Biotransformation of lipids Figure 9. 4 Some examples of side chain degradation of 6B and 19 oxidosterols a: 3B-acetoxy-5a-bromo-6B, 19-0xidocholestane b: 5a-bromo-6B, 19-oXidoandrostane-3, 17-dione c∴:3β acetoxy-5α chloro6,19 oxidocholestane, 眠 19-oxidoandrost-4-ene-317-dione 19-oxido-4-cholestene-3-one f: 6B, 19-oxido-9a-hydroxyandrost-4-0ne-3, 17-dione .3.2 Use of enzyme inhibitors Earlier we icated that an alternative strategy to prevent ring cleavage is to use selective inhibition of the enzymes which catabolise the ring structure. The inhibitors used include: chelating agents metal ions with similar ion radii which will displace Fe
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+;
Chapter 9 inorganic -sH rea auto-oxidisable redox dyes A list of some examples of these is given in Table 9.2. Are these compounds likely to be toxic to cells? Since they inhibit metabolism, they are likely to be toxic to cells. It is, therefore, usual to add these compounds after the culture has grown and to subsequently add the sterol to be metabolised ring deavage Of the compounds listed in Table 9.2,8-hydroxyquinoline, ax, a -dipyridyl and 1, 10-phenanthroline have found most use. They are usually used in the concentration range 0.1 mmol r. It is, however, essential to use an optimal concentration of these agents in order to get high yields. echanism。 f actlon Chelating agents for Fe Diethyldithiocarbamate a,a'-Dipyrie Dipheny thiocarbazone 8-Hydroxyquinoline Isonicotinic acid hydrazide 5-Nitro-1, 10-phenanthroline 1. 10-Phenanthroline Tetraethylthiuram-disulphide Metal ions replacing iron or blocking N+ Co2+Pb+ SH-functions SeO,AsOh Redox dyes Methylene blue Resazurin Table 9.2 Compounds used to inhibit steroid ring degradation(based on Martin CKA Sterols in Biotechnology Volume 6a Edited by Kieslich K 1984. Verlag Chemie, Weinheim) 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
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~~. Itis, 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 optimal Achieving optimal concentrations is difficult because components in the medium and inhibi tory the biomass itself may neutralise the inhibitory effects of these reagents. This is concentration especially true if complex, non-defined media, such as cornsteep liquor, are us This approach has been used with a wide variety of organisms, although it is more commonly employed using Arthrobacter simplex, Brevibacterium lipolyticum, Corynebacterium spp and certain strains of nocardia ∏ 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 androstadiene-3, 17-dione androstene 3, 17-dione and other androstane and testosterone-related 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 reduction of In some cases, the chelating agents used to trap Fe to reduce g-hydroxylase activity are toxicity toxic. The toxicity may be reduced by using absorbants such as styrene-divenylbenzene copolymers. Although this reduces the toxicity of the chelating agent it does not seem to prevent the chelating agent from trapping Fe ions. This increases yields, especially of 1 A-androstadiene-3, 17-dione. Yields can be further increased by the addition of oils: linseed and soya oils are most effective Typically, sterol concentrations of 3 to 4 gr are used and incubation DOh. Yields are dependent upon the species and substrates used. Some the yields of 1A-androstadiene-3, 17-dione from various sterols and 如c cultures of Arthrobacter simplex are reported in Table 9.3
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
hapter 9 Substrate Yleld of 1 4-androstadiene-3, 17-dIone (as of substrate) lithocholic acid βs 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. simplex(data from Martin CKA Sterols in Biotechnology Vol 6a. Edited by Klesllch K 1984 Verlag Chemie, Weinheim) ∏ The data shown in Table 9.3 indicate that highest yields of A-androstadiene-3, 17-dione are obtained using lithocholic adid as substrate However, this substrate is not necessarily the one of choice for the commercial production of 1A-androstadiene-3, 17-dione using A. simplex. List factors that uld determine the choice of substrate Lithocholic acid costs 2 or 3 times more than cholesterol. 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 electing a substrate we need to consider more than simply the conversion efficiency and the cost of the substrate
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 SAQ 9.1 Assume that you have a culture of a Mycobacterium sp which is able to use cholesterol or B-sitosterol 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 。/ 2) HOH2c 3) HOCH a)Predict where or not and to what extent the organism will grow on these bitrates b)Predict the likely metabolic products from incubating this organism with these substrates
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