U. S. Food and Drug Administration
Center for Food Safety and Applied Nutrition
Office of Food Additive Safety
October 3, 2002

Lori Gregg
Novozymes North America, Inc.
77 Perry Chapel Church Road
Box 576
Franklinton, NC 27525

Re: GRAS Notice No. GRN 000106

Dear Ms. Gregg:

The Food and Drug Administration (FDA) is responding to the notice, dated April 29, 2002, that you submitted in accordance with the agency's proposed regulation, proposed 21 CFR 170.36 (62 FR 18938; April 17, 1997; Substances Generally Recognized as Safe (GRAS); the GRAS proposal). FDA received the notice on April 29, 2002, filed it on April 29, 2002, and designated it as GRAS Notice No. GRN 000106.

The subject of the notice is a glucose oxidase enzyme preparation from Aspergillus oryzae carrying the gene encoding glucose oxidase from A. niger. The notice informs FDA of the view of Novozymes North America, Inc. (Novozymes) that the glucose oxidase enzyme preparation is GRAS, through scientific procedures, for use in baking applications as an enzyme, at minimum levels necessary in accordance with good manufacturing practice. Novozymes estimates that the glucose oxidase enzyme preparation would be used at the following levels: 0.25-5 g per 100 kilogram (kg) flour (corresponding to 25-500 glucose oxidase units (GODU)/kg flour).

In an amendment dated July 19, 2002, Novozymes notes that there are additional applications for the glucose oxidase enzyme preparation in cheese, beer, carbonated beverages, and fruit juice. These additional uses would require the use of a catalase enzyme preparation in combination with the glucose oxidase enzyme preparation. In an amendment dated August 28, 2002, Novozymes further notes that the estimated use levels for the glucose oxidase enzyme preparation in these other applications would fall within the range estimated for the baking applications.

Commercial enzyme preparations that are used in food processing typically contain an enzyme component, which catalyzes the chemical reaction that is responsible for its technical effect, as well as substances used as stabilizers, preservatives or diluents. Enzyme preparations may also contain constituents derived from the production organism and constituents derived from the manufacturing process, e.g., components of the fermentation media or the residues of processing aids. Novozymes' notice provides information about each of these components of the glucose oxidase enzyme preparation from A. oryzae.

Novozymes describes the glucose oxidase enzyme that is the subject of its notice as a protein transcribed and translated from the A. niger gene coding for glucose oxidase. The glucose oxidase encoding sequence from A. niger was incorporated into the DNA of the A. oryzae production strain without modification. Novozymes states that the subject glucose oxidase is catalytically and functionally equivalent to the glucose oxidase from A. niger. Novozymes concludes that the A. niger glucose oxidase expressed in A. oryzaev is the same enzyme as the glucose oxidase from A. niger that has been in use in food production since 1957.

Novozymes identifies the glucose oxidase by the following classification numbers: EC 1.1.3.4, CAS Registry No. 9001-37-0. Novozymes describes the enzymatic activity of the glucose oxidase as catalyzing the oxidation of beta-D-glucose to hydrogen peroxide and D-glucono-1,5-lactone, which spontaneously hydrolyzes to gluconic acid. In an amendment dated June 21, 2002, Novozymes notes that the primary intended use of this glucose oxidase is in baking applications to modify gluten. The hydrogen peroxide produced from the enzyme-catalyzed reaction oxidizes free sulfhydryl groups of gluten protein. In an amendment dated July 19, 2002, Novozymes further notes that glucose oxidase may be used in other non-baking applications, such as in the manufacture of cheese, beer, carbonated beverages, and fruit juice. For these applications, glucose oxidase would be used in combination with a catalase enzyme preparation to remove oxygen from the food product. Glucose oxidase would generate hydrogen peroxide, which would be removed by the action of catalase to yield oxygen and water.

In assessing the safety of the enzyme itself, Novozymes discusses the history of safe use of glucose oxidases in food processing. Novozymes cites published articles reporting the use of fungal glucose oxidases in food production since 1957. Novozymes describes specific glucose oxidase enzyme preparations that have been used in food, including glucose oxidase from A. niger. This preparation is one of several enzymes in a GRAS affirmation petition (GRP 3G0016), which was filed in 1973. In GRP 3G0016, the petitioner requested that FDA affirm GRAS status through experience based on common use in food, as evidenced by published articles that discussed the pre-1958 uses of the enzymes in the petition. In 2001, the petitioner requested that FDA partially convert GRP 3G0016 to a GRAS notice. As a result, the glucose oxidase from A. niger was one of several enzymes that were the subject of GRAS Notice No. GRN 000089. The uses of glucose oxidase enzyme preparation described in the articles originally provided in GRP 3G0016 focused on uses of glucose oxidase enzyme preparation in combination with catalase to remove glucose or oxygen from food.

In assessing the safety of the host microorganism, A. oryzae strain BECh2, Novozymes describes the host as a derivative of a well-known industrial production strain of A. oryzae (Ahlburg) Cohn. Novozymes obtained the strain, designated IFO 4177 or A 1560, from the Institute for Fermentation, Osaka, Japan. Novozymes considers A. oryzae to be nontoxigenic and nonpathogenic based on published criteria for the assessment of the safe use of microorganisms used in the manufacture of food ingredients.

Novozymes provides information about the components of the glucose oxidase expression plasmid pHUda107 that was introduced into the host strain BECh2 by transformation. Novozymes cites published scientific articles to support its view that all of the DNA sequences that were used in the construction of the production strain are well-known, well-characterized, and commonly used. Novozymes assessed the identity and stability of the introduced DNA using the technique of Southern hybridization and concluded that the DNA is integrated into the A. oryzae chromosome as expected and is not prone to genetic transfer to other organisms. The resulting production strain meets the criteria for Good Industrial Large-Scale Practice published in the Organization for Economic Co-operation and Development's 1992 report entitled "Safety Considerations for Biotechnology."

Novozymes describes the manufacturing process for glucose oxidase preparation, which is produced by submerged, fed-batch pure culture fermentation of the A. oryzae production strain. The enzyme is secreted into the fermentation broth and separated from the cells using filtration. The enzyme preparation is concentrated by ultrafiltration and evaporation. The enzyme preparation is then preserved and stabilized with the addition of sodium chloride. Novozymes follows standard industry practices and uses a quality management system that complies with the requirements of ISO 9001. Novozymes cites several published sources to support the conclusion that the production and control methods used are generally accepted methods that are commonly used for the production of microbial enzyme preparations.

Novozymes describes unpublished toxicity studies performed on its glucose oxidase preparation. The test article for these studies was a glucose oxidase preparation known as Gluzyme™. Novozymes describes Gluzyme™ as a liquid enzyme concentrate, predominately with glucose oxidase activity, but also a minor catalase side activity. Gluzyme™ is produced by submerged fermentation of a strain of A. oryzae expressing the glucose oxidase gene from A. niger. These studies include a 13-week oral gavage study in rats and tests for genetic toxicity, including an Ames test and a chromosome aberration test with human lymphocytes. Novozymes concludes that these toxicity studies showed no treatment related toxicity and no induction of gene mutation in bacteria or chromosomal aberrations in cultured human blood lymphocytes.

Based on the information provided by Novozymes, as well as other information available to FDA, the agency has no questions at this time regarding Novozymes' conclusion that glucose oxidase enzyme preparation from Aspergillus oryzae carrying the gene encoding glucose oxidase from Aspergillus niger is GRAS under the intended conditions of use. The agency has not, however, made its own determination regarding the GRAS status of the subject use of this glucose oxidase preparation. As always, it is the continuing responsibility of Novozymes to ensure that food ingredients that the firm markets are safe, and are otherwise in compliance with all applicable legal and regulatory requirements.

In accordance with proposed 21 CFR 170.36(f), a copy of the text of this letter, as well as a copy of the information in the notice that conforms to the information in proposed 21 CFR 170.36(c)(1), is available for public review and copying on the homepage of the Office of Food Additive Safety (on the Internet at http://www.cfsan.fda.gov/~lrd/foodadd.html).