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OFAS/Office of Food Additive Safety
August 19, 2002
Novozymes North America, Inc.
77 Perry Chapel Church Road
Franklinton, NC 27525
Re: GRAS Notice No. GRN 000103
Dear Ms. Gregg:
The Food and Drug Administration (FDA) is responding to the notice, dated March 25, 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 March 27, 2002, filed it on April 24, 2002, and designated it as GRAS Notice No. GRN 000103.
The subject of the notice is a lipase enzyme preparation from Aspergillus oryzae carrying a gene constructed from a modified Thermomyces lanuginosus lipase gene and a portion of the Fusarium oxysporum lipase gene. The notice informs FDA of the view of Novozymes North America, Inc. (Novozymes) that the lipase enzyme preparation is GRAS, through scientific procedures, for use as a processing aid in bakery products, egg yolks, whole eggs, and fats and oils at minimum levels necessary in accordance with good manufacturing practice. Novozymes estimates that the lipase enzyme preparation would be used at the following levels: 0.5 grams (g) per kilogram (kg) flour, 1 kg to produce 250 kg of modified egg yolk/whole egg, 1 kg to produce 400 kg of modified lecithin, and 1 kg to produce 20 tons of de-gummed oil.
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 lipase enzyme preparation from A. oryzae.
In assessing the safety of the enzyme itself, Novozymes discusses the history of safe use of lipases in food processing. Novozymes cites published articles reporting the use of microbial lipases in food production since 1952. Novozymes describes specific lipase enzyme preparations that have been used in food, including the following:
- Animal lipase, which FDA affirmed as GRAS for use as an enzyme to hydrolyze fatty acid glycerides (21 CFR 184.1415)
- Lipase enzyme preparation derived from Rhizopus niveus, which FDA affirmed as GRAS for use as an enzyme for the interesterification of fats and oils (21 CFR 184.1420)
- Esterase-lipase derived from Mucor miehei (now known as Rhizomucor miehei), which FDA approved for use as a flavor enhancer (21 CFR 173.140)
- Lipase enzyme preparation from A. oryzae carrying a gene encoding lipase from T. lanuginosus, which Novozymes determined to be GRAS for use in dough, baked goods, and the fats and oil industry (GRAS Notice No. GRN 000043)
- Lipase enzyme preparation from A. oryzae carrying a recombinant gene encoding lipase from Fusarium oxysporum, which Novozymes determined to be GRAS for use as a processing aid in the modification of fats and oils, and in baking applications (GRAS Notice No. GRN 000075)
- Lipase enzyme preparation from Candida rugosa, which Amano Enzymes, Inc. determined to be GRAS for use in the modification of fats and oils (GRAS Notice No. GRN 000081).
Novozymes describes the lipase enzyme that is the subject of its notice as a 339 amino acid hybrid protein derived from two other lipase enzymes that are used in food. The N-terminal 284 amino acid residues are derived from the T. lanuginosus lipase, with three amino acid substitutions, and the remainder of the hybrid protein (amino acids 285-339) is derived from the F. oxysporum lipase. Novozymes notes that the lipases used to construct the hybrid protein have been the subject of previous GRAS notices (i.e. GRN 000043 for the lipase from T. lanuginosus and GRN 000075 for the lipase from F. oxysporum).
Novozymes describes the catalytic activity of the lipase as hydrolyzing ester bonds in triglycerides, resulting in the formation of free fatty acids, diglycerides, monoglycerides, and glycerol. The enzyme also catalyzes the hydrolysis of the sn-1 ester bond of diacylphospholipids to form 2-acyl-1-lysophospholipid and a free fatty acid. Compared to other lipases, the subject lipase has a higher preference for long chain fatty acids in position 1 in the substrate, has increased activity towards phospholipids, and has a higher efficacy when used for the modification of egg, hydrolysis of lecithin, and vegetable oil degumming. The lipase enzyme is identified by the following classification numbers: Chemical Abstracts Service Registry No. 9001-62-1, Enzyme Commission No. 22.214.171.124, European Inventory of Existing Commercial Chemical Substances No. 232-619-9.
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 describes the donor microorganisms, T. lanuginosus and F. oxysporum, as nonpathogenic, nontoxigenic fungi.
Novozymes provides information about the components of the lipase expression plasmid pCaHj559 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 lipase enzyme 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 lipase preparation. The test article for these studies was prepared according to the standard production method, except that stabilization and standardization were omitted. These studies include two-week and 13-week oral gavage studies 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 lipase enzyme preparation from Aspergillus oryzae carrying a gene constructed from a modified Thermomyces lanuginosus lipase gene and a portion of the Fusarium oxysporum lipase gene 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 lipase enzyme 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).
Alan M. Rulis, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition