Subtilisin detergent enzyme

The oxidative stability of subtilisin has been extensively studied and improved stability has been engineered. In subtilisin BPN two methionines, MET " and MET are especially susceptible to oxidation. To prevent the negative influenee eaused by the formation of methioiune sulfoxide the MET can be substituted with ALA, SER or LEU, without loosing more than 12-53% of the activity. One such mutant MET222. ALA is currently in use as a commercial detergent enzyme Durazyme (Riisgard, 1990). 

In 1989, two enzymes based on genetic engineering techniques were introduced, ie, a cloned alkaline protease (IBIS) and a protein engineered Subtilisin Novo (Genencor, California). Lipase and cellulase types of detergent enzymes have also begun to appear. 

Extracellular proteases are of commercial value and find multiple applications in various industrial sectors. A good number of bacterial alkaline proteases are commercially available, such as Subtilisin Carlsberg, subtilisin BPN and Savinase, with their major application as detergent enzymes. 

World War II spurred much advancement in the production of penicillin, particularly the advent of submerged fermentation processes. This new technique was soon adapted to the large-scale production of bacterial proteases. The first modern detergent protease, isolated from Bacillus licheniformis, was introduced in 1962. This enzyme, a particular type of protease called a subtilase, was stable at higher temperatures, had broad substrate specificity and worked well in alkaline conditions. The appearance of this enzyme and others similar to it (i.e., subtilisins) opened up the detergent enzymes market, and by 1969, 50% of the laundry detergent products sold in the United States and Europe contained enzymes. ... 

Protease is by far the most widely used of all detergent enzymes. Introduced in the 1960s, it has since become one of the more important components of detergent formulations [6]. Proteases aid in the removal of many soils commonly encountered by the consumer, such as food stains (cocoa, egg yolk, meat), blood, and grass. This enzyme hydrolyzes or breaks up the peptide bonds found in proteins resulting in the formation of smaller and more soluble polypeptides and amino acids. Since most enzymes have to function under high pH conditions, subtilisin, a bacterial alkaline protease, is commonly used in laundry detergents. This particular protease does not hydrolyze any specific peptide bond in proteinaceous stains but cleaves bonds in a somewhat random manner. 

Synonyms Alcalase Bacillus subtilis Carisberg Detergent enzyme Maxalase Protease, bacterial, alkaline Proteinase Proteolytic enzymes Subtilisin Sublili-sin Carisberg Subtilisins ClassiTication Enzyme... 

Subtilisins are a group of serine proteinases that are produced by different species of bacilli. These enzymes are of considerable commercial interest because they are added to the detergents in washing powder to facilitate removal of proteinaceous stains. Numerous attempts have therefore recently been made to change by protein engineering such properties of the subtilisin molecule as its thermal stability, pH optimum, and specificity. In fact, in 1988 subtilisin mutants were the subject of the first US patent granted for an engineered protein. 

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. 

Unlike many other enzymes, the subtilisins are fairly stable towards e.g. organic solvents, anionic surfactants, high temperatures and high pH. This makes the subtilisins very suitable as detergent proteases. But despite this fact, stabilization of these protease enzymes in liquid detergents remains a major issue. 

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). 

In order to prove enzyme engineering feasibility, it was important to develop a model system. One of the prime considerations for any model would be the commercial potential of the model. Table I lists the major commercial enzymes and the market size in US dollars (5). The alkaline proteases (subtilisins) are clearly the major single class of enzymes in commercial use today, representing 25% of the total enzyme market of 600 million. The primary use of subtilisins is as additives in laundry detergents to aid in the removal of proteinaceous stains from cloth. 

MW 27,500) with no cofactors or metal ions reqnirement for its function, it displays Michaelis-Menten kinetics and it is secreted in large amounts by a wide variety of Bacillus species. Subtilisin is also among the most important industrial enzymes due to its use in laundry detergents. Protein engineering strategies for subtilisin have focused on a number of aspects, namely catalysis, substrate specificity, thermal and oxidative stability and pH profile. We will describe briefly each of these aspects. 

Narhi et al. (1991) recently reported an enhancement in the thermal stability of aprA-subtilisin by three point mutations. The mutations were ASNi. SER and ASN. SER to prevent cyclisation with the adjacent glycines and ASN . ASP in the Ca binding loop. The mutant form also exhibits improved stability to detergent denaturation with little dependence on calcium concentration. Subtilisin 8350 (derived from subtilisin BPN via six site-specific mutations) was found to be 100 times more stable than the wild type enzyme in aqueous solution and 50 times more stable than the wild type in anhydrous dimethylformamide (Wong et al, 1990)... 

An example of this kind of work involves the enzyme subtilisin, frequently used as an additive in laundry detergents because it attacks the proteins that soil clothing. The problem, however, is that subtilisin is easily destroyed by bleaches with which a detergent is often used. Research showed that subtilisin is sensitive to bleach because a single amino acid in its primary structure—a methionine at position 22—is destroyed by bleach. By replacing this methionine with an amino acid that is not sensitive to attack by bleach, then, researchers were able to synthesize a new form of subtilisin that did not degrade in the presence of bleach for use in laundry detergents. 

Numerous methods have been used to successfully identify new enzymes for detergent applications. Historically, the method of choice has been to screen for new enzymes from naturally occurring microbes. The most commonly used protease, subtilisin, was initially isolated in this manner. More recently, researchers have resorted to exploring extreme environments, such as hot springs, to more closely match... 

Last but not least, it should be mentioned that a couple of peptidases have industrial importance. In particular, since subtilisins have a broad substrate specificity and are highly stable at neutral and alkaline pH they are of considerable industrial interest as protein-degrading additives to detergents. These reasons combined with their large data base make subtilisins attractive for protein engineering. Extensive engineering studies have been carried out on the Bacillus subtilins and more than 500 site-directed mutants have been produced to alter specific enzyme properties, such as pH profile, thermal stability or substrate specificity (see e. g. references[37 391). 

ISynonyms/Trade Names Bacillus subtilis, Bacillus subtilis BPN, Bacillus subtilis Carlsburg, Proteolytic enzymes, I Subtilisin BPN, Subtilisin Carlsburg [Note Commercial proteolytic enzymes are used in laundry detergents.] ... 

Alcalase is mainly used as additive in detergents for the degradation of proteinogenic impurities, its major enzyme component is subtilisin Carlsberg (alkaline protease A). 

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