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Production of enzymes

The document discusses the production of enzymes through pharmaceutical biotechnology, detailing the steps from organism selection to enzyme recovery and purification. Key processes include fermentation techniques such as submerged liquid culture and solid-state fermentation, as well as various methods for enzyme isolation and purification like chromatography. Additionally, it covers specific types of amylases and their commercial production methods using bacteria and fungi.

Education◦
SJM College of Pharmacy, Chitradurga Prepare By, Adarsh Patil Ass Professor(Pharmacognosy) SJM College of Pharmacy 1 PHARMACEUTICAL BIOTECHNOLOGY
Production of Enzymes: Steps Involved: 1. Selection of organisms 2. Formulation of medium 3. Production process 4. Recovery & Purification of enzymes
1. Selection of organism: • The most important criteria for selecting the microorganism are that the organism should produce the maximum quantities of desired enzyme in a short time while the amounts of other metabolite produced are minimal. • Once the organism is selected, strain improvement for optimising the enzyme production can be done by appropriate methods (mutagens, UV rays). From the organism chosen, inoculum can be prepared in a liquid medium.
2. Formulation of medium: • The culture medium chosen should contain all the nutrients to support adequate growth of microorganisms that will ultimately result in good quantities of enzyme production. • The ingredients of the medium should be readily available at low cost and are nutritionally safe. Some of the commonly used substrates for the medium are starch hydrolysate, molasses, corn steep liquor, yeast extract, whey, and soy bean meal. Some cereals (wheat) and pulses (peanut) have also been used. • The pH of the medium should be kept optimal for good microbial growth and enzyme production.
3. Production process: • Industrial production of enzymes is mostly carried out by submerged liquid conditions and to a lesser extent by solid-substrate fermentation. • In submerged culture technique, the yields are more and the chances of infection are less. Hence, this is a preferred method. • However, solid substrate fermentation is historically important and still in use for the production of fungal enzymes e.g. amylases, cellulases, proteases and pectinases.
• The medium can be sterilized by employing batch or continuous sterilization techniques. The fermentation is started by inoculating the medium. The growth conditions (pH, temperature, O2 supply, nutrient addition) are maintained at optimal levels. The froth formation can be minimised by adding antifoam agents. • The production of enzymes is mostly carried out by batch fermentation and to a lesser extent by continuous process. • The bioreactor system must be maintained sterile throughout the fermentation process. The duration of fermentation is variable around 2-7 days, in most production processes. Besides the desired enzyme(s), several other metabolites are also produced. The enzyme(s) have to be recovered and purified.
4. Recovery and purification of enzymes: • The desired enzyme produced may be excreted into the culture medium (extracellular enzymes) or may be present within the cells (intracellular enzymes). Depending on the requirement, the commercial enzyme may be crude or highly purified. • Further, it may be in the solid or liquid form. The steps involved in downstream processing i.e. recovery and purification steps employed will depend on the nature of the enzyme and the degree of purity desired.
• In general, recovery of an extracellular enzyme which is present in the broth is relatively simpler compared to an intracellular enzyme. For the release of intracellular enzymes, special techniques are needed for cell disruption. • Microbial cells can be broken down by physical means (sonication, high pressure, glass beads). The cell walls of bacteria can be lysed by the enzyme lysozyme. For yeasts, the enzyme β- glucanase is used. However, enzymatic methods are expensive. • The recovery and purification (briefly described below) steps will be the same for both intracellular and extracellular enzymes, once the cells are disrupted and intracellular enzymes are released. The most important consideration is to minimise the loss of desired enzyme activity.
5. Removal of cell debris: Filtration or centrifugation can be used to remove cell debris. 6. Removal of nucleic acids: Nucleic acids interfere with the recovery and purification of enzymes. They can be precipitated and removed by adding poly-cations such as polyamines, streptomycin and polyethyleneimine. 7. Enzyme precipitation: Enzymes can be precipitated by using salts (ammonium sulfate) organic solvents (isopropanol, ethanol, and acetone). Precipitation is advantageous since the precipitated enzyme can be dissolved in a minimal volume to concentrate the enzyme. 8. Liquid-liquid partition: Further concentration of desired enzymes can be achieved by liquid-liquid extraction using polyethylene glycol or polyamines.
9. Separation by chromatography: There are several chromatographic techniques for separation and purification of enzymes. These include ion-exchange, size exclusion, affinity, hydrophobic interaction and dye ligand chromatography .Among these, ion- exchange chromatography is the most commonly used for enzyme purification. 10. Drying and packing: • The concentrated form of the enzyme can be obtained by drying. This can be done by film evaporators or freeze dryers (lyophilizers). The dried enzyme can be packed and marketed. For certain enzymes, stability can be achieved by keeping them in ammonium sulfate suspensions. • All the enzymes used in foods or medical treatments must be of high grade purity, and must meet the required specifications by the regulatory bodies. These enzymes should be totally free from toxic materials, harmful microorganisms and should not cause allergic reactions.
1. Amylase: • Amylases are important hydrolase enzymes which have been widely used since many decades. • These enzymes randomly cleave internal glycosidic linkages in starch molecules. • To hydrolyze them and yield: (dextrins ) (oligosaccharides)
TYPES: 1. α-Amylase: •α-Amylase is a hydrolase enzyme that catalyses the hydrolysis of internal α-1, 4- glycosidic linkages in starch to yield products like glucose and maltose. • It is a calcium metalloenzyme i.e. it depends on the presence of a metal co factor for its activity. •The optimum pH for activity is found to be 7.0 •he substrate that α-amylase acts upon is starch. • Starch: is a polysaccharide composed of two types of polymers – amylose and amylopectin.
• Amylose constitutes 20-25% of the starch molecule. • It is a linear chain consisting of repetitive glucose units linked by α-1,4-glycosidic linkage. • Amylopectin constitutes 75-80% of starch and is characterized by branched chains of glucose units. • The linear successive glucose units are linked by α-1, 4- glycosidic linkage. • while branching occurs every 15-45 glucose units where α-1, 6 glycosidic bonds are present.
2. β-Amylase: • an exo-hydrolase enzyme that acts from the nonreducing end of a polysaccharide chain by hydrolysis of α-1, 4-glucan linkages to yield successive maltose units. • Since it is unable to cleave branched linkages in branched polysaccharides such as glycogen or amylopectin. • the hydrolysis is incomplete and dextrin units remain. • Primary sources of β-Amylase are the seeds of higher plants and sweet potatoes. • The optimal pH of the enzyme ranges from 4.0 to 5.5. • β-Amylase can be used for different applications on the research as well as industrial front. • It can be used for structural studies of starch and glycogen molecules produced by various methods.
3. γ-Amylase: • γ-Amylase cleaves α(1-6)glycosidic linkages, in addition to cleaving the last α(1- 4)glycosidic linkages at the nonreducing end of amylose and amylopectin, unlike the other forms of amylase, yielding glucose. • γ- amylase is most efficient in acidic environments and has an optimum pH of 3.
Sources: • α-Amylase can be isolated from plants, animals or microorganisms. • The enzyme has been isolated from barley and rice plants. • It has been found that cassava mash waste water is a source of α-Amylase. • In the recent past, there has been extensive research on microbial production of α- Amylase.
There are 2 major reasons for the increasing interest in microbial sources: 1) The growth of microorganisms is rapid and this will in turn speed up the production of enzyme. • Microorganisms are easy to handle when compared to animals and plants. • They require lesser space and serve as more cost effective sources. 2) Microorganisms can be easily manipulated using genetic engineering or other means. • They can be subjected to strain improvement, mutations and other such changes by which the production of α-Amylase can be optimized. • α-Amylase is produced by several bacteria, fungi and genetically modified species of microbes.
Bacterial Sources: • Thermostable α-Amylase:  B.subtilis,  B.stearothermophilus,  B.licheniformis  B.amyloliquefaciens. • Temp:(45°C to 55°C) • PH:(6.0–7.0) • Incubation :48_72 hr Halophilic amylases:  Chromohalobacter sp.,  Halobacillus sp.,  Haloarcula hispanica,  Halomonas meridiana • Substrate : • Carbon source: maltose, Glucose,& Oil cakes
Fungal Sources: Aspergillus species A.oryzae, A.niger, A.kawachii Temp (30-90°c) PH:(5.0-6.0) Incubation 96 hr Penicillium species: P.fellutanum, P.roquefortii, P.chrysogenum Temp:(30-55°C) PH(6.0 to 7.0) Incubation : 72 hr Nitrogen source: Inorganic nitrogen sources ammonium sulphate, ammonium chloride and ammonium hydrogen phosphate. organic sources peptone, yeast extract and soyabean meal.
Production:  There are mainly two methods which are used for production of α-Amylase on a commercial scale.  These are: 1) Submerged fermentation (SMF) 2) Solid State fermentation (SSF)  1) Submerged fermentation (SMF):  Submerged fermentation (SmF) employs free flowing liquid substrates, such as molasses and broths.  The products yielded in fermentation are secreted into the fermentation broth.  This fermentation technique is suitable for microorganisms such as bacteriathat require high moisture content for theirgrowth.  SmF is primarily used for the extraction of secondary metabolites that need to be used in liquid form .
 This method has several advantages.SmF allows the utilizationof genetically modified organisms to a greater extent than SSF.  The sterilization of the medium and purification process of the end products can be done easily.  Also the controlof process parameters like temperature, pH, aeration, oxygen transfer and moisture can be done conveniently.
2. Solid State Fermentation:  Solid state fermentation is a method used for microbes which require less moisture contentfor their growth.  The solid substrates commonly used in this method are bran, bagasse, and paper pulp.  The main advantage is that nutrient-rich waste materials can be easily recycled and used as substrates in this method  Other advantages that SSF offers over SmF are simpler equipments, higher concentration of products and lesser effluent generation.  For several such reasons SSF is considered as a promising method for commercial production of enzymes.
 3 methods 4.1. Dinitrosalicylic Acid Method (DNS):  In the dinitrosalicylic acid method, aliquots of the substrate stock solution are mixed with the enzyme solution.  Followed by 10 min of incubation at 50°C,  DNS reagent is added to the test tube and the mixture is incubated in a boiling water bath for 5 min.  After cooling to room temperature, the absorbance of the supernatant at 540 nm is measured.
 In the NS method, an aliquot of stock solution of substrate is heated at 50°C for 5min.  Preheated (50°C for 5 min) enzyme solution is added to the substrate.  This reaction mixture is incubated at 50°C and the reaction is carried out for 10min.  After incubation Somogyi copper reagent is addedto terminate the reaction.  This is then incubated in boiling water bath for 40 min & cooled to room temperature.  Finally water is added and the mixtureis centrifuged at 13,000 rpm for 1 min and absorbance of supernatant is read at 610 nm .
 The hydrolytic activity of α-Amylase can be determined based on the principle that starch and iodine react to form a blue colored complex.  On hydrolysis of starch this complex changes to a reddish brown colored one.  The absorbance can be read after the enzyme substrate reaction has been terminated.  This gives a measure of the extent of hydrolysis of starch by α-Amylase.
 This can be followed by any of the chromatographic techniques like ion exchange,  Gel filtration and affinity chromatography for further separation and purification of the enzyme.  the enzyme was precipitated was followed by dialysis and then column chromatography
Production of enzymes