Lytic enzyme system

Many microorganisms produce enzymes that lyse the cell wall of yeast. The most extensive work has been done with the lytic system from Arthrobacter sp., Cytophaga sp., Oerskovia, Bacillus circulans, Rhizopus, Tri-choderma, Penicillium, Pellicularia sp., Rhizoctonia, and Streptomyces sp. (3-9). 

Gupta, MN, Mattiasson B (1992) Unique applications of immobilized proteins in bioana-lytical systems. In Suelter CH (ed) Bioanalytical applications of enzymes. Wiley 36 1... 

Microbial cell-wall-lytic enzymes are widely used in the laboratory for cell breakage, proto-plasting of yeasts and bacteria, and for studies of the structure and composition of microbial cell walls (J ). Recently lytic systems have come under consideration as a specific and chemically mild way to rupture microbial cells on an industrial scale (2 ). There appear to be attractive commercial applications of lytic systems for the recovery of enzymes, antigens and other recombinant products accumulated within cells, for upgrading of microbial biomass for food and feed uses (4 5) and for the manufacture of functional biopolymers from cell wall carbohydrates (6). 

Lytic enzyme systems provide a chemically mild, low-shear and catalytically specific alternative to mechanical cell disruption. Depending on the particular lytic system employed and its purity, the enzymes may be engineered to attack cell wall components alone, without product damage. The enzyme lysozyme, active against some bacterial cell walls, has been used to harvest bovine growth hormone granules from 12. coli (8), and a membrane-associated hydroxylase complex from . putida (11) use of other bacteriolytic enzymes from a variety of microbial sources have also been reported (3). 

Enzymes of the lytic system. Microbial yeast-lytic enzyme systems are widely distributed in nature, and have been isolated from Rhizoctonia sp., (4), Bacilus circulans (21), Coprinus macrorhizus (22), and Cytophaga sp. (23), among other sources. 

Work continues in two areas purification of the lytic system to allow protease and glucanase levels to be controlled independently and investigation of the release of site-specific yeast enzymes and subcellular fractions by enzymatic lysis. 

Figure 8 shows a simulation of enzyme recovery from the wall, cytosol and mitochondria. The concentrations of recoverable enzyme are normalized to the initial amount of enzyme present in the cell site. The curves rise as enzyme is released from a site, then fall as it is hydrolyzed. It may be seen that the lytic system is usable even as a crude preparation to recover wall linked yeast enzymes in 60 to 80% yield. The yield of yeast wall enzyme depends on... 

These simulations suggest an additional test for the structured model that is, to compare its predictions to data on release of site-linked enzymes in yeast. Tests for cytoplasmic and mitochondrial enzyme release will be aided by preparation of a low-protease lytic system. 

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). 

Tissue Plasminogen Aetivator (tPA). While streptokinase and urokinase can effectively induce clot dissolution in the majority of patients if given early, they lack clot specificity. Treatment with these enzymes results in a systemic lytic state attributable to their degradative action on circulating fibrinogen. Tissue plasminogen activator (tPA) was developed to achieve rapid and specific thrombolysis. 

The lytic enzyme systems, active against yeast cell walls, usually contain l,3-/ -glucanases, proteases, mannanases, chitinases, and 1,6-) -glucanases. The proportion of those enzyme activities, their action pattern, synergism, and dependence on inhibitors constitute the activity profile... 

En me Preparation. The four 1,3-j -glucanases, GI, GII, GIV, and GVIII were purified from lytic enzyme system produced by Streptomyces sp. 1228 using methods previously described (17). 

The experiment based on the two-level factorial design as described by Box and Wilson (19) was carried out in order to check the influence of the individual glucanases (g ) of Streptomyces sp. 1228 lytic enzymes system on the degree of yeast cell lysis (y). 

Enzyme Treatment. There are a number of enzymes which hydrolyze the microbial cell wall constituents. Enzymes exhibiting these activities include lysozyme, enzyme from snail extract and lytic enzyme systems of microbial origin composed of proteases,... 

Anistreplase (streptokinase- plasminogen complex) streptococci protein streptococci plaminogen, which is then cleaved to form plasmin. Plasmin mediate fibrinolysis. Systemic lytic state and immunogenicity may limit its use. Because antistreplase is aheadly lined to plaminogen, the onset of fibrinolytic is faster. These enzymes are not fibrin-specific. 

Immobilization onto a solid support, either by surface attachment or lattice entrapment, is the more widely used approach to overcome enzyme inactivation, particularly interfacial inactivation. The support provides a protective microenvironment which often increases biocatalyst stability, although a decrease in biocata-lytic activity may occur, particularly when immobilization is by covalent bonding. Nevertheless, this approach presents drawbacks, since the complexity (and cost) of the system is increased, and mass transfer resistances and partition effects are enhanced [24]. For those applications where enzyme immobilization is not an option, wrapping up the enzyme with a protective cover has proved promising [21]. 

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