Australasian Biotechnology (backfiles) AusBiotech ISSN: 1036-7128 Vol. 9, Num. 2, 1999

Biotransformations and Bioconversions in New Zealand: Past Endeavours and Future Potential

by Anne M Collins and Max J Kennedy, Industrial Research Limited, PO Box 31-310, Lower Hutt, New Zealand (tel: +64 4 569 0000, fax: +64 4 569 0132, email: m.kennedy@irl.cri.nz)

Code Number:AU99006

The area of biocatalysis which was of only limited interest 20-30 years ago, has now become a highly active area of research in chemistry. This paper describes the advantages of biotransformation processes over traditional chemistry, the technology available to overcome some of the inherent problems and examples of some commercially successful processes. Biocatalysis is an under exploited area in New Zealand and areas of biotransformation research have been suggested that could utilise some of New Zealand's natural resources.

Introduction and definitions

A biotransformation can be defined as the specific modification of a defined compound to a defined product with structural similarity, by the use of biological catalysts (Lilly 1994). Bains (1993) describes the biological catalyst as an enzyme, or a whole, dead microorganism (that contains an enzyme or several enzymes): however "resting whole cells" maybe added to this definition. There is a subtle difference between a biotransformation and a bioconversion. A bioconversion utilises the catalytic activity of living organisms and hence can involve several chemical / reaction steps. A living microorganism will be continuously producing enzymes and hence bioconversions often involve enzymes which are quite unstable. The properties of biotransformations and bioconversions are very similar and in many cases the terms are cited as interchangeable (Walker and Cox 1995). Fermentation, where the product of metabolic activity often bears no structural resemblance to the pool of compounds given to the microorganism, is significantly different from biotransformations and bioconversions (Lilly 1994).

Advantages of biotransformations:

The advantage of biocatalysis lies in the ability to operate at near neutral pH, ambient temperatures and atmospheric pressures; industrially useful chemistry often requires extremes of these conditions. More importantly biocatalysts are highly reaction specific, enantiomer-specific and regio-specific. In light of recent developments in pharmaceutical policies and the thalidomide catastrophe, this enantiomeric characteristic of biotransformations is highly desirable (thalidomide was produced as a racemate, unfortunately only one enantiomer was the effective, safe antinausea agent, the other enantiomer producing teratogenic side effects in pregnant women). The US Food and Drug Administration (FDA) policy allows pharmaceutical companies to market chiral compounds as racemic mixtures or single enantiomers; although the majority of companies have now decided to develop single enantiomers (Stinson 1995). Not only does this protect against any adverse side-effects of an undesired enantiomer but concentrates the activity of a compound with only one active enantiomer. An agricultural example of this is found in the phenoxypropionic acid herbicides; herbicide activity of these compounds lies in the (R) enantiomer, the (S) enantiomer being inactive. Zeneca BioProducts (formerly ICI BioProducts and Fine Chemicals, Huddersfield, UK) have been operating a plant for the resolution of racemic 2-chloropropionic acid by enantiospecific dehalogenation, using a mutant Pseudomonas putida strain. The (R) enantiomer can be synthesised using (S) 2-chloropropionic acid as a chiral intermediate (Holt 1992).

Another stereo-specific biotransformation carried out by Zeneca BioProducts (Billingham, UK) is the production of aromatic dihydrodiols (cis-glycols), an intermediate in the formation of high molecular weight polymers (polyphenylenes). Polyphenylenes are used for the design of advanced liquid crystal displays for computers, construction of light emitting diodes (Ballard et al. 1994) and used in the aerospace industries (Grund et al. 1994), amongst others. The hydroxylation reaction is carried out by the whole cell biocatalyst Pseudomonas putida, a mutant capable of only the first stage in the aromatic degradation pathway and unable to further metabolise the cis-glycol to the catechol; hence the accumulation of cis-glycol. Pseudomonas putida is able to hydroxylate other aromatics to their corresponding cis-glycol and is also able to tolerate a large diversity of substitutes; but only the cis-enantiomer is formed (Ballard et al. 1994). The hydroxylation of aromatic hydrocarbons to their corresponding cis-glycols is only possible by a biological route. It is the ability of biocatalysts to carry out reactions not possible (or involving several steps and adverse conditions) by chemical synthesis that gives biotechnology a lead over traditional chemical industries.

Move away from animal sources

There is a move away in industry from the use of animal sources for the production of natural products. This is in part due to the perception amongst consumers of the transmission of animal based diseases such as the encephalopathies (bovine spongiform encephalopathy (BSE), scrapies, Kuru and Creutzfeld-Jacob Syndrome). Growth hormone, purified from cadaver pituitaries was used to treat dwarfism but has been identified as the cause for the high occurrence of Creutzfeld-Jacob syndrome in patients (growth hormone is now produced by a recombinant Escherichia coli) (Walker and Cox 1995). The recent developments in the UK that have shown that BSE can be directly transmitted to humans, highlights the need to move away from animal derived products. The production of compounds by plant and microbial source is therefore of great interest to the biological industries. There is therefore a huge potential for biotransformation processes, using microbial and plant enzymes to produce "nature-identical" compounds, replacing traditional animal based products.

Disadvantages of biotransformations:

Overcoming the existence of well-developed traditional technology however is an inherent challenge to biotransformation processes. Often there is no financial incentive for implementing a new process when old technology is known and investment in plants have been paid for (Faber and Franssen 1993). It is unlikely therefore that biotechnology will automatically replace existing chemical technology, unless of course compounds of choice cannot be made by chemical means or process economics are dramatically in favour of the new process.

Twenty to thirty years ago there were high expectations for biotechnology and many saw it as the technology of the future. Some of these expectations have not materialised as anticipated, but as can be seen from the examples discussed there is great scope for biotransformation processes. The use of biocatalysts to carry out useful chemistry is often difficult: involving the challenges of reactant and/or product toxicity or inhibition, high dilution and the use of pH and temperature labile biocatalysts. However, biological and process solutions do exist to many of these problems and methods to compare strategies and techniques for biotransformation operation are being developed (Lilly 1994, Woodley and Lilly 1994).

Table 1 summarises the principle advantages and disadvantages of biotransformations compared with traditional chemical processes.

Table 1: Advantages and disadvantages of biotransformation and bioconversion processes

Technology to enhance the usefulness of biotransformations

It has not just been the market-pull but also the technological-push that has made biotransformations a viable substitute for conventional chemical synthesis (Table 2). These technological advances can best be described as two areas; the biocatalyst and biochemical engineering.

Recombinant DNA

The term biocatalyst can refer to enzymes in various forms ie. pure enzyme, crude enzyme, immobilised enzyme, whole cell, immobilised whole cell, catalytic antibodies, designer enzymes and recently cross-linked enzyme crystals. Arnold (1990) has looked at methods of enhancing biocatalysis by enzyme engineering and similarly the industrial applications of protein engineering have been discussed by Recktenwald et al. (1993). However the biggest advance in biotransformation operation has to be recombinant DNA technology. Not only is it possible to over express particular genes but it is also possible to express them in another organism, or to create unique catalysts. For example, a plasmid containing genes over expressing trehalase has been inserted into Escherichia coli. The enzyme accumulated in the periplasm of the Escherichia coli and this made it easy to extract it by osmotic shock (Tourinho-dos-Santos et al. 1994). It is now anticipated that within a few years more than half the world's enzymes will be produced commercially using recombinant DNA technology (Lilly 1994). More recently it has been shown that enzyme crystals grown and crosslinked with a bifunctional agent (eg glutaraldehyde) exhibit higher kinetic tolerance to high temperatures, near-anhydrous organic solvents and mixed aqueous - organic solvents than both soluble and conventionally immobilised enzymes (St. Clair and Navia 1992). It is coming to the stage now where the biochemical engineer will be able to describe the characteristics of the preferred biocatalyst and the molecular biologist will be able to produce it.

Immobilisation

It was the technology to extract intracellular enzymes on a large scale (Hetherington 1971) and the subsequent biocatalyst immobilisation that made biotransformation an alternative to chemical synthesis. Prior to the advent of large scale high-pressure homogenisation it was only extracellular enzymes or those extracted by chemical lysis that could be used for biotransformation. Immobilisation techniques allow the water soluble enzymes to be recovered and reused in a solid form. Lilly (1992) and Katchalski-Katzir (1993) have presented good reviews of the advantages and disadvantages of biocatalyst immobilisation, the successes and the failures. Immobilisation of the biocatalyst, enzyme or whole cell, is not only carried out for processing reasons but also used to modify enzyme properties, and to alter and regulate cell product formation (Clarke 1994).

Two Phase Systems

Often useful chemistry involves reactants and products that are poorly water-soluble and from this evolved the techniques to operate biotransformations in organic solvents (Tramper and Vermue 1993). The organic phase can be the sole liquid phase, or part of a two-liquid (aqueous - organic) phase system. In the latter case the organic phase can be the reactant (eg. the conversion of cholesterol to cholest-4-ene-3-one by Nocardia rhodochrous (Buckland et al. 1975)) or a reservoir for the poorly water-soluble and/or toxic reactant and/or product (eg. hydroxylation of toluene to toluene cis-glycol, by Pseudomonas putida, using tetradecane as a second liquid phase (Collins etal. 1995)). The addition of a second organic liquid phase has also allowed kinetically unfavourable reactions to be carried out (eg. reverse ester hydrolysis (Klibanov 1986)). A summary of the general thermodynamics of multiphase systems has been presented by Halling (1994) and Woodley and Lilly (1992) have discussed the process engineering aspects of operating a two-liquid phase biotransformation.

High fructose syrups

One of the earliest biotransformation processes to employ immobilised enzyme technology was the production of high fructose glucose syrups, by glucose isomerase, which has been operating now since 1972. High fructose glucose syrup is used in the soft drinks and confectionary industries as it has health advantages over the conventional sugar, sucrose (fructose is twice as sweet, lower in calories and not absorbed into the bloodstream as quickly as sucrose). More than 8 x 106 tonnes of high fructose glucose syrups are produced annually. On a smaller scale (but a higher value product), 7.3 x 103 tonnes of 6-amino penicillanic acid, the precursor for the synthesis of all semi-synthetic penicillins are produced annually. This process, using the immobilised penicillin acylase, ( amidase) has been operational since 1973. These examples show that biotransformations can be efficient and commercially competitive.

Flavours and fragrances

Biotransformations are being used in speciality chemical manufacture of flavours and fragrances (Cheetham 1993) (again in the area where only one isomer or stereo isomer has the required biological activity and the other is inactive or has undesirable activity). For example, the (L) enantiomer of carvone tastes of spearmint whilst the (D) enantiomer tastes of dill / caraway. Also only (L)-monosodium glutamate has taste enhancing properties and only (L)-menthol (out of eight isomers) has the desired combination of mint taste and cooling sensation. The role of biotransformation technologies in the flavours and fragrances industry are becoming more prominent and not just for the ability to produce single enantiomers. There is an increasing demand by consumers for natural, environmentally friendly and healthy products, made from natural, renewable sources; for use in both food ingredients and in personal care products. As modern market demand for these "natural" products increases so there is a decline in the availability of traditional raw materials eg. ambergris, animal musk's and many plant derived materials and essential oils. A biotransformation process has now been developed for Ambrox7 which is one of the most important aroma components of ambergris (a naturally occurring excretion product of the sperm whale) (Cheetham 1993). It is in this area, the production of "natural products" that holds potential for New Zealand industries.

Animal products transformed

The use of biotransformation techniques in New Zealand has principally been limited to the primary industries (meat and dairy industries). This is illustrated by Table 3 which highlights the uses of biocatalysts in New Zealand industries. Tallow is a major waste product of the meat industry and is exported (to produce fat based products) for low prices ($57.5M for tallow from a total meat industry export value of $3790M (Wright 1995)). Work at Industrial Research Limited (Stevenson 1994) has looked at the use of an immobilised lipase to produce a tallow derivative enriched with monoglyceride. Monoglycerides are used as emulsifiers in food and cosmetics and as antistaling agents in bread. Immobilised lipases can also be used to rearrange fatty acids in triglycerides and to modify the composition of the fat (Stanley and Armstrong 1995). Therefore potentially it will be possible to produce tasty, monosaturated oil from tallow or to make tallow less prone to raise cholesterol levels when used in cooking (MacKenzie and Stevenson 1995). New Zealand Pharmaceuticals Limited manufactures bulk pharmaceuticals and fine chemicals from a range of natural raw materials eg. biochemicals derived from sheep and cattle bile for use in producing corticosteroids, gallstone dissolving agents, liver protection preparation and dyspectic formulations (Yorke 1993). Steroid transformation work (by biocatalysis) in relation to the bile acid production facility has proved successful, but unfortunately at the time there was no commercial application (Maddox 1993).

Table 3:Biotransformation processes used commercially in New Zealand (information taken from Kennedy and Davies (1994))

Cheese production

In the dairy industries, the utilisation of enzymes and microorganisms has had a vast impact in the areas of cheese starter technology, cheese ripening and the modification and enhancement of cheese flavour products (by lipase addition) (Coolbear and Holland 1994). Other research in this area has concentrated on the enzymic modification of milk fat and milk proteins, lipase catalysed milkfat for concentration of flavours and the production of natural dairy emulsifiers (Ravenhall and Davies, personal communication). The advantages of using biotransformations for these types of reactions is the ability to operate under mild conditions, hence retaining the traditional properties of the dairy products. Enzymes that can remove "sticky dextrins" during baking have also been investigated at Food and Crop Research (Every 1994).

Bioremediation

Another use of biotransformations, the use of microorganisms in the treatment of sewerage and waste water, has been used for many years. Recent advances have seen the use of enzymes and genetically engineered bacteria for biological waste water treatment processes (Freeman and Fullerton 1994). At the Waste Technology Research Centre in Otago, research has focused on the use of biocatalysts in the degradation of toxic compounds and pollutants. The bioremediation of contaminated environments, as opposed to the use of high temperature incineration is relevant in situations where the contamination is widespread and/or just above permissible levels. Not only does the use of microorganisms require cheap nutrients but the by-products of the biotransformation (water, CO2 etc) are relatively environmentally harmless (Hauber 1993, Thiele 1994).

Malolactic conversion for wine

In the wine industry, Leuconostoc oenos is used to convert malic to lactic acid, during the maturation of Chardonnay. Without this conversion, which reduces its acidity, the wine is less drinkable. The conditions in the wine are not conducive to microorganism growth and as a result this process can take up to a year. At DSIR, Industrial Development (now Industrial Research Limited) the Leuconostoc oenos was grown up, under favourable conditions and subsequently immobilised on oak chips. The immobilised biocatalyst was packed into a column and the wine was passed through the column in a continuous process. This demonstrates a classic example of how the use of biotransformation processes can be employed to the advantage of traditional New Zealand industries (Janssen et al. 1993).

Potential areas for biotransformations in New Zealand industry

Biotransformations offer opportunities to produce compounds that cannot be produced by chemical means in a highly reaction-specific and stereo-specific manner. More importantly, they offer the chance to produce large quantities of "nature identical" compounds (which are biodegradable) from raw materials, which is important owing to the consumer increasingly demanding "natural products".

Biotransformation technology is an area that has not been fully exploited in New Zealand. From the examples given, the conversion of compounds by biological means has focused on the primary industries and the conversion of New Zealand raw materials into high value products, by the use of commercially available enzymes. One aspect of this not fully investigated to date is the utilisation of native New Zealand plants. Analogues and derivatives of chemicals from native plants formed by biocatalysts would be optically active structures; useful for investigation into drug development and fine chemical production (van Klink 1995). This is especially relevant as a recent directive from the WHO (World Health Organisation) indicates that there is a need to find new antibiotics to fight currently resistant strains of micro organisms (Beardsley 1996). Other NZ raw materials that are under utilised are Kiwifruit and apple and grape pomace; there is potential that transformation of some of their components could produce interesting flavour and fragrance products (Kennedy 1994). New Zealand offers an abundant supply of potential biocatalysts in its diverse ecosystems; ranging from hot pools to alpine terrain and native forest. Adams et al. (1995) have recently stated that for every enzyme now in a commercial application, a more stable version is available from a microoganism inhabiting the appropriate environment. At the University of Waikato, genes for enzymes from thermophilic bacteria have been inserted into other microorganisms, to simplify purification techniques (Daniel 1994) and thermophilic enzyme bleaching in the paper pulp industry is being investigated at the University of Auckland (Berquist 1995). However there is more potential for research into this unique enzyme source for the production of novel chemicals. For example, the use of extremophiles for biotransformation processes would have beneficial effects on process compatibility. In areas where reactants have high viscosity and operation at higher temperatures would be prefered, biocatalysts from thermophiles would be advantageous. Similarly enzymes from halophiles have been cited as more stable in organic solvents, which would be benefical when carrying out a reverse hydrolysis reaction (Klibanov 1986) or reactions where the reactant / product is toxic to the biocatalyst (Hack et al. in preparation) and operation in an organic solvent is preferred. One of the most active areas of biotechnology research in New Zealand is in the area of molecular biology. Currently this has yet to reach industries but is poised to do so in the next 10 years (Kennedy and Davies 1994). Therefore alongside a screening programme to further identify potential biocatalysts, there appears a need to develop the fundamental technology and a basic backbone of biotransformation techniques in order for New Zealand to harness its developing technologies and natural inheritance. Table 4 identifies avenues for biotransformations in New Zealand, highlighting potential biocatalysts, raw materials and niches for research. The use of biotransformation processes would enhance New Zealand's reputation as an environmentally friendly nation.

Table 4: Potential areas for biotransformations to contribute further to the New Zealand biotechnology industry

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