Subtilisin properties

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. 

Thermolysin acts simultaneously at several sites on the Ca -ATPase without accumulation of large fragments this property proved useful in the sequence analysis of the Ca -ATPase [78,79,82,83], and in the isolation of SH-group-containing peptides [257]. Small fragments also accumulate after treatment of sarcoplasmic reticulum with subtilisin [256]. 

Initially, the sol gel compositions were optimized using Congo red dye as the dopant because of its optical properties. This facilitates monitoring of the release process by optical spectroscopy. Next, the gels were evaluated for their stabilization and release of subtilisin. These sol gel matrices bring about controlled release of the encapsulated enzyme molecules as a response to a change in the water content of the medium (Figure 2.20).15... 

Subtilisins are a class of related serine endo proteases produced by members of the Bacillus genus. The B.amvloliauefaciens subtilisin (BPN ) is well-characterized with regard to its DNA sequence 4 protein sequence (5), X-ray crystal structure (6) and kinetic properties (7). With this wealth of information available, BPN was chosen as the model enzyme for our recombinant approach. 

An entirely different property of subtilisin was affected by substituting leucine at the 222 location. Native BPN is extremely sensitive to the presence of oxidation agents, showing rapid inactivation when incubated in the presence of 0.3% H2O2 (Figure 4). The Leu-222 variant, in contrast, was found to be totally stable under the same oxidation conditions. The data clearly show that single amino acid alterations can have dramatic effects upon the activity of the enzyme. Similarly, other changes have been shown to affect catalytic properties, substrate specificities and thermostability (7,2,9). 

Lipase, which is highly useful for kinetic resolution, however, has a limitation for use in DKR in that it carmot be used for (S)-configuration products. For this purpose, subtiHsin, a protease from Bacillus licheniformis, can replace lipase since it provides complementary enantioselectivity (Scheme 1.4). Subtilisin, however, has been much less frequently employed in resolution compared to lipase because it displays poor catalytic performance in organic media. Subtilisin is inferior to lipase in several properties such as activity, enantioselectivity and stability. Accordingly, the use of the enzyme usually requires some special treatments for activation and stabilization before use. For example, the treatment of subtilisin with surfactants has enhanced substantially its activity and stability up to a synthetically useful level. 

Subtilisins Primary Structure, Chemical and Physical Properties Francis S. Markland, Jr. and Emil L. Smith... 

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... 

E Tuchsen, M Ottesen. Kinetic properties of subtilisin type Carlsberg in the crystalline state. Carlsberg Res Commun 42 407-420, 1977. 

The plot of the stabilities and activities of clones from the first generation S41 random mutant library shows once again that most mutations are detrimental to stability and activity (Fig. 14). However, compared to the esterase library (Fig. 7), there are more mutants with improvements in both properties, suggesting that the two enzymes have different adaptive potentials. This may be due to the relatively poor stability of S41, or it may reflect constraints intrinsic to the three-dimensional structures of the two proteins. Evidence for the former can be found by comparing the results for the first generations of the psychrophilic sub-tilisin S41 and the mesophilic subtilisin E. Screening 864 mutants of S41 yielded nine thermostabilized variants (a hit rate of approximately 1%) (Miyazaki and Arnold, 1999) in contrast, screening 5000 subtilisin E mutants identified five thermostable variants (a hit rate of only 0.1%) (Zhao and Arnold, 1999). 

The shuffling of twenty-six subtilisins and over twenty human interferons are two recent examples that demonstrate the power of molecular breeding to generate high quality and functionally diverse libraries of useful proteins that serve as rich sources of hits when screened for desired properties (Chang et al., 1999 Ness et al., 1999). 

Subtilisin 26 sequences, 56%-99% DNA identity, 1 round, up to 4-fold improvement in 5 properties Ness, Welch et at, 1999... 

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