allowing the thermostable enzyme 


-amylase to work at 95?C for up to two hours. After this, during the saccharification step, the mixture has to be returned to pH4.2. If the 


-amylase liquefaction enzyme could be persuaded to work at pH4.2 then a complex pH step-change could be eliminated.  

However, even with the extraordinary diversity of extremophile enzymes, such an enzyme has not been found in the natural world. It is here that the cutting edge of biotechnology meets the strange world of the extremophiles. 

Using a technique called DNA shuffling (see box), a synthetic 


-amylase has been created with just these properties and will soon be entering testing and ultimately production. 

The potential of archaeal extremophile enzymes is colossal. New techniques for better and faster screening of potentially useful types are being developed, including screening the metagenome - effectively a technique for testing all the enzymes present in an environmental sample. Recent tests on materials derived from the Sargasso sea in the central Atlantic ocean revealed the presence of 148 species of previously unknown bacteria and no fewer than 1.2 million previously unknown genes.  

With Chikyu about to set sail to probe this strange world there is no doubt that the vast library of archaeal genes - the oldest and therefore most abundant on the planet - will provide us with a transgenic future of literally undreamed of chemical possibility.  

Richard Corfield is a freelance science writer 

DNA shuffling

DNA shuffling was invented by Californian biotechnologist William Stemmer in 1994 and relies on the principle that genes (sequences of DNA that actually make something) or fragments of genes are more important than fiddling around with base pairs alone. Similarly, it is easier to build a new car out of prefabricated components, engines, tyres, brake discs etc than it is to build these from scratch.  

Stemmer created a process where he could break up and then reassemble genes and gene fragments in a test tube. Using the enzyme deoxyribonuclease 1, he breaks existing strands of DNA down into random 10 to 50 base pair fragments that are then heated up to separate the two strands. These then reanneal (pair up again) in the presence of a DNA polymerase, an enzyme that synthesises more DNA.  

These newly separated stands of DNA pair up only with homologous DNA, where all the base pairs are complementary. Additionally, a shorter sequence of DNA, when paired with a longer one, will automatically act as a template for assembling the rest of the sequence of complementary bases until the strand of DNA is restored.  

The essential point here is that, although the DNA base pair duality is immutable, the fragments of DNA reanneal randomly, with DNA polymerase switching between templates many times during reassembly. The result is that many new varieties of the original DNA strand - so-called recombinants - are generated automatically and rapidly. In this way literally thousands of varieties can be tested - in the case of modified 


-amylase 19 000 - and the version that works can be isolated and amplified by phage injection followed by conventional polymerase chain reaction (PCR) techniques.  

Indeed DNA shuffling is very like PCR. The difference is that, while PCR works by faithfully copying strands of DNA using individual bases floating free in the mixture, DNA shuffling relies on unfaithful replication by using the larger building blocks of partially assembled DNA strands. 

DNA shuffling is not without its detractors. Fears centre around the fact that DNA shuffling is essentially hyper-accelerated evolution, with millions of variants generated in a tiny fraction of the time taken by natural evolution. With this vastly compressed timescale there is no leeway for nature’s policeman - natural selection - to weed out unfit variants and make sure they have time to integrate with pre-existing ecosystems.