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Samples - Sculpting perfect biocatalysts (Splendid Science 2008)


Nitrile hydratases are a class of enzymes with a broad industrial application. Scientists at the Biotechnology Department are developing ways to produce these biocatalysts and to adapt them to industrial demands.

Nienke Beintema

Nitriles are organic compounds that contain a triple-bonded carbon-nitrogen group. They are precursors of some industrially very important chemicals, such as acrylamide and nicotinamide. Acrylamide is used in the paper industry, as well as in the production of polyacrylamide gels and many other useful compounds. Production worldwide is over 500,000 tonnes per year. Production of nicotinamide (vitamin B3), widely used as a human and animal food additive, as well as a component in pharmaceuticals and cosmetics, is over 11.500 tonnes per year.   

Greener processes

“There are three different ways to catalyse the nitrile conversion needed to produce these two chemicals,” says Sander van Pelt, PhD student at the Biotechnology Department. “One option is traditional acid-base catalysis. This, however, requires a high reaction temperature, it is not very specific, and it yields a large amount of chemical waste.” A second option is copper catalysis. This too needs to be done at high temperatures, and the reaction is not complete: nitrile needs to be recovered from the product mixture, and then recycled. Moreover, copper catalysis yields quite a lot of acid by-product, and the desired product is contaminated with copper traces.

“By far the most attractive is the third option,” says Van Pelt, “which is biocatalysis, carried out by micro-organisms that contain the necessary enzymes inside their cells. They work at low temperatures, their conversion is 100 percent, their reactions are highly selective, and they practically eliminate the need for downstream processing.” Furthermore, the catalyst is biodegradable, and reactions can often be carried out using water as a solvent. Biocatalysis, as Van Pelt highlights, thus offers an attractive industrial alternative that is far more clean and sustainable than traditional types of nitrile hydration. It represents an effective strategy towards meeting the fast-growing demand for ‘greener’ technologies and industrial processes.

Scientists like Van Pelt, however, always aim to optimise chemical processes. Nitrile hydration carried out by micro-organisms may seem near-perfect, but improvements are possible – and needed, if biocatalysis is to consolidate its role in the industrial arena. “Biocatalytic nitrile conversion in general is still hampered by some serious limitations,” says Van Pelt, “one of which, paradoxically, is that it is carried out inside living cells. In addition to the enzymes that they use for nitrile conversion, they contain a multitude of enzymes for many other biochemical reactions, which may result in undesired reactions involving your product. Or in some cases the micro-organisms may use part of the product for their metabolism.” To overcome this problem, scientists would like to use these enzymes, which are called nitrile hydratases, or NHases, outside the living cells. Purified, cell-free NHases would not be subject to the limitations mentioned above. “However, these have some problems of their own,” laughs Van Pelt, “and these are the problems that we are trying to solve.”

One of these problems is the fact that cell-free NHases turn out to be very unstable. Inside the living cells, hydrophobic/hydrophilic interactions ensure that the active parts of the enzyme molecules stay firmly together. In the artificial watery environment used by the scientists, relatively high substrate and product concentrations affect these useful interactions and the enzymes quickly dissociate.

Van Pelt and his colleagues have found a charming solution to this problem. They discovered a way to stabilise cell-free enzymes by first aggregating them using ammonium sulphate, and then building bridges between them. These bridges are organic molecules containing two or more aldehyde groups. The bridges connect to the various amino groups at the enzymes’ surface. The resulting ‘cross-linked enzyme aggregates’, or CLEAs, are sufficiently stable in a cell-free environment to perform biocatalytic reactions under industrial conditions. Van Pelt: “This was quite an achievement in itself. We patented the CLEA concept, which is now being exploited and further developed by a spin-off company in our group called CLEA Technologies. My research showed that the CLEA concept also works for NHases.”


The Delftscientists acquire their NHases by extracting them from micro-organisms. This, however, is not as self-evident as it may seem. “We are very lucky to be cooperating with competent microbiologists and enzymologists here in Delft,” says Van Pelt. “For people in normal organic chemistry labs, NHases are very difficult to obtain, particularly in their purified form. It takes a special expertise to grow these organisms.” This is a second factor that limits the wider application of biocatalytic nitrile conversion – and a second challenge that Van Pelt has taken upon himself.

“Basically we are looking for new NHases,” he explains. This endeavour starts with natural micro-organisms – but not necessarily the classical lab bacteria that are in use today. “I have a Russian colleague,” Van Pelt elaborates, “who specialises in isolating new micro-organisms from extreme natural environments, such as soda lakes and soils. He uses the traditional way of discovering new micro-organisms: collecting microbial samples in the field, in this case in inner Russiaand Mongolia.” These samples may contain millions of microbial species that are unknown to science – and that may or may not be useful for industrial application.

To find out whether or not the sample contains nitrile-converting micro-organisms, the scientists attempt to grow these bacteria on a diet of pure nitrile. The ones that survive and multiply are apparently able to convert nitrile, and these are used as a starting point for further research. Among other things, the scientists still need to confirm whether or not these bacteria actually convert nitrile by use of the desired enzymes – in this case NHases. “It is truly exciting to be working with entirely new organisms,” says Van Pelt. “We have managed to isolate a few NHase-producing species, and we are currently conducting more detailed studies with these.”

Directed evolution

A more modern way of ‘mining’ these desirable properties from nature is taking a soil sample, extracting all of the DNA of all of the micro-organisms that it contains, and screening this DNA for sequences that code for NHase activity. These sequences can then be transplanted into the DNA of a model organism, such as the bacterium E. coli, which can easily be grown in a lab. “This type of genetic modification – creating what we call a ‘plug bug’ – is still not very successful for NHases,” says Van Pelt. “And even if we could get it to work, it would still be a challenge to actually bring these DNA sequences into expression in the right way. In other words, to make sure that the organism produces the enzyme correctly.”

Van Pelt’s ultimate dream would be to combine this technology with so-called directed evolution – a well-established technique for optimising enzymes for industry. “Directed evolution uses various sections of DNA that code for different NHase-like enzymes,” he explains. “These you cut into many small pieces. If you mix them, they will reassemble in tens of thousands of random combinations. You then transplant these new pieces into separate E. coli bacteria. Each of them will produce a new variation of the enzyme. You test each of those, and select for very specific characteristics, such as selectivity and stability. If you want, you can repeat all of these steps to make your enzymes even more perfect.”

As Van Pelt underlines, this directed evolution will pave the way for a true revolution in NHase biocatalysis. However, there are still many challenges to be overcome. New scientific questions will arise, which will evoke further research. Van Pelt hopes, for instance, to increase the enantioselectivity of these enzymes: their selectivity for only one of two possible mirror images of the same substrate molecule. This is a highly desirable characteristic for application in the production of pharmaceuticals and pesticides. “We have plenty of ideas, but only limited time,” he concludes. “I am keeping my fingers crossed that I will be able to witness some of these major breakthroughs during my PhD project. But this field is developing so incredibly fast that I wouldn’t be surprised.”

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