ASTM D3048-89(2016)

Standard Test Method of Assay for Alkaline Protease

ABSTRACT
This test method covers the assay of alkaline protease enzymes. This procedure is applicable to enzyme preparations with high activity but is inapplicable to formulated detergent products or air samples. The apparatus and reagents used in testing the assay for alkaline protease enzymes are presented.
SCOPE
1.1 This test method covers the assay of alkaline protease enzymes. This procedure is applicable to enzyme preparations with high activity but is inapplicable to formulated detergent products or air samples.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Material Safety Data Sheets are available for reagents and materials. Review them for hazards prior to usage.

General Information

• Status: Published
• Publication Date: 30-Jun-2016
• ICS: 71.100.40 - Surface active agents
• Technical Committee: D12 - Soaps and Other Detergents
• Drafting Committee: D12.12 - Analysis and Specifications of Soaps, Synthetics, Detergents and their Components

Relations

• Refers: ASTM D459-16 - Standard Terminology Relating to Soaps and Other Detergents
• Effective Date: 01-Jul-2016
• Referred By: ASTM D8179-18 - Standard Guide for Characterizing Detergents for the Cleaning of Clinically-used Medical Devices
• Effective Date: 01-Jul-2016

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: D3048 − 89 (Reapproved 2016)
Standard Test Method of
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Assay for Alkaline Protease
This standard is issued under the fixed designation D3048; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3.1.2 standardized enzyme—an enzyme preparation of
known activity for calibrating the sample enzyme in terms of a
1.1 This test method covers the assay of alkaline protease
3,4
gravimetric standard of enzymatic activity.
enzymes. This procedure is applicable to enzyme preparations
3.1.3 The terms “alkyl benzene sulfonate (ABS)” and “lin-
with high activity but is inapplicable to formulated detergent
ear alkylate sulfonate (LAS)” in this method are defined in
products or air samples.
accordance with Terminologies D1129 and D459:
3.1.3.1 alkyl benzene sulfonate (ABS)—the generic name
1.2 The values stated in SI units are to be regarded as
applied to the neutralized product resulting from the sulfona-
standard. No other units of measurement are included in this
tion of an alkylated benzene.
standard.
3.1.3.2 linear alkylate sulfonate (LAS)—a form of alkyl
1.3 This standard does not purport to address all of the
benzene sulfonate (ABS) in which the alkyl group is linear
safety concerns, if any, associated with its use. It is the rather than a branched chain.
responsibility of the user of this standard to establish appro- 3.1.4 nonionic surfactant—a mixed C -C linear primary
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alcohol containing 65 % ethylene oxide.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use. Material Safety
3.1.5 For definitions of other terms used in these methods,
Data Sheets are available for reagents and materials. Review
refer to Terminology E131.
them for hazards prior to usage.
4. Summary of Test Method
2. Referenced Documents
4.1 This test is based on the hydrolysis of casein at 50°C for
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15 min at pH 9. The trichloroacetic acid-soluble hydrolysate is
2.1 ASTM Standards:
assayed by the spectrophotometric determination of the absor-
D459 Terminology Relating to Soaps and Other Detergents
4,5
bance at approximately 275 nm. The results are correlated
D1129 Terminology Relating to Water
with the absorptivity of tyrosine or the absorbance of hydro-
D1193 Specification for Reagent Water
lysate from standardized enzyme. Results are reported asAPB,
E131 Terminology Relating to Molecular Spectroscopy
which is defined in Section 3, or in micrograms of pure
crystalline enzyme per gram of sample.
3. Terminology
5. Apparatus
3.1 Definitions:
3.1.1 APB unit—that amount of enzyme which releases in 1
5.1 Water Bath, constant-temperature, maintained at 50 6
min under the conditions of the test a casein hydrolysate that 0.2°C.
has the same absorbance as 1 µg of tyrosine in an equivalent
5.2 Ultraviolet Spectrophotometer, suitable for liquid mea-
volume. The number ofAPB units per gram of a preparation is
surements at a wavelength of approximately 275 nm.
called the APB of the preparation.
5.3 Absorption Cell, silica, 10-mm light path.
5.4 pH Meter.
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This test method is under the jurisdiction of ASTM Committee D12 on Soaps
and Other Detergents and is the direct responsibility of Subcommittee D12.12 on
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Analysis and Specifications of Soaps, Synthetics, Detergents and their Components. The sole source of supply known to the committee at this time is National
Current edition approved July 1, 2016. Published August 2016. Originally Institute of Occupational Safety and Health, 1014 BroadwayAve., Cincinnati, Ohio
approved in 1972 as D3048 – 72 T. Last previous edition approved in 2009 as 45202.
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D3048 – 89 (2009). DOI: 10.1520/D3048-89R16. If you are aware of alternative suppliers, please provide this information to
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For referenced ASTM standards, visit the ASTM website, www.astm.org, or ASTM International Headquarters. Your comments will receive careful consider-
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contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM ation at a meeting of the responsible technical committee, which you may attend.
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Standards volume information, refer to the standard’s Document Summary page on Bailey, J. L. “Techniques in Protein Chemistry,” Elsevier Publishing Co., New
the ASTM website. York, NY. Chapter 11, 1967, pp. 340–352.
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D3048 − 89 (2016)
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