Agency Response Letter
GRAS Notice No. GRN 000126

David S. Hanselman, Ph.D.
Director of Regulatory Affairs, Environment, Health and Safety
Innovase LLC
5501 Oberlin Drive
San Diego, CA 92121

Re: GRAS Notice No. GRN 000126

Dear Dr. Hanselman:

The Food and Drug Administration (FDA) is responding to the notice, dated March 24, 2003, that Keller and Heckman LLP submitted on behalf of Innovase LLC (Innovase) 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 1, 2003, filed it on April 3, 2003, and designated it as GRAS Notice No. GRN 000126.

The subject of the notice is alpha-amylase enzyme preparation from Pseudomonas fluorescens Biovar I expressing a gene encoding a hybrid alpha-amylase derived from three microorganisms within the order Thermococcales (referred to as BD5088 alpha-amylase). The notice informs FDA of the view of Innovase that this alpha-amylase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in the hydrolysis of edible starch to produce various starch hydrolysis products and to produce fermentable sugars for use in the production of distilled ethanol for alcoholic beverages. Innovase estimates that the alpha-amylase enzyme preparation would be used at a level of 500 grams per metric ton of starch (i.e., 500 milligrams per kilogram).

As part of its notice, Innovase includes the report of a panel of individuals (Innovase's GRAS panel) who evaluated the data and information that are the basis for Innovase's GRAS determination. Innovase considers the members of its GRAS panel to be qualified by scientific training and experience to evaluate the safety of substances added to food. Innovase's GRAS panel evaluated information related to the hybrid alpha-amylase enzyme, host and production strains, introduced genetic material, manufacturing process, dietary exposure, toxicity studies, and genotoxicity studies. Based on this review, Innovase's GRAS panel concluded that the alpha-amylase enzyme preparation manufactured in accordance with current good manufacturing practices and meeting the relevant food grade specifications is GRAS, through scientific procedures, for its intended use.

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 host microorganism and constituents derived from the manufacturing process (e.g., components of the fermentation media or the residues of processing aids). Innovase's notice provides information about each of these components of its alpha-amylase enzyme preparation.

Innovase describes general information about the use of alpha-amylases during the past several decades to hydrolyze 1,4-alpha-glucosidic linkages in alpha-1,4,-linked polysaccharides containing three or more glucose units (such as amylose or amylopectin) to soluble dextrins and oligosaccharides. Innovase also describes specific information about the use of thermostable alpha-amylases in the corn wet milling process for the production of high fructose corn syrup. Innovase sought to develop a thermostable alpha-amylase that would have a lower pH optimum and require the addition of less calcium than other alpha-amylases for use in the corn wet milling process. Innovase provides a published article that describes how it used the process of "directed evolution" to screen DNA from a variety of unusual natural environments, including those with extremes of pH, temperature, and other physical parameters, for alpha-amylases with desired pH optima and temperature stability. The DNA that Innovase screened was either obtained from available cultures of microorganisms (including some proprietary strains) that had been isolated from such environments, or obtained by screening the environments without an intervening step of actually isolating or cultivating the microorganisms.

From this DNA screening, Innovase selected three enzymes for further investigation. Two of these alpha-amylases (BD5031 and BD5064) were derived from proprietary microbial strains that Innovase screened. These two strains are procaryotes that are distinct from bacteria and are classified within the order Thermococcus. The third alpha-amylase (BD5063) derives from an isolate from an extreme environment; the characteristics of this alpha-amylase are consistent with an Archaeal source within the order Thermococcus.⁽¹⁾. Innovase provides a published article that describes how it used a "gene reassembly" process to isolate a series of fragments from each of these three enzymes and then combine fragments to obtain a hybrid enzyme (designated BD5088) that contains two fragments from BD5031, three fragments from BD5064, and four fragments from BD5063. The hybrid enzyme is a thermostable enzyme with a molecular weight of 49,770 Da. Its amino acid sequence and activity are comparable to those of commercially available alpha-amylases, except that it is active at a lower pH and requires less calcium than some commercially available, thermostable alpha-amylases. Innovase identifies its hybrid alpha-amylase enzyme by the following classification numbers: Chemical Abstracts Service Registry No. 9000-90-2, Enzyme Commission No. 3.2.1.1, European Inventory of Existing Commercial Chemical Substances No. 232-565-6.

Innovase describes the host microorganism (P. fluorescens Biovar I strain MB101) that it transformed with DNA encoding the hybrid alpha-amylase. P. fluorescens Biovar I strain MB101 is a natural isolate obtained from lettuce leaves. It is Gram-negative, aerobic, saprophytic, and nonpathogenic. It is ubiquitous in soil, water, and plant environments. Innovase notes that P. fluorescens has rarely been implicated in opportunistic infections of animal species and was used as the host in the construction of microbial strains that have been bioengineered to produce proteins that are toxic to the larvae of certain species of moths and butterflies. Innovase also reports that published studies conducted in mice fed P. fluorescens showed no evidence of pathogenicity or toxicity.

Innovase describes the construction of the production strain (P. fluorescens Biovar 1 strain DC88) that contains the gene encoding the hybrid alpha-amylase. Innovase developed two plasmids and introduced them into P. fluorescens Biovar I strain MB101 by electroporation. One plasmid (pMYC5088), a 12.02 kb self-replicating derivative of the Pseudomonas plasmid RSF1010 encodes the hybrid alpha-amylase under the regulation of the tac promoter. The tac promoter is repressed by binding of the Escherichia coli lac repressor, encoded by the lacI gene on the second (7.06 kb) plasmid (pCN51lacI). Using this two-plasmid system, the microorganism can be grown to an appropriate level while the expression of the hybrid alpha-amylase is repressed by high levels of the lac repressor. After optimal growth is achieved, the expression of the hybrid alpha-amylase enzyme is induced by the addition of isopropylthio-beta-D-galactopyranoside (IPTG). Innovase notes that it followed domestic and international guidelines for the use of recombinant DNA products in developing these two plasmids. The plasmids remain as extrachromosomal DNA - i.e., they are not incorporated into the genome of P. fluorescens. Plasmid pMYC5088 encodes resistance to the antibiotic tetracycline; plasmid pCN51lacI encodes resistance to the antibiotic kanamycin. Both plasmids are well-characterized and poorly mobilizable. Innovase has found that the plasmids remain stable through forty-five generations even in non-selective media.

Innovase describes the manufacturing process for its alpha-amylase enzyme preparation, which is produced by submerged, fed-batch pure culture fermentation of the bioengineered production strain, P. fluorescens Biovar 1 strain DC88. During the growth phase, Innovase uses the antibiotics tetracycline and kanamycin to provide selective pressure for maintenance of the plasmids. Following the growth phase, Innovase uses IPTG to induce enzyme production. Following optimal alpha-amylase production, the culture is pasteurized to kill the production organism and is transferred to the dissolution tank for recovery. The pH is then adjusted to lyse the cells and enhance extraction and solubilization of the intracellular alpha-amylase enzyme, which is present as an inclusion body.⁽²⁾ Innovase separates cell debris from the solubilized enzyme fraction using membrane microfiltration, which removes much of the DNA. Next, the enzyme is concentrated to the desired level of activity using ultrafiltration, which excludes the antibiotics and other small molecules. The resulting material is transferred to a DNA treatment and formulation tank where intact DNA is hydrolyzed by the addition of acid and/or endonuclease. Following DNA removal, appropriate stabilizers, diluents, and preservatives are added. Because the plasmids and the cells that harbor them are discarded after production and any DNA in the enzyme preparation is eliminated by treatment with an endonuclease or acid, Innovase concludes that DNA encoding antibiotic resistance would not be transmitted from its alpha-amylase enzyme preparation to bacteria that reside in the intestinal tract or to intestinal tract cells.

The enzyme preparation complies with the general and additional requirements for enzyme preparations set forth in the Food Chemicals Codex, 4th edition, 1996. The enzyme preparation also conforms to the general specifications and considerations for enzyme preparations used in food processing provided by the Joint Food and Agriculture Organization/World Health Organization's (FAO/WHO) Expert Committee on Food Additives (JECFA) in the Compendium of Food Additive Specifications (2001). Additional specifications include absence of the production organism and absence of detectable antibiotic activity.

Innovase discusses published studies conducted with its alpha-amylase enzyme preparation in various animal species. For these studies, the investigators utilized concentrated enzyme preparations that lack the stabilizers, diluents, and preservatives described above. Innovase concludes that acute and subchronic oral toxicity studies conducted in rats showed no treatment-related toxicological effects at any dose tested and that genotoxicity studies conducted in vivo or in vitro demonstrated no mutagenic effects. Innovase also discusses tests to assess the allergencity potential of its alpha-amylase enzyme preparation, including pepsin digestibility and sequence comparison to known allergens.

Based on the information provided by Innovase, as well as other information available to FDA, the agency has no questions at this time regarding Innovase's conclusion that its alpha-amylase enzyme preparation 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 alpha-amylase enzyme preparation. As always, it is the continuing responsibility of Innovase 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 your 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).

Sincerely,    /s/ Laura M. Tarantino, Ph.D. Acting Director Office of Food Additive Safety Center for Food Safety      and Applied Nutrition

References

⁽¹⁾ Woese, C. R., Kandler, O., and Wheelis, M. L. 1990. Towards a natural system of organisms: Proposal for the domains Archaea, Bacteria, and Eucarya. Proc. Natl. Acad. Sci. USA 87: 4576-4579

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⁽¹⁾Innovase describes information about contemporary taxonomic schemes that are based on the relatedness of DNA sequences encoding ribosomal RNA (rRNA) for the small ribosomal subunit of different organisms (Ref. 1). These data have been used to identify a small group of procaryotes, including methanogens, halophiles, and extreme thermophiles, that form a group of organisms that are distinct from both bacteria and eucaryotes. These procaryotic organisms are currently referred to as Archaea and are considered to be a "domain" that is above the level of kingdom in other taxonomic systems. The Archaea have many fewer known or characterized organisms than the other two domains in this taxonomic system (i.e., the Bacteria and the Eucarya) and, thus, has a simplified taxonomic scheme in which several taxonomic levels have a single component. Archaea are not known to be human pathogens or to produce substances that are toxic to humans, and some Archaea reside in the human gut as commensals.

⁽²⁾When heterologous proteins are expressed at high levels in Gram-negative bacteria, these proteins may not fold properly in the bacteria, resulting in aggregation and the formation of insoluble structures called inclusion bodies. Inclusion bodies generally can be denatured and solubilized by the addition of either 8 M urea or 6 M guanidine hydrochloride, then renatured in milder buffers.