Special Food / 2001-08281-03.pdf
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Alcalase Fo Fo o d Grade Description Alcalase is a proteolytic enzyme produced by submerged fermentation of a selected strain of Bacillus . The main enzyme component, Subtilisin Bacillus licheniform is A (= Subtilisin Carlsberg), is an endoproteinase extensively described in the literature. Table 1 summarizes some of the biochemical properties of the proteinase in Alcalase.The optimal conditions for Alcalase are temperatures between 55°C (131°F) and 70°C (158°F), depending on the type of substrate, and pH values between 6.5 and 8.5.
Generic name Ty Type of action tion Nature of catalytic site
Subtilisin Subtilisin Carlsberg Carlsberg Endope opeptid tidase Serine
Inhibition by: DFP & PMSF1) PM SF1)
+
EDTA EDTA2) & phosphate
0
Soya Soya bean trypsin inhibi inhibitor tor
0
Bonds attacked in the oxidized B-chain of insulin3)
4-5, 9-10, 11-12, 15-16, 26-27
Molecular weight (approx.) (approx.)
27,300
1)
: DFP = Di-isopropyl fluorophosphate, PMSF = Phenylmethylsulphonylfluoride
2)
: EDTA = Ethylenediamine tetra-acetic acid
NH2-Phe-Val-Asn-Gln-His-Leu-CySO3H-Gly-Ser-His-Leu-Val-Glu-Ala-Leu1 5 10 15 Ty Tyr-Le -Leu-Va -Val-Cy l-CyS SO3H-Gly -Gly-Glu -Glu-Ar -Arg g-Gly -Gly-Ph -Phe-Phe -Phe-Ty -Tyr-Th -Thr-P r-Pro-L o-Lys-Al -Ala a 17 20 25 30 (from Johans Johansen, en, J.T. J .T. et al., C.R. C.R. Trav Trav. Lab. Carlsberg 36 36,, 365-384,1968) Biochemical properties of Alcalase.
Product Properties Product Type Alcalase 2.4 L is a brown liquid and is readily soluble in water at all concentrations. The colour may vary from batch to batch and colour intensity is not an indication of product strength. Activity Alcalase Food Grade is standardized in Anson Units per g (AU/g). Alcalase 2.4 L....................Declared activity: 2.4 AU/g The proteolytic activity is determined according to an analytical standard using the DMC method. See the Analytical Method for further information.
Food-grade status The product complies with the recommended purity specifications for foodgrade enzymes issued by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the Food Chemicals Codex (FCC). Packaging See the standard Packaging List for more packaging information.
Application Alcalase Food Grade is a highly efficient bacterial protease developed for the hydrolysis of all kinds of proteins. More detailed recommendations with respect to the various applications are given in separate leaflets, which are available on request.
Reaction Parameters Activity By analyzing Alcalase at various pH values using a modified Anson/haemoglobin method, its optimum activity has been found to be at a pH of between 6.5 and 8.5 (Figure 1). Similarly, under analytical conditions Alcalase has its temperature optimum around 60°C (140°F) (Figure 2).
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100
80
60
40
20
6
8
10
12
pH
Fig. 1. Influence of pH on the activity of Alcalase. Enzyme concentration: Temperature: Method:
0.06 AU/litre 60°C (140°F) Modified Anson/ hemoglobin analysis 10 minutes
Reaction time:
100
80
60
40
20
20
40
60
80
ºC
Fig. 2. Influence of temperature on the activity of Alcalase. Enzyme concentration: pH: Method: Reaction time:
0.06 AU/litre 8.0 Modified Anson/ hemoglobin analysis 10 minutes
Figure 3 illustrates the productivity of Alcalase at various temperatures on the substrates whey protein and soya protein, respectively. The productivity is depicted in relative values and measured as the % Degree of Hydrolysis (DH) obtained in 4 hours. DH is defined as the percentage of peptide bonds cleaved.
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100
80
60
40
20
whey protein, 8% soy protein, 8%
50
60
70
80
ºC
Fig. 3. The productivity of Alcalase 2.4 L measured at various temperatures on whey and soy protein. Protein %: Enzyme dosage (on protein): pH: Reaction:
8.0 0.51% 8.0 4 hours
Inactivation Alcalase can be inactivated in 30 minutes at 50°C (122°F) or higher when the pH is 4, and in 10 minutes at 85°C (185°F) or higher when the pH is 8. However, the inactivation is very much dependent on the substrate (substrate concentration, pH, etc.). Thus, the documentation for efficient elimination of Alcalase must be based on actual analysis for the detection of residual activity. See the Method for the detection of residual protease activity in protein hydrolysate for further information.
Safety Enzymes are proteins and inhalation of dust or aerosols may induce sensitization and may cause allergic reactions in sensitized individuals. Some enzymes may irritate the skin, eyes and mucous membranes upon prolonged contact. The product may create easily inhaled aerosols if splashed or vigorously stirred. Spilled product may dry out and create dust. Spilled material should be flushed away with water (avoid splashing). Left-over material may dry out and create dust. A Material Safety Data Sheet is supplied with all products. See the Safety Manual for further information regarding how to handle the product safely
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Storage Enzymes gradually lose activity over time depending on storage temperature. Cool conditions are recommended. When stored at 5°C (41°F), the product will maintain its declared activity for at least 1 year. When stored at 25°C (77°F), the product will maintain its declared activity for at least 3 months. Extended storage and/or adverse conditions, including higher temperatures, may lead to a higher dosage requirement.
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2001-08281-02 05 10 2002 © Novozym es A/S
