293 Biotransformation of lipids 9.1 Introduction 9.2 The structure, roles and abundance of sterols and steroids 9.3 Selective degradation of the sterol side chain 9.4 Specific steroid interconversions and reactions 9.5 Transformation of other terpenoids 9.6 Chemical conversion of miscellaneous organic compounds 9.7 Production and use of fatty acids and their derivatives 9.8 Selection of production systems for the biotransformation of lipids Summary and objectives 294 295 298 309 321 326 329 337 339 294 Chapter 9 Biotransformation of lipids 9.1 Introduction In this chapter we will examine how cells and enzymes are used in the transformation of lipids. The lipids are, of course, a very diverse and complex series of molecular entities including fatty acids, triglycerides, phospholipids, glycolipids, aliphatic alcohols, waxes, terpenes and steroids. It is usual to teach about these molecules, in a biochemical context, in more or less the order given above, since this represents a logical sequence leading from simple molecules to the more complex. Here, however, we have adopted a different strategy. Clearly, the technical and commercial aspects of industrial lipid transformation are both diverse and complex. We have, therefore, to be selective in what we can include in a single chapter. We have decided to begn the chapter by discussing the transformation of sterols and steroids since these transformations are illustrative of the potency of biocatalysts in bringing about selective and stereospecific chemical transformation of quite complex molecules. This part of this chapter is a logical extension of the issues discussed in the previous chapter. We have also elected to focus on the technical rather than the commercial issues although attention is drawn to the importance of commercial criteria in the selection of strategies for production. The reader should be aware, however, that the production of steroids for pharmaceutical use and as contraceptives is a large market. Estimates of annual sales of these materials vary widely (WHO 1990 Year Ekmk $3~10' annum-' - $10'' annum-') reflecting the difficulty of accessing details on such a diverse group of compounds. After discussing the biological capability to transform steroids, we briefly examine the biotransformation of other terpenoids to ensure that the reader develops an awareness of the potential of biotechnology to modify or produce derivatives of a wide range of natural materials that are of tremendous potential, commercial value in the food and health care sectors. We also include a brief consideration of the use of biocatalysts to transform a range of other hydrocarbon compounds. The bulk of the chapter is therefore concerned with highly speafic reactions arising to produce molecules of known structure. However, in a chapter on lipid transformation, we should not miss the use of general lipases to change the composition of triglycerides. Although the replacement of one set of fatty acids in trigycerides is, from a chemical/biochemical point of view, not as stimulating as the biotransformation of steroids, it is of mapr commercial value in the food industry. The biotransformation of triglycerides (and phospholipids) to produce food materials which have desirable organoleptic properties (eg melt in the mouth feel) potentially dwarfs the steroid market in terms of volume and turnover. The placing of this topic towards the end of the chapter does not imply that it is of limited commercial value. The final part of the chapter briefly explores the potential of using non-aqueous solvents for lipid transformation. bids i~~~ea wide^$^^! types Biotransformation of lipids 295 Despite the technical emphasis of this chapter, we have included some examples of industrial processes. 9.2 The structure, roles and abundance of sterols and steroids Some examples of sterols and steroids are given in Figure 9.1. Also included in this Figure are some examples of bile salts. You should realise that the structures shown are only a few of the many hundds of compounds which occur in nature. All of these compounds include the steroidal ring structure which is numbered as shown below. steroidal ring smare - Substituents are designated as in the a configuration if they are below the plane of the steroidal nucleus, and as 0 if above the plane: Thus is 3a hydroxy - whilst is 38 hydroxy - Examine Figure 9.1 and see if you can distinguish between the roles of the sterols n (at the top of the figure) and the C18, (219, Cn and CN steroidal compounds. In general, the sterols perform a structural function, for example as components of the lipid layers of membranes. The Cis, Ci9 and C21 steroids mainly perform an endocrine function. In other words they are hormones. The bile salts (Czlr-steroids) fulfil a functional role in digestion in animals. 296 Chapter 9 Bearing this in mind, which of the three groups are likely to occur a) in greatest n amounts, b) in lowest concentration in biological systems? You should have predicted that the sterols are present in greatest quantity in biological systems. Your knowledge of biology should have enabled you to identify the steroid hormones as being present in lowest concentrations since hormone, in general, are effective at very low concentrations. Figure 9.1 Some examples of sterols, bile salts and steroids. Biotransformation of lipids 297 Although representatives of all of the classes of sterols and steroids are essential to humans, the biological (pharmacological) activities of the CW, CI~ and Cn steroids make these potentidy very usef~l as therapeutic agents. It has long been realid that variations on the structures of naturally occurring steroids lead to products with greatly modified biological activities. Thus we can visualise the situation in which steroids may be modified to produce substances which have enhanced or reduced activities. This has far reaching implications in the healthcare sector. For example, natural and modified corticosteroids have applications as anti-inflammatory agents and may be used where the immune response needs to be moderated. Similarly the ability of CM and CI~ steroids to modulate reproductive capabilities makes them useful as fertility agents and as contraceptives. Which of the groups of compounds shown in Figure 9.1 is (are) likely to be of n greatest commeraal (and social) value? Again, the answer should be fairly obvious. The potential therapeutic value of the steroid hormones makes these of tremendous commercial value. The commercial market for these is of the order of hundreds of millions of dollars per year. There! is no comparable market for sterols and bile salts. We are faced with the interesting situation, therefore, that sterols are relatively abundant in natural sources but of relatively low commeraal value, whilst steroids occur naturally at very low concentrations but are of great commeraal value. Although there are tremendous variations amongst different products, steroids with desirable properties command market prices that are (ten to one thousand fold) greater than their sterol counterparts. Sbdds have @mamutical value steroids are of gFt commeraal ValUe Bearing in mind the relative abundance of sterols and steroids and their chemical structures, which of the following strategies for producing steroids is most likely to be commercially successful? n 1) Extraction from animals. 2) Total chemical synthesis. 3) Partial chemical synthesis starting from a natural product. 4) Total biosynthesis. 5) Enzymatic transformation of natural products. Below we have considered each of these strategies in turn. 1) Although animals produce steroids, the low concentrations of these compounds does not make these commercially (nor ethically) attractive sources of these substances. Furthermore, they could only serve as sources of naturally occurring steroids. Thus we would not have selected this option. The steroid ring structure is complex and contains many chiral carbons (for example at positions 5,8,9,10,13,14 and 17) thus many optical isomers are possible. (The actual number of optical isomers is given by 2" where n = the number of chiral carbons). From your knowledge of biochemistry you should have realised that only one of these optical isomers is likely to be biologically active. Synthesis of such a complex chemical structure to produce a single isomeric form is extremely difficult, especially when it is realised that many chemical reactions lead to the formation of racemic mixtures. Thus, for complete chemical synthesis, we must anticipate that 298 Chapter 9 we would need a multistage reaction and that the desired isomer would only be a small fraction of the final product We would also be presented with the difficulty of isolating the desired isomer from its isomeric partners. We, therefore, conclude that, although technically feasible, this approach is not a realistic commercial option. 3) Partial chemical synthesis is perhaps more realistic. If, for example, we wish to slightly modify the structure of a naturally OcCuRing substance, then this rmght be possible using chemical processes. The problem here is to identify reagents and reactions which will be specific, both in terms of the site of attack on the natural product and the stereospecificity of the reaction. We must anticipate, therefore, that chemical reactions may be used in some cases, but this is not a universally applicable strategy. 4) The natural systems that produce steroids do so in quantitatively small amounts. Although in principle the cells producing these might be isolated and cultivated in mho, the quantities produced will still be small and the costs of cultivation are high. This approach is, therefore, not generally commercially viable. You may have considered the option of transferring the genes, which ende for the enzymes involved, into an easy to cultivate system (for example a yeast or bacterium) and to control the expression of these genes using strong promoters. Although this approach is theoretically possible using the techniques of genetic engineering, the difficulties in isolating the necessary genes and the multiplicity of the enzyme steps needed for steroid biosynthesis makes the development costs of this approach extremely high. In the longer term, this may become a realistic option, but is not, currently, commercially viable. 5) The enzymatic transformation of natural products is by far the most attractive option. In this approach, it can be envisaged that sterols, which are relatively abundant, may be selectively modified to produce desired products. The diversity of enzyme activities, their reaction specificity, regiospecificity and stereospeaficity are all features which could contribute to carrying out the desired changes. This does not mean, however, that transformations using enzyme systems are simple. Nevertheless, biotransformations have become of vital importance in the production of steroids. In the following sections we will explain some applications of enzymes (and cells) in the transformation of sterols and steroids. You should realise, however, that for each process a decision has to be made whether to use an enzyme-mediated transformation or to use a chemical reaction. In many instances the biotransformation process is the most attractive but, as we will see later, this is not always the case. 9.3 Selective degradation of the sterol side chain Re-examine the structures shown in Figure 9.1 and see if you can identify the n fundamental difference between sterols and the steroid hormones. Although there are many differences between these two groups of molecules, the fundamental difference between them is that the steroids do not possess the long side chain attached to position 17 that occurs in sterols. Thus, if we are to use sterols as the starting point for producing steroids, then we need to selectively remove this side chain. Biotransformation of lipids 299 miaOorgE4lliSmS may selectively degrade he side cham use of mutants modification of Ihe substrate Fortunately many micro-organisms can be used to selectively remove the side chain of abundant, naturally OcCuRing sterols such as cholesterol, &sitosterol and compesterol. These organisms include members of the genera Nmdia, Pseudomonas, Mywhzderium, Corynebacterium and Arthrobacter. They are capable of using sterols as their sole source of carbon. Unfortunately, the natural occurring organisms catabolise both the side chain and the ring structure of the sterols. The catabolism of these two components may occur simultaneously. Therefore, methods have to be found to prevent ring structure catabolism whilst allowing the degradation of the side chain. n See if you can idenhfy two strategies for achieving this objective. In practice, several strategies have been used. In one, mutants are produced which are defective in the enzymes involved in ring structure catabolism but still retain the enzymes involved in side chain catabolism. n Would such mutants grow on a) cholesterol b) testosterone? a) We would anticipate that such mutants would grow, albeit slowly, on cholesterol as they could still derive carbon and energy from catabolising the side chain. b) The mutants would probably not grow on testosterone as their is no side chain for them to catabolise. We could use these differences to identdy putative mutants with the desired metabolic block. A second strategy is to find a way of inhibiting an enzyme involved early in the catabolism of the ring. One such enzyme is a Sa-hydroxylase (it hydroxylates carbon 9). This enzyme has an absolute requirement for Fez+ ions. By adding chelating agents which complex with these ions, the enzyme can be inhibited. A third option is to modify the ring structure of the sterol so that it no longer serves as a substrate for the ring-catabolising enzyme. In this approach, a chemical reaction is used to modify the ring-structure and the product is subsequently incubated with the catabolising organism. For example, hydroxylation at C-19 prevents ring cleavage. Some examples in which modified sterols have been used for selective side chain degradation are given in Table 9.1. This table also indicates the nature of the products formed and the organisms used. We would not expect you to remember all of the details of these substrates, products and organisms. We will, however, examine some examples in more detail to illustrate the principles involved. 300 Chapter 9 Substrate Produd Mlcro-organlsm 1 9-hydroxysterols, 1 9-norsterols, 3-hydro~y-19-nor-A’~~~~- sterols 19-hydro~y-A~~~-steroIs, 9-hydro~y-l9-nor-A’~~*~~’- sterols Sp,lO-oxidostenones, 3p-acetoxyda-chloro(fluoro)- Sp, 19-oxidosterols 3p-acetoxy-5a-bromo-Gp, 1 9- Dxidosterols 5a,5a-cyclosterols 3a,5a-cyclo-6p, 1 O-oxido- jterols sterol-3-oximes I-hydroxycholestenone masterone A estrone Nocardia restrictus ATCC 14887 Nocardia sp ATCC 191 70 Arthrobacter simplex I AM 166( Corynebacterium sp M ycobaderia Corynebacterium simplex Nocardia mbra Sp, 19-oxido-4-androstene-3, Nocardia sp ATCC 191 70 17dione Mycobacteria 5a-bromo-Gfi,19-oxidoandros- Nocardia sp ATCC 191 70 tane3,17dione 3s5a-cycloandrostane-17-one Mycobacterium phlei 3a,5a-cyclo-6p, 1 9-oxido-an- Arthrobacter spp drostane-17-0ne Corynebacteria 4-androstene-3,17-dione Mymbacterium sp (after hydrolysis) 38 hydroxy-5a-androstane-4, Mycobactenurn phlei 17dione 3a-hydroxy-5a-androstane-4, 17dionq 3p,4u4i hydroxy-5a-andro- stane-17-One 1 ,4-androstadiene-3,1 Gdione, Fusarium solani 20a-hydroxy-4pregnene-3,16- Verticillium theobromae dione Stachylklium bicolor 3a,l1 p,20a-trihydroxy-5a- pregnane-16-one rubrosterone Fusarium lini 9593 equilin, equilenin, estrone Mycobacterium sp Table 9.1 The use of modified sterols to allow selective cleavage of the side chain (based on Martin, CKA Sterols in Biotechnology Volume 6a Edited by Kieslich K 1984 Verlag Chemie, Wein heim). 9.3.1 Use of modified sterols First, let us briefly examine the mute of side chain degradation in micro-organisms. ’Ihe pathway is illustrated in Figure 92. Biotransformation of lipids 301 Figure 9.2 Generalised metabolic sequences of sterol side chain degradation by micro-organisms. Fit carbon 27 is hydroxylated and oxidised to a carboxylic acid. The resulting acid is then cleaved to release propionic and acetic acids and a second propionic acid. The final reaction in this sequence results in the formation of a keto pup at C-17. side chain degdaam 302 Chapter 9 If a hydroxyl group is introduced into position C-19 then complete breakdown of the ring structure is prevented, although it may be sub- to some modification. In the exampl? shown in Figure 9.3, the incubation of 19-hydroxysterols with Nocardia restn'ctus ATCC 14887 or Nocardia sp ATCC 19170 leads to the production of estrone. In these cases, you will notice that ring A has become modified but the ring structure is not broken. The yields of estrone using these substrates and organisms are of the order of 30%. hydroxylation ate19 ~~ ~~~~ ~~ Figure 9.3 The figure shows the degradation of the side chain of sterols which have substitutions at C-19. Removal of the C-19 methyl group (eg 19 norcholesta-l13,5 (1 0) triene-3-01) also prevents ring breakdown. Note, however, hydroxylation of C-19 does not prevent all ring modifications. a: 19-hydroxychoIesterone, b: 1 9-hydroxyslosterone, c: 3p-acetoxy-l9-hydroxy-5cholestene, d: estrone. Other modifications which restrict ring cleavage are the formation of 6fi,19-oxido derivatives and 3a, 5a cyclo-derivatives. The structures of some of these are given in Figure 9.4. Biotransformation of lipids 303 Figure 9.4 Some examples of side chain degradation of Sp and 19 oxidosterols. a: 3p-acetoxy-5a-bromo-6p, 1 9-oxidocholestane, b: 5a-bromo-6p, 1 !3-oxidoandrostane-3,17dione, c: 3~acetoxy-5a-chloro-6p, 19 oxidocholestane, d: Sp, 19-oxMoandrost-4-ene-3,l irdione, e: Sp, 19-0xido-4-cholestene-3-0ne, f: Sp, 19-0xido-9a-hydroxyandrost-4-ene-3,17dione. 9.3.2 Use of enzyme inhibitors Earlier we indicated that an alternative strategy to prevent ring cleavage is tu use selective inhibition of the enzymes which catabolise the ring structure. The inhibitors used include: 0 chelating agents; 0 metal ions with similar ion radii which will displace Fez+; 304 Chapter 9 inorganic -SH reagents; 0 auto-oxidisable redox dyes. A list of some examples of these is given in Table 9.2. n Are these compounds likely to be toxic to cells? Since they inhibit metabolism, they are likely tu be tuxic to cells. It is, therefore, usual to add these compounds after the culture has grown and to subsequently add the sterol to be metabolised. Of the compounds listed in Table 9.2, 8-hydroxyquinoline, a,a'-dipyridyl and 1,lO-phenanthroline have found most use. They are usually used in the concentration range 0.1 mm011~~. It is, however, essential to use an optimal concentration of these agents in order to get high yields. ring cleavage Uechanism of action Compound Zhelating agents for Fe2+ Cupferron Diethyldithiocarbamate a,a' -Dipyridyl Dipheny It hiocarbazone 8-Hydroxyquinoline lsonicotinic acid hydrazide 4-lsopropyltropolone 5-Nitro-l , 1 O-phenanthroline 1,l O-Phenanthroline @Phenylenediamine Tetraeth ytthiuramdisulphide Xanthogenic acid Metal ions replacing iron or blocking Ni2+, Co2+, Pb2' SH-functions w-, Asor 3edox dyes Methylene blue Resazurine Table 9.2 Compounds used to inhibil steroid ring degradation (based on Martin CKA Sterols in Biotechnology Volume 6a Edffed by Kieslich K 1984. Verlag Chemie, Weinheim). n What will happen if the concentration of the inhibitor is a) too low b) too high? a) If it is too low, complete degradation of the substrate may occur. Biotransformation of lipids 305 b) If it is too high, inhibition of other enzymes, including those involved in side chain degradation, may occur. Achieving optimal concentrations is difficult because components in the dum and the biomass itself may neutralise the inhibitory effects of these reagents. This is especially true if complex, nondefined media, such as comsteep liquor, are used. This approach has been used with a wide variety of organisms, although it is more commonly employed using Arthrobacter simplex, Brmibacterium Ziplyticum, Corynebacterium spp and certain strains of Nocardia. WSmd i*MtOrV concentrations Because of the difficulties in creating optimal inhibitory concentrations, do you think that incubation of a culture of sterol degrading organisms in the presence of cholesterol and inhibitor will lead to the production of a single steroidal The answer is that a mixture of compounds is usually produced because the inhibition is somewhat imprecise and therefore ‘leaky”. The major product is usually 1,4- androstadiene3,17dione. n product? CAndrostene3,17dione and other androstane and testosteronerelated steroids are often also produced. The key to successfully using the inhibitor approach to convert sterols like cholesterol to steroids, is to reduce the further metabolism of the C-17 keto steroid as it accumulates. In some cases, the chelating agents used to trap Fez+ to reduce 9-hydroxylase activity am toxic. The toxicity may be reduced by using absorbants such as styrenedivenylbenze copolymers. Although this reduces the toxicity of the chelating agent it does not seem to prevent the chelating agent from trapping Fez+ ions. This increases yields, especially of 1,4-androstadiene3,17dione. Yields can be further inmased by the addition of oils: linseed and soya oils are most effective. Typically, sterol concentrations of 3 to 4 g I-’ are used and incubation times of about 100h. Yields are dependent upon the species and substrates used. Some data relating to the yields of 1,4-androstadiene-3,17-dione from various sterols and steroids using cultures of Arthmbacter simplex are reported in Table 9.3. reduction of bxicitV 306 Chapter 9 Substrate Meld of 1,4-androstedlene3,17-dlone (as % of subarate) campesterol 38 cholesterol 58 cholestanol 33 7dehydrocholestrol 16 ergosterol 5 lithocholic acid 63 psitosterol 39 Table 9.3 Yields of 1,4 androstadiene-3,17, dione using a variety of sterols and steroids as substrates and employing cultures of A. sinlplex (data from Martin CKA Sterols in Biotechnology Vol6a. Edited by Kieslich K 1984 Veriag Chemie, Weinheim). The data shown in Table 9.3 indicate that highest yields of 1,4-androstadiene-3,17dione are obtained using lithocholic acid as substrate. However, this substrate is not necessarily the one of choice for the commercial production of 1,4-androstadiene3,17dione using A. swZex. List factors that could determine the choice of substrate. n Lithocholic acid costs 2 or 3 times more than cholesteml. Thus, although the yields are slightly lower with cholesterol, it is cheaper to use it Furthermore, cholesterol is more widely available and in greater quantities than lithocholic acid. These two factors tend to favour the use of cholesterol. Lithocholic acid does have the advantages, however, of being more water soluble and is, therefore, more easily supplied to cultures in aqueous media. The costs of recovery of the desired product from the reaction brew are also commercially important. The point we are making in this in-text activity is that in selecting a substrate we need to consider more than simply the conversion efficiency and the cost of the substrate. Biotransformation of lipids 307 Assume that you have a culture of a Mywbaderium sp which is able to use cholesterol or psitosterol as its sole source of carbon and energy. You incubate aliquots of this culture with samples of media containing one of the following as the sole carbon source. a) Predict where or not and to what extent the organism will grow on these b) Predict the likely metabolic products from incubating this organism with substrates. these substrates. 308 Chapter 9 A medium containing bile salts such as lithocholic acid along with carbohydrates and peptone has been used to isolate gut organisms. One such isolate has been shown to completely degrade the lithocholic acid (structure given in Figure 9.1). In an experiment in which a student was examining the tolerance of this organism to Pbz+ ions, it was observed that the organism grew quite well in 0.1 mmol 1-I Pb2+ but that the lithocholic acid was only partially catabolised yielding a range of products, including the following: -I I _. . main products minor product What is the likely explanation for this observation? 9.3.3 Use of mutants A wide variety of mutants with modified sterol (steroid) meta;bolism has been produced using conventional mutagens such as N-methyl-N'-nitroscFguanidine and ultraviolet light. The organism most commonly, but not exclusively, used is Mycobacterium forhritum. Wild type members of this species can catabolise a range of sterols. Mutants have been produced that are blocked at various stages of the catabolism, giving rise to a wide range of products. Some of the produds are used by a variety of commercial organisations as intermediates in the manufacture of medically valuable steroids. We have reported a few of the many possible examples in Table 9.4. We would not expect you to recall the details of these. You should, however, realise that this is a very valuable commercial market and companies involved in this area hold extensive patent rights protecting these processes. The strains described in Table 9.4 are all of commercial value since they produce compounds which are either pharmacologically active or can be converted to pharmacologically important compounds. For example, the production of 1,4-androstadiene3,17-dione from &sitosterol provides material which can be read$ converted to estrone, while 4-androstene3,17-dione can be converted to testosterone. Pmt ah& Biotransformation of lipids 309 Organism Prlnclple Product USer Mycobacterium fottuitum 9a-hydroxyandrostQ-ene-4,17- Upjohn NRRL B-8119 dione M. fortuitum 4-androstene-3,17dione NRRL 8-1 1359,11045 M. fortuitum M. fortuitum FERM Pa09 NRRL B-8119 9a-hydroxy-3-oxo-23,24- dinorchol-4-ene-22-oI Upjohn Upjohn 3,9dihydroxy-9,1O-secoandrosta Mitsubishi -3,5-triene-17-one Mycobacterium parafortuitum 9u-hydroxyandrost4-ene-3,17- Mltsubishi FERM P-4926 dione M. parafortuitum 4-androstene-3,17dione MCI 0807 M. parafortuitum 3-oxo-23,24dinorchola-l,4- MCI 061 7 diene-22-01 Mitsubishi Mitsubishi Mycobacterium sp 1,4-androstadiene-3, 1 7-dione Searle NRRL 8-3683 Mycobacterium sp 4-androstene-3,17dione NRRL 8-3805 Searle Rhodocoms corallinus 3p-hydroxy-23,24dinorchol-5- Mitsubishi FERM P-4812 9n9-22-0k2 acid Arthrobader simplex 3-hydroxy-9-oxo-9, 1 0-sec FERM P-4477 pregna-1.33 Corynebacterium equi 9-a hydroxy-3-oxo-23,24- triene90carboxylic acid dinorchoIa-4,17diene-22-oic acid Mitsubishi Mitsubishi Table 9.4 Some examples of mutants used industrially for the removal of the side chain of sterols. 9.4 Specific steroid interconversions and reactions In section 9.3, we discussed in general terms the use of microbial metabolism to selectively remove the side chain from sterols to produce steroids. This removal may also be accompanied by some modification to the ring structure. We did not, however, discuss in any detail any specific reactions. In this section we will focus on some specific reactions. Before we begm this discussion, see how many types of enzyme catalysed reactions you can think of that may be used to carry out speafic modification of sterols/steroids. (Do this without looking at Table 95). n 310 Chapter 9 Table 9.5 Some readion types commonly encountered in sterokteroid interconversions. In Table 95 we have listed a large number of rraction types. For many of these reaction types you may be able to think of examples from central metabolism. For example, the oxidation of alcohols to ketones is a very commonly encountered reaction. Thus: Biotransformation of lipids 31 1 Similarly, the introduction of double bonds, isomerisation or hydrolysis are also frequently encounted reactions in central metabolism. Many of these reactions have their analogues in sterol/steroid intermnversions. Below we will confine ourselves to a limited number of examples. Consider the following reaction sequences. Using the terms given in Table 95, name the types of reactions shown as 1,2,3 and 4. 9.4.1 Hydroxylations Many examples of microbial hydroxylation of sterols/steroids have been reported. These hydroxylations usually involve mixed function oxidases which utilise molecular oxygen and cytochrome P-450. The reaction can be represented by: n Which positions of the steroid rings could be hydroxylated? 312 Chapter 9 Virtually all positions of the steroid nucleus may be hydroxylated. Of course, any particular enzyme is fairly specific. Any particular organism may carry out single or multiple hydroxylations. Where di-hydroxylations take place, the usual combinations are hydroxylations at positions 6 and 11 or 12 and 15. You should also note that not only are the positions of hydroxylation specific, the orientation of the added hydroxyl groups is specific as well. Thus hydroxylation at position 6 is often in the configuration, while at position 11 the hydroxyl group is often added in the a configuration. Multiple hydroxylations usually involve more than one enzyme. Thus addition of hydroxyl groups in position 68, involves a different enzyme from that which inserts hydroxyl groups in position lla. Frequently substrates containing an lla hydroxyl group are required to induce the enzyme which catalyses the introduction of a hydroxyl group at position 68. The key questions are: can hydroxylations be carried out in vitro at desirable positions on the steroid nucleus, and can this be done to achieve the desired configuration? The answer is yes in both cases. A very large number of hydroxylating enzymes have been identified and it is theoretically possible to hydroxylate any of the 19 carbon atoms of the steroid nucleus. However, these enzymes will each only utilise a specific range of substrates. The sources of some hydroxylating enzymes and the reactions they catalyse are given in Table 9.6. We have been selective in compiling this table, giving only a single example for each hydroxylation. Again we would not expect you to remember all of the details given in Table 9.6; we have provided these data primarily as an illustration of the potential of using enzymes to bring about specific chemical transfoxmations. Biotransformation of lipids 313 Reaction Substrate Product Micro-organism 1 a-Hydroxylation 1 p-Hydroxylation 2a-Hydroxylation 2p-Hydroxylation 3u-Hydroxylation 3p-H ydroxylation 4p-Hydroxylation 5u-Hydroxylation Sp-H ydroxylation 6u-H ydroxylation 6p-H ydroxy lation 7u-Hydroxylation 7p-Hydroxylation 8p-H ydroxylation Sa-H ydroxylation 9&H ydroxylation Androst-4-ene-3,17- 1 a-Hydroxyandrost-4- Penkillium sp. dione ene3,17dione Androst-4-ene-3,17- 1 p-Hydroxyandrost-4- Xylans sp. dione ene-3,17dione Androstane-7,17- 2a-Hydroxyandrostane Wojnowkia graminis dione -7,17dione Androst-4-ene-3,17- 2p-Hydroxyandrost-4- Penkillium sp. dione ene-3,17dione Androstane-7,17- 3a-Hydroxyandrostane Diaporthe celastrinia dione -7,17dione 1 7p-Hydroxyandro- 3$,17$-Di hydroxy- Wojnowicia graminis stan-1 1 -one androstan-1 1 -one 17a-Methylestra-l,3,5 1 7a-Methylestra-l,3,5 Aspergillus flaws (1 O)-triene-3,17gdiol (1 O)-trieneS,4,17ptriol Nor-5a-Pregnane-2, 5a-Hydroxy-nor- Cokeromyces 20-dione pregnane-2,2O-dione recurvatus 3p,14-Dihydroxy-5p, 3p,5,14-Trihydroxy-Sp, Absklia orchidis 14pcard-20(22)- 14pcard-20(22)- enolide enolide Androstane-1 ,17- 6u-Hydroxyandrostane Calonectria decora dione -1,17dione 1 7p-Hydroxyestr-4- 6$,17p-Di hydroxyestr- Helminthosponum 0ne-3-0ne 4-ene-3-0110 rasonoi Androst-4-ene-3,17- 7u-Hydroxyandrost- Mucor griseocyanus dione 4-ene-3,17dione Androst-dene-3,17- 7&Hydroxyandrost-4 Xylaria sp. dione -ene3,17dione 17,21 -Dihydroxypregn- 8#3,17,21 -Trihydroxy 4-ene-3,20dione pregnQ-ene-3,20- Androst-dene-3,17- 9a-Hydroxyandrost-4 Nomrdia corollina dione -ene3,17dione 9p,1 Oa-Pregn-4-ene- S&Hydroxy-Sp,l Oa- Cephalofheciom 3,204ione pregnQ-ene-3.20- roseum Cercospora melonis dione dione Table 9.6 Examples of hydroxylation of steroid nudei by micro-organisms. (Data derived from Neidleman, SL "Industrial Chemicals: Fermentation and lmmobilised Cells" in "Biotechnology the Sdence and the Business", edited by Moses V and Cape RB, published by Hamood Academic, London 1991). 314 Chapter 9 1 Op-H ydroxylation 1 1 a-Hydroxylation 11 p-Hydroxylation 12p-Hydroxylation 14a-Hydroxylation 15a-Hydroxylation 1 5B-Hydroxylation 1 6a-Hydroxylation 16p-H ydroxylation 17a-Hydroxylation 1 7p-Hydroxylation 1 8p-Hydroxylation 19-Hydroxylation 21 -Hydroxylation 1 7p-Hydroxyestr-4- ene-3-0ne Progesterone 11 -Deoxycortisone ene-3-one Androst-4-ene-3,17- dione 1 7p-Hydroxyestr-4- 1 1 a-Hydroxy-%- pregnane-3,20- dione Androst-4-ene-3,17- diore Androst-4-ene-3,17- dione 1 7p-Hydroxyestr-4- ene-3-one Pregn-4-ene-3,20- dione Androstane-3,ll- dione 1 1 p,21 -Dihydroxy- pregn-4-ene-3,20- dione 1 0$,17$-Dihydroxyestr Botrytis paeoniae -4-en 9-3-0 ne 1 1 a-Hydroxyproges- Rhizopus terone Hydrocortisone Curvularia lunata 1 2p,17p-Dihydroxyestr Colledotrkhum -4-ene-3-one d8ffidiS 14a-Hydroxyandrost-4 Dematlaasae -ene-3,17-dione Strain M202 11~,15a-Dihydroxy-5a Calonectria obcura -pregnane3,20dione 1 5pHydroxyandrost Xflm’a sp. -4-ene-3,17dione 1 6a-H ydroxyandrost streptomyces -4-ene-3,17dione roseochrogene sp. 1 6p, 1 7p-Dihydroxyestr Mycosphaerella -4-ene-3-0ne latebrosa 1 7a-H ydroxypreg n- Cqhalothecium 4-ene-3,20dione roseurn 1 7gHydroxyandro- WojnoWida graminis stane3,ll dione 1 1 p, 18,21 -Trihydroxy- pregn-4-ene-3,20- cassiicola dione Corynespora 6u-Hydroxyandrostane 6a,lg-Dihydroxyandro- CalOneCtra decors -3,17dione stane-3,17dione Nor-pregn-3-ene-2,20 21 -Hydroxy-nor-pregn Aspergillus niger dione -3-ene-2,20dione Table 9.6 ......... Continued From your knowledge of the st~ctum of pharmacologically active steroids such as the corticosteroids, which of the hydroxylations shown in Table 9.6 are likely to be of most commercial value? (You may find Figure 9.1 helpful). You probably concluded that hydroxylation at position 11 is of potential value. This is in fact true. However, hydroxylation at position 16 is also important. The three commercially most important hydroxylations are lla, llf) and 16a hydroxylations. lla-Hydroxylation of progesterone is used by Upjohn, whilst 1lf)-hydroxylation of various substrates is used by a number of companies including Gist Brocades, Pfizer and Wering AG. 16a Hydroxylation of 9-fluorohydrmrtisone is used by Squibb. n commerciaiiy hWmt Biotransformation of lipids 31 5 The first microbial hydroxylation to be exploited was the lla-hydroxylation. This reaction is catalysed by a wide variety of organisms but, after a considerable amount of screening, the preferred species is Rhizopus nigrim ATCC 622%. This filamentous fungus is used to hydroxylate progesterone. Normally large bioreactors are inaculated with a large inoculum and the culture allowed to grow for about 12-20h with moderate aeration. The media used are usually rich in carbohydrates (usually 5% dextrose) and corn steep liquor (3%) although there are many variations. The pH is maintained at about pH 4.24.7 and the incubation is carried out at 28°C. In practice, the actual composition of the medium used depends on the current economics and availability of the feedstocks. 11 a-hydroxy- After the initial growth, progesterone (05-5 g 1-l) is added as a powder or as an acetone solution. If a powdered form is used, it is wetted with a small amount (0.01%) of Tween to facilitate its dissolution into the reaction mixture. A single addition of progesterone at a concentration of about 5g 1” enables about 86% hydroxylation to take place within about 50h. The remaining progesterone remains unaltered. Alternatively, hydroxylation may be carried out in a continuous process. In this the organism is first cultured in one vessel and then transferred to a production vessel. Progesterone is fed into this second vessel at a concentration of 05g 1-’. Over 50% of the progesterone is hydroxylated when the residence time in the production vessel is about m. Bearing in mind that hydroxylation requires molecular oxygen and that Rtnzopus is a filamentous fungus, what factors in the reaction vessel do you anticipate are critical to the successful hydroxylation of progesterone? You should have anticipated that aeration and mixing are of critical importance. The mycelial nature of Rhizopus restricts the rate at which we can use impeller mixing. High impeller speeds will disperse air within the medium but will also cause breakage of the mycelium. Thus fairly low impeller speeds have to be used. This, of course, restricts the distribution of air (oxygen) within the system. This could, in principle, be circumvented by using high aeration rates but this, in turn, may cause foaming problems. If anti-foaming agents are used, these may interfere with the subsequent collection and purification of the product. In practice a compromise has to be achieved between impeller speeds and aeration rates which produces tolerable damage to mycelia without slowing the hydroxylation too greatly. Considerable attention has to be paid to optimising and controlling these factors. n the conversion of substrate to product, in the process described above? Both of these factors are difficult. Obtaining representative samples of filamentous organisms from cultures is difficult and both substrate and product are relatively insoluble. Thus it is difficult to monitor these processes. Nevertheless, in practice satisfactory procedures have been developed to generate data that are sufficiently accurate to enable reasonable monitoring of cultures. n darnageto rnymlm Can you foresee any major problems in monitoring the health of the culture and 316 Chapter 9 Figure 9.5 Outline of the recovery of 1 la-hydroxyprogesterone from cultures of Rhizopus nigricans. The recovery of the product is desaibed in outline in Figure 95. Essentially the process involves separating the broth and mycelium by filtration, extracting the mycelium with acetone and methylene chloride. Combining these extracts with the broth and re-extracting with methylene chloride. The extract is washed with 2% sodium bicarbonate, evaporated and re-dissolved in methylene chloride. The product is allowed to crystallise from the methylene chloride. Biotransformation of lipids 31 7 n Would you expect the product to be pure lla-hydroxyprogesterone? A single crystallisation is unlikely to lead to the isolation of pure crystals. In practice the product recovered in this process contains about 90% 11 a-hydroxypmgeterone with low levels of other products (especially 5 a-pregnane-3,20-dione and 6g, lla-dihydroxyprogesterone). An example of a manufacturer who uses microbial lla hydroxylation is Upjohn; progesterone is used as substrate. 11s-Hydroxylation is achieved in a process analogous to that described above for lla-hydroxylation. In this case, however, the organism of choice is Cum2mia lunata NRRL 2830. The use of this organism is particularly attractive as it will carry out the hydroxylation using a wide variety of substrates, including steroids that have already been substantially modified. This has, however, a serious draw back: 11s-hydroxylations by C. lunata suffer from the production of a wide variety of other products, especially other hydroxylated steroids. Substrate purity, incubation times and recovery processes are, therefore, of crucial importance. Commercial 11s-hydroxylations are carried out by Pfizer, Gist-Brocades, Shering AG and Me& Darmstad t. 16a-Hydroxylation is used in the production of triamcinolone and fluocinolone. The organism of choice is Streptomyces mechrmgenes. In commercial use, this organism is used to catalyse the lk-hydroxylation of 9a-fluorohydrmrtis. The product is subsequently dehydrogenated, as shown in Figure 9.6. This two stage process replaced a fourteen-step chemical-based process. 1 1 p- hydm)(@tim lea-hydroxy- lation Figure 9.6 An example of 16u-hydroxylation catalysed by Streptomyces roseochromogenes. Commercial 16u-hydroxylations are carried out by Squibb. 318 Chapter 9 We will leave the hydroxylation of the steroid nucleus at this stage. You should however appreciate that the examples we have described are but a very small part of the potential of enzymatic chemical transformations. Considerable efforts are being made to find easier (cheaper/mre efficient) ways of using biocatalysis. This may involve developing new strains, exploring the possibilities of using immobilised cells or using extracted (cell-free) enzymes. Although microbial steroid hydroxylation has been achieved using cell-free extracts (see Table 9.7), there are many problems to be overcome before these systems become commercially applicable. Thus, although currently there is only limited commercial interest in such systems, developments in enzymology, especially the application of site directed mutagenesis to produce enzymes with desirable characteristics, may rekindle this area of development. I E- source Substrate Site@) of hydroxylation Fungi Aspergillus niger progesterone Aspergillus ochraceus various Curvularia lunata various Rhuopus nigrans progesterone Prokaryotes Bacillus megaterium progesterone Nocatdia restrktus progesterone Streptomyces progesterone roseochromogenes 1 la 1 la lop, lip, 14a 6p, 1 la, 17a 1- 158 9a 16a ~ Table 9.7 Examples of steroid hydroxylation using cell-free extracts. (Data cited by Martin CKA, Steroids in Biotechnology Vol6a. Edned by Kieslich K 1984 Verlag Chemie, Weinheirn). So far we have focused on hydroxylation in the steroid nucleus. From what you read earlier, where else on sterol molecules may hydroxylation n take place? You should have recalled that the breakdown of the side chain involves an initial hydroxylation (see Figure 9.2). Hydroxylation most frequently occurs on the terminal or side chain methyl groups. Biotransformation of lipids 319 Select the most appropriate organism from the list below to catalyse each of the fnllowing conversions. List of organisms: saccharo?mJces cerevisiae Steptomyces meochromogene Rhizopus nigrans Curvularia lunata Corynebacterium sp L 9.4.2 Alcohol - ketone interconversions The interconversion of alcohols to ketones is a common biochemical reaction. The introduction of hydroxyl groups into the steroid nucleus and side chain creates a variety of secondary alcohols. Some of these, especially at positions 3, 7, 11 and 17 are frequently oxidised to ketones. We have illustrated this type of reaction by showing the conversion of a 11 a-hydroxy-steroid to a ketone. 320 Chapter 9 11 a-hydroxy-steroid 11 keto-steroid The mapr commercial application of this type of reaction is the reduction of 17-keto steroids to the corresponding secondary alcohol. The manufacturers Wering AG use enzymes from Sacchuromyces sp to reduce secosteroid (rac-3-methoxyd, 14, secoestra-1,3,5(10),9(1 l)-tetraene-l4,17dione) and androst+ene, 1,17 dione. 9.4.3 Desaturation Although there are many potential places around the steroid nucleus where desaturation may occur, only a few are of commercial value. The most important is the introduction of a second double bond in ring A already containing a double bond at position 4. Thus: reduction of 17-keto seroids The desaturation takes place by the stereospecific removal of hydrogens from C-1 and C-2 (in fact it is the la and the 28 hydrogens that +e removed). Dehydration at position one often leads to ring A aromatisation. Thus: rir~ aromalisabon -T The use of microbial ldehydrogenations is essential to the manufacture of corticosteroids, as chemical dehydrogenation processes are commercially noncompetitive. A variety of enzymes are available from various organisms including Arth~obucter simplex, Bacillus cydwxydm, B. lentus, B. sphaericus, Mycobacterium srobifane and Septomym uffinis. Arthrobacter simplex is particularly useful as it will dehydrogenate a range of sterols/steroids. Other dehydrogenation processes show varying specifiaties. The Shering Corporation uses Arthobacter simplex to ldehydrogenate hydrocortisone, whilst Upjohn employs Septomyxu uffinis to l-dehydrogenate dienodiol (1 18, 21 dihydroxypregna-4,17(20)-diene-S-one). organisms Biotransformation of lipids 321 9.4.4 lsomerisation Although many isomerisations are A5-3 ketones to A4-3-ketones. ssible, relatively few are of commercial value. Most involve the movement of doub f" e bonds, typically illustrated by the conversion of movement of double bonds The isomerase (EC 5.3.3.1) from Pseudomonas testosteroni has been studied in detail. This enzyme transfers a hydrogen from position 4 to the 6fLposition. Although several isomerases have been detected, their presence is often seen as presenting problems as they frequently lead to product diversification. 9.5 Transformation of other terpenoids In section 9.4 we described some of the transformations that are employed in the manufacture of valuable steroids. Sterols and steroids are, however, only a small fraction of the total range of terpenoids produced within the biosphere. Others include monoterpenoids based on the structures: monoterpenads These include a wide variety of compounds used as flavours and fragenm. Others, including citronellal, citral and limonene, are used as starting points for the production of more valuable terpenoids. As an illustration of the use of microbial transformation of monoterpenoids we can cite the production of menthol from citronellal. Various organisms, including Pseudomonas ueruginosa and Penicillium digitdurn have been used for this conversion. High yields (6% for P. ueruginosa; 93% for P. digitaturn) have been claimed. We have illustrated these processes in Figure 9.7. men*d 322 Chapter 9 ~ Figure 9.7 Examples of the microbial transformation of citronellal to menthol. Cyclical monoterpenes such as limonene have also been used as substrate for the production of valuable products. A good example is the conversion of limonene to u-terpineol by Cladosporium sp. Thus, ionones Related to the monoterpenes are the ionones based on the structure Using a variety of organisms, a range of chemical modifications may be carried out. We have summarised these in Figure 9.8. Biotransformation of lipids 323 Figure 9.8 Typical enzyme-mediated modifications to ionones. A wide variety of products are, therefore, possible. Many find commercial use as constituents of essential oils. It is beyond the scope of this text to examine all of these processes in depth. Much literature is available, but we would recommend Volume 6a of Biotechnology, edited by Kieslich K (published by Verlag Chemie, 1984) as a good source if you wish to pursue this aspect further. Before we leave the enzymatic modification of terpenoids, we should point out that enzymes are also employed to resolve racemic mixtures of terpenoids. The principles of this are similar to those employed in the resolution of racemic mixtures of amino acids (see Chapter 8). See if you can suggest a straw for resolving a racemic mixture of DL-menthol to produce L-menthol. Assume that the DL-menthol is produced as the succinate ester. You probably came up with the suggestion that by using an esterase which selectively hydrolyses the ~ucci~te ester of L-menthol, you would be able to isolate L-menthol from the mixture. This is in essence the way the process is carried out commercially. We can represent this process by: DL-menthyl SUCC~M~~ L - specific L-menthol + D-menthyl succinate resdution of racemic mi*res n > esterase The process uses cells of RhodotoruZa minuta entrapped in polyurethane. These cells selectively hydrolyse the L-ester. The remaining D-menthyl succinate is then hydrolysed and the D-menthol racemised via D-menthone and then “cycled. We have represented this process in Figure 9.9. 324 Chapter 9 Figure 9.9 Resolution of DL-menthol using an esterase from Rhodotorula minuta var fexensis. The resolution of DL-menthol is important industrially. L-Menthol has a mint taste and gives a cooling sensation. It finds use in a number of important products including toothpaste and confectionary. D-Menthol does not have the same taste nor the same cooling properties. DL-menthol can be produced relatively simply using a variety of chemical routes. Biotransformation of lipids 325 Using terms given in the list below, identify the nature of the following biotransformations. Term list reduction; hydrogenation; epoxidation; oxidation; hydroxylation. 326 Chapter 9 Candida cyZindricae produces a lipase which will esterify L-menthol using !%phenyl valeric acid. Explain how this enzyme may be used to resolve a racemic mixture of DL-menthol. Assume you want to prepare both D-menthol and L-menthol. 9.6 Chemical conversion of miscellaneous organic compounds In the previous sections, we have given illustrative examples of biologically mediated chemical transformation of various terpenoids, particularly emphasising their application in chemically modifymg sterols, steroids and monoterpenes. Analogous reactions are available for a whole range of organic molecules, be they of biological or chemical origin. In this section, we will briefly examine the application of biologically mediated chemical transformation to these compounds. Again we have been very selective, choosing a few examples simply for illustrative purposes. To give a comprehensive review of all of the possibilities would require a series of texts. 9.6.1 Biotransformation of alicyclic compounds n Consider that you have a racemic mixture of the following compound. See if you can come up with an extensive range of possible applications of biological transformations that may facilitate separation and use of the racemates. (Think of the application of enzymatically mediated transformations which we describe earlier, this should help you to corn up with several ideas). The list of possibilities you could have come up with is quite extensive. You might, for example, have suggested that enzymatic processes may be used to resolve the racemic mixture. This has, for example, been achieved using strains of Sacchmomyces cerevisk. These strains reduce the ketone to a secondary alcohol only if the chlorine on position 2 is in the R configuration. Thus: duuionof ketone Bdransfotmation of lipids 327 1 S hydroxy 2R 2,S-chloro 2 chbrohexanone chbro cyclohexane cyclohexanone With this single example we have in fact described two uses of enzymes in alicyclic chemistry, the reduction of ketone groups and the resolution of racemic mixtures. Other possible transformations that are theoretically feasible a: the introduction of double bonds in the ring by using dehydrogenation reactions; specific hydroxylations. The products of these reactions may be further modified by, for example, epoxidation or esterification. Using this simple example, we have illustrated how the pMciples we established in discussing steroid transformation may be applied to a much wider range of organic materials. In Figwe 9.10 we have illustrated a few compounds. It would be instructive for you to take a separate sheet of paper for each compound, draw the compound at the top of the sheet and then draw out some products that may be produced from each of these using enzymatidy mediated transformations. For each, you should consider the possibilities that arise from: n hydroxylation reaction; dehydrogenation/hydrogenation reaction; alcohol/ ketone interconversions; 0 esterification; 0 amidation; epoxidation. 328 Chapter 9 Figure 9.10 Compounds that are to be used for the in-text activity. If you have carried out this in-text activity, you should be impressed by the enormous range of possible products that could be made. Whether or not these products are of commercial value, or the enzymatic conversion can be conducted in a commercially viable manner is another matter. Nevertheless, chemists have, over the past few decades, turned increasingly to microbial systems to mediate desirable chemical changes. An extensive review of the biotransformation of alicyclic and heteroalicyclic compounds is given by A. Kergomard in Biotechnology - Vol6a edited by Kieslich K (published by Verlag Chemie, Weinheim 19841, for those who would like to examine this area in greater detail. Kergomard not only reviews the chemical modifications that may be carried out by biological systems, but also lists examples of organisms which will carry out these modifications. For example, he lists 21 species which will hydroxylate substituted phenylcyclohexanes. Such lists also indicate that the organisms that may be of value are drawn from diverse genera and include both prokaryotic and eukaryotic species. In the same text, P. R. Wallniifer and G. Engelhardt have reviewed the application of biotransformation of aromatic and heterocyclic compounds, again describing a wide range of chemical transformation and organisms. The metabolic processes underpinning the catabolism of aliphatic and aromatic compounds are described in the BIOTOL text "Energy Sources for Cell". Before we leave the chemical transfoxmation of these compounds we draw your attention to the fact that these transformations may be of value not only to produce desirable compounds, but also as a means of removing undesirable materials from the environment. This aspect is examined in the BIOTOL text "Biotechnological Innovations in Energy and Environmental Management". wide range Of b.anSfOlTIWtiOnS chemicai ad wanisms Biotransformation of lipids 329 9.7 Production and use of fatty acids and their derivatives No discussion of the use of biotransformation in lipid chemistry would be complete without some mention of chemical transformation relating to fatty acids. Fatty acids are a major component of the lipid fraction of organisms. They are mainly found as components of triglycerides and phospholipids, although they may occur in smaller quantities as free fatty acids or as esters of other moieties. Fatty acids, either as free acids or as esters, are valuable commodities in the food and cosmetics industries. They may also serve as precursors of a variety of other compounds. 9.7.1 Production of desirable triglycerides Much of the efforts in the food industry centres on creating oils/fats with desirable characteristics, especially fats that will melt at about body temperature. These give the desired "melt in the mouth" feel. The melting point of triglycerides is governed by the degree of saturation and chain length of the fatty acids which esterifies the glycerol. We have illustrated the composition of cocoa butter and palm oil in Table 9.8. Palm oil, with a high unsaturated fatty acid content, has a low melting point (hence it is an oil). The fatty acids of cocoa butter are, on average, longer chained and more saturated and hence these triglyceride are solid at ambient temperature. Triglyceride Palm oil mid-fraction Cocoa butter degree of ad chain length (% dry welght) (% dry weight) StStSt 5 1 POP Post StOSt StLnSt 58 13 2 9 16 41 27 8 st00 4 6 Others 2 1 stearate POP stearate E stearate StStSt palmitate oleate E palmitate palmitate oleate E stearate post stearate linoleic E stearate StLnSt StOSt stearate oleate E stearate stearate oleate E oleate st00 Table 9.8 Triglyceride composition of palm oil and cocoa butter. 330 Chapter 9 Cocoa butter has the desired "melt in the mouth property and is of high commercial value in comparison with palm oil. On the other hand, palm oil is more abundant than cocoa butter. The question is, can we convert palm oil to a product which has the desired properties of cocoa butter? The answer is yes, by using lipases. Selection of lipases The enzymes used for modification of oils and fats are extracelluar microbial lipases. They are excreted by micro-organisms into the growth medium to catalyse the degradation of lipids, and can be produced on a large scale by fermentation. Lipases catalyse the hydrolysis of oils and fats to give diglycerides, monoglycerides, glycerol and free fatty acid. The reaction is reversible, and consequently microbial lipases also catalyse the formation of glycerides from glycerol and free fatty acid. Because of the reversibility of the lipase reaction, hydrolysis and re-synthesis of glycerides occur when the enzymes are incubated with oils. These cause an exchange of fatty acid groups between triglyceride molecules giving interesterified products (Figure 9.11). lipase action Figure 9.1 1 Mechanisms of lipasecatalysed interesterifications. Microbial lipases are catalytically active in a predominantly organic environment containing very small amounts of water. Under these conditions the hydrolytic action of the enzymes is restricted by the limited availability of water, and high yields of interestenfied triglycerides can be obtained. Mixtures of triglycerides and free fatty acids can be used as reactants for lipase catalysed interesterifications. In these cases, free fatty acid exchanges with the fatty acyl groups of the triglycerides to fonn new triglycerides enriched in the added fatty acid. The substrate specificities of lipases are crucial to their application as catalysts for modification of triglycerides. The enzymes can show specificity with respect to both the fatty acid and glycerol parts of triglycerides. However, most extracelluar microbial lipases are not highly speafic with respect to the fatty acid groups found in the oils and fats used as raw materials for the edible fats industry, although reaction rates can vary with the chain length, and extent and position of maturation of the fatty acid group. liPmsadve m bdrO*Obic enviroments Biotransformation of lipids 331 With regards to the glycerol part of triglycerides, the speaficities of lipases are of technical significance. Some microbial lipases are not specific and catalyse reactions at all three positions of glycerol. When lipases of this type are used as catalysts for interesterification of triglyceride mixtures, products containing a random distribution of fatty acid groups are obtained. These products have similar composition to those obtained by chemical interesterification. smreospeclficity lipase stability readon mixture enzyT”e adsorpbon onto macropamus partides A second group of lipases catalyse reactions specifically at both the outer (1- and 3-) positions of glycerol. These enzymes are said to show regiospecificity. This specificity can be exploited to produce triglycerides which are difficult to obtain by conventional chemical pdures. Regiospecific lipases catalysing reactions selectively at either the 1- or 3-positions of triglycerides would have very useful commercial applications. Microbial lipases show stereospedicity (specificity for stereoisomers) in reactions with many types of esters, but unf~rtu~tely stereospecificity in reactions with triglycerides has not been detected to date. In addition to having the required specificity, lipases employed as catalysts for modification of triglycerides must be stable and active under the reaction conditions used. Lipases are usually attached to supports (ie they are immobilised). Catalyst activity and stability depend, therefore, not only on the lipase, but also the support used for its immobilisation. Interesterification reactions are generally run at temperatures up to 70°C with low water availability. Fortu~tely many immobilised lipases are active and resistant to heat inactivation under conditions of low water availability, but they can be susceptible to inactivation by minor components in oils and fats. If possible, lipases resistant to this type of poisoning should be selected for commercial operations. Interesterification catalysts The reaction systems used for modification of triglycerides usually consist of a lipase catalyst and a small amount of water dispersed in a bulk organic phase containing the reactants and, if required, a water immiscible solvent. The small amount of water in the reaction system partitions between the catalyst and the bulk organic phase. Lipases catalyse reactions at interfaces, and to obtain a high rate of interesterification the reaction systems should have a large area of interface between the water immiscible reactant phase and the more hydrophilic phase which contains the lipase. This can be achieved by supporting the lipase on the surface of macroporous particles. Highly active catalysts have been produced by adsorption of lipases onto macropomus acrylate beads, polypropylene particles and phenol-formaldehyde weak anion exchange resins. protein is bound, presumably essentially as a monolayer, within the pores of the particles. The large surface area of the particles (lorn2 g”) means that substantial amounts of pmtein can be adsorbed, and the pores are of sufficient size to allow easy access of reactants to this adsorbed protein. When choosing a support for an immobilised enzyme, what other factors (apart from activity and access to the substrate) do you think need to be considered? (Think about the cost of producing the immobilised system and how it will be Used). n 332 Chapter 9 As well as being active, the immobilised enzyme also needs to be stable (active for a long period) and the support must promote this. The support must also have appropriate mechanical characteristics: it should not disintegrate if used in a stirred tank reactor; it should produce even flow (without channelling) in a packed bed reactor. The cost of the support is also important. The interesterification processes Mixtures of triglycerides, triglycerides plus free fatty acids or triglycerides plus fatty acid alkyl esters are used as reactants in fat modification processes. These mixtures are exposed to lipases supported on macroporous particles in the presence of a small amount of water. Liquid substrates (oils) can be reacted without use of a solvent, but with solid reactants (fats) it is necessary to add a solvent to ensure that the reactants and products are completely dissolved in the organic phase. Various water immiscible solvents can be used, but hexane is preferred for commercial operation because this solvent is already used industrially for the processing of oils and fats. The fat modification processes can be operated either in batches using stirred tank reactors or continuously with packed bed reactors. Which process do you think would be preferred: stirred tank reactors operated batch-wise, or packed bed reactors operated continuously? (Before reading on consider the likely overall yields of each type of operation and the cost of operation. Then make your decision). Continuous reactors are likely to give the greatest overall specific productivity (quantity of product formed in a given time from a given quantity of enzyme), and therefore could be most cost-effective. In addition, in batch systems, the longer residence time involved can result in side reactions, leading to a decrease in the yield of triglycerides (the triglycerides are degraded). Continuous systems are also easier to monitor and to regulate by automation. In a typical reaction, a feedstream consisting of refined palm oil and stearic acid dissolved in petroleum either, is almost saturated with water (water content 0.06961, and then pumped through a bed of regiospecific lipase from Mucor miehei supported on diatomaceous earth. High catalyst activity as measured by an increase in the stearoyl content of the triglycerides can be obtained throughout 300 hours of continuous operation. Analysis of the triglyceride products shows that steamy1 groups are incorporated exclusively intu the 1- and 3-positions, mostly in exchange for palmitoy1 groups. This stearoyl incorporation results in the formation of 1(3jpalmituyl-3(1)- steamyl-2-oleoylglycerol (Post) and,l,3distearoyl-2-oleoylglycerol (Stost). Post and StOSt are the major triglycerides of cocoa butter, a valuable confectionary fat. n productivity Thus when palm oil is incubated in this way its composition shifts and becomes more like cocoa butter (see Table 9.9 and compare with Table 9.8). Biotransformation of lipids 333 us8 of high vakre and low Value produds bemr supports for enzyme irnrnobiliition nahr ral soufc6s StStSt 5 3 I POP post stost stlnst st00 Others 58 13 2 9 4 2 16 39 28.5 8 4 1.5 I Table 9.9 The triglyceride content of palm oil pre- and post-incubation with lipase and stearic acid as described in the text. Other reactants have been used for the production of cocoa butter equivalents by the enzyme technique. For example products enriched in StOSt and Post can be produced by reaction of olive oil, high oleate safflower and sunflower oils, sal fats and shea oleine with stearic and palmitic acids or their esters. It is also possible to use lipases speafic at 1,3 sites to produce triglyceride mixtures having useful functional properties in products such as margarines, low-calorie spreads and bakery fats. An example is the formation of triglycerides containing two long chain saturated fatty acid groups and one medium or short chain fatty acid group. These fats are effective hardeners for margarines and other spreads. It has been shown that lipase-catalysed reactions can be used for the largescale production of modified triglycerides. At present the technology is being targeted to the production of comparatively high-value products such as confectionary fats. Wider application of the reactions to lower-value, higher-tonnage products will be dependent on the development of cheaper processes using more-productive and/or cheaper catalysts. Fortunately there are indications that immobilised lipase catalysts will become more efficient and cheaper in the future. In the past, because of low fermentation yields, lipases have been expensive in comparison with the other main groups of extracelluar microbial enzymes such as proteases. Application of gene transfer technology to lipases could make them available at lower cost in the future. Considerable attention is also being given to the development of more effective supports for enzyme immobilisation. A range of organic and inorganic materials are being investigated as potential enzyme supports, and parameters which affect the activity expressed by imbilised enzymes are being studied. 9.7.2 Production of fatty acids and related compounds The fatty acids commonly encountered in biological systems are straight chained alkanoic or alkenoic acids, containing an even number of carbon atoms (usually CICW. In general, these fatty acids can be produced readily by extraction of the lipids from natural sources and saponifymg the neutral triglycerides. This is satisfactory providing a mixture of fatty acids is acceptable. Purification of specific fatty acids from the saponification mixture increases the costs considerably. 334 Chapter 9 Considerable interest arose during the 1970's and 1980's in the use of micrmrganisms to produce useful fatty acids and relabid compounds from hydrocarbons derived from the petroleum industry. Dunng this period, a large number of patents were granted in Europe, USA and Japan proteding processes leading to the production of alkanols, alkyl oxides, ketones, alkanoic acids, alkane dioic acids and surfactants from hydrocarbons. Many of these processes involved the use of bacteria and yeasts associated with hydrocarbon catabolism. Let us see if we can first establish some principles. bacteriaand yeasts If you were attempting to produce the alkanol, C16aH, what would be a suitable n substrate? We hope you would suggest a Cl6 alkane. If you were attempting to produce a GO alkanoic acid, what would be a suitable n substrate? Again, we hope you would suggest CIO alkane. The point we are trying to establish is that to some extent we control the nature of the product by selecting a particular substrate. Do you think we need to select a different organism for each product we would n like to produce? The answer is, generally, no. Most of the enzymes involved are specific in terms of the reaction they catalyse, but will work with a range of substrates. Some Crmdida sp for example contain an enzyme system which will convert n-alkanes into alkane dioic acids: (313(CH2)nCH3 > HOOC(CH2)n COOH choice of substme Thus if provided with CIZ the product will be CI~ dioic acid, whilst if provided with CIS the product will be CIS dioic acid. If supplied with a mixture of alkanes, then a mixture of dioic acids will be produced. There are, however, some restrictions on this and not all alkanes will be oxidised equally. Thus the Candida sp generally use ClZ-& alkanes, whilst Corynebacteria will preferentially utilise C~Z-CZ alkanes. We should not, however, mislead ou into believing that the product is always predictable. Strains of Candida topaz& (FERM P3291) will preferentially produce CIO dioic acid even if a mixture of C1I~lS alkanes are used. In Table 9.10 we have listed some examples of substrates, products and organisms that have been cited in the patent literature for the production of various alkanols, alkanoic acids, ketones and dioic acids. These are meant to act as illustrations, we would not expect you to remember them all. Which type of organism, prokaryotic or eukaryotic, seem to dominate the list of n organisms which appear to be useful for the chemical modification of alkanes? Most of the organisms listed in Table 9.10 are prokaryotes. The exceptions are strains of Gzndida and Torulopsis. Biotransformation of lipids 335 hllenob 21-OH c1 Methylocms &OH C2H4-CslI3 Methylosinus trtchospdum C2H444H8 Methylosinus MchospOriUm %-CirOH c&16 Methylosinus trichosptium &CeOH -6 Methylosinus sp Methylococcus sp Methylobacter sp alkyloxkles &oxide &oxide 24-oxide &oxide 28Hl6-oxide Ketones 2346 ones kc2 ones kc3 ones 2.644 ones s6-cp ones Alkonolc ad& S10-C20-oiC c8-cl6 c11420 c10-c20 Aanetobader sp Arthrobacter sp Brevibacterium sp Corynebaderium sp Mmus sp Nocardia Sp Methylocystis sp Methylomonas sp Methylobacter sp Pseudomon8s oleovorans Methyl8rnonas Sp Wide range of methanotrophic bacteria An?hrobacter sp Arthrobacter sp Arthrobacter sp Nocardia sp Wide range of organisms especially Candda sp, Tomlopsis sp, Arthrobader sp, Coryn668ctedum sp Note that, by using alkane derivatives such as alkyichloride, corresponding derivatives of the alkanoic acids may be produced nilem dlolc a- 4 Cxdioic acid cx (x can be between 10-22) Wde range of organisms induding Candd sp, Torulapsis sp, Corynebacterium sp. Table 9.1 0 Examples of microbial products produced from alkanes and related compounds. At the examples cited are the subject of patents. 336 Chapter 9 Prostaglandins Prostaglandins are important derivatives of unsaturated fatty acids. Below we have drawn the structure of prostaglandin E2 as an illustration of the general structure of the prostaglandins. prostaglandin (PGE,) There are however a very large number of prostaglandins and these show differing pharmacological properties. They have therefore different applications in health care. These compounds are, however, only produd in very small quantities by natural systems. Therefore a variety of strategies have been developed to produce these compounds in larger quantities in vitro. This usually involves a mixture of chemical and biological procedures. For example, biological catalysis is used to: 0 synthesis chiral reactants for subsequent chemical modification; 0 resolve racemic mixtures produced by chemical modification; undertake stereo specific reactions. Since you are now familiar with the types of chemical modifications that may be mediated by biological systems, you should be able to answer the following SAQ regarding the modification of prostaglandins. biobgical mtaWB Biotransformation of lipids 337 Consider the structure of prostaglandin PGEz Prostaglandins belonging to the PGF, group have the following ring structure: _. . whilst prostaglandins belonging to the PGFp group have the ring structure: -8 I Assume you have a plentiful supply of PGE2, suggest two strategies for producing samples of PGF, and PGFp prostaglandins, using PGEz. 9.8 Selection of production systems for the biotransformation of lipids Generally speaking we consider that most micro-organisms live and grow in aqueous environments, and that the cytoplasm within ells in which enzymes function is also aqueous. On the other hand, most lipids are only sparingly soluble in aqueous media. Cholesterol, for example, has a solubility of less than 2 mg 1-' (equivalent to a concentration of less than 5 pol 1-l). Even at much lower concentrations (2!j-40 mol 1-') it tends to aggregate into micelles. There is, therefore, a general problem of how to supply lipid substrates at sufficient concentration to produce reaction kinetics that are appropriate for industrial purposes. n See if you can write down at least two ways that this pmblem may be overcome. The two most commonly used strategies are: duction of the aggregation of lipids in aqueous solvents by using non-ionic sufface active agents, such as Tween. To achieve an even, finely dispersed distribution of the substrate, this is usually dissolved in an organic solvent (acetone, ethanol, dimethyl sulphoxide) prior to being mixed with the culture broth containing Tween; sdubiri proMem sewion of sohmt yam 338 Chapter 9 use of the enzyme in a non-aqueous solvent or in a finely dispersed aqueous emulsion within an organic solvent containing the substrate. The industrial application of enzymes using non-aqueous system has been discussed in detail elsewhere in the BIOTOL text 'Technological Applications of Biocatalysts", so we will not elaborate on this aspect here. In addition to selection of solvent system, we also have a choice of using freely suspended enzymedorganism or imrnobilised system. Increasingly, attention is being paid to imbilised systems because of their potential advantages (cost, purity and ease of recovery of products). Again, this aspect of industrial enzymology is discussed in detail in the BIOTOL book 'Technological Applications in Biocatalysts", so we will not discuss this aspect further. immobiri '-' Biotransformation of lipids 339 Summary and objectives In this chapter, we have examined the use of cells and enzymes to chemically transform lipids. We have had to be selective and have predominantly focused attention on the transformation of sterols and steroids. We first explained why these compounds were commercially important and why they only occur in low concentrations in natural systems. We pointed out that a very large number of reaction types are possible, but those which have found greatest use include stereospecific hydroxylations, alcohol/ketone intemnversion, hydrolysis, conjugation and isomerisation. We included some discussion of other terpenoids and other organic molecules, including aliphatic and alicyclic materials. We also considered the use of enzymes in producing triglycerides with desirable characteristics. Now that you have completed this chapter you should be able to: explain why steroids are of value and why they only occur in low concentrafiofls in nature; describe the mapr molecular differences between sterols and steroids; list the options for producing steroids commercially and explain the advantage and disadvantages of these options; explain, using examples, the options available for selectively removing the side chains of sterols, including using modified sterols, selective enzyme inhibition and mutants; list the important reaction types used in sterol/steroid in terconversions; identify suitable organisms for conducting particular chemical transformations for a number of substrates, but especially for steroids and other terpenes; idenbfy a wide range of reaction types; explain how enzymes may be used to produce lipids, especially triglycerides, with desirable characteristics; list some examples of how hydrocarbon-utilising organisms may be used to inWuce functional groups into hydmcarbons.