Proteases in detergents

Industrial enzymes must be tested and formulated for the maximum degree of safety in handling. As enzymes are proteins they may cause allergic reactions in susceptible people. For instance, early attempts to use proteases in detergents foundered because... 

Examples of Le.used commercially are proteases (in detergents, and for making cheese and other food products), carbohydrases (for hydrolysing starch, con-... 

The food, beverage, detergent, textile, leather, and paper industries are by far the major commercial users of enzymes. In some cases, enzymes appear in the final products, for example, proteases in detergents, (3-galactosidase as a digestive aid to mitigate lactose intolerance or as animal feed components. In other... 

Bulk Enzymes. Enzymes such as proteases, amylases, glucose isomerases, and rennin are used in food processing. Similarly proteases and Hpases are used in detergents. CeUulases and xylanases are used in the paper pulp industry. The genes for most of the enzymes used in the various commercial processes have been cloned and overexpressed. Rennin (chymosin) produced from E. coli and A. nigerhas been approved by FDA for use in the dairy industry. 

Many enzymes have been the subject of protein engineering studies, including several that are important in medicine and industry, eg, lysozyme, trypsin, and cytochrome P450. SubtiHsin, a bacterial serine protease used in detergents, foods, and the manufacture of leather goods, has been particularly well studied (68). This emphasis is in part owing to the wealth of stmctural and mechanistic information that is available for this enzyme. 

AH detergent proteases are destabilized by linear alkylbenzenesulfonate (LAS), the most common type of anionic surfactant in detergents. The higher the LAS concentration and wash temperature, the greater the inactivation of the enzyme. The presence of nonionic surfactants, however, counteracts the negative effect of LAS. Almost aH detergents contain some nonionic surfactant therefore, the stabHity of proteases in a washing context is not problematic. 

Pig. 7. Storage stabHity of proteases in European powder detergent with activated bleach system. ( ) Traditional protease (--) protein-engineered... 

Proteases are hydrolytic enzymes with important application in industries, in particular, in detergent and in the food industry. A metagenomic study in which 100 000 plasmid clones were screened for proteolytic activity found one positive done, which was determined to be novel by sequencing analysis [84]. 

The first choice of enzyme to add to a detergent is practically always a protease. The proteases in modem detergents are subtilisins which are microbial enzymes from Bacillus. The subtilisins consist of approximately 270 amino acids and are heart-shaped molecules with a binding cleft and a binding pocket to which substrates such as protein stains can be bound by non-covalent forces. 

Figure 3 Typical efficiency of 4-FPBA in liquid detergent containing lipase and protease. In this case the efficiency is around 100 times that of boric acid...

A critical consideration in the development of biocatalytic systems is the form in which the enzyme or enzyme system is going to be used. There are two general approaches. One is to use isolated enzymes. If these are inexpensive, they can be used as disposable biocatalysts, as is the case for glucose isomerase, ° which is the key biocatalyst in the production of high-fructose corn syrups from starch, or the lipases and proteases that are present in detergents. Alternatively, if enzymes are expensive to produce, they can be immobilized and used repeatedly by recovering the enzyme particles after each use. 

A practical enzymatic procedure using alcalase as biocatalyst has been developed for the synthesis of hydrophilic peptides.Alcalase is an industrial alkaline protease from Bacillus licheniformis produced by Novozymes that has been used as a detergent and for silk degumming. The major enzyme component of alcalase is the serine protease subtilisin Carlsberg, which is one of the fully characterized bacterial proteases. Alcalase has better stability and activity in polar organic solvents, such as alcohols, acetonitrile, dimethylformamide, etc., than other proteases. In addition, alcalase has wide specificity and both l- and o-amino acids that are accepted as nucleophiles at the p-1 subsite. Therefore, alcalase is a suitable biocatalyst to catalyse peptide bond formation in organic solvents under kinetic control without any racemization of the amino acids (Scheme 5.1). 

BPN is a 275 amino acid protease with a serine in the active site. Since it is functional in an alkaline environment it has potential use in detergent applications. Our program was to change specific characteristics of BPN to make it more effective in certain applications. Two main activities were targeted pH range and oxidative stability (since bleaches are often components of detergents). 

Since the mid-60s, the use of enzymes in detergents has been the largest of all enzyme applications. Over half of all detergents presently available contain enzymes, in particular proteases, amylases, lipases and ceUulases. Besides improved washing efficiency, the use of enzymes allows lower temperatures and shorter wash periods (of agitation) to be employed, often after a preliminary period of soaking. Further in this chapter (section 3.3) the detergent enzymes are worked out in more detail. 

In many applications several quite different technical criteria must be successfully fulfilled before the product will work , and even then cost and scale-up and commercial criteria must be met. A good example is detergent enzymes where scientists had to discover proteases that would be active and stable under conditions of high pH and temperature and in the presence of oxidising and surface active agents. The same criteria exists for lipases for use in detergents. However, suitable lipases proved rather more difficult to find than the B. subtilis proteases that are used... 

Mukherjee, A. K., Adhikari, H., Rai, S. K. (2008). Rroduction of alkaline protease by a thermophilic Bacillus subtilis under solid-state fermentation (SSF) condition using Imperata cylindrical grass and potato peel as low-cost medium Characterization and application of enzyme in detergent formulation. Biochem. Engg. J.,39, 353-361. 

Genencor International, Inc. received a patent for a process to make bleach-resistant protease enzymes to use in detergents. 

Protease is used in detergents to remove protein-based stains such as blood, mucus, feces, and various foods such as egg and gravy. These substances are almost insoluble and they tend to adhere to textiles and other surfaces. The protease hydrolysis products are peptides which are readily dissolved or dispersed in the washing liquor. 

All proteolytic enzymes described are fairly non-specific serine endoproteases, cleaving peptide chains preferentially at the carboxyl side of hydrophobic amino acid residues. The enzymes convert their substrates into small, readily soluble fragments which can be removed easily from fabrics. Only serine protease can be used in detergent formulations, as thiol proteases such as papain would be oxidized by the bleaching agents, acidic proteases are not active at common laundry conditions, and metalloproteases such as thermolysin would lose their metal cofactors because of complexation with the water-softening agents or hydroxyl ions. 

The selective enzyme-catalyzed acylation of carbohydrates is of great interest, as of carbohydrates fatty acid esters of carbohydrates have important applications in detergents, cosmetics, foodstuff, and pharmaceuticals because of their surface-active properties. Monoacylated sugars have been synthesized by lipase-catalyzed transesterifications of activated esters in pyridine and by protease-catalyzed esterifications in DMF. A most remarkable new development... 

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. 

The alkaline serine protease of Bacillus licheniformis, also known as Subtilisin Carlsberg, is the preferred protease in most nonionic and anionic detergents. It attacks many peptide bonds and easily dissolves proteins. It may be used at temperatures up to 65°C, and its pH optimum is close to 9.0, the pH normally used in washing fluids. 

A few other proteases are also used in detergents. The serine protease of Bacillus amyloliq-uefaciens has found applications, presumably because of its substantial content of a-amylase, which may be advantageous for these applications. 

Enzymes. A number of different enzymes have been developed for use in detergent systems to remove common soils and stains. Protease enzymes are used to cleave the protein-based soils into smaller pieces more easily removed by the detergent system. Lipase enzymes are used to cleave lipid bonds resulting in fatty acid and glycerol. Lipase enzymes work better at elevated temperature and tend to become active in the... 

So far, one member of the S2P family has been purified to homogeneity with preservation of proteolytic activity (17). The purification of this protease, the E. coli YaeL/RseP, should allow a more rigorous and direct determination of substrate sequence requirements. Moreover, the ability to purify an I-CLiP and reconstitute activity (in appropriate detergent and usually in the presence of added lipids) is an important step toward studying these unusual membrane-embedded proteases in terms... 

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