Special Food / 2001-08281-03.

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Product Sheet
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Alcalase Food Grade

Description
Alcalase is a proteolytic enzyme produced by submerged fermentation of a selected strain of Bacillus licheniformis. The main enzyme component, Subtilisin 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 55C (131F) and 70C (158F), depending on the type of substrate, and pH values between 6.5 and 8.5. Alcalase Generic name Type of action Nature of catalytic site Inhibition by: DFP & PMSF1) EDTA2) & phosphate Soya bean trypsin inhibitor Bonds attacked in the oxidized B-chain of insulin3) Molecular weight (approx.)
1)

Subtilisin Carlsberg Endopeptidase Serine

+ 0 0 4-5, 9-10, 11-12, 15-16, 26-27 27,300

: DFP = Di-isopropyl fluorophosphate, PMSF = Phenylmethylsulphonylfluoride : EDTA = Ethylenediamine tetra-acetic acid

2)

NH2-Phe-Val-Asn-Gln-His-Leu-CySO3H-Gly-Ser-His-Leu-Val-Glu-Ala-Leu1 5 10 15 Tyr-Leu-Val-CySO3H-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala 17 20 25 30 (from Johansen, J.T. et al., C.R. Trav. Lab. Carlsberg 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 60C (140F) (Figure 2).

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100 80 60 40 20

10

12 pH

Fig. 1. Influence of pH on the activity of Alcalase. Enzyme concentration: Temperature: Method: Reaction time: 0.06 AU/litre 60C (140F) Modified Anson/ hemoglobin analysis 10 minutes

100 80 60 40 20

20

40

60

80 C

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 C

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 50C (122F) or higher when the pH is 4, and in 10 minutes at 85C (185F) 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 5C (41F), the product will maintain its declared activity for at least 1 year. When stored at 25C (77F), 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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Novozymes A/S Krogshoejvej 36 2880 Bagsvaerd Denmark Tel. +45 8824 9999 Fax +45 8824 9998 info@novozymes.com www.novozymes.com
Laws, regulations and third party rights may prevent customers from importing, processing, applying and/or reselling certain products in a given manner. It is the responsibility of the customers that their specific use of products from Novozymes does not infringe relevant laws and regulations and, furthermore, does not infringe patents or other third party rights. The contents of this document are subject to change without further notice. 2001-08281-02 05 10 2002 Novozymes A/S