Proteases subtilisin Carlsberg

For single-tryptophan proteins there is some correlation between blue-shifted fluorescence emission maximum and phosphorescence lifetime (Table 3.2). Another correlation is that three of the proteins which exhibit phosphorescence, azurin, protease (subtilisin Carlsberg), and ribonuclease Tlt are reported to show resolved fluorescence emission at 77 K. Both blue-shifted emission spectra and resolved spectra are characteristic of indole in a hydrocarbon-like matrix. 

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). 

Khmelnitsky et al. were the first to observe the activating effects salt showed on enzymes in the nonaqueous environment [88]. As shown in Figure 3.7, the transesterification activity of the serine protease subtilisin Carlsberg in anhydrous solvents is strongly dependent on the KC1 content in a lyophilized enzyme preparation and increases sharply as the salt content is increased. This increase in activity was determined to be a result primarily of an increase in kcat and not a decrease in Km, as shown in (Table 3.4). 

An illustrative example of a change in chemoselectivity is the inversion of substrate specificity of the serine protease Subtilisin Carlsberg in the transesterification reaction of ethyl esters of A-acetyl-L-serine and A-acetyl-L-phenylalanine with 1-propanol, measured in twenty anhydrous organic solvents. The enzyme-catalysed reaction with the serine substrate is strongly favoured in dichloromethane, while the reaction with the phenylalanine substrate is preferred in t-butylamine, with a 68-fold change in substrate specificity [313]. 

Modification of Ultrafiltered versus Acid Precipitated Soy Protein. When the retentate obtained from the ultrafiltration of soybean extract is subjected to an enzymatic hydrolysis as described earlier (2) for acid precipitated protein, a hydrolysis curve (DH versus time) may be drawn. A comparison of such hydrolysis curves is shown in Fig. 2 for acid precipitated soy protein isolate and ultrafiltered soy protein isolate. The curves are drawn on the basis of the same hydrolysis parameters. The enzyme used is the microbial alkaline protease subtilisin Carlsberg (ALCALASE ). 

Kinetic Resolution of Diester by Protease Subtilisin Carlsberg from Bacillus sp. (E.C. 3.4.21.62)[55 561... 

Protease, Subtilisin Carlsberg. Origin Bacillus licheniformis... 

Kinetic Resolution of Diester by Protease Subtilisin Carlsberg from... 

The use of an immobilized protease for transesterification reactions and the procedure for the immobilization have been described by FMC Corporation [121]. The company applied the protease subtilisin Carlsberg embedded into a dehydrated hydrocolloid polymer gel consisting of kappa-carageenan for the... 

The company applied the protease subtilisin Carlsberg embedded into a dehydrated hydrocolloid polymer gel consisting of kappa-carageenan for the... 

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. 

Naturally occurring Upases are (R)-selective for alcohols according to Kazlauskas rule [58, 59]. Thus, DKR of alcohols employing lipases can only be used to transform the racemic alcohol into the (R)-acetate. Serine proteases, a sub-class of hydrolases, are known to catalyze transesterifications similar to those catalyzed by lipases, but, interestingly, often with reversed enantioselectivity. Proteases are less thermostable enzymes, and for this reason only metal complexes that racemize secondary alcohols at ambient temperature can be employed for efficient (S)-selective DKR of sec-alcohols. Ruthenium complexes 2 and 3 have been combined with subtilisin Carlsberg, affording a method for the synthesis of... 

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. 

Immobilization in a sol-gel matrix accelerated the propanolysis of N-acetyl-i-phenylalanine ethyl ester in cyclohexane for several serine proteases compared to the non-immobilized lyophilized enzymes 31-fold for Subtilisin Carlsberg, 43-fold for a-chymotrypsin, and 437-fold for trypsin (van Unen, 2001). The activity yield upon immobilization was 90% (a-chymotrypsin). The rate enhancement effect of immobilization on the enzyme activity is highest in hydrophobic solvents. 

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. 

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. ... 

For the two proteases trypsin and subtilisin Carlsberg (Table 2, Entries 6 and 7), only the latter showed some activity under these conditions, also with some selectivity for the longer acyl chains similar to HLE. (3-Galactosidase ((3-Gal, Table 2, Entry 5), did not, however, show any activity, and control experiments with bovine serum albumin (BSA, Table 2, Entry 8) resulted in no hydrolysis products. 

A similar resolution has also been achieved on large scale <20040PD22>. The KR of racemic isoxazoline 312 catalyzed by enzymes was studied. The best result was obtained with lipase B from Candida antarctica (CALB), which hydrolyzed the ethyl ester of (—)-312 to the corresponding monoacid (—)-313. The reaction, which was run in 0.1 M phosphate buffer/acetone at room temperature, spontaneously stopped at 50% conversion to yield monoacid (—)-313 and the residual ester (- -)-312 with ees higher than 99% <2004TA3079>. The C-5 epimer of 312 underwent enantioselective hydrolysis (>99% ee) of the methyl ester linked to C-5 in the presence of the protease proleather (subtilisin Carlsberg), whereas CALB and other lipases were not able to resolve it (Equation 53). 

Subtilisin Carlsberg serine protease and protease inhibitor phenylmethylsulphonyl fluoride (PMSF) are from Sigma (St. Louis, MO). 

Catalytic activities of proteases like a-chymotrypsin [21, 22] and subtilisin Carlsberg [23] have been also studied in ILs. Laszlo and Compton [24] examined the transesteriflcation reaction of A-acetyl-L-phenylalanine ethyl ester with 1-propanol catalysed by a-chymotrypsin in ionic liquids ([bmim ][PF ] and [omim ][PF "]) and organic solvents (isooctane, acetonitrile and n-hexane). They found that the transesteriflcation rates using a-chymotrypsin freezedried with KjHPO in ionic liquids, at 1% water content, were comparable with that obtained in organic solvents. The protease subtilisin in free form did not show any significant transesteriflcation activity in ILs [25]. However, modified subtilisin was able to catalyse the transesteriflcation of iV-acetyl-L-phenylalanine ethyl ester in [emim ][NTfj ], [bmim ][PF ] and [bmim ] [BF,-] [23,25]. 

It appears from Fig. 2 that the ultrafiltered soy protein isolate is hydrolyzed considerably more slowly than the acid precipitated protein. This is due to the compact molecular structure of the ultrafiltered protein, which is still in the native state. That the degree of denaturation of a protein substrate has a profound influence on the kinetics of the proteolysis has been known for long, see Christensen W. It should be noted that subtilisin Carlsberg is not inhibited by the protease inhibitors present in native bean protein (7 ). 

As Subtilisin Carlsberg was, and still is, one of the cheapest enzymes available on the market, no further proteases were tested. Four Subtilisin Carlsberg preparations from Solvay Enzymes [9] and Novo Nordisk [10] were tested successfully Solvay-Protease M 440 (solid form) and L 660 (liquid form) as well as the Novo enzymes Alcalase 2.0 T (solid) and Alcalase 2.5 L (liquid). 

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