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In this article we will discuss about:- 1. Introduction to Biotransformation 2. Microorganisms Employed in Biotransformation 3. Isolated Enzymes 4. Types 5. Methods 6. Range 7. Applications. [with importance and examples]
Contents:
- Introduction to Biotransformation
- Microorganisms Employed in Biotransformation
- Isolated Enzymes for Biotransformation
- Types of Biotransformation Reactions
- Methods of Biotransformation
- Range of Biotransformation
- Applications of Biotransformation
1. Introduction to Biotransformation:
Biotransformation is the process by which an organism or its enzyme bring out chemical changes on compounds that are not part of their metabolism and they result in the formation of novel or useful products that are often difficult or impossible to obtain by conventional chemical means. The total chemical transformation of one steroid to another not only requires many stages but an expensive process although provide only low yield.
The biotransformation is used for the preparation of products of defined chemical structure that are related to the substrate or starting material for the reaction by only a small number of chemical changes and in many cases the changes are brought about by the action of only a single enzyme. Biotransformation reactions reported in the chemical literature of nineteenth century was developed as part of the synthetic routes for the production of L-ascorbic acid (vitamin C) and ephedrine.
Oxidation of alcohol to acetic acid by bacterium Acetobacer xylinum; oxidation of glucose to gluconic acid by Acetobacter acetii, sorbitol to sorbose by Acetobacter but were not fully utilized. Biotransformation is realised beyond doubt in early part of 20th century when the conversion of D-sorbitol to L-sorbose by Acetobacter suboxidans and benzaldehyde to phenyl (lactyl carbinol) by yeast. Mamoli and Vercellone (1937) were the first to demonstrate the oxidation of nuclear hydroxyl group of steroid and reduction of nuclear double bond of steroid by yeast.
Welsch and Hongshem (1948) have not only confirmed but also enlarged the above result by employing a streptomces sp. Kramli and Horvath (1949) could oxidize cholesterol to hydroxyl cholesterol by Pencillium roseum and Azatobacter sp. Hench and his associates (1949) demonstrated the curative effect of cortisone on rheumatoid arthritis was possible only by introducing O2 at its 11th carbon atom with the help of Rhizopus arrhizus which chemically was very difficult.
Subsequently several people could accomplish this task by using different fungi, actinomycetes and bacteria. Presently varieties of biocatalysts are in use for carrying biotransformation reaction.
They are listed in table 17.1.
The distinguishing feature of biotransformation is its use for the preparation of products of defined chemical structure that are related to the substratum. These biotransformations are being effected either by isolated enzymes or microorganisms (whole cell), plant cells and catalytic antibiotics. The general goals of biotransformation, are specific modification of the substrate structure via selective transformation reactions. Partial degradation of substrates into desirable metabolites by means of controlled microbial reactions or reaction pathways extension of the substrate structure by the use of biosynthetic reactions to artificial structures.
Parallel with the growth of steroid hydroxylation during 1950 and 1960, was the discovery and development of other whole cell catalyzed biotransformation reactions notably the side chain degradation of steroids, Baeyer – villeger oxidation of ketones to esters, redox reactions resulting in the interconversion of alcohol and carbonyl groups, dehydrogenation reactions, hydrolytic reactions of esters and amides particularly those applicable to the production of β-lactum antibiotics which are illustrated in Fig. 17.1.
Seres (1992) and Kelley (1998) have given detailed account of different types of biotransformations achieved with the help of variety of microorganisms and their enzymes.
The interest in this area is growing because of the following reason:
1. The ease with which the biological approach can be exploited with every day organic chemical laboratory equipment.
2. The commercial availability of enzymes, baker’s yeast and a variety of microorganisms.
3. The ease with which one can straight away get into complex chiral products for assessing their biological, industrial and medicinal utility.
4. Generally enzymes (biocatalysts) are more efficient catalyst than chemical catalysts. Very low concentration (10-3 to 10-4 mol % of catalysts) of biocatalysts is required for an enzymatic reaction as compared to the chemical catalyst (0.01 -1 mol %).
5. The availability of guides to procedures for selection of the most suitable microorganism for desired conversion.
6. Microbial transformations take place under mild conditions, often at a room temperature and close to neutral pH thus minimizing the problems of isomerisation, racemization, epimerization and rearrangement that often plague the traditional chemical method.
7. Enzyme systems are highly efficient, very selective in terms of the type of reactions catalyzed and with respect to structure and stereochemistry of the substrate. Use of enzymes minimizes undesired side reactions such as decomposition, isomerization, racemization and rearrangement which can be a problem in chemical reactions.
8. Contrary to the stereospecific enzymatic conversions which are easy, the stereospecific chemical conversions of even a simple organic compound needs a complex organometallic reagent which is expensive, difficult to synthesize in simple laboratory and quite not possible for industrial exploitation. Enzymes can distinguish between enantiomers of a racemic substrate. Therefore, only one enantiomer is attacked leading to the formation of one selective product.
9. Biotransformations exhibit reaction specificity, regiospecificity, stereospecificity and can be carried out under mild reaction conditions. The substrate molecule is usually attacked at a specific site, even if several groups of equivalent or similar reactivity centres are present.
10. Most biocatalysts show catalytic activity in both aqueous and organic solvents, hence suitable over conventional chemical reactions.
11. Enzymes, mediators of biotransformations, are environment friendly. They can degrade completely in the environment, unlike chemical catalysts which often require special processes for their disposal, e.g. disposal of toxic heavy metals.
Disadvantages with the Use of Biotransformation Processes:
1. The necessary expenditures for the development of a biotransformation process including the product isolation are usually high.
2. In most cases the reaction time is rather long.
3. The substrate/product concentrations are low and the stability of the biocatalysts is limited.
With the advances in fermentation technology and the advent of cheaper enzymes, the majority of biotransformation reactions using isolated enzymes, are being adopted from the post 1970 era. The range of reactions currently known to be catalyzed by isolated enzymes is vast, apparently limited only by the ease of isolation, the stability and the cost of these biocatalysts.
Influence of genetic engineering, recombinant DNA techniques in this field hold the potential to facilitate production of expensive enzymes and perhaps to partially or completely remove many of the current limitations on the application of isolated enzymes for biotransformation.
For instance chemical transformation of bile steroid to cortisone required 37 steps, whereas the same is accomplished under simple conditions with biotransformation within a short time under simple conditions. Since 1970 there is steady progress in number of biotransformations during 1982-1992 (Fig. 17.2).
The biotransformations can be accomplished by employing number of biocatalysts. Probably this is the reason why there was rapid growth in the field of biotransformation since 1970. Further, interest in this area has been recognised all over the globe (Fig. 17.3).
2. Microorganisms
Employed in Biotransformation:
Some of the important microorganisms employed in biotransformation are precised in table 17.2.
The importance of baker’s yeast is attributable to its ease of availability and use with commercial dried preparation often being used without any other processing. Baker’s yeast, pseudomonas putida and Aspergillus niger are for 50% of the usage of whole cell biocatalyst. Similarly Beaveria bassiana (B. sulfurastum) and Sporotrichum sulfuricans are probably more versatile of the known biocatalyst in terms of the number of different chemical reactions that they can perform.
These can perform biotransformation of more than 300 substrates involving wide range of reactions such as hydroxylation of saturated, unsaturated carbon compounds, aromatic carbon ketone alcohol redox reactions, Baeyer villager oxidations, conjugate reactions, epoxide, hydrolysis, the hydrolysis of esters, amides and other functional groups.
3. Isolated Enzymes
for Biotransformation:
Biotransformations catalysed by isolated enzymes are dominated by the use of various lipases for the hydrolysis or formation of esters. Hydrolytic enzymes constitute the most frequently used biocatalysts which account for about 30% biotransformations. Some of the isolated enzymes mediate biotransformations are precised in table 17.3.
Use of plant cells for biotransformation is not common. However, cells of Datura carnata, Nicotiana tabacum and Catharanthus roseus are being used for relatively specialized applications. Catalystic antibodies raised in response to a small molecule transition state analogue such as antigen has received considerable attention in the past decade. However, its application for preparative biotransformations is still in early stages of development.
4. Types of Biotransformation Reactions
:
Some of the reactions, which can be catalysed by microorganisms or their enzymes, cover nearly all types of chemical reactions as detailed below:
Some of the reactions which can be catalysed by microorganisms or their enzymes covers nearly all types of reactions and are precised in table 17.4 and table 17.5.
(i) Oxidation:
Hydroxylation, epoxidation, dehydrogenation of C-C bonds, oxidation of alcohol and aldehydes, oxidative degradation of alkyl, carboxyalkyl or ketoalkyl chains, oxidative removal of substituents, oxidative deamination, oxidation of heterofunctions and oxidative ring fission.
(ii) Reductions:
Reduction of organic acids, aldehydes, ketones and hydrogenation of C-C bonds, reduction of heterofunctions, dehydroxylations and reductive elimination of substituents.
(iii) Hydrolysis:
Hydrolysis of esters, amines, amides, lactones, ethers, lactams etc.
(iv) Condensation:
Dehydration, O- and N-acylation, glycosidation, esterification, lactomization and amination.
(v) Isomerization:
Migration of double bonds or oxygen functions, racemization, rearrangements, formation of C-C bonds or hetero-atom bonds.
(vi) Mixed Reactions:
Hydroxylation with reduction; Hydroxylation with oxidation; hydroxylation with side chain degradation; rupture of C-C linkages with oxidation of side chain.
5. Methods of Biotransformation
:
Transformation of organic compounds may be accomplished by use of microorganism, isolated enzyme, immobilization techniques and solvent selection. The submerged fermentation is carried out in a stainless steel tank with minimal nutritional quantities to allow maximum transformation and use of easy extraction and purification of transformation product.
The microorganisms are grown in a suitable medium for 12-72 hrs depending on bacterium and fungus at optimum temperature, pH, aeration and agitation.
The fermentation is carried out in two phases:
1. Growth phase
2. Product formation phase
At the end of suitable incubation period (growth phase), measured quantity of organic compound to be transformed is added to the growing culture. The enzyme produced by the microorganism act upon the organic compound and does the desired function (product formation phase). At the end of suitable incubation period, the microbial biomass is separated from the fermentation broth. The broth is subjected to separation of both added substratum and product formed by the transformation.
For analysis, if the product samples are obtained at regular intervals upto end of incubation period (1-5 days), which are analysed by using TLC, paper chromatography, gas chromatography or HPLC technique. The extraction of product is done by appropriate organic solvents such as methylenechloride, chloroform, ethylacetate and methyl isobutylketone. Product obtained from cell and substratum should be extracted separately. Different factors like pH, temperature, addition of steroid and mineral content are reported to influence biotransformations.
Use of isolated enzymes for biotransformations is limited by the availability of an enzyme for the desired transformation and subjective optimization of parameters such as temperature, pH and substrate protecting groups (if appropriate). These are most conveniently performed at milligram to gram scale. However, some are amenable to scale up, to process tens or even hundreds of grams of substrate.
Procedures for using isolated enzyme are dictated by the nature of the enzymes, the biotransformation reaction to be catalysed and the requirements (if any) for cofactors or cofactor recycling. For preparative uses, an immobilized form of the enzyme is often desirable to facilitate catalyst recovery and product isolation.
Whereas use of an organic solvent or co-solvent for the reaction may be indicated when dealing with substrates or products of low water solubility and is necessary in the application of hydrolytic enzymes for ester or amide formation.
Immobilization of an isolated enzyme or whole cell leads to increased ease of biocatalyst recover, thus facilitates reuse and often results in greater stability leading to an increase in the durability of the catalyst. In some instances immobilization can result in a catalyst of lower activity.
Conversion time is related to the type of reaction, the substrate conversion and the microorganisms involved. Oxidation and dehydration reactions using bacteria are often completed in a few hours, while yeasts and fungi require several days. Hydrolytic reactions with most of the microorganisms can be accomplished in a few hours.
Biotransformations in a large scale are carried out under sterile conditions in aerated and stirred fermenter. The conversion process is being monitored chromatographically or spectroscopically. The process is terminated when a maximal titer is reached. Sterility is required because contamination can suppress the desired reaction, induce the formation of faulty conversion products or cause total substrate break down.
If enzyme induction by the added substrate is not necessary, resting cells may be used. This has the considerable advantage that growth inhibition by the substrate is eliminated. High cell densities, which promote increased productivity, may be used and at the same time risk of contamination is reduced.
Since the transformation reaction occurs predominantly in the buffer solution, the recovery of the product is relatively easy. A number of transformation processes can be carried out continuously and cells can be used over and over again. Immobilized bacterial cells are being used commercially in the production of L-aspartic acid, L-alanine and malic acid. Microorganisms are very convenient enzyme source which can easily be manipulated to produce increased amount of enzyme either through environmental factors or genetic manipulations.
6. Range of Biotransformation:
Wide ranges of reactions are being catalysed through biotransformation. Almost 50 of such biotransformations reported since 1950 are concerned with only two reactions – hydroxylation at saturated carbon and the reduction of carboxyl groups.
Stereochemical Features:
Biotransformations are used for a variety of chemical purposes many of which rely on the regioselectivity (Fig. 17.4), diastereo selectivity or enantio selectivity inherent in enzyme catalysed reactions for the formation of a product with defined stereochemistry.
Progesterone 11-α hydroxy progesterone.
Majority of biotransformations involve some aspect of stereochemical selectivity. This may be expressed as mesoselectivity, prochiral selectivity enantio selectivity in either substrate utilization or product formation, diastereo selectivity in either substrate utilization or product formation.
Green Chemistry:
The conversion of acrylonitrite to acrylamide by Rhodococus modrochrous, the hydroxylation of nicotinic acid and related compounds at C6 by pseudomonas, other bacteria and the conversion of indole to indigo by E.coli expressing naphthalene dioxygenase gene are important examples of valuable biotransfomations with regiochemical but no explicit stereochemical features.
Such biotransformations represent green alternatives to conventional chemical procedures involving extreme conditions of temperature and pressure or harsh reagents, as such they exemplify the current emphasis placed on the development of environmentally responsible industrial process.
Unique Reagents:
Biocatalysts are frequently used to perform reactions for which no analogous chemical method is available. Biotransformations are also used for production of intermediates in the environmental biodegradation or mammalian metabolism of other xenobiotic compounds. Some of the fungi carry out biotransformation of polycyclic aromatic compounds resulting in the production of oxidized intermediates present in the biodegradation pathway.
Large Scale Applications:
Large scale application of biotransformations for enzymatic processes are summarized in Table 17.6.
These are restricted to the use of inexpensive enzymes with high activity for desired conversion, almost invariably hydrolytic enzymes with no factor requirements. Similarly these are applied to whole cell catalyzed transformations (Table 17.7).
7. Applications of Biotransformation:
Although a vast array of biotransformations have been described, only a few of these processes have found industrial applications either because of insufficient yields or market is too limited. However, in future wide range of applications is expected to arise as most cost effective process.
Transformation of Steroids:
Steroids are a group of organic compounds which have four membered ring, are produced by testis, ovaries, adrenal cortex and placenta. The steroids which differ in the nature of functional group or side chain, exhibit different biological properties. Naturally occurring steroids have hormonal properties. Adrenal cortex hormones androgens, estrogens and progesterone’s are some such steroids.
These steroids are widely used medically as anti-inflammatory agents, anesthetics, antifertility agents and in the treatment of sterility. Cortisone is useful because of anti-inflammatory action in arthritis and skin diseases. By introducing bond in ring A of cortisone or Cortisol is converted to prednisolone or prednisone with increased anti-inflammatory action.
Microorganisms help in biotransformation of steroids as follows:
1. Rhizopus arrhizus and R. nigricans hydroxylates progesterone to produce 11-α hydroxy progesterone.
2. Cunninghamella blakesleeana hydroxylates cortexotene at 11 carbon atom.
3. Corynebacterium simplex dehydroxylates cortisone to produce prednisone. It can also bring about dehydrogenation of hydrocortisone or Cortisol to produce prednisolone.
4. Nocardia restrictus biotransforms cholestene-19-hydroxy-3-one into estrone.
5. Androstenodione is converted into testosterone by yeast.
6. Peterson and his associates have demonstrated that compounds like progesterone and Reichstein compound S may be biotransformed to testolactone through action of A.flavus and P. adametzi.
Some of the biotransformations resulted from progesterone involving microorganisms are illustrated in Fig. 17.4.
Microorganisms are capable of carrying out a wide variety of other steroid transformation reactions besides hydroxylations (table 17.8).
In recent times thousands of modified steroids produced by a combination of chemical and microbial reaction steps have been tested for their therapeutic effectiveness. Production of cortisone and its 1-dehydro-derivatives from diosgenin via Reistein’s S (11-deoxycortisol) is illustrated in Fig. 17.5.
Products of microbial transformation of steroids proved to be of great economic importance (table 17.8).
Biotransformations involving different types of chemical reactions in steroids mediated by different microorganisms are given below.
Δ4: Actinomycete
Δ16: Rhizopus nigricans
Ketone: Streptomyces lavendulae, Bacillus Putrifaciens, Epicoccum oryzae
Aldehyde: R. nigricans
Didymella lycopersici, Alternaria sp., Calonectria decora, Fusarium solani and Corynebacterium simplex.
Mucor giseocyanas, M. parasiticus, Cunninghamella blakesleana and Helicostylum pyriiforme are able to transfer epoxile group.
4. Cleavage:
Gliocladium catenulatum, Pycnodothis sp, Streptomyces lavendulae, Fusarium solani and Cephalothecium subverticellatum.