Enzyme Explorer

Protease Type XIV

Isolated from Streptomyces griseus
Synonyms: Actinase E, Pronase

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Physical Properties
Protease Type XIV is a mixture of at least three caseinolytic activities and one aminopeptidase activity. The caseinolytic enzymes were named as Streptomyces griseus Protease A, Streptomyces griseus Protease B and Streptomyces griseus Trypsin.3 The amino acid sequences and molecular weights have been reported: 18,093 for Protease A4, 18,629 for Protease B5, and 22,918 for S. griseus trypsin.6 Properties of this trypsin have also been reported.7 Values of 16,000 and 18,000 for two different proteolytic activities have been reported8; molecular weights, usually determined by gel filtration, range from 16,000 to 27,000.1,2 Detailed references concerning isolation, properties and structure are noted.14

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Specificity, Stability and Kinetics
This protease mixture is so nonspecific that it can digest casein to the extent of >70% as mono-amino acids.1,2 It was shown to be much more effective in digestion of casein than trypsin, chymotrypsin and several other proteases.9

Protease Type XIV is highly stable in the pH range 5.0 to 9.0, but fairly unstable below pH 4 and above pH 10.1 The neutral components in the enzyme mixture are stable at pH 5-9 with calcium present; the alkaline components are stable over pH 3-9, with optimal activity at pH 9-10. The aminopeptidase and carboxypeptidase components are stable at pH 5-8, in the presence of calcium ion.2 The optimum activity will be at pH 7-8.2

The product dissolves in 0.01 M sodium acetate with 0.005 M calcium acetate at pH 7.5 at 37°C; at 0.2 mg/mL the clear solution ranges from colorless to light tan. Calcium ion is recommended for protection from autolysis. The activity of a dilute enzyme solution containing 0.01 to 0.1 M calcium ion was stable over 24 hours at neutral pH at 2-8°C. Protease Type XIV is stable at 4°C for at least six months. Stock solutions of 5 to 20 mg/mL in water are usually stored at 20°C.2

The product can be completely inactivated by heating above 80°C for 15-20 minutes.1 Some components of the mixture are inactivated more quickly than others. Adding excess EDTA results in irreversible loss of aprrox. 70% of the original activity. The mixture retains activity in 1% SDS (w/v) and 1% Triton (w/v).

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Inhibitors
No single substance will inhibit all the different proteases in the mixture (see structure). DFP, PMSF and EDTA have been used with some success.2

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Applications

  • DNA isolation: 2,13 Protease Type XIV is usually prepared as a stock solution, and prior to storage at -20°C, the solution is first heated to 56°C for about 15 minutes, then incubated at 37°C for 1 hour. This encourages self-digestion, to eliminate DNAse and RNAse contamination. The enzyme is added to a DNA sample (in the presence of 0.5-1% SDS to disrupt DNA protein interactions) typically at 250-500 µg protein/mL, then incubated at 37°C for 1-4 hours.
  • For protein hydrolysis, dissolve about 0.2 micromole of protein in 0.2 mL of 50 mM ammonium bicarbonate buffer at pH 8 (or phosphate buffer pH 7). Add Protease Type XIV to 1% (w/w) and incubate at 37°C for 24 hours. It may be necessary to add aminopeptidase M at 4% (w/w) and incubate at 37°C for another 18 hours.
  • Hydrolysis of amino acid amides10
  • Pretreatment of liver tissue sections to enhance the intensity of immunostaining11
  • Regeneration of certain types of affinity columns12

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Products
Product #  Product Name Add to Cart
P5147 Protease from Streptomyces griseus, Type XIV, ≥3.5 units/mg solid, powder
P8811 Protease from Streptomyces griseus, ~4 units/mg solid, powder, mouse embryo tested
P6911 Protease from Streptomyces griseus, Molecular Biology Reagent, DNase, RNase, and nickase, none detected (No RNase.)

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References

  1. Supplier information
  2. Enzymes of Molecular Biology (Methods in Molec. Biol., Vol. 16), M.M. Burrell, ed., Chapter 14, pp. 271-276.
  3. Jurasek, L., Johnson, P. et al., Canadian J. Biochem., 49, 1195 (1971).
  4. Johnson, P. and Smillie, L.B., FEBS Letters, 47, 1-6 (1974).
  5. Jurasek, L. and Carpenter, M.R. et al., Biochem. Biophys. Res. Commun., 61, 1095-1100 (1974).
  6. Olafson, R.W. and Jurasek, L. et al., Biochemistry, 14, 1168 (1975).
  7. Olafson, R.W. and Smillie, L.B., Biochemistry, 14, 1161-1167 (1975).
  8. Wahlby, S., Biochim. Biophys. Acta, 185, 178-185 (1969).
  9. Nomoto, M. et al., J. Biochemistry, 48, 593-602 (1960).
  10. Yamskov, I.A. et al., Enzyme Microb. Technol., 8, 241 (1986).
  11. Litwin, J.A. et al., Histochemistry, 81, 15-22 (1984).
  12. Holroyde, M.J. et al., Biochem. J., 153, 351-361 (1976).
  13. Molecular Cloning: A Laboratory Manual, Sambrook et al., eds. (Cold Spring Harbor Press, 1989), p. B.16.
  14. Nomoto, M. and Narahashi, Y., J. Biochemistry, Papers I - VII: 14a. 46, 653 (1959); 14b. 46, 839 (1959); 14c. 46, 1481 (1959); 14d. 46, 1645 (1959); 14e. 48, 453 (1960); 14f. 48, 906 (1960).

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