Detergent enzymes recovery

To begin, enzyme products can be inside the cell (intracellular), loosely associated with the cell, or secreted (extracellular). Each of these products requires a different approach on how to purify the enzyme. The majority of presently commercialized detergent enzymes is of the extracellular variety and can be recovered directly from the fermentation broth. Thus, the primary recovery challenge involves removing the cells from the broth, aptly called cell separation. Three different techniques are commonly used to achieve this goal filtration, microfiltration, and centrifugation. 

After recovery, purification of the enzyme is the next step. This can be achieved in many ways, through precipitation with salts, crystallization, chromatography, and aqueous two-phase extraction. Many of these methods are associated with substantial capital cost, low throughput, or low yields and are not commonly used for detergent enzymes. However, authors have reported the use of crystallization and aqueous two-phase extraction for large-scale preparations. These methods are also effective in concentrating enzyme broths. 

Recovery. The principal purpose of recovery is to remove nonproteinaceous material from the enzyme preparation. Enzyme yields vary, sometimes exceeding 75%. Most industrial enzymes are secreted by a microorganism, and the first recovery step is often the removal of whole cells and other particulate matter (19) by centrifugation (20) or filtration (21). In the case of ceU-bound enzymes, the harvested cells can be used as is or dismpted by physical (eg, bead mills, high pressure homogenizer) and/or chemical (eg, solvent, detergent, lysozyme [9001 -63-2] or other lytic enzyme) techniques (22). Enzymes can be extracted from dismpted microbial cells, and ground animal (trypsin) or plant (papain) material by dilute salt solutions or aqueous two-phase systems (23). 

Polymers and resins Water purification, including removal of phenol, chlorophenols, ketones, alcohols, aromatics, aniline, indene, polynuclear aromatics, nitro- and chlor-aromatics, PCB, pesticides, antibiotics, detergents, emulsifiers, wetting agents, kraftmill effluents, dyestuffs recovery and purification of steroids, amino acids and polypeptides separation of fatty adds from water and toluene separation of aromatics from ahphatics separation of hydroquinone from monomers recovery of proteins and enzymes removal of colours from symps ... 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

Recovery/purification reagents (salts, detergents, urea enzymes, filter aids, etc.)... 

Alternative protocols are described elsewhere (Ribeiro-Neto efal., 1985 Kopf and Woolkalis, 1991 Carty, 1994). ATP, phospholipids and small amounts of certain ionic and nonionic detergents (i.e. SDS, CHAPS, Lubrol PX) promote dissociation of the A and B protomers (Moss et a/., 1986). We do not use SDS since it denatures solubilized G proteins very easily. DTT or 3-ME are necessary to break the disulfide bonds of the A protomer. Supplementation with BSA (final concentration approx. 0.9 mg/ml) helps prevent loss of enzyme through adsorption to the walls of the tube, and ensures recovery of proteins following precipitation with sodium chloride/acetone, trichloroacetic acid (TCA), or chloroform/methanol. Furthermore, when samples are subjected to SDS-PAGE, intensities of the stained 67 kDa BSA protein bands allow rough estimation of incomplete recovery of the precipitated sample (see section 4.5). Preactivated PT should be used immediately, and enzyme left over from an experiment should be discarded, since reduced toxin has been shown to lose activity rapidly (Kaslow et a/., 1989). 

Surfactant aggregates (microemulsions, micelles, monolayers, vesicles, and liquid crystals) are recently the subject of extensive basic and applied research, because of their inherently interesting chemistry, as well as their diverse technical applications in such fields as petroleum, agriculture, pharmaceuticals, and detergents. Some of the important systems which these aggregates may model are enzyme catalysis, membrane transport, and drug delivery. More practical uses for them are enhanced tertiary oil recovery, emulsion polymerization, and solubilization and detoxification of pesticides and other toxic organic chemicals. 

The Eprs of B. subtilis and related bacilli are also very relevant for commercial applications. In particular, these enzymes are employed in the manufacture of detergents, tanning of leather, management of industrial and household wastes, bioprocessing of X-ray or photographic films for the recovery of silver, protein hydrolysate preparation in the food industry, synthesis of aspartame, and other applications [102]. Recently, the fibrinolytic activity of Vpr was discovered. Accordingly, Vpr has the potential to work as a thrombolytic agent in medical applications [103, 104]. 

Most enzymes for detergent formulations, which are produced by genetically modified microorganisms, are monocomponents, that is, only one enzyme protein contributes to the overall activity. In a few cases, like cellulases, the enzyme product can be a multicomponent mixture of several enzyme proteins. In these cases, the recovery process must be designed to ensure that the ratio between the enzyme proteins important for the application is maintained throughout the recovery process. 

After recovery and purification of the enzymes, the third important process step is to make a final formulation of the enzymes. The enzyme concentrate, whether it is present in an aqueous solution or in a dried form, has to be transformed into a stable form that fulfils the detergent producers requirements. Obviously, the chosen formulation needs to be compatible with the detergent and a range of different quality parameters should be defined. 

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