No shortage of inspiration

Biotechnology has a long and venerable history in Japan, where micro-organisms are being used to produce a wide diversity of pharmaceutical compounds, says Stuart Nathan

Japan, famously, lacks natural resources. Although it has coal reserves, it has no oil, no gas; it's even short of flat land. However, this hasn't held it back from industrial development; it's just had to find different ways of achieving its goals.

A recent mission organised by the UK Department of Trade and Industry's GlobalWatch organisation found that the Japanese willingness to find alternatives and maximise its particular advantages may hold valuable lessons for the British process sector.

Although Japan is poor in fossil fuels, it is rich in other resources. In particular, it has one of the highest diversities of micro-organisms of any region. This is because of its huge range of different climatic and environmental conditions.

The Japanese archipelago is more than 1000 miles long and stretches across 20 degrees of latitude temperate in the north, near-tropical in the south. It has mountains up to 3776m high, and marine trenches plunging 8km deep. And as industrial chemists in the US and Europe are finding, micro-organisms are increasingly valuable tools in many branches of the process industry.

developing microbes

Need to carry out a tricky reaction? There's likely to be a microbe that does something similar. Toxic waste requiring treatment? There's probably a bacterium that views it as a tasty snack. And in Japan, where every gramme of soil contains between 10m and 100m micro-organisms, there's a fair chance that the bacterium you need lives in the soil outside your laboratory window, or halfway up the mountain overlooking your university.

For the past five years, Japan's government has been sponsoring research programmes to develop the use of microbes in the chemical industry. The main goal of the programmes is energy reduction Japan is almost completely reliant on imported fuel for energy generation, and biologically-mediated processes tend to need milder conditions (and therefore less energy) than classic inorganic-catalysed industrial chemistry.

energy points

A study led by Kyoto University estimated that the chemical industry consumed 13% of all energy in Japan, but if 30% of chemical production were to be achieved by enzyme-mediated processes, total energy consumption would fall by one percentage point.

Japan is one of the highest energy consumers in the industrialised world, using 50% more energy than the UK, and with the world's fourth-highest emissions of carbon dioxide; a 1% cut would present a considerable saving. Moreover, the study estimated that the market for products generated by these processes was worth $70bn per year.

The proposed use of bioprocesses didn't stop there: the study also recommended that using specialised micro-organisms to convert biomass into gas could meet a fifth of energy needs. The initial project set a target of 2007 for the enzymatic processes goal, and 2010 for the biogas target. Japan's industrial ministry, METI, estimates that 10% of chemical production through enzymes by 2007 is probable, and 15% is achievable.

These goals might sound ambitious, but biological processes are nothing new to Japanese industry. In fact, they're ingrained in the culture. Japan's national drink, sake, is produced by a complex fermentation process involving the culturing of microbes which change the carbo-hydrates in rice into glucose, and yeasts, which then turn the glucose into alcohol. These processes occur at the same time in the same vat. Similar techniques are used to make two staples of Japanese food, soy sauce and the soya bean paste called miso.

'We've lived with microbes since ancient times,' explained Professor Sakayu Shimuza, of the Hakko Laboratory at Kyoto University. 'They've always been essential for us. We extend our thanks to them.' So great is the regard for these microscopic workhorses that the university has a monument dedicated to the bacteria that died as a part of its biotechnology research a quintessentially Japanese touch.

Shimuza is one of the leading figures in Japanese biotechnology. Between 1984 and 2000, his laboratory developed 29 enzyme-mediated industrial processes, working with companies from Japan, Korea and Singapore. These produced compounds ranging from amino acids, sweeteners, edible oils and vitamins to pharmaceutical intermediates, through to bulk chemicals more generally associated with large conventional chemical plants.

The processes also span the range of bioprocesses: some used membrane bioreactors, where colonies of whole, live micro-organisms carry out the conversions, while others were purely enzymatic processes, where the enzyme is isolated from its original host organism and used in the same way as a conventional catalyst.

useful activities

Much of the work of the Hakko Laboratory involves screening micro-organisms for industrially useful activity. This is tedious, repetitive work, which Shimuza acknowledges would not be economical for a commercial company. However, the university has an important advantage - students. 'Young students enjoy tedious and time-consuming screening,' he says. 'Their fresh, careful and patient observation sometimes brings serendipitous results - and their will is more important than their skill.'

The laboratory uses two kinds of screening process. The conventional low-tech screening - the preserve of Shimuza's enthusiastic students - involves looking at a large number of naturally-occurring organisms and enzymes. The other, a high-tech advanced screening method, depends on genetic modification techniques.

Researchers subject only one, or a few, enzymes or genes to methods such as random mutagenesis, DNA shuffling, site-directed mutagenesis, tailoring the biological processes to produce the required products from the available raw materials.

biocatalyst advantages

For companies producing compounds for the pharmaceutical sector, biocatalysts have several clear advantages, says Andy Wells, a principal scientist with AstraZeneca and one of the GlobalWatch team. They are highly stereo-, chemo- and regioselective. Moreover, they are not limited to the reactions they would normally perform.

'In nature, an enzyme may not catalyse a chiral process, but it may well do with an unnatural substrate,' he says. They are also very efficient, allowing for high turnover rates, and are economical, both in terms of the relatively simple equipment needed and in the mild conditions they need for reaction.

The latter has another advantage: as extreme conditions are not needed, substrates can be used that would normally not tolerate such conditions.

The most common types of enzymes used in industry catalyse hydrolytic reactions. The enzymes include amidases, esterases and lipases. These account for about 80% of all industrial biocatalysed reactions, Wells says.

Their popularity stems from their stability, their robustness, their tolerance of oxygen and some common organic solvents, and their ability to work without any need for expensive organic cofactors or metals. They will usually catalyse reactions using unnatural substrates, and can often be made to work reversibly that is, catalysing syntheses rather than the hydrolyses.

One example of this is the manufacture of D-(-)-hydroxyphenylglycine, figure 1, an intermediate in the synthesis of amoxycillin. The Kaneka Corporation has devised a synthesis that uses a dynamic deracemisation process to separate out the desired enantiomer of its precursor, with a selective hydantoinase as a catalyst, in water at pH9.0.

The unwanted enantiomer undergoes spontaneous hydrolysis back to the racemate, while the target enantiomer can be treated with a carbamoylase to form the D-(-)- hydroxyphenylglycine. The yield is close to 100%, and enantiomeric excess above 99%, Wells says.

considerable rewards

Extensive process development was needed, because the wild-type carbamoylase that was the starting point for the research was not stable under process conditions. However, the rewards were considerable. 'The process takes a cheap, readily available racemate and converts it to a high-value single enantiomer,' he adds.

The carbamoylase step replaces a deprotection stage in the conventional synthesis, which requires the use of nitrous acid and its attendant safety and environmental risks. 'The enzymatic process needs no recovery stage and no further processing.'

Less common - but becoming more so - is the use of redox biotransformations. Richard Lloyd, a senior research chemist with Dowpharma, estimates that only 10% of industrial bioreactions are reductions, because of their need for expensive cofactors, organic intolerance and so on. However, some progress has been made in this area, with Shimuza's laboratory carrying out process development on the production of 4-chloro-3-hydroxybutyrate esters (CHBE) through a reduction reaction, figure 2.

Kaneka, Daicel and Mitsubishi are now running a commercial process to make S-CHBE, and Daicel is additionally piloting an R-CHBE process.

The techniques aren't confined to pharmaceutical chemistry, however. Hakko Laboratory collaborated with Mitsubishi Rayon for a process to make acrylamide, an intermediate in acrylic resin production, from acrylonitrile, using cells derived from R. rhodochorus bacteria as a catalyst. Using conventional chemistry, this isn't a difficult reaction, says Wells.

'If you ask an industrial chemist, he'd say that you need a concentrated sulphuric acid and copper ion catalyst, and you'd need to do it at 90°C. You'll get pretty poor selectivity - lots of side-products, including acrylic acid - and incomplete conversion, so you'd have to put in some recovery steps. And you'd end up with a huge amount of sodium sulfate and copper salts in the waste.

'But do it with a biocatalyst, and you're using supported cells as the catalytic material, you need temperatures of 5°-10°C, you get 99.9% selectivity for acrylicamide, 100% conversion, and your only waste product is water.'

Efficiency advantage isn't the only environmental factor. As the GlobalWatch team leader, Arnold Black of C-Tech Innovation, points out, producing conventional inorganic catalysts often involves dealing with metals like platinum and palladium, and these have to be obtained by mining, with further processing to purify the metal and produce the complex organometallic molecules used in catalysis.

'The amount of waste you make producing 1kg of platinum is frightening,' he says. The enzymatic approach, though requiring as much, if not more initial research, is far less damaging.

So why don't European and American companies use more biotransformations? Part of the answer is in the rigid working methods in the West, says Wells.

'In Japan, it's not unusual for fermentation specialists to work with microbiologists, synthetic chemists and engineers, all in one team, from the start of the project,' he says. 'In the UK, it's still quite rare for the organic chemists to talk to the engineers.' The conservatism of the engineers is also a factor. 'They know about continuous stirred tank reactors, they know inorganic catalysts, it's all fully costed.'

Changing to a biological paradigm is a big step, - and it's one that alarmingly few engineers seem willing to take at the moment.