Microbial proteases

Plasteins ate formed from soy protein hydrolysates with a variety of microbial proteases (149). Preferred conditions for hydrolysis and synthesis ate obtained with an enzyme-to-substrate ratio of 1 100, and a temperature of 37°C for 24—72 h. A substrate concentration of 30 wt %, 80% hydrolyzed, gives an 80% net yield of plastein from the synthesis reaction. However, these results ate based on a 1% protein solution used in the hydrolysis step this would be too low for an economical process (see Microbial transformations). 

Prior to the bating process, the hides are delimed with ammonium sulfate and/or ammonium chloride. Proteases are then appUed. The early preparation proposed by Rn hm was pancreatic trypsin. The use of a bating enzyme makes the hides soft and supple to prepare them for tanning. A new microbial protease, Pyrase 250 MP (82) (Novo Nordisk A/S) has been found to be a promising substitute for pancreatic trypsin [9002-07-7] which is more expensive because it must be extracted from pancreatic glands. 

Kuehler and Stine (43) studied the functional properties of whey protein with respect to emulsifying capacity as affected by treatment with three proteolytic enzymes. Two microbial proteases and pepsin were examined. The emulsion capacity decreased as proteolysis continued, suggesting that there is an optimum mean molecular size of the whey proteins contributing to emulsification. 

Fermentation by microorganisms accounts for a two-thirds share of commercial protease production in the world (Kumar, 1999). Depending on their pH optimum and active-site characteristics, microbial proteases are classified as aspartyl proteases (E.C. 3.4.23, acidic), metalloproteases (E.C. 3.4.24, neutral), cysteine or sulf-hydryl proteases (E.C. 3.4.22, alkaline), or serine proteases (E.C. 3.4.21, also alkaline) (Kalisz 1988 Rao, 1998). Their commercial uses are listed in Table 10.4. 

Rao, M. B., A. M. Tanksale, M. S. Ghatge, and V. V. Deshpande. 1998. Molecular and biotechnological aspects of microbial proteases. Microbiology and Molecular Biology Reviews 62 597-635. 

Alkanhal, H.A., Frank, J.F., Christen, G.L. 1985. Microbial protease and phospholipase C stimulate lipolysis of washed cream. J. Dairy Sci. 68, 3162-3170. 

Microbial proteases with widely different properties are produced commercially. Bacillus protease, however, represents more than 95 percent of the sale of all proteases. The most important use of Bacillus proteases is in detergents. Proteinaceous dirt often precipitates on clothes and it coagulates during the normal washing process. The addition of proteolytic enzymes to the detergent can easily dissolve such proteinaceous stains, which are otherwise difficult to remove. 

Proteases for Accelerating Cheese Ripening. Proteolysis plays such a major role in developing the flavor and texture of hard cheese that the addition of exogenous microbial proteases has been attempted using every commercially available protease preparation. 

The only way for microbes to enter a healthy plant is via the stomata or at sites of injury, inflicted by herbivory, wind, or other accidents. At the site of wounding, plants often accumulate suberin, lignin, callose, gums, or other resinous substances which close off the respective areas (4.17). In addition, antimicrobial agents are produced such as lysozyme and chitinase, lytic enzymes stored in the vacuole which can degrade bacterial and fungal cell walls, protease inhibitors which can inhibit microbial proteases, or secondary metabolites with antimicrobial activity. 

Miso and natto are traditional Japanese foods made from soybeans by a fermentation process. These foods are produced by a mixed fermentation process using a characteristic microorganism and ripening for a given time. In these processes, the hydrolysis of proteins by microbial proteases results in the production of free... 

In addition, Z-Asp(OAl)-OAl is converted into Z-Asp(OAl)-OH in quantitative yield by papain.t l Some Z-protected Glu and Asp diesters are regioselectively hydrolyzed by several microbial proteases, yielding the co-protected derivatives. 

The ulcer may be sterile. Stromal breakdown and progressive ulceration is largely driven by matrix metalloproteinases (MMPs) and serine proteases derived from local comeal cells and from leukocytes sequestered in the cornea in response to the initial injury (Fig. 13.1). Inoculation and replication of pathogenic bacteria at the site of minor epithelial injury releases exotoxins and microbial proteases that potentiate the initial processes of comeal breakdown and amplify endogenous stromal hydrolysis. However, once started, an ulcer can evolve its own biochemical momentum based... 

The first two enzymes are well documented to occur extracellularly. We have studied urease and have also found evidence that this enzyme is on the cell surface of at least one phytoplankton species. Possibly with the exception of the proteases, these enzymes contain essential metal cofactors which are necessary for their activity. There are reports, however, which document the inactivation of microbial proteases by chelating agents, suggesting that they too are metal-loenzymes (Matsubara and Feder, 1971). 

Kumar, D., and Bhalla, T.C. (2005) Microbial proteases in peptide synthesis approaches and applications. Appl. Microbiol. Biotechnol., 68,726-736. 

In a directed evolution approach pursued in our laboratories, the sequence specificity of a microbial protease was systematically altered away from its natural substrate to match the activation site of plasminogen... 

In addition to studies on enzymic solubilization of solvent-extracted FPC, the relative effectiveness of more than 20 commercially available proteolytic enzymes in the production of hydrolysates from a specially prepared haddock protein substrate has been determined (13). The concentration of enzyme, at its optimum temperature and pH, required to effect a 60% digestion in a 24-hr period was the inverse measure of enzyme activity. Pronase exhibited the greatest activity per unit weight. In general the microbial proteases ranked low in relative activity. Porcine pepsin, papain, and pancreatin combined good activity with moderate cost. 

Although the general proteolytic activities of various milk clotting enzymes may vary, their milk clotting activities are apparently predicated on the same specific cleavage of the Phe-Met bond in -casein. Apparently rennin, pepsin, chymotrypsin, a microbial protease, proteases from Endo-thia parasitica, Mucor pusillus, and Mucor miehei exert the same type of activity on -casein (2, 169). Enzymes that are currently used commercially for cheesemaking in the United States include rennin, rennin-pepsin mixtures, and microbial proteases from Endothia parasitica, Mucor pusiUus, and Mucor miehei. 

Use of Immobilized Proteases to Clot Milk on a Continuous Basis. Immobilized proteases might be valuable in continuous coagulation of milk for cheese manufacture. Since the immobilized enzyme would not remain in the product, it may be possible to substitute a less expensive, less desirable, but more readily available enzyme which normally cannot be used, such as undesirable microbial proteases, instead of commercially available milk-clotting enzymes. 

Proteases are enzymes that break down protein molecules through peptide bond hydrolysis [1]. They are commercially employed in many industrial processes. In foods, proteases have two main applications in the processing of traditional food products and in the processing of new protein-based ingredients called functional foods [2]. Proteases are also used in other industrial segments such as leather industry, pharmaceutical, waste management, and the detergent industry. Currently, microbial proteases make up approximately 40% of total enzyme sales [3, 4]. 

Pepsin derived from the mucosa of hogs has found use in chillproofing beer and as a digestive aid. Microbial proteases (and papain) can replace pepsin in chillproofing, but there are no commercially available microbial proteases which show the low pH optimum (1.8-2.2) exhibited by pepsin. 

Rennet is used extensively in the production of cheese. This enzyme is found in the fourth stomach of the calf it converts casein into paracasein, which, in the presence of calcium, precipitates to form an elastic curd. Much commercial rennet is contaminated with pepsin which tends to act strongly on casein and thereby produces a weak curd with off flavor. Many microbial proteases can clot milk, but only recently have microbial preparations been produced which can replace calf rennet to prepare cheeses of good flavor. Microbial preparations from Endothia parasitica and Mucor pusillus var. Lindt seem to be the most promising, and these have been used commercially. The new sources of rennet were reviewed recently (Sardinas, 1969). 

Kumar D, BhaUa T (2005) Microbial proteases in peptide synthesis approaches and appUcations. 

Maeda H. Role of microbial proteases in pathogenesis. Microbiol Immunol 1996 40 685-699. 

J. et al (2013) Recent patents on microbial proteases for the dairy industry. Recent Adv. DNA Gene Sequences, 8, 44 - 55. 

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