Methods in Molecular Biology • 16 Enzymes of Molecular Biology

Pronase 275 W~ihlby, S. and Engstrrm, L. (1968) Studies on Streptomyces griseus protease. Amino acid sequence around the reacti ...

276 Sweeney and Walker Awad, W. M., Soto, A. R., Siegei, S., Skiba, W. E., Bernstrom, G. G., and Ochoa, M. S. (1972) The proteo ...

CHAPTER 15 Proteolytic Enzymes for Peptide Production Patricia J. Sweeney and John M. Walker Introduction There are three main ...

(^278) Sweeney and Walker Table 1 Major Cleavage Sites of Some Proteolytic Enzymes Enzymes Cleavage site Chymotrypsin Clostripai ...

Peptide Production 279 A Asp- ,,o C-O'- .......... H_NT. H-O-Ser 195 102 \ / i c c I H His 57 I B o II Asp-- C-- O7--'+ H- N N'H ...

280 Sweeney and Walker also been reported C terminal to Met, Ileu, Ser, Thr, Val, His, Gly, and Ala residues. The nature of neig ...

Peptide Production 281 Chymotrypsin is a basic protein, and hence, it sticks easily to glass. Therefore, dilute solutions of the ...

282 Sweeney and Walker 2.2.3. Molecular Mass The enzyme has a mol mass of approx 50,000 Da. The amino acid composition has been ...

Peptide Production 283 forming assays, it should be noted that citrate, borate, Veronal, and Tris anions partially inhibit ester ...

284 Sweeney and Walker 2.3.6. Stability The enzyme is active in 0.1% SDS. The lyophilized enzyme is stable indefinitely at-20°C ...

Peptide Production 285 substrate, cleavage occurred at most arginine residues, but one Arg-- Val bond and one Arg--Arg bond were ...

286 Sweeney and Walker 2.5. Endoproteinase Asp-N 2.5.1. General Information This enzyme has been isolated from the culture filtr ...

Peptide Production 287 presence of 5 mM CaC12 appears to stabilize the enzyme somewhat with residual activity still remaining at ...

288 Sweeney and Walker 2.6. 5. Assay The assay is based on the hydrolysis of casein. The reaction condi- tions consist of 1% cas ...

Peptide Production 289 Z Endoproteinase Lys-C (EC 3.4.99.30) 2. 7.1. General Information This enzyme is a serine protease isola ...

290 Sweeney and Walker 2.8. Pepsin (EC 3.4.23.1) 2.8.1. General Information The term "pepsin" refers to a group of gastric prote ...

Peptide Production 291 1 U is defined as the amount of enzyme that hydrolyzes 1 gmol of substrate/min at pH 2.0 and 37°C. 2.8.6. ...

292 Sweeney and Walker 2.9.4. pH Optimum Thermolysin has optimal activity in the pH range 7.0-8.0, but can be used up to pH 9.0 ...

Peptide Production 293 Oxalate, citrate, and phosphate also inhibit the enzyme, so phosphate buffers should be avoided. Mercuric ...

294 Sweeney and Walker absorbance at 255 nm at 25°C is measured, and the enzyme concentration calculated using an extinction coe ...