Industrial enzymes Enzymes have been used for centuries in industrial processes such as tannery, brewing, bakery, dairy etc. Depending on the process, enzymes have been used in soluble form – such as proteases from animal origin in tannery – or through microbial cells in fermentation processes (brewing and baking). The role of microorganisms in fermentation was well established by Pasteur in 1876, while enzyme mechanisms and kinetic behavior began to be established in the beginning of the 20th century. Invertase and amylase were the first enzymes to be isolated and produced at an industrial scale. Production strategies As all commercially valuable enzymes are proteins, they can be produced through processes using similar unit operations, i.e., extraction from a source (animal, vegetal or microbial), filtration, centrifugation, precipitation, purification, drying, stabilization, standardization, and packaging. In biotechnology, all unit operations between filtration and packaging are called downstream processing. Enzymes of animal and plant origins are produced by macerating tissues, organs, leaves, and fruits – often residual materials from cattle breeding and agriculture activity –, followed by extraction with water or organic solvents. On the other hand, microbial enzymes are obtained from either prokaryotic (bacteria) or eukaryotic (yeasts, fungi, mainly) cells cultured in a liquid or a semi-solid medium and carried out in a special reactor called fermenter. This process is known as fermentation. The semi-solid culture: used in large scale by the first time in 1894 for the production of amylase from Aspergillus oryzae grown in humidified cooked rice mass for one week (“Koji Process”) – occurs as follows: a) the semi-solid medium (corn, wheat, soy, rice, or barley bran) is subjected to vapor jets for cooking and sterilization. In the submerged culture: the microorganism is kept in suspension through constant agitation under controlled growing conditions (pH, temperature, nutrients, oxygenation etc.). The medium is an aqueous solution comprised of low-cost substances such as starch hydrolyzate, molasses, corn steep liquor, whey, and many cereals. At the completion of the fermentation, the enzyme may be present within the microorganism or excreted into the medium. When inside the cell, the suspension is centrifuged or filtered, the supernatant or filtrate is discharged, and the cell cake is collected; otherwise, the cell cake is discarded and the liquid phase is collected. Downstream processing of industrial enzymes: Downstream are several unit operations aiming a concentration and purification of an enzyme often present in a raw extract. The main unit operations used are cell disruption (if needed), filtration, centrifugation, sedimentation, flocculation, coagulation, ultrafiltration, precipitation, chromatography, crystallization, evaporation, drying, standardization, and packaging. 1. Filtration The rate of passage of a liquid through a filter depends on the pressure difference applied, the resistance of the filter material, the viscosity of the liquid, and the resistance produced by the cake already present. Thus, the effectiveness of a filter will initially be high, but will fall as the material accumulates and perhaps compresses. Filter aids, such as diatomaceous earth, retain finer particles and are valuable in enzyme isolation, but they tend to occlude the liquor containing the enzyme and will damage downstream equipment if allowed to pass into the filtrate. The commonest forms of industrial filter are plate and frame press and rotary drum filter. In this filter vacuum is applied to the inside of a hollow drum rotating in a trough containing the material to be filtered. Sediment accumulates on a filter cloth from which it may be removed by a multiplicity of methods. 2. Centrifugation and Sedimentation These unit operations are both based on density differences between insoluble particles and the surrounding fluid. Sedimentation relies on gravity, settling to achieve solid-liquid separation, and is generally performed in rectangular or circular flow tanks. Centrifugation involves the mechanical application of a centrifugal force to obtain a solid concentrate and clarified supernatant. Centrifugation has become a widespread technique for the removal of solids, particularly under circumstances where filtration is unwanted or ineffective (such as removing gelatinous or colloidal material). There are several types of centrifuges for enzyme isolation. The tubular bowl centrifuge, multichamber centrifuge, disc bowl centrifuges, solid-bowl scroll centrifuge, and perforate bowl basket centrifuge are largely used. The rate at which the particles move depends on this force, the shape, size, and density of the particles, and on the density and viscosity of the suspending medium. 3. Flocculation and Coagulation Coagulation is the result of a direct adherence among very small particles in a medium due to neutralization of charges of the particles by adding polyvalent ions of opposite charge. Flocculation refers to the formation of very open aggregates, in which the flocculating agent (gelatin, charged or uncharged synthetic polymers) acts as an extended bridge between particles. Inorganic ions cannot cause flocculation, although they may be used to neutralize charges and assist in flocculation. However, organic polyelectrolytes may cause simultaneous coagulation and flocculation. These techniques have been applied to whole cells, cell debris, and insoluble proteins. 4. Cell disruption If the desired product is an intracellular substance (for example, glucose 6-phosphate dehydrogenase from Saccharomyces cerevisiae), cell disruption is an important early step in product recovery because the cell envelopes (cytoplasmic membrane and cell wall) are natural barriers to the liberation of macromolecules into the culture medium. Methods used for disrupting microorganisms can be classified as mechanical (freeze/thaw, ultrasound, dyno and colloid mills, Gaulin/Manton and French presses, ball mill) and nonmechanical (treatment with alkali, detergents or organic solvents, osmotic shock, freeze-drying, enzymatic digestion etc.). The effectiveness of a particular disruption technique is usually assessed in terms of the degree of cell breakage and/or the level of enzyme activity recovered, or the total protein dissolved in the disrupted suspension. 5. Extraction Extraction is a generic term meaning isolation of an enzyme from a crude cell free extract. The isolation can be carried out through ultrafiltration – a direct separation procedure; solvent extraction or aqueous two-phase systems. Extraction of lipophilic substances with water-immiscible organic solvents is a well-established separation process of the chemical industry, which can be performed on a large scale. Extraction also plays a major role in the isolation of antibiotics. A typical antibiotic extraction involves transfer of the solute from a clarified fermentation broth into an organic phase, followed by re-extraction of the concentrated product in an aqueous buffer. The two-step process thus combines product concentration with purification. Final recovery is often achieved by precipitation, crystallization, or evaporation. 6. Precipitation It is a procedure in which the addition of a reagent or a change in conditions causes proteins to leave the solution and form insoluble particles. There are several ways to carry out the precipitation of a macromolecule : a) salting-out: precipitation of proteins by using neutral salt – mainly ammonium and sodium sulfate - at a high concentration; b) pH variation: aims to precipitate the protein at its isoelectric point (the pH in which the net overall charge of the macromolecule is zero); c) temperature variation: the temperature is increased slowly in order to promote the denaturation of unwanted proteins, and the desired protein remains dissolved; d) precipitation by organic solvents (ethanol, methanol, acetone or diethylether); e) precipitation by high-molecular-weight polymer: it is based on the polymer’s influence on the interaction of the protein with its aqueous environment. The phenomenon occurs due to the insertion of the polymer in the protein-water interface. The 6000 MW polyethylene glycol (PEG 6000), mixed with water at 50% (wt/wt) concentration, is an effective precipitant and stabilizer of proteins at room temperature. f) precipitation by manganese salts or streptomycin sulfate: this procedure aims to remove nucleic acids from the cell-free extract in isolating intracellular enzymes because the presence of nucleic acids (MW ranging from 25x103 to 1x106) increases the medium viscosity, which reduces the yield of separation in fractional precipitation and/or chromatography. 7. Chromatography Chromatography can be defined as the uniform percolation of a fluid through a column filled with a finely divided substance that selectively retards certain components of the fluid. For enzyme isolation, the great majority of chromatographic separations are performed in a completely aqueous environment. There are several chromatographic methods for the separation or purification of biological products: a) ion-exchange chromatography: the molecular electric charge is the basis of separation; its effectiveness is remarkable at early downstream stages, where large volumes are handled; resolution, retention capability and speed depend on the matrix used; it can be used either in batch or with continuous procedures; b) hydrophobic interaction chromatography: following Van der Waals forces and steric interactions between solute and matrix (phenyl agarose, for instance); resolution, retention capacity and speed are relevant; it can be applied at any stage of the downstream protocol, although the greatest separation occurs when the ionic strength of the medium is high, mainly after salting-out; c) gel filtration: the separation capability is based on the size and shape of the solutes regarding the gel spherical beads diameter, so that the small solute molecules enter the beads and traverse a greater effective retention volume while the largest molecules surround the beads emerging first from the gel column; the flux across the column is high, but has a moderate resolution for fractionation; it is efficient for desalting and buffer exchanging; d) affinity chromatography: based on biological affinity, whose selectivity, retention capacity and speed depend on the type of ligand used (monoclonal antibody, specific inhibitor, cofactor, modified substrate etc.); if the ligand is immobilized by a covalent attachment to an insoluble matrix (cellulose or polyacrylamide), the protein of interest, in displaying affinity for its ligand, becomes bound and immobilizes itself. Once the contaminant proteins are removed by filtration and wash the matrix profusely, the protein of interest is eluted from the matrix by the addition of high concentrations of the free ligand in solution; though it can be used at anystage of the downstream protocol, the recommendation is to use it when protein loads and fouling substances have been sharply reduced by other cheaper procedures. 8. Crystallization Crystallization is a process – carried out in either batch or continuous crystallizers – in which a substance crystallizes from a supersaturated solution. This can be achieved by either gradually cooling the solution below the saturation temperature or by evaporating the solvent at a constant temperature above the saturation concentration of the substance. Crystallization is the preferred method of forming a final product because a very high purity is feasible. Pure enzyme crystals for crystallographic analysis or for pharmaceutical use (lysozyme, hyaluronidase, superoxide dismutase, urokinase, uricase etc.) can be obtained from the solution previously sterilized or not by ultrafiltration. The crystallization is a final purification procedure that allows attaining end products (proteins, enzymes, fine chemicals etc.) with an exceptional purity, ease of handling, long shelf-life, and a pleasant appearance. The detailed three-dimensional structures of some eight hundred proteins have been established by x-ray crystallography (beams of x-rays are passed through a crystal of protein). 9. Drying Before handling, packaging and storing, pure bioproducts must be free from residual water or organic solvents. This can be accomplished by spray-drying and freeze drying. Both techniques allow gently removing water or other solvent with a minimal increase in temperature. The heat transfer may be by conduction, convection, radiation or a combination of them. Spray-drying is a convective drying method carried out using spray-driers in which pressure or centrifugal atomizers or gas-liquid jets are used to generate a fine spray of solution droplets in continuous contact with cyclonic flowing hot gas in a conical chamber (100-300°C). The main advantages of this process are the continuous operation, resulting powdered bioproducts, and handling heat-labile materials without significant loss of their main features such as molecular structure (for biopolymers such as DNA, RNA, polysaccharides, proteins and lipids), catalytic activity (for enzymes), surfactant capability (for non-catalytic proteins), and pharmacological activity (for drugs). Freeze-drying aims the transformation of a biological material from active into a non-active form by freezing (cooling rates between 0.5°C/min and 5°C/min) followed by the removal of water under low pressure (often between 0.1 and 0.5 mbar) and controlled temperature (often between 15°C and 30°C). Freeze-drying is based on the fact that frozen water sublimates to water vapor under low pressure. The heat is transferred to the frozen solid primarily by conduction from a heated plate, the water vapor condenses at low temperature (the condenser maintained at -40°C), and the solid temperature is regulated by controlling the pressure in the drying chamber. This process can be divided in primary and secondary drying. Primary drying removes the free water (duration between 10 and 20 h), and the secondary drying (duration about one-third of the primary drying time) removes the moisture (for example, water of crystallization or water dispersed in a glassy material). 10. Formulation Formulation is the way an enzyme preparation - either in solution or powder forms that can be marketed. The nature and the purity of the preparation will be determined by the requirements of the industry. The enzymes used in industry require a minimum of purification and, in the case of microbial extracellular catalysts, a lyophilized cell-free supernatant treated to remove the colored components is acceptable. For industrial enzymes, the downstream protocol is designed for increasing catalytic potency per weight and not to achieve purification – as in industry reducing cost is pivotal, the enzyme purity must be minimal.
