Subtilisin enzyme catalyst

High yields and S -O-regioselectivity can be obtained in the acylation of libonucleosides using subtilisin as catalyst if pyridine is used as solvenL When the acetoxime esters of Cbz-glycine and Cbz- -alanine were used as acyl donors, Candida antarcHca lipase catalysed the formation of S -O-aminoacyl derivatives of ribo- and 2 -deoxyiibonocleosides, whilst use of Pseudomonas cepacia lipase gave reaction at 0-3 in 2 -deoxyribonucleosides (the same selectivities as observed in previous work see Vol. 27, p. 266). The enzymes would not accq>t donaminoacid derivatives could not be used. ... 

The KR of secondary alcohols by some hydrolytic enzymes has been well known. The combinations of these hydrolytic enzymes with racemization catalysts have been explored as the catalysts for the efficient DKR of the secondary alcohols. Up to now, lipase and subtilisin have been employed, respectively, as the R- and 5-selective resolution enzymes in combination with metal catalysts (Scheme 2). 

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... 

DKR of secondary alcohol is achieved by coupling enzyme-catalyzed resolution with metal-catalyzed racemization. For efficient DKR, these catalyhc reactions must be compatible with each other. In the case of DKR of secondary alcohol with the lipase-ruthenium combinahon, the use of a proper acyl donor (required for enzymatic reaction) is parhcularly crucial because metal catalyst can react with the acyl donor or its deacylated form. Popular vinyl acetate is incompatible with all the ruthenium complexes, while isopropenyl acetate can be used with most monomeric ruthenium complexes. p-Chlorophenyl acetate (PCPA) is the best acyl donor for use with dimeric ruthenium complex 1. On the other hand, reaction temperature is another crucial factor. Many enzymes lose their activities at elevated temperatures. Thus, the racemizahon catalyst should show good catalytic efficiency at room temperature to be combined with these enzymes. One representative example is subtilisin. This enzyme rapidly loses catalytic activities at elevated temperatures and gradually even at ambient temperature. It therefore is compatible with the racemization catalysts 6-9, showing good activities at ambient temperature. In case the racemization catalyst requires an elevated temperature, CALB is the best counterpart. 

Figure 3.8 (a), (b) Initial rates of subtilisin Carlsberg-catalyzed transesterification of N-Ac-L-Phe-OEt with n-PrOH in hexane as a function of active enzyme in different salt-enzyme preparations. Catalyst composition (a) 98% (w/w) KCl, 1% (w/w) SC, and 1% phosphate buffer (average of three preparations), (b) 99% (w/w) subtilisin and 1% phosphate buffer (average of five preparations) [99]. 

Besides these rather complex coenzyme-dependent enzymes, the none-coenzyme requiring protease subtilisin is the most extensively mutated enzyme. The substrate specificity of the enzyme as well as its dependence on pH and its stability were altered by site-directed mutagenesis [72-78]. As the knowledge about exact details of the structure and active site of the enzyme is essential for the application of this method, progress in this field is difficult to achieve. Site-directed mutagenesis as a means of catalyst improvements will be used only after extensive application of conventional optimization procedures. 

Little was done in the area of cross-linked enzyme crystals over the next 10 years. In 1977, the kinetic properties of CLCs of the protease subtilisin were reported by Tuchsen and Ottesen [3], They reported that cross-linked enzyme crystals of subtilisin were highly effective catalysts with increased thermal stability and increased stability toward acid compared to the soluble enzyme. They further reported that the CLCs of subtilisin showed essentially no autodigestion at 30°C. Like Quiocho and Richards before them, Tuchsen and Ottesen found... 

A very straightforward approach in route C (Fig. 2) would have been the direct enzyme-catalyzed peptide formation (cf. Chen et al. [18]) by enantioselective aminolysis of diester 9 with (S)-tert-leucine methylamide 13 or even racemic 13. This would combine three synthetic objectives the resolution of (rac)-9, the resolution of (roc)-13 and the coupling step. In orientating experiments monoester 10 was tested as a model substrate. It was contacted with an equal amount of (S)-amine 13 in the presence and absence of an organic solvent. Solid or liquid subtilisin Carlsberg preparations (Alcalase 2.0 T or Alcalase 2.5 L, respectively) were used as the catalyst. Only with the liquid enzyme preparation was the formation of minor amounts of one of two possible diastereoisomeric peptides observed [19], whereas most of the ester was hydrolyzed to the acid. Likewise, a few selected lipases also provided negative results. 

Bakker et al. [25] replaced the zinc in thermolysin with anions snch as molybdate, selenate, or tungstate to give an enzyme that catalyzed the nonenantioselective oxidation of thioanisoles with hydrogen peroxide. Van de Velde et al. [26] added a vanadate ion to the active site of phytase to create a catalyst for enantioselective oxididation of thioanisole to the sulfoxide in 66% ee. Selenosubtihsin (protease subtilisin where a selenoserine replaces the active-site serine) catalyzes the enantioselective reduction of hydroperoxides [27] with enantioselectivity >100 for one substrate. 

Enzymes are highly specific catalysts. The nature of this specificity is believed to result from structural and electrostatic complementarity between the enzyme and its substrate. The serine protease, subtilisin, is being extensively studied as a model system to explore the effects of single amino acid substitutions on its structure and function Q). The gene for Bacillus amyloliquefaciens subtilisin has been expressed and secreted in B. subtilis 12).A site-directed mutagenesis scheme, cassette mutagenesis ( ), has been used to produce a series of subtilisin variants that are more resistant to oxidants (4), and have altered stability ( ), specificity, and specific activity. 

Goto et al. 1998). None of them probably reflects properly the enzyme activity over the real substrate, so it will be a matter of judgment and experience to select the most pertinent assay with respect to the actual use of the enzyme. Hydrolases are currently assayed with respect to their hydrolytic activities however, the increasing use of hydrolases to perform reactions of synthesis in non-aqueous media make this type of assay not quite adequate to evaluate the synthetic potential of such enzymes. For instance, the protease subtilisin has been used as a catalyst for a trans-esterification reaction that produces thiophenol as one of the products (Han et al. 2004) in this case, a method based on a reaction leading to a fluorescent adduct of thiophenol is a good system to assess the transesterification potential of such proteases and is to be preferred to a conventional protease assay based on the hydrolysis of a protein (Gupta et al. 1999 Priolo et al. 2000) or a model peptide (Klein et al. 1989). 

Among many other peptide splitting enzymes such as (bacterial) subtilisin and thermolysin, (vegetable) papain, ficin and bromelain, (mammalian) cathepsin and others, the yeast enzyme carboxypeptidase Y finally deserves special mention. The enzyme is an exopeptidase, like carboxypeptidase A i.e. it catalyzes, rather unspecifically, the hydrolytic fission of the carboxy-terminal a-amino acids from a peptide chain. J.T. Johansen and his associates at the Carlsberg laboratory in Copenhagen showed about 10 years ago that CPD-Y is an effective catalyst of peptide bond synthesis [36]. 

In order to obtain (5 )-selective DKR of secondary alcohols, an enzyme with a complementary (5 )-stereoselectivity was needed, since the lipase-catalysed DKR provides only (R)-products. In this context, Park et al. reported, in 2003, the use of subtilisin instead of lipase, but the commercial form of subtilisin was not applicable to DKR, due to its low enzymatic activity and instability in non-aqueous medium. However, these authors succeeded in enhancing its activity and stability by treating it with a surfactant before use. In these conditions, the combination of subtilisin with an analogue of Backvall s catalyst and trifluoroethyl butanoate as the acylating agent... 

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