INTRODUCTION TO BIOGECHEMICAL TRANSFORMATIONS IN SOIL : MINERALIZATION AND IMMOBILIZATION OF ELEMENTS

 Erth is a closed system, where the over all quantity of matter remains constant. Microorganisms need electron, energy and nutrients to grow. They are responsible for cyclic transformation of compounds, and therefore they are called biogeochemical agents. They carryout transformation of carbon, nitrogen, sulphur, phosphorus, iron etc. This cycling of elements is called biogeochemical cycling. Both biological and chemical process are involved in biogeochemical cycling.

The oxidation reduction reactions are mainly responsible for biogeochemical cycling of compounds. This changes the chemical and physical characteristics of different compounds. These cyclic turnover of elements are brought about by different types of microorganisms resulting into continuous change in chemical states of matter.

The life of earth depends on cyclic conversion of chemicals from inorganic state to organic (complex state) to the elemental state. The break in the cycle at any point would dramatically affect all life forms. Various processes carrying out these transformation include :

Mineralization : 

It is a process of conversion of complex organic compounds to simple inorganic forms. Many heteroprophic microbes play role in mineralization.
The resultant simple compounds are made available to plants and microbes. Energy is released in the process. The process of mineralization is very important as it increases soil fertility.

Carbon mineralization :

  • Organic carbon is mineralized to inorganic state.
  • Under aerobic condition the main products of carbon mineralization are CO₂ and water.
  • In absence of O₂ organic carbon is incompletey metabolized to produce organic acids, alcohols and gases.

Assimilation :

  • It is the process of conversion of substrate elements to protoplasmic elements. 
  • Microbes take up the simple materials from the environment (soil) and convert them into cellular materials. It is known as assimiliation or biosynthesis.
  • In this process synthesis of energy and cellular material takes place. Through assimilation microbes store the excess of simple inorganic chemicals, and prevent their loss due to erosion.

Immobilization :

It is a process in which the quantity of plant available nutrients are reduced in soil by microorganisms. The nutrient assimilation is an important method of immobilization.
The uptake of various elements like carbon, nitrogen, phosphorus, sulphur, etc. cause immobilization.

  Intermediary substances accumulate abundant quantities of CH4 and smaller amount of H₂ is evolved. The factors affecting mineralization are level of organic matter, temperature, moisture, pH, depth and aeration.
  All these factors affect the growth and metabolism of microbes and the process of mineralization.

  • In nitrogen mineralization ammonium, nitrite and nitrates are accumulated from organic nitrogenous compounds like proteins, nucleic acids, etc.
  • In phosphorus mineralization organic phosphorus present in nucleic acid, phytin, lecithin, etc. are converted to inorganic phosphorus.
  • Sulfur mineralization involves aerobic breakdown of sulfur containing amino acids : cystine, cysteine, methionine, and vitamins, thiamine, biotin, thioctic acid releasing sulfates. Whereas in absence of oxygen , H₂S and odoriferous mercaptans accumulate in soil.

Lytic cycle: Multiplication of T4 Bacteriophage

T4 coliphage is a phage that infect coliform bacteria especially E.coli. T4 is a double stranded DNA phage, it has a contractile sheath and an unique base 5-hydroxyl methyl cytosine (5-HMC). It is the best known member of large virulent phages.

The lytic cycle of T4 phage involve following steps: ADSORPTION
PENETRATION
BIOSYNTHESIS
ASSEMBLY
RELEASE

1]. Adsorption :

  • The lytic cycle begins when a bacteriophage comes in contact with a susceptible host cell by random collision.
  • Phage possess an adsorption organs or anti-receptors and host cells possess receptors.
  • Host cell surface components -Flagella, Pilli , Teichoic acids, Proteins, Carbohydrates , LPs and Lipopolysaccharides serves as receptors.
  • Phage components such as tail fibers, tail proteins and spikes serves as adsorption organs or anti-receptors.
  • Each phage has its specific receptor to which it adsorbs.
  • Adsorption takes place only when the anti-receptor is chemically complementary to the receptor.
  • T4 phage possess tail fibers that serves as an adsorption organ or anti-receptor.
  • Normally, tail fibers are present in folded form around the tail.
  • Whiskers hold the tail fibers in folded form.
  • When phage come in contact with host, tail fibers unfold.
  • Unfolding of fibers requires tryptophan & co-factors - Mg++ & Ca++.
  • Thus, the phage and host binding is favoured by ionic environment.
  • T4 host E.coli possess outer membrane protein C (OmpC) – lipopolysaccharide complex as receptor.
  • Initial attachment occurs when tail fibers attach to the OmpC- lipopolysaccharide complex.
  • Initial adsorption is weak and reversible.
  • It becomes irreversible when tail pins attach to lipopolysaccharide.

2]. Penetration :

  • Once attached, the bacteriophage injects DNA into the bacterium.
  • Bacteria possess rigid cell wall and therefore the phages directly cannot penetrate into the bacterial cells.
  • They inject only their nucleic acids inside the host cell.
  • In the T-even phage, irreversible binding of the phage to host results in the contraction of the sheath and the hollow tail tube is inserted through host cell wall.
  • Some phages have enzymes like lysozyme that digest the cell wall components of the bacterial cell.
  • The penetration of T4 phage DNA occurs when -
  1. There is irreversible attachment of phage to host cell,
  2. Contraction of sheath, pushing tail tube through cell envelope
  3. Injection of DNA into cell like injection of vaccine/drug by a syringe

3]. Biosynthesis :

Biosynthesis divided into three steps:
  1. Formation of immediate early and delayed early protein
  2. Replication of phage genome
  3. Formation of late proteins

i) Formation of immediate early and delayed early protein :

  • Part of phage DNA is immediately transcribed by host RNA polymerase to form immediate early m-RNAS.
  • These early m-RNA translate to following enzyme proteins -
  • a) Nucleases - Breaks down host DNA & make nucleotides available for its own synthesis.
  • b) α-subunit modifying enzyme - modifies α-subunit of host RNA polymerase.
  • Modified host RNA polymerase transcribes part of viral genome to delayed early m-RNAS.
Delayed early mRNAs are translated to following enzymes-

  •  a) Phage enzymes that produce 5-hydroxyl methyl cytosine (5-HMC), a unique base in phage DNA
  • b) Polymerases and ligases - that play role in phage DNA replication and recombination.
  • c) Glucosylation enzyme-adds glucose to HMC & protects phage DNA from host restriction endonuclease
  • d) σ-subunit modifying enzyme - modifies σ-factor of RNA polymerase so that is transcribes late mRNAs.

ii) Replication of Phage Genome :

  Two modes have been proposed for the replication of T4 phage DNA.
By bi-directional mode - at early stage
By rolling circle mode - at later stage

  • Initial replication is bi-directional and semi-discontinuous.
  • Leading strand is synthesized continuously and lagging strand is synthesized discontinuously leading to the formation of eye structure.
  • Bi-directional replication is initiated at several origins along the DNA and is catalyzed by phage coded enzymes.
  • In the rolling circle mode of replication, a cut is made in one of the DNA strands by a specific endonuclease and 3'end is made free.
  • DNA polymerase extends the free 3'OH end by adding complementary bases.
  • Intact strand serves as template for addition of complementary bases.
  • Due to extension of 3'OH end, the 5'end is displaced.
  • Displaced strand is synthesized discontinuously by adding Okazaki fragments.
  • This mechanism produces multi-genome length molecules.
  • Such molecules are referred to as concatemers.
  • The concatemers are later cleaved to head sized molecules by headful cutting mechanism.
III] Formation of late proteins 
  • Soon after the replication of phage DNA, transcription of late m-RNAs occur.

  • These late m-RNAs translate to different proteins.
  • These proteins include the structural proteins.
  • They are proteins involved in phage assembly and an enzyme lysozyme that degrades the peptidoglycan layer of bacterial cell wall.
  • For example - head (capsid) proteins, tail tube protein, sheath proteins, collar, whiskers, base plate, tail fiber, tail pins, lysozyme etc.

4. Assembly of Phages :

  • Assembly of new phage particles begins after accumulation of structural proteins and nucleic acid molecules in the cell.
  • Process of assembling phage particles is known as known as maturation.
  • There are four different pathways that lead to the formation of phage particles.
  • These include base plate, tail tube & tail sheath, tail fibers and head.
  • About 50 genes take part in the morphogenesis of T4 phage.
  • Subunits of base plate assemble to form a base plate.
  • Then tail tube and sheath subunits polymerize on base plate to form mature tail.
  • The subunits of head assemble together to form prohead and then DNA is inserted in the prohead to form complete head.

5. Release :

  • The release of newly synthesized phages occurs by sudden explosion or bursting (lysis) of bacterial cell.
  • Lysis begins after about 22 minutes.
  • One of the gene products involved in the process include lysozyme.
  • It cleaves glycosidic bonds in the peptidoglycan making the cell wall susceptible to the rupture.
  • There is another protein termed as holin that make holes in the cell membrane and makes the way for lysozyme action.

Eijkman Test Principle and Procedure

 The IMViC test has two drawbacks. The first, It has many controversial procedures and second is test results do not give satisfactory differentiation between fecal and non-fecal coliforms. In 1904 Eijkman proposed another test to differentiate fecal and non-fecal coliform.

Principle of Eijkman Test

  • Only fecal coliforms of warm blooded animals grow at 46°C and ferment lactose with Acid & Gas production
  • Most strains of fecal E.coli can ferment lactose in a special buffered broth when incubated at 45°C, where as very few or less frequently the Enterobacter aerogenes do so.
  • The test is called as Eijkman test or elevated temperature test.

Procedure of Eijkman Test

  • A buffered tryptose lactose broth in tubes with inverted Durham's tube is inoculated with a culture of coliforms.
  • It is then incubated in water jacketed incubator at 45°C for 48 hours.
  • Gas production after incubation constitutes a positive test for fecal coliforms.
  • Another method, instead of tryptone lactose broth, buffered boric acid lactose broth (BALB) medium is used.
  • The advantage is that, the medium used is selective for fecal Escherichia.
  • It selectively inhibits growth and gas production by Enterobacter and other intermediate members of coliforms.
  • Sterile medium is first warmed to 37°C and then inoculated with culture & incubated at 45°C for 48 hrs.
  • Gas production indicates positive test.
  • Eijkman test gives better result than IMViC tests.
  • Therefore, it is generally preferred in water examination.

IMViC Biochemical Tests: Principles Procedures and Results

IMViC tests are a group of individual tests used in microbiology lab testing to identify an organism in the coliform group. In this the first test is Indole test, the second one is Methyl Red, the third one is Voges Proskauer and the fourth one is Citrate Utilization test.

  • In the quantitative test for coliforms if completed test is positive, further testing is essential to differentiate fecal and nonfecal coliforms.
  • Escherichia coli and Enterobacter aerogens are the important contaminants of water respectively.
  • Escherichia coli is a fecal coliform as it is mainly found in human feces while Enterobacter aerogenes is considered as non fecal as it also occurs in soil & plant material.
  • They closely resemble each other in their morphological and cultural characteristics. 
  • Therefore, the biochemical tests are performed to differentiate them.
  • Tests are collectively designated as the IMViC tests.
  • The name was coined by Parr from the first letters of the four tests namely - I for Indole, M for Methyl Red, V for Voges Proskauer and C for Citrate Utilization test.
  • The letter i between V and C is added solely for euphony.

Indole Test

  • Indole Test is used to detect indole production from amino acid tryptophan.
  • E. coli has the ability to breakdown the tryptophan by enzyme tryptophanase with release of indole, pyruvic acid and ammonia.
  • Enterobacter doesn't produce enzyme tryptophanase. Therefore, they are not producing indol from an amino acid tryptophan.
  • Test is performed by inoculating the test organism in 1 % tryptone water or 2 % peptone water, incubation at 37°C for 24 hrs.
  • Indole production can be detected by adding few drops of xylene and Kovac's or Ehrlich's reagent which contains p-dimethyl aminobenzaldehyde.
  • This p-dimethyl aminobenzaldehyde reacts with the indole and produce a cherry red(pink) coloured compound. This is a reduction type of reaction.
  • Xylene extracts the indole in upper layer of the medium.

Methyl Red Test

  • Methyl Red Test is carried out to detect acid production ability of test organism from glucose.
  • It is performed by inoculating test organism in glucose phosphate broth tube and incubating at 37°C for 24 hrs
  • Methyl red indicator is then added to detect acid production which gives red colour in positive reaction and yellow in negative.
  • Escherichia coli rapidly ferments glucose with production of acids and reduce the pH to about 5.0
  • This pH / acidity prevents further growth of E.coli in glucose phosphate broth tube.
  • Enterobacter aerogens initially produce acids but later on it is converted to non acid products such as ethanol, acetyl-methyl-carbinol (acetoin) and 2, 3- butane-di-ol ( reduction product of acetoin).
  • Due to this, E.aerogens continues to grow without producing its limiting pH.
  • Thus, Escherichia coli gives positive methyl red test while Enterobacter aerogeys gives negative test.
  • Methyl red is a pH indicator which is red at pH 4.4 while yellow at pH 6.2

Voges Proskauer

  • The test used for the detection of acetyl methyl carbinol (acetoin) production from glucose, by the test organisms.
  • It is also performed by inoculating the test organisms in glucose phosphate broth medium and incubating at 37°C for 24 hrs.
  • This is followed by addition of 40 % potassium hydroxide and 5 % a-naphthol solution with the shaking of the tube.
  • For the Detection of acetoin requires its further oxidation to diacetyl, In presence of catalyst α-naphthol, alkali(KOH) and air, acetoin is further oxidised to diacetyl.
  • Diacetyl in presence of peptone, gives a red colour.
  • The constituent of peptone responsible for red colour is guanidine nucleus of the amino acid arginine.
  • Thus, a positive test is indicated by development of red colour.
  • Enterobacter aerogenes produces acetoin from pyruvic acid(Positive test) while Escherichia coli doesn't produce it(Negative test).

IMViC tests Results

Citrate Utilization Test

  • Citrate Utilization test is carried out to detect the ability of organism to utilize citrate as a sole source of carbon and energy.
  • The utilization of citrate depends upon the enzyme citrate permease that facilitates citrate transport into the cell.
  • E. aerogenes produce citrate permease and are able to utilize citrate as the sole source of carbon while E.coli do not produce
  • The test is performed by inoculating the test organisms in Koser's citrate medium which sodium citrate as the sole source of carbon; and incubating at 37°C for 24 hrs.
  • Ability to use citrate is indicated by the development of turbidity in medium.
  • Citrate Utilization test can also be performed by inoculating the test organisms on the Simmon's citrate agar slant and incubating at 37°C for 24 hrs.
  • The Simmon's citrate agar is the modified processed agar media which contains bromothymol blue as a pH indicator
  • Enterobacter converts citrate to oxaloacetate and acetic acid by enzyme citrase.
  • These products are further converted to pyruvic acid and CO2.
  • The CO2 reacts with sodium and water to form sodium carbonate
  • Sodium carbonate is an alkaline product and it raises the pH of medium Bromothymol blue (pH indicator) is blue in alkaline and green in acidic condition.
  • The change in colour of slant from green to blue indicates positive test.

 Results :


The IMViC test has two drawbacks as- 
  1. It has many controversial procedures 
  2. Test results do not give satisfactory differentiation between fecal and non-fecal coliforms.

Microbial Metabolism

  Every living organism has the fundamental capability to grow and synthesize new cell material. This requires processing of the nutrient molecules taken up by the cell and involves a series of biochemical transformations. The set of biochemical reactions occurring in cell includes degradation, synthesls as well as modification of the molecules.

   These chemical reactions, which operate by the living cells are collectively referred to as metabolic reactions and the phenomenon is called metabolism. Thus, metabolism is defined as the sum total of all biochemical reactions carried out by a living cell.

The metabolic reactions are further categorized as

  1. Catabolism
  2. Anabolism
  3. Primary metabolism
  4. Secondary metabolism
  5. Intermediary metabolism

Catabolism

Catabolism includes the set of biochemical reactions which involve degradation of the molecules taken up by the cell and generation of substances essential for biosynthesis of cell constituents. The products of catabolism are catabolites.

  They, generally, involve formation of various precursor metabolites, energy rich compounds and reducing power. Hence, these metabolic reactions are often called as fuelling reactions, which provide essential fuels required for cellular synthesis. The precursor metabolites provide basic carbon skeleton for the synthesis of building blocks of the called cellular macromolecules.

  The energy rich compounds are ATP, GTP, CTP, TTP, UTP and acetyl CoA, which provide necessary form of biochemical energy required to drive various energy requiring blochemical reactions. On the hydrolysis of high energy bond of these compounds, necessary free energy is available for the purpose.
 
Reducing power, generated during the catabolism. is in form of reduced pyridine compounds. NADH and NADPH. They provide essential reducing conditions required for several blosynthetic as well as assimilatory reactions of the cell.

Anabolism

Anabolism includes the set of blochemical reactions which involve synthesis cellular molecules.
These include blosynthesis of
  1. Building blocks of cellular macromolecules e.g. amino acids, nucleotides, fatty acids, sugars etc.
  2. Vitamins and coenzymes, which are essential for driving various enzyme catalyzed reactions.
  3. Cellular macromolecules such as proteins, lipids, nucleic acids, polysaccharides as well as synthesis of cell structural compounds.
These anabolic reactions are fuelled by the products of catabolism.

Primary metabolism

  The part of cellular metabolism which is very much essential for cell growth is termed as the primary metabolism. The products of primary metabolism are called primary metabolites.
 
  The primary metabolism includes the metabolisms associated with generation of energy rich compounds, reducing power, precursor metabolites as well as the synthesis of bullding blocks of cellular macromolecules.

The products of primary metabolism include

  1. Energy rich compounds such as ATP and others.
  2. Organic acids such as lactic acid, citric acid, acetic acid etc.
  3. Organic alcohols and solvents such as ethanol, glycerol. acetone, butanol etc.
  4. Amino acids, vitamins coenzymes, nucleotides etc.

The primary metabolism, in general, is found operative lin the cell during log phase of the growth.

Secondary metabolism

  The part of cellular metabolism, which is not essential for cellular growth is called secondary metabolism. Products of secondary metabolism are called secondary metabolites.

  It becomes active during late log phase and stationary phase of the growth. It involves utilization of the excess remains of carbon precursors and energy for synthesis of new molecules which may have secondary role in growth of a cell.
e.g. Antibiotic synthesis, which is not essential for growth. but does help the organisms to survive in the environment in the presence of various antagonistic organisms by destroying them.

Intermediary metabolism

The part of cellular metabolism which occurs after the entry of nutrients into the cell, leading to the synthesis of bullding blocks of the cellular macro molecules is generally referred to as the intermediary metabolism.

Central metabolic pathways

The central metabolic pathways are basically catabolic in nature. These pathways Include Glycolysis, pentose phosphate pathway and TCA Cycle.They participate in generation of energy, reducing power and precursor metabolites required for cellular synthesis.

Role of reducing power in metabolism

All elements, except phosphorus, present in cell are in reduced form. Carbon exists in organic form. Nitrogen is present as amino group. sulfur as úSH group etc. Therefore, all these elements must be reduced at the cellular level of reduction, before they are assimilated in to cell during biosynthetic reactions.

  Most of these elements exist in oxidized state in nature. Therefore, before they are assimilated in the cell, they must be reduced. This requires avallability of sultable reducing power. NADPH serves as the principle reducing power in all such assimilatory and biosynthetic reactions of anabolism. In addition to NADPH, flavin coenzyme, FADH2 can also serve as immediate reducing agent in biochemical reactions.

Generation of reducing power :

  Reducing power is generated on oxidation of a suitable electron donor during catabolic reactions of metabolism. These electron donors can be either organic or inorganic or both depending on the type of organism. NADPH acts as the reducing power in all anabolic reactions. In addition to NADPH, flavin coenzyme, FADH2 can also serve as reducing power in certain biochemical reactions.

  NADPH is generated during oxidative pentose phosphate pathway, where glucose 6 PO4 is oxidized to pentose phosphate in all most all living organisms. However in arche bacteria, where pentose phosphate pathway does not function, alternate glycolytic pathway function to generate NADPH.

NADPH can also be generated in cell by transhydrogenase reaction, where reduced NAD will participate in reduction of NADP. This reaction also helps in maintaining adequate cellular levels of NADH / NADPH ratio. NADH mainly participates In ATP formation, by transferring electron through electron transport chain.

Role of precursor metabolites

  Precursor metabolites are the intermediate molecules in the metabolic pathways. They are produced during operation of catabolic pathways. The precursor metabolites can
  1. Provide basic carbon skeleton for the synthesis of all the building blocks required to synthesize macromolecules.
  2. Undergo oxidation via catabolic pathways to provide ATP and other energy rich compounds that fuel anabolic pathways.

About 150 different low molecular. weight compounds are required for cellular synthesis. They include
1. Building blocks for synthesis of cellular macromolecules. They include

  • Amino acids for synthesis of proteins.
  • Fatty acids for synthesis of lipids.
  • Monosccharides for synthesis of polysaccharides.
  • Purines and pyrimidines for synthesis of Nucleic acids
2. Soluble molecules required for cellular metabolic activities. They include vitamins, co-enzymes and polyamines.

There are only 12 compounds, which act as precursor metabolites. They are virtually the same in all living organisms. They include

  • Acetyl CoA
  • Pyruvate
  • Phospho enol pyruvate (PEP)
  • 3 phospho glyceraldehydes (3PGAL)
  • Dihydroxy acetone phosphate (DHAP)
  • Glucose 6 Phosphate
  • Fructose 6 Phosphate
  • Erythrose 4 Phosphate
  • Ribose 5 Phosphate
  • Xylulose 5 Phosphate
  • Aplha Keto glutaric Acid (Alpha KG)
  • Succinate

The precursor metabolites are the intermediates of three Indispensible pathways of catabolism

  1. TCA cycle
  2. Glycolytic or gluconeogenic pathways
  3. Pentose phosphate pathway

Role of energy rich compounds

To perform all cellular activities, a suitable form of blochemical energy is required by the cell. This biochemical energy is obtalned as the energy rich compounds, which possess high energy rich chemical bonds.
  The necessary energy required to drive a blochemical reaction is released on hydrolysis of this energy rich bond. In living cells, a variety of energy rich compounds are formed, which are utilized for general purpose or to drive a specific biochemical set of reactions.

Energy rich compounds of cell

  There are mainly two classes of energy rich compounds formed in the cell, which satisfy need of energy requiring reactions. They are

  1. Compounds having high energy anhydrous phosphoester bond.
  2. Compounds having high energy thiolester bond.

Compounds having high energy anhydrous phosphoester bond
Most energy rich compounds of the cell belong to this category. They are obtained as nucleoside triphosphate derivatives. These include

  1. ATP Adenosine triphosphate
  2. GTP Guanosine triphosphate
  3. CTP Cytidine triphosphate
  4. TTP Thymidine triphosphate
  5. UTP Uridine triphosphate

ATP and its role

  ATP is considered as one of the most commonly used energy currency of the cell. It possesses two energy rich anhydrous phosphoester bonds.

Hydrolysis of each of this energy rich bond releases 7.3 Keal energy.

ATP + H₂O ➞ ADP + Pi + 7.3 Kcal.
ADP + H₂O ➞ AMP + Pi + 7.3 Kcal.
AMP + H₂O ➞ Adenosine + Pi+ 4 Kcal.
ATP is most commonly required for

  1. Uptake of nutrients
  2. Activation of most substrate molecules so that they are able to enter the cell metabolism
  3. Biosynthesis of most cellular molecules, nucleic acids, chromosome replication and cell division.

Other energy rich compounds and their role

  Apart from ATP, various other kinds of energy rich compounds are formed in the cell. They have specialized role in cellular metabolism. Their specialized utilization in specific metabolic reactions may be considered useful for adequate supply of energy for the concerned biosynthetic reactions, so that they can operate at optimal level in the cell. These energy rich compounds and their role are summarised in below table.

Energy rich compounds and their role in metabolism.

Mechanism and Specificity of Enzyme Action

  Enzymatic reactions include the formation or the destruction of chemical bonds. When two or more substrate molecules are Joined, chemical bonds are formed. When a complex molecule is split into simpler compounds chemical bonds are destroyed.

Both the formation and destruction of bonds generally requires the prior stretching or bending of existing bonds, creating a transition (Intermediate) state. The energy required to acquire transition state is called activation energy. Enzymes acts as catalyst and lowers the requirements for this activation energy. Therefore, the reaction can occur even at normal temperature and pressure.

Enzyme catalyzed reaction requires a lower activation energy as compared to uncatalyzed reaction.

Enzymes act by reacting with substrate through its active site. They are specific in action. Only a specific substrate can bind at the active site of the enzyme to form ES complex, which finally yields product. This can be expressed as under.
E+S ➞ ES ➞ ES* ➞ EP ➞ E + P.

  1. First the substrate binds to the active site of enzyme to form ES complex.
  2. Now the ES complex gets structurally Induced in such a manner that it is able to convert into product and forms EP complex. This involves a chemical change. The reaction energy required for this chemical conversion is lowered dowm by the enzyme.
  3. Now, the product dissociates from the complex and the enzyme molecule is made free. So that It is able to react with another substrate molecule again.

Specificity of enzyme action :

 Enzymes are highly specific in their action. They are specific for the substrate on which they attack and the reaction they catalyze. The basis of this specificity is their active site. The enzyme specificity, therefore, can be divided into two types as under:
1. Substrate specificity
2. Reaction specificity

Substrates specificity

Enzymes are specifie for the substrate on which they attack. This substrate specificity may further be categorized in to different types.
1. Absolute specificity
2. Group specificity
3. Stereo specificity

Absolute specificity

  When the enzyme possesses specificity for the entire substrate molecule, the specificity is called absolute specificity.
e.g. Urease acts on urea to degrade It into CO2 and NH3.

Group specificity

  Enzymes may be specifie for a particular group or chemical bond within the substrate and attack on them. This kind of specificity is called group specificity.
e.g. Transaminase acts only on (NH2) group of substrate and cause Its transfer.

Stereo specificity

Almost all enzymes are able to recognize orientation of groups within the structure of molecule. Therefore, they are able to distinguish structural as well as optical Isomers and attack them specifically. Such specificity is called stereo specificity.
e.g. Alanine recemase. It causes isomerization of alanine and is able to convert L-alanine to D-alanine.

Reaction specificity

  Enzymes are also specific for the reaction they catalyze. One particular enzyme will catalyze one particular type of blochemical reaction only. Such specificity is called reaction specificity. This kind of specificity is given prime importance for enzyme classification by IUB. Accordingly, they are divided Into six classes:

  1. Oxidoreductases
  2. Transferases
  3. Hydrolases
  4. Lyases
  5. Isomerases
  6. Ligases

What is Sterilization in Place (SIP)

 Most pharmaceutical GMP systems available today incorporate some form of sterilization using steam. In situ sterilization of equipment using a medium called Pure Saturated Steam. The installation comprises of one or more pieces of processing equipment

Example • Fermentor or a centrifugal separator to handle harvests, connected by rigid stainless steel or flexible Teflon-lined piping

Why is there a need for sterilization?

  • To increase the level of sterility assurance associated with those products made by aseptic processing.
  • The size and configuration of large number of process equipment utilized in the production of Biological/parenteral restricts them to be placed inside an autoclave for sterilization.
  • To assure a higher degree of sterility assurance for these items, they should be sterilized in situ rather than sanitized.
  • SIP is required as a result of desire for enhanced sterility assurance for aseptically produced materials.
  • Steam SIP is in daily use in the Biological & parenteral industry.

What makes SIP a critical process?

  • Steam-in-place sterilization enables the entire processing system to be sterilized as a single entity thereby reducing the need for aseptic connections.
  • Criticality of system design to achieve sterility with SIP is considered more closely than the steam sterilizer.
  • SIP employs the same moist heat mechanism as steam sterilization in autoclaves.

SIP Fundamentals

  • SIP differs only slightly from steam sterilization in autoclaves.
  • To achieve effective SIP, the elements necessary for process effectiveness inherent in the autoclave design and operation must be provided in the SIP system.

What is a saturated pure steam?

  • Saturated steam is a steam-water mixture in which the vapour phase (steam) is in equilibrium with the liquid phase (water or condensate).
  • The addition of heat to saturated steam can result in its de-saturation (or superheating).
  • The loss of heat from saturated steam will result in its condensation.

SIP Design Considerations

  • Complete displacement and elimination of entrapped air.
  • Constant bleeds of steam at all low points to eliminate condensate build-up Heating large masses of stainless steel from ambient temperature to 121°C results in the creation of large quantities of condensate.
  • Strict adherence to the sterilization procedures.
  • Proper maintenance of the sterility after the process.

  • SIP system design concepts is achieved through a review of the equipment elements that make up that system.
• The Components of an SIP system:
   Pressure Vessels
   Piping system
   Filters

  • The system must be designed in a way that condensate can be readily removed.
  • To achieve this objective, system is sterilized in multiple patterns, in which each pattern sterilizes a portion of the larger system.
  • Some portions of the system must be sterilized more than once to assure that all portions of the system are fully covered.