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Steroid transformation, Bioreactor and Bioprocess

Engineering

Ritasree Sarma

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Steroids

Steroids are small organic molecules with a characteristic molecular structure containing four rings of carbon atoms synthesized in steroidogenic tissues

It include many hormones, alkaloids, and vitamins

It act on target sites to regulate a cascade of physiological functions

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Types of steroids

Sex hormones. These are the male hormones, including testosterone, which together are called androgens, and the female hormones, including estradiol, a type of estrogen.

Corticosteroids. Hormones include cortisone and cortisol. They are thought to have a role in the immune system.

Mineralocorticoids. These hormones maintain the balance of sodium and potassium in the body and include aldosterone.

Bile salts or bile acid. These steroids are made in the liver. They don't function as hormones, but are necessary for digestion and absorption of fats.

Sterols. The most commonly known of these is cholesterol. Other sterols help your body to make vitamin D from sunlight and to build cell walls.

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Steroidogenic EnzymesCommon name "Old"

nameCurrent name

Side-chain cleavage enzyme; desmolase

P450SCC CYP11A1

3 beta-hydroxysteroid dehydrogenase

3 beta-HSD 3 beta-HSD

17 alpha-hydroxylase/17,20 lyase

P450C17 CYP17

21-hydroxylase P450C21 CYP21A2

11 beta-hydroxylase P450C11 CYP11B1

Aldosterone synthase P450C11AS CYP11B2

Aromatase P450aro CYP19

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Steroid hormones and their derivatives have been used for a wide range of therapeutic purposes

Utilization as immunosuppressive, anti-inflammatory, anti-rheumatic, progestational, diuretic, sedative, anabolic and contraceptive agents

Recent applications of steroid compounds include the

treatment of some forms of cancer, osteoporosis, HIV infections and treatment of declared AIDS

The pharmaceutical industry has great interest in the transformation of steroids for the production of steroid hormones.

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Transformation of steroids

Transformation of steroids means conversion of precursor steroids to important drug intermediates and further conversion of these intermediates to active compounds by simple chemical or microbial processes.

The chemical synthesis and transformations of steroids requires multiple steps and makes the use of reagents that have health risks and cause serious environmental disposal problems.

Alternative is microbial transformation

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Transformation of steroids

Microbial transformation These involves simple, chemically defined reactions catalyzed by enzymes

present in the cell. Microbial cells provide the enzymes to catalyze the transformation reactions.

The microorganisms have got the ability to chemically modify a wide variety of organic compounds. These microbes during the bioconversion provide enzymes which act upon and convert the organic compound into other compounds or modify it.

Microbial transformations cleave the complex side chains of precursor steroids in one single step and incorporate desirable modifications in steroid nucleus.

Microbial transformations are regiospecific and stereospecific, whereby organic compounds are modified into desirable isomers of products involving simple chemically defined reactions catalyzed by the enzymes in the microbial cells.

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TYPES OF STEROIDAL TRANSFORMATION

Oxidation Hydroxylation Dehydrogenation Epoxidations Oxidation to ketone through hydroxylation Ring A Aromatization Degradation of steroid nucleus

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Transformation of steroids

Oxidation Oxidation of alcohols to ketone: 3β-OH to 3-keto Side chain cleavage of steroids Decarboxylation of acids

Hydroxylation

• Hydroxylation involves the substitution of hydroxyl group directly for the hydrogen at the position, be it or , in the steroid with a retention of configuration.

• The oxygen atom in the hydroxyl group is derived form molecular oxygen (gaseous), not from water, and the hydroxyl group thus formed always retains the stereochemical configuration of the hydrogen atom that has been replaced.

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Fungi are the most active hydroxylating microorganisms, but some bacteria particularly the Bacilli, Nocardia and Streptomyces show fair good activity.

The hydroxylation at the 11-position of progesterone was one of the first hydroxylation described

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Dehydrogenation

• Dehydrogenation with the concomitant introduction of a double bond has been reported for all four rings of the steroid nucleus

• The introduction of unsaturated bonds in Ring A is the only reactions of commercial importance.

Example :• In 1955, Charney and co-worker observed that they could greatly enhance

the anti-inflammatory properties of cortisol by causing the compound to be dehydrogenated at 1st position by Corynebacterium simplex.

The resultant product, prednisolone, was 3-5 times more active than the parent compound and produced fewer side effects.

cortisol prednisolone

Corynebacterium simplex

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Epoxidation

• The epoxidation of steroidal double bonds is arare example of biological epoxidation. The 9,11-epoxidation of 9(11)-dehydro-compound , and the 14, 15-epoxidation of 14(15)-dehydro compounds, using Curvalaria lunata

CH3CH3

OCurvalaria lunata

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Ring A Aromatization

• The microbial aromatization of suitable steroid substrates can lead to ring A aromatic compounds, particularly the estrogens which constitutes an important ingredient in oral contraceptives drugs and play important role in replacement therapy for menopause treatment

• Cell free extracts of Pseudomonas testosteroni could transform 19-nor-testosterone into estrone with small quantities of estradiol-17.

19-nortestosterone Estrone Estradoil-17

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Reduction

Reduction of aldehydes and ketones to alcohols

OH

Estradiol

Streptimyces

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Hydrolysis

Hydrolysis of esters

Flavobacterium dehydrogenans contain a specific enzyme acetolase which hydrolyses the steroidal acetates

OAc

OH

EstradiolFlavobacterium dehydrogenans

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Esterification

Usually involve acetylation

O

OAndrostenedione

OAc

OTestosteron acetate

Sacromyces fragilis

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Steroid Ring Degradation

HO

O

O

O

O

O

OOH

O

HO

O

Cholesterol Androstenedione androstadiendione

9 ydroxy-androstadiendione

androstatriendione

Degradation of cholesterol by mycobacteria

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Fermentation condition of some steroids

M/O Steroid substrate

Steroid product

Length of incubation , temperature, aeration

Alcaligenes faecalis

Cholic acid Ketocholic acids (90-100%)

2 days (monoketo acid)4 days (diketo acid)6 days (triketo acid)37-39̊ ,surface culture

Fusarium solani Progesterone 1,4- androstadiene-3, 17-dione(85%)

4 days , 25̊ C , rotary shaker (100 rpm)

Corynebacterium mediolanum

21-acetoxy -3 β- hydroxy -5-pregnen-20-one

21-hydroxy-4-pregnene-3, 20-dione (30%)

6 days , 36-37̊ C , pure oxygen with agitation

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Advantages

Microorganisms have great potential for inducing new or novel enzyme systems capable of converting foreign substrates.

Microorganisms are capable of producing unique enzymes which are stable toward heat, alkali and acid. A combination of microbial transformation and chemical transformations (chemo-enzymatic synthesis) can be exploited for partial, as well as the total synthesis of the organic compounds

Disadvantages• If the substrate is toxic, it can kill the microorganisms. Hence no

transformation will be observed.• Very low chemical yields are obtained due to the involvement of a

complex biological system

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Bioreactor and Bioprocess Engineering

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Bioprocess engineering

 Bioprocess engineering is a conglomerate of mathematics, biology and industrial design and consists of various spectrums like designing of bioreactors, study of fermentors (mode of operations etc.)

It also deals with studying various biotechnological processes used in industries for large scale production of biological product for optimization of yield in the end product and the quality of end product.

Bioprocess engineering may include the work of mechanical,

electrical, and industrial engineers to apply principles of their disciplines to processes based on using living cells or sub component of such cells.

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Contd..

Process for developing useful products by taking advantage of natural biological activities.

Classical example include making alcoholic beverages – the yeast cells and nutrients (cereal grains) formed a fermentation system in which the organisms consumed the nutrients for the growth and produced by-products (alcohol).

Today's modern bioprocess technology is based on the same principle: combining living matter (whole organisms or enzymes) with nutrients under the conditions necessary to make the desired end product

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GENERALIZED VIEW OF BIOPROCESS

GENERALIZED VIEW OF BIOPROCESS

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Bioreactor Bioreactor: device, usually a vessel, used to direct the activity of a

biological catalyst to achieve a desired chemical transformation

To reach its’ necessary goals, the biotechnological process has usually 3 major stages:

1. Preparation of nutrient media for the cultivated microorganism and the cultivation process

2.The course of the microorganism reproduction process in bioreactors (called also fermenters) or in other equipment;

3. Obtaining of the final product or substance from the cultivated medium. This stage includes operations such as separation, purification and other technologies, which are connected with obtaining the commodity form.

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Typical Bioprocess Flow Sheet

RAW MATERIASNutrients and Reactants

in Aqueous Solution(may contain insoluble

organic and/or inorganicmaterials)

Air

CELL SEPARATION

1). CELL DISTRUPTION2). PRODUCT EXTRACTION

PRODUCT CONCENTRATION

PROCESS

FINAL PRODUCT

DRYING

PURIFICATION

PRODUCT SEPARATION

PREPARATIONOF BIOMASS

Innoculum StagesFOAM CONTROL

Antifoam AdditionpH CONTROL

Acid-Alkali Addition

Extracellularproduct

Intracellularproduct

STERILIZATION

BIOREACTOR

Free Cells,Immoblized Cells

or Enzyme Bioreactor

PRODUCT RECOVERY

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Design of a bioreactor

The basic points of consideration while designing a fermenter: Productivity and yield Fermenter operability and reliability Product purification Water management Energy requirements Waste treatment

Other significant factors to be taken in account: Design in features so that process control will be possible over

reasonable ranges of process variables. Operation should be reliable Operation should be contamination free

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Types of bioreactor

Bioreactors are generally classified into two broad groups;

1. SUSPENDED GROWTH BIOREACTORS

The reactors use microbial metabolism under aerobic, anaerobic, or sequential anaerobic/aerobic conditions to biosorb organic compounds andbiodegrade them to innocuous residuals.

The microbial activity in the systems produces biomass that is removed by gravity sedimentation, with a portion of the settled biomass recycled to maintain a desired mixed liquor suspended solids concentration in the bioreactor.

Eg Batch reactors, CSTR’S, Plug-flow reactors etc

2. BIOFILM BIOREACTORS

In biofilm reactors most of the microorganisms are attached to a surface, and in this manner kept within the reactor.

The different kinds of biofilm reactors include membrane, fluidized bed, packed bed, airlift, reactor.

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The batch bioreactor: A typical batch reactor consists of a tank with an agitator and

integral heating/cooling system. These vessels may vary in size from less than 1 litre to more than 15,000 litres.

They are usually fabricated in steel, stainless steel, glass lined

steel, glass or exotic alloy.

Liquids and solids are usually charged via connections in the top cover of the reactor. Vapors and gases also discharge

through connections in the top. Liquids are usually discharged out of the bottom.

Advantages of the batch bioreactor

Easy operation and absence of mechanical pumps Versatility; a single vessel can carry out a sequence of different operations

Disadvantages where mixing is a critical parameter , they are not the ideal solution

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THE CONTINUOUS STIRRED TANK REACTOR:

The liquid or slurry stream is continuously introduced and liquid contents are continuously removed from the reactor.

The basic characteristic of the ideal CSTR is that the concentration of the substrate and microorganisms are the same everywhere through out the reactor.

• Advantages :The rate of many chemical reactions is dependent on concentration, continuous reactors are generally able to cope with high concentrations due to their superior heat transfer capabilities

•Disadvantages:Consumption of more power due the presence of mechanical pumps

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THE PLUG FLOW REACTOR

The liquid or slurry stream continuously enters one end of the reactor and leaves at the other end.

The concentration of substrates and microorganisms vary throughout the reactor. Concentrations of substrates are highest at the entrance of the reactor, which tends to make rates there quite high

ADVANTAGES:1. Can run for long periods of time without

maintenance.2. The heat transfer rate can be optimized by

using more, thinner tubes or fewer, thicker tubes in parallel.

DISADVANTAGES:1. Temperatures are hard to control and can

result in undesirable temperature gradients2. Expensive to maintain.

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Packed bed bioreactors

• A bed of solid particles usually with compressing walls constitute packed bed.

• Biocatalyst is supported in porous or non porous bed.

• Fluid comprising of dissolved nutrient and substrate flows through the solid bed. The Flow rate and in term the residence time of substrate is manipulated to increase or decrease substrate contact with the bed (microorganisms)

• Commonly packed bed reactors are used for aerobic treatment of waste waters and are known as tricking filters and or biological towers

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Fluidized Bed Reactor

• In this type of reactor, a fluid (gas or liquid) is passed through a granular solid material at high enough velocities  to suspend the solid and cause it to behave as though it were a fluid. This process, known as fluidization

• ADVANTAGES: 1. Uniform particle mixing2.Uniform temperature gradients3. The ability to operate reactor in continues state.

• DISADVANTAGE:1. Increased reactor vessel size2. pumping requirements and pressure drop

• Typical application of these reactor is in waste treatment.

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Lab scale bioreactorPilot scale bioreactor

38Industrial scale bioreactor

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