Bacterial Alkaline Protease for Protein Hydrolysis

Description

Deterzyme®, a bacterial alkaline protease (E.G. 3.4.21) produced by the controlled fermentation of Bacillus licheniformis, is an endopeptidase capable of hydrolyzing the interior peptide bonds of protein molecules. The broad substrate specificity of Deterzyme® enables the enzyme to effectively hydrolyze most proteins. Hemoglobin, casein, egg yolk, soy, gelatin, fish, and other proteins are hydrolyzed to lower molecular weight peptides. Deterzyme® demonstrates excellent stability and activity at elevated temperatures in alkaline solutions.

Product	Activity	Comment
Deterzyme® APL-440	440 DAPU/g	Liquid
Deterzyme® APL-660	660 DAPU/g	Liquid
Deterzyme® APUG-380	380 DAPU/g	Granules

Properties

	Liquids	Granules
Form	Low viscosity liquid	Dust free granules
Color	Amber to light brown	Tan
Odor	Free of offensive odor	Free of offensive odor
Solubility	Readily mixable	Completely soluble

Activity

One Detergent Alkaline Protease Unit (DAPU) is that activity which will liberate four micromoles (4 x 10-6 moles) of tyrosine per minute under the conditions of the assay. A copy of the Detergent Alkaline Protease Unit procedure is available upon request.

Inhibitors

Deterzyme® is a serine hydrolase and is inhibited by organophosphorous reagents, such as diisopropylphosphofluoridate and diisopropyl fluorophosphate, which react with the amino acid serine. Oxidizing agents that release active chlorine also inhibit the action of Deterzyme®.

Effect of pH

In the presence of 0.6% (w/v) casein and 0.03% (w/v) (STPP), Deterzyme® exhibits maximum activity at pH 9 - 10 and 60 - 70C. (140 -158F). Deterzyme® effectively hydrolyzes casein over the pH range of 6.0 to 11.0. The enzyme is stable in dilute solutions over the range of pH 6.0 to 10.0 at temperatures below 50C (122F). At temperatures above 50C (122F) and pH above 10.0, the enzyme is rapidly inactivated.

Optimum pH range	pH 9.0 to 1 0.0
Effective pH range	pH 7.0 to 1 0.0
pH stability	pH 6.0 to I 0.0

Effect of Temperature

In the presence of 0.6% (w/v) casein and 0.03% (w/v) STPP, Deterzyme® exhibits maximum activity at 60 - 70C. (140 -158F). Deterzyme® is very stable in dilute solutions at temperatures up to 50C (122F).

Maximum activity	60 - 70C. (140 - 158F)
Effective range	up to 70C (158F)
Stability	up to 50C (122F)

Inactivation

Deterzyme® may be inactivated by raising the temperature to 80 - 85C (176 -185F) and holding for 5 to 10 minutes. If high temperature inactivation is undesirable, the enzyme may be inactivated by adjusting the pH below 4.0 and holding for 30 minutes at 50C (122F.) or by adjusting the pH above 10.0 and holding 60 minutes at 60C.

Substrate

The physical state of protein is important with respect to susceptibility to protease hydrolysis. Native globular proteins are generally resistant to proteolytic attack because of their compact tertiary structure. Susceptibility of proteins to hydrolysis can be improved by denaturing the protein substrate and thereby exposing peptide bonds to enzymatic hydrolysis. Heat, extremes of acidity or alkalinity, urea, and reducing or oxidizing agents promote protein denaturation and subsequent proteolysis.

Deterzyme® bacterial protease has a broad substrate specificity and randomly hydrolyzes the inner peptide bonds of denatured proteins. The enzyme efficiently hydrolyzes casein, hemoglobin, egg yolk, gelatin, soy, fish and other plant and animal proteins. The proteolytic activity yields lower molecular weight peptides and amino acids.

Applications

Feed Supplement	To improve animal feed utilization and efficiencies.
Laundry	Detergents, presoaks, and spot remover.
Leather	Dehairing and bating of hides.
Paper	Removal of protein-coated broke.
Photographic	Removal of gelatin from film and silk screens.
Textile	Removal of warp sizes.
Waste Treatment	Solubilization of protein in septic tank and other waste disposal systems.
Misc.	Cleaning protein processing equipment: i.e., extruders, etc.

Safety

Deterzyme® is formulated to provide maximum safety when properly handled. In certain individuals, exposure of enzymes to skin, eyes or mucous membranes may result in irritation. The Material Safety Data Sheet should be reviewed with all personnel involved for proper precautions.

Use Level

Enzyme requirements are generally dictated by processing conditions. They will very with protein substrate concentration, degree of desired protein hydrolysis, pH, temperature, and time. In general, increasing the Deterzyme® bacterial protease concentration will increase the rate of protein hydrolysis and shorten the time necessary for hydrolysis. Initial evaluations of Deterzyme® are made at a concentration of 200 to 400 DAPU per kilogram of protein. Trial applications, however, should be made to determine the final usage level.

Storage Stability

In sealed containers, under cool, dry conditions, the loss of activity is normally less than 10% in six months. Storage life can be extended by storing under refrigeration at 5C (41F).

References

Keay, L. 1971. Microbial Proteases, Process Biochemistry, (August) pp. 17-20.

Kelly, C.T., Fogarty, W.M. 1976. Microbial Alkaline Proteases, Process Biochemistry, (July/August) pp. 3-9.

Mihalyi, E. 1978. Application of Proteolytic Enzymes to Protein Structure Studies, 2nd., CRC Press, West Palm Beach, FL.