Subtilisins commercial

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 lipase-catalyzed DKRs provide only (/ )-products to obtain (5 )-products, we needed a complementary (5 )-stereoselective enzyme. A survey of (5 )-selective enzymes compatible to use in DKR at room temperature revealed that subtilisin is a worthy candidate, but its commercial form was not applicable to DKR due to its low enzyme activity and instability. However, we succeeded in enhancing its activity by treating it with a surfactant before use. At room temperature DKR with subtilisin and ruthenium catalyst 5, trifluoroethyl butanoate was employed as an acylating agent and the (5 )-products were obtained in good yields and high optical purities (Table 3)P... 

DNA polymerase I has been purified to homogeneity. When the pure enzyme is treated with subtilisin, a proteolytic enzyme from Bacillus subtilis, the polymerase is cleaved into two pieces. The small fragment retains the 5 to 3 nuclease activity, whereas the larger piece, called a Klenow fragment, has both polymerase activity and the 3 to 5 exonuclease activity. The Klenow fragment is sold commercially for use in labeling DNA for use in detecting recombinant DNA. 

H) Resolution of amino acid esters with subtilisin The commercial Prt Alcalase from... 

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. 

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 contrast to the R-preference displayed by CALB, quite impressive S-preference toward several 1-aryl-l-ethanols can be achieved by using a protease, the commercially available subtilisin Carlsberg, as catalyst, and isopropenyl pen-tanoate as the acyl donor in THF in the presence of sodium carbonate [74]. To achieve a successful resolution, the protease has to be treated with a mixture of two surfactants, octyl-P-D-glycopyranoside and Brij 56 [the monocetyl ether of polyoxyethylene (10)], 4/1/1 by weight, at pH 7.2, and then lyophilized before use. 

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. 

An example is described for the UAA ( Wert-leucine (44) (Scheme 19.24).207 It uses the commercially available Lipozyme (Mucor miehei) from Novozymes to hydrolyze the racemic 2-phenyl-4-tm-butyl-oxazolin-5-(4/f)-one (45) to the (S)-./V-benzoyl-tert-leucine butyl ester (46) followed by Alcalase (subtilisin, Bacillus licheniformis from Novozymes) treatment to hydrolyze the butyl ester, which on debenzoylation yields (S)-tert-leucine (44) with an ee of 99.5% and a yield of 74%. 

The enzymes used were Alcalase(R) 0.6 L, a liquid, food-grade preparation of subtilisin Carlsberg, and Neutrase(R) 0.5 L, a liquid, food-grade preparation of a B. subtilis neutral protease. Both enzymes are commercially available from Novo Industri A/S (7 ). All other reagents were analytical grade laboratory chemicals. ... 

The first step in setting up a successful directed evolution protocol is the development of an efficient expression system using an appropriate bacterial host. This is not a trivial task, in particular when overexpression is to be coupled to enzyme secretion. Fortunately, some proteins can easily be overexpressed and secreted by using commercially available systems [27 - 29], a prominent example being subtilisin of Bacillus subtilis [30]. However, many enzymes of interest are not amenable to such systems examples include a variety of different lipases from Pseudomonas species. 

Commercial bacitracin comprises more than 30 different substances, but the major antibiotic isoforms A and B account for about 60% of the mixture. An impurity has been identified in some but not all bacitracin lots (13). The impurity is a powerful subtilisin-type protease capable of cleaving many proteins, including protein disulfide isomerase, myosin, and a variety of artificial substrates. Investigators using bacitracin are therefore... 

Subtilisin Carlsberg exhibited virtually absolute specificity ( >200 [35]) for the (S)-ester that corresponds to the L-form of a natural amino acid and provided the desired acid (S)-2 in high enantiomeric and chemical purity. Various commercial enzyme preparations from Bacillus lichmiformis were available the cheaper solid preparations Optimase M 440 from MKC [32] and Alcalase 2.0 T from Novo Nor-disk [33] as well as the more expensive liquid forms Alcalase 2.5 L and Optimase L 660. 

A big factor for the success of the process was the close similarity of the substrate to the natural substrates of Subtilisin Carlsberg, an enzyme with extraordinary properties. Since cheap commercial sources for this enzyme were available on the market, the enzyme could be discarded. A major disadvantage of the present chemoenzymatic route was the fact that no satisfactory racemization procedure for the unwanted enantiomer (R)-2 could be established due to elimination and/or hydrolysis side reactions. 

An efficient enzyme-catalyzed reaction for the large-scale preparation of (R)-2-iso-butyl succinic acid 4-ethyl ester (R)-2a, a key intermediate in the synthesis of collagenase inhibitor R00319790 (1), is described (Fig. 1). The corresponding racemic diethyl ester substrate 9 is applied at 20% concentration and hydrolyzed re-gio- and enantioselectively ( >100) at the sterically more hindered secondary ester group using a cheap commercial subtilisin preparation. The desired (R)-monoacid is separated from the remaining antipodal diester (S)-9 by means of extraction and obtained in > 99% ee and > 43% yield. The development of the reaction from process research to the pilot-scale is described. 

Several types of enzymes have found uses in LADD compositions [4,48], Most common are proteases, amylases, and lipases, which attack proteinaceous, starchy, and fatty soils, respectively. Proteases work by hydrolyzing peptide bonds in proteins. Proteases differ in their specificity toward peptide bonds. The typical protease used in LADD formulations, bacterial alkaline protease (subtilisin), is very nonspecific. That is, it will attack all types of peptide bonds in proteins. In contrast to proteases, amylases catalyze the hydrolysis of starch. They attack the internal ether bonds between glucose units, yielding shorter, water-soluble chains called dextrins. Lipases work by hydrolyzing the ester bonds in fats and oils. Often, combinations are used because of the specificity of each kind to one type of soil. The commercially available enzymes are listed in Table 9.6. 

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.] ... 

Proteases can also catalyse the esterification of carbohydrates. Thus 1 -O-butyl sucrose was prepared Irom sucrose and trichloroethyl butyrate with subtilisin in anhydrous DMF [14]. This strategy has heen extended to commercially available insoluble crosslinked forms of subtilisin to prepare regiospecifically I -O-acylsucroses from fatty acids and vinyl esters in high yields [15]. 

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