Subtilisin-BPN

Engineering the pH Proji/e of Subtilisin. The activity of subtilisin BPN increases between pH 6 and 8 as His64 7.2) is deprotonated (68). Changes in... 

Drenth, J., et al. Subtilisin novo. The three-dimensional structure and its comparison with subtilisin BPN. 

Figure 7.11 Linear free energy correlation plots for inhibition of subtilisin BPN mutants by wild type (open circles) and mutant (close circles) chymotrypsin inhibitor 2. Left panel Correlation between AGbinding for the inhibitor and AGm. Right panel Correlation between AGbinding for the inhibitor and AGES.

Matthews, D.A. Alden, R.A Birktoft, J.J. Freer, S.T. Kraut, J (1975) X-ray Crystallographic Study of Boronic Acid Adducts with Subtilisin BPN (Novo). J. Biol. Chem. 250, 7120-7126. 

A. A. Kossiakoff, M. Ultsch, S. White, C. Eigenbrot, Neutron Structure of Subtilisin BPN Effects of Chemical Environment on Hydrogen-Bonding Geometries and the Pattern of Hydrogen-Deuterium Exchange in Secondary Structure Elements , Biochemistry 1991, 30, 1211-1221. 

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. 

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)... 

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). 

Carter, P., Nilsson, B., Bumier, J.P., Burdick, D. and Wells, J.A. (1989) Engineering subtilisin BPN for site-specific proteolysis. Proteins, 6, 240-248. 

Rheinnecker, M., Eder, J., Pandey, P. S. Fersht, A. R. (1994). Variants of subtilisin BPN with altered specificity profiles. Biochemistry, 33, 221-5. 

Wild-type subtilisin BPN with the mutation Ser — Cys-24 has a kcat value of 59 s 1 and a KM value of 200 fxM with the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, compared with a rate constant of 1.1 X 10 8 s 1 for its spontaneous hydrolysis under the same conditions. Replacement of Asp-32, His-64, and Ser-221 one at a time by alanine reduced the value of kcat by factors of 3 X 104, 2 X 106, and 2 X 106, respectively. Converting all three to alanine also decreases activity by 2 X 106. The value of KM increases only by a factor of two on all these mutations.34 It is unlikely that the residual activity results from the presence of a small amount of wild-type active site in the thiol mutants... 

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. 

Adsorption of the enzymes subtilisin BPN and lysozyme onto model hydrophilic and hydrophobic surfaces was examined using adsorption isotherm experiments, infrared reflection-absorption spectroscopy (IRRAS), and attenuated total reflectance (ATR) infrared (IR) spectroscopy. For both lysozyme and BPN, most of the enzyme adsorbed onto the model surface within ten seconds. Nearly an order-of-magnitude more BPN adsorbed on the hydrophobic Ge surface than the hydrophilic one, while lysozyme adsorbed somewhat more strongly to the hydrophilic Ge surface. No changes in secondary structure were noted for either enzyme. The appearance of carboxylate bands in some of the adsorbed BPN spectra suggests hydrolysis of amide bonds has occurred. 

Subtilisin BPN was prepared through a series of protein purification steps applied to the fermentation broth. These steps included ultrafiltration ethanol precipitation DEAE (diethyl-aminoethyl) Tris Acryl batch anionic exchange SP (sulfopropyl) Tris Acryl column cationic exchange and, concentration with an Amicon stirred cell. The enzyme purity was determined to be -951 via spectroscopic assays that measure the ratio of active enzyme to total protein. In addition, purity was verified via HPLC and SDS-page (sodium dodecyl sulfate polyacrylamide gel electrophoresis). 

In order to test whether our CIRcle cell spectra were dominated by adsorbed protein or protein in solution, we ran spectra of a series of lysozyme solutions ranging in concentration from 0.12 to 102. The IR response of the amide I and II bands at 1653 and 1543 cm-1 is nearly linear with concentration between 5 and 102 lysozyme. However, the IR intensities change very little between 0.1 and 12, strongly suggesting that most of the signal we observe at 0.12 concentration is due to adsorbed lysozyme. Since our study of subtilisin BPN was done at 0.012, we are almost certainly observing only adsorbed species in our ATR spectra. 

Figure 3. A series of spectra of 100 ppm subtilisin BPN from 6 to 180 minutes after injection into the CIRcle cell. The solvent spectrum has been subtracted.

Figure 5. Adsorption of 100 ppm subtilisin BPN onto hydrophobic and hydrophilic Ge IREs as determined by the amide II band absorbance after subtraction of the solvent spectrum.

Figure 7. Overlay of the Gram-Schmidt (GS) and functional group (FG) profiles of the adsorption of 100 ppm subtilisin BPN on hydrophobic Ge (uncoated o-rings).

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