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WHO FOOD ADDITIVES SERIES: 52

alpha-Amylase From Bacillus Licheniformis Containing A Genetically Engineered alpha-Amylase Gene From B. Licheniformis

First draft prepared by

Mrs I.M.E.J. Pronk
Centre for Substances and Integrated Risk Assessment, National Institute for Public Health and the Environment, Bilthoven, Netherlands;

and Dr C. Leclercq
National Research Institute for Food and Nutrition, Rome, Italy

Explanation

Construction of the production strain

Biological data

Biochemical aspects

Toxicological studies

Acute toxicity

Short-term toxicity

Studies of long-term toxicity and carcinogenicity

Genotoxicity

Reproductive toxicity

Observations in humans

Dietary intake

Comments

Evaluation

References

1. EXPLANATION

The enzyme preparation under evaluation contains the enzyme LE399 alpha-amylase from the genetically modified Bacillus licheniformis. LE399 alpha-amylase has not been evaluated previously by the Committee. The enzyme is thermostable and active at a relatively low pH and low calcium concentration. These characteristics make the enzyme particularly suitable for use in starch hydrolysis conducted at high temperatures, for example, for the liquefaction of starch used in the production of nutritive sweeteners.

LE399 alpha-amylase is produced by pure culture fermentation of a strain of B. licheniformis that is nonpathogenic and nontoxigenic and which has been genetically modified to carry a genetically engineered gene coding for alpha-amylase. The enzyme is subsequently partially purified and concentrated, resulting in a liquid enzyme concentrate (LEC). In the final preparation, this LEC is stabilized and standardized and formulated with methionine, sodium chloride, and glucose and sucrose.

alpha-Amylases break down starch into soluble dextrins and oligosaccharides via endohydrolysis of 1,4-alpha-glucosidic linkages in amylose and amylopectin. This results in a rapid reduction of the viscosity of gelatinized starch. The LE399 alpha-amylase can operate at lower pH and lower concentrations of calcium ions than conventional heat-stable alpha-amylases.

Concerning amino acid sequence and enzymatic activity, the LE399 alpha-amylase from the genetically modified B. licheniformis is substantially equivalent to other alpha-amylases including wild-type B. licheniformis alpha-amylase (affirmed in USA as "generally recognized as safe" (GRAS)) and wild-type B. amyloliquefaciens alpha-amylase (also affirmed as GRAS). LE399 alpha-amylase has been developed to have improved specificity and specific activity, and partly to improve stability at low pH and low concentrations of calcium ions at high temperatures. The wildtype alpha-amylase enzymes require calcium ions for stability.

The activity of the heatstable LE399 alpha-amylase enzyme preparation is determined relative to that of a commercial standard alpha-amylase preparation of declared strength. The activity is stated in terms of kilo novo units of alpha-amylase (Termamyl) or KNU(T). At present, Liquozyme is available as two variants: Liquozyme EX has a standardized activity of 100 KNU(T)/g while Liquozyme X has a standardized activity of 200 KNU(T)/g. The typical composition of the two variants is given in Table 1.

Table 1. Typical composition of two available commercial variants of LE399 alpha-amylase, Liquozyme EX and Liquozyme X

 

Liquozyme EX

Liquozyme X

Total organic solids

Approximately 4%

Approximately 8%

Water

Approximately 52%

Approximately 52%

Sucrose/glucose

Approximately 29%

Approximately 25%

Sodium chloride

Approximately 15%

Approximately 15%

Methionine

0.6%

0.6%

The enzyme preparation Liquozyme is used in the food industry as a processing aid in the liquefaction of starch. In the starch industry it is used, for example, in the production of syrups, in the alcohol industry for thinning of starch in distilling mashes, in the brewing industry for liquefaction of the adjunct, and in the sugar industry for breaking down the starch present in cane juice, thereby reducing the starch content, which facilitates filtration. For starch and alcohol applications the recommended dosages are up to 100 KNU(T) per kg of solid substrate, corresponding to 0.1% Liquozyme EX per kg substrate or 0.05% Liquozyme X per kg substrate. For brewing, the recommended dosages are up to 500 mg per kg of raw fruit.

Toxicological studies have been performed with a liquid enzyme concentrate (batch PPY 7075), omitting formulation, stabilization and standardization. The composition of test batch PPY 7075 given in Table 2.

Table 2. Composition of LE399 alpha-amylase, test batch PPY 7075

Composition of batch PPY 7075

Contents

Enzyme activity, KNU(T)/g

287

Water (% w/w)

86.5

TOS (% w/w)

9.6

pH

8.9

Ash (600 °C) (% w/w)

3.9

Density (g/ml)

1.06

From Elvig-Jørgensen & Pedersen (2002); Nielsen (2002a, b)

KNU(T), kilo novo units of alpha-amylase (Termamyl)

TOS, total organic solids

1.1 Construction of the production strain

The alpha-amylase protein was developed by changing four amino acids in the polypeptide chain of another genetically engineered thermostable alpha-amylase, "Termamyl LC". These modifications were accomplished by introducing appropriate mutations into the DNA sequence encoding the Termamyl LC alpha-amylase. The engineered gene, designated as the LE399 alpha-amylase gene, was introduced into the host strain SJ5550.

The host strain was developed from a parent strain DN2717, a derivative of a natural B. licheniformis isolate. The DN2717 strain was genetically engineered to inactivate the following native genes: the apr gene encoding the "Alkalase" protease; the amyL gene encoding the Termamyl alpha-amylase; the xyl gene encoding xylose isomerase; and the gnt gene encoding gluconate permease. The inactivated amyL, xyl, and gnt genes were replaced with three copies of the LE399 alpha-amylase gene. In a separate step, the gene encoding C-component protease was deleted. The resulting strain was designated as MOL2083 and used as a production strain. The aim of these genetic modifications was to produce the LE399 alpha-amylase, to prevent the synthesis of proteases that might hydrolyse the LE399 alpha-amylase, and to avoid the production of the Termamyl alpha-amylase.

The genetic material introduced into the production strain has been well characterized and does not contain any sequences that would encode for proteins resulting in the production of toxic or undesirable substances. The LE399 alpha-amylase gene is stably integrated into the B. licheniformis chromosome. The production strain does not contain genes encoding proteins that inactivate antibiotics.

2. BIOLOGICAL DATA

2.1 Biochemical aspects

The LE399 alpha-amylase was assessed for potential allergenicity by amino acid sequence comparison with known allergens listed in publicly available protein databases. No immunologically significant sequence homology was detected.

2.2 Toxicological studies

2.2.1 Acute toxicity

No information was available.

2.2.2 Short-term toxicity

Rats

According to a summary report, a 14-day range-finding study in rats was performed (Elvig-Jørgensen & Pedersen, 2002). However, this study was not submitted.

Groups of 10 male and 10 female Sprague-Dawley rats (aged 6 weeks) were given water containing LE399 alpha-amylase (batch PPY 7075) at a dose of 0, 1, 3.3, or 10 ml/kg bw per day (equivalent to a dose of 0, 304, 1004, or 3040 KNU(T)/kg bw per day, or 0, 102, 336, or 1020 mg/kg bw per day expressed as TOS (total organic solids from the fermentation; mainly protein and carbohydrate components)) by gavage for 13 weeks. The study was performed according to OECD test guideline 408 (1998), and was certified for compliance with good laboratory practice (GLP) and quality assurance. All animals were observed twice daily for morbidity and mortality and daily for clinical signs and reactions to treatment. Ophthalmoscopy was performed before treatment on all animals and during week 13 on all animals in the control group and in the group receiving the highest dose. All animals underwent functional and behavioural tests during weeks 4 and 13. Body weight and food consumption were recorded weekly. Food conversion efficiency was calculated. Food and water were freely available. At termination of treatment, haematology and clinical chemistry were performed in all animals. Absolute and relative (to body weight) weights of 12 organs were determined. All animals were examined macroscopically. Microscopy was carried out on about 35 organs/tissues from all animals in the control group and the group receiving the highest dose, and on all macrospically abnormal tissues.

No effects on survival were seen. Ophthalmoscopy was normal. Functional and behavioural tests did not reveal any abnormalities. Body-weight gain and food consumption were normal. Haematology and clinical chemistry did not show abnormalities, organ weights were normal and macroscopy did not reveal any effects related to treatment. Microscopy of the stomach revealed minimal to slight dilatation of the fundic glands (crypt dilatation) in males and females in both the control group and the group receiving the highest dose. When compared with the control animals, the incidence and severity of this effect were similar in treated females, but slightly increased in treated males. As this effect is a very common finding in rats, its incidence and severity increasing with age, the Committee did not consider the slight variation in the incidence and severity between control and treated males to be toxicologically significant. It can be concluded that in this 13week study in rats treated orally, the NOEL for LE399 alpha-amylase (batch PPY 7075) was the highest dose, 10 ml/kg bw per day (equivalent to 3040 KNU(T)/kg bw per day, or 1020 mg/kg bw per day, expressed as TOS) (Christ, 2002; Elvig-Jørgensen & Pedersen, 2002).

2.2.3 Studies of long-term toxicity and carcinogenicity

No information was available.

2.2.4 Genotoxicity

The results of two studies of genotoxicity in vitro with LE399 alpha-amylase (batch PPY 7075) are summarized in Table 3. Both studies followed OECD test guidelines, 471 (1997) and 473 (1997), respectively, while only the test for chromosomal aberration was certified for compliance with GLP and quality assurance.

Table 3. Results of studies of genotoxicity with LE399 alpha-amylase (batch PPY 7075)

Endpoint

Test object

Concentration

Results

References

In vitro

 

 

 

 

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA

156–5000 mg/ml for Salmonella strains and 156–5000 mg/ plate for E. coli. Solvent: sterile water

Negativea

Pedersen (2002); Elvig-Jørgensen & Pedersen (2002)

Chromosomal aberration

Human lymphocytes

In first experiment: 3200, 4000, and 5000 mg/ml, ±S9. In second experiment: 1000, 1100, and 1500 mg/ml S9, and 3613, 4250, and 5000 mg/ml +S9. Solvent: sterile water

Negativeb

Whitwell (2001); Elvig-Jørgensen & Pedersen (2002)

S9, 9000 × g supernatant of rat liver homogenate

a

In the presence and absence of metabolic activation from S9; no cytotoxicity was seen. Owing to the presence of free amino acids (e.g. histidine and tryptophan) in the alpha-amylase preparation, the growth of Salmonella strains requiring histidine was significantly increased after direct-plate incorporation. Therefore, the Salmonella strains were exposed to the alpha-amylase preparation in a phosphate-buffered nutrient broth in liquid culture ("treatandplate assay") at six concentrations (highest dose, 5 mg/ml) for 3 h. After incubation, the test substance was removed by centrifugation before plating. Stimulation of growth of E. coli strains requiring tryptophan was only weak and insignificant

b

In the presence and absence of metabolic activation from S9. In the first experiment, cells were treated for 3 h in the absence and presence of S9 and were harvested 17 h later. Mitotic inhibition was 0–18%. In the second experiment, cells were exposed continuously for 20 h in the absence of S9 and then harvested (mitotic inhibition, 54% at 1500 mg/ml), or treated for 3 h in the presence of S9 and harvested 17 h later (mitotic inhibition, 28% at 5000 mg/ml)

2.2.5 Reproductive toxicity

No information was available.

2.3 Observations in humans

No information was available.

3. DIETARY INTAKE

alpha-Amylases have been used for many years by the starch, sugar and alcoholicbeverage industries (Association of Manufacturers and Formulators of Enzyme Products, 2003). In Australia and New Zealand, there are currently a number of approved sources for alpha-amylases listed as processing aids in Standard 1.3: Processing Aids (Food Standards Australia New Zealand, 2003b).

The Food & Drug Administration of the United States received GRAS notices for alpha-amylase produced by a strain of Bacillus licheniformis in 2001 (Food & Drug Administration, 2003). A complete and comprehensive list of enzymes and their uses in food manufacturing in the European Union is not available, but an inventory of enzyme use in nine Member States was compiled for scientific cooperation (SCOOP) Task 7.4 (European Commission, 2000). A number of alpha-amylase enzyme preparations were reported to be in use and, in particular, the specific alpha-amylase enzyme preparation under evaluation was reported to be used in France in biscuits, beer, pulse juices, fruit juices, concentrated fruit juices, dehydrated fruit juices, fruit nectar and syrups. In Germany, Greece and Spain, the specific alpha-amylase enzyme preparation was reported to be used in beverages, cereals and starch, sugar and honey.

The alpha-amylase preparation is intended for use in starch liquefaction in the production of sweetener syrups, as well as in the production of alcoholic beverages and beer. The absence of the alpha-amylase protein in the final (purified) syrup has been experimentally confirmed. No LE399 alpha-amylase or other organic solids are expected to be present in alcoholic beverages that have been distilled because ethanol is removed from the fermentation mash by distillation. In the brewing of beer, the LE399 alpha-amylase is active in the mash. It is subsequently inactivated during wort boiling and removed during beer purification. Therefore, no residual LE399 alpha-amylase is expected to be present in food processed using this enzyme preparation. The reviewer was not aware of any other uses for the LE399 alpha-amylase in which the enzyme might persist in the final product. Nevertheless, very conservative estimates of intake were perfomed.

Recommended dosage and TOS content of the enzyme preparation were provided by the sponsor (Nielsen, 2002a, 2000b).

A "worst-case" scenario for human intake from sugar and sweeteners was estimated on the basis of the following assumptions:

According to the budget method, the upper physiological intake of calories is 100 kcal/kg bw per day, corresponding on average to an intake of 50 g of food per kg bw per day (Hansen, 1979). For this "worst-case" scenario, it was assumed that 20% of these calories derive from sugar, corresponding to a sugar intake of 5 g/kg bw and thus leading to an intake of 0.2 mg of TOS/kg bw per day (1000× 4% × 0.005), i.e. 12 mg of TOS per day for a 60-kg person.

A "worst-case" scenario for intake from beer was estimated on the basis of the following assumptions:

According to the budget method, the upper physiological intake of liquid is 100 ml/kg bw per day (6 l for a 60-kg person) (Hansen, 1979). A "worst-case" scenario is that of ingestion of 50 ml/kg bw per day of beer, leading to an intake of 0.05 mg of TOS/kg bw per day (500 × 350 × 4% × 0.05/6660), i.e. 3 mg of TOS per day for a 60-kg person.

A "worst-case" scenario for intake from alcohol in spirits was estimated on the basis of the following assumptions:

A "worst-case" scenario is that of ingestion of 0.5 l of spirit, leading to an intake of 0.18 mg of TOS/kg bw per day ([0.5/60] × 0.35 × 4% × 1000 × 1000/640), i.e. 10.9 mg of TOS per day for a 60-kg person.

Therefore, the use of extremely conservative estimates of daily intakes based on the assumption that all TOS would persist in sugars and syrups, in beer and in spirits, and considering very high intakes of these products, lead to an estimated cumulative intake of 0.43 mg of TOS/kg bw per day. Compared with the NOEL of 1.02 g of TOS/kg bw per day from the 13week study of oral toxicity, the margin of safety is >2000.

4. COMMENTS

Toxicological studies were conducted on the LEC. The materials added to the LEC for stabilization and formulation and standardization have either been evaluated previously by the Committee or are common food constituents and do not raise safety concerns.

In a 13-week study in rats, no significant treatment-related effects were seen when the LEC was administered by oral gavage at doses of up to and including 10 ml/kg bw per day, the highest dose tested. Therefore this highest dose (equivalent to an intake of 1.02 g of TOS/kg bw per day) was considered to be the NOEL. The LEC was not mutagenic in an assay for mutagenicity in bacteria in vitro and was not clastogenic in an assay for chromosomal aberrations in mammalian cells in vitro.

The alpha-amylase preparation is intended for use in starch liquefaction in the production of sweetener syrups, alcoholic beverages and beer. The absence of the alpha-amylase protein in the final (purified) sweetener syrup has been confirmed experimentally. In the spirits industry, no LE399 alpha-amylase or other organic solids are expected to be carried over to the final product because ethanol is removed by distillation from the fermentation mash containing the enzyme preparation. In the brewing of beer, the enzyme preparation is added during the mashing process and is denatured and inactivated during the subsequent wortboiling stage. The beer filtration process is likely to remove the denatured enzymes along with other insoluble materials. In conclusion, no residual LE399 alpha-amylase is expected to be present in food processed using this enzyme preparation.

Very conservative estimates of daily intakes were performed on the basis of the assumption that all TOS would persist in the final products. These gave an estimated daily intake of 12 mg of TOS/day (equivalent to 0.2 mg/kg bw per day) for sugar and syrups, 3 mg/day (equivalent to 0.05 mg/kg bw per day for a 60-kg person) for beer and 10.8 mg/day (equivalent to 0.18 mg/kg bw per day) for spirits. Compared with the NOEL of 1020 mg of TOS/kg bw per day derived from the 13-week study of oral toxicity, the margin of safety is >2000.

5. EVALUATION

The Committee allocated an ADI "not specified" to alpha-amylase from this recombinant strain of B. licheniformis, used in the applications specified and in accordance with good manufacturing practice.

6. REFERENCES

Association of Manufacturers and Formulators of Enzyme Products (2003) Enzymes used in food (http://www.amfep.org/enzymes/enz3.html).

Christ, M. (2002) Alpha-amylase LE 399—13 week oral (gavage) toxicity study in the rat. Unpublished report No. 412/002 from MDS Pharma Services, L’Arbresle Cedex, France. Submitted to WHO by Novozymes A/S, Bagsvaerd, Denmark.

Elvig-Jørgensen, S. & Pedersen, P.B. (2002) Summary of toxicity data. Alpha-amylase LE399 PPY 7075. Unpublished report No. 2002-49708-01 from Novozymes A/S, Bagsvaerd, Denmark. Submitted to WHO by Novozymes A/S, Bagsvaerd, Denmark.

European Commission (2000) Report on task for scientific cooperation (SCOOP). Report of experts participating in Task 7.4. Study of the enzymes used in foodstuffs and collation of data on their safety (available at http://www.europa.eu.int/comm/food/fs/scoop/ index_en.html).

Food & Drug Administration (2003) List of the substances that are the subject of each GRAS Notice (http://www.cfsan.fda.gov/~rdb/opagras.html).

Food Standards Australia New Zealand (2003a) Approved genetically modified processing aids and food additives and their use. Canberra (http://www. foodstandards.gov.au/whatsinfood/gmfoods/approvedgmprocessing1031.cfm).

Hansen, S.C. (1979) Conditions for use of food additives based on a budget for an acceptable daily intake. J. Food Protect., 42, 429–434.

Nielsen, K.R. (2002a) An alpha-amylase from Bacillus licheniformis (Liquozyme). Data relevant to the specification for the identity and purity of a protein engineered alpha-amylase expressed by Bacillus licheniformis. Unpublished report No. 2002-49722-01 from Novozymes A/S, Bagsvaerd, Denmark. Submitted to WHO by Novozymes A/S, Bagsvaerd, Denmark.

Nielsen, K.R. (2002b) Alpha amylase enzyme preparation from Bacillus licheniformis (Liquozyme). Data relevant to the specification for the identity and purity of a protein engineered alpha amylase expressed by Bacillus licheniformis. Unpublished report No. 2002-50717-01 from Novozymes A/S, Bagsvaerd, Denmark. Submitted to WHO by Novozymes A/S, Bagsvaerd, Denmark.

Pedersen, P.B. (2002) Alpha Amylase (batch number: PPY 7075): Test for mutagenic activity with strains of Salmonella typhimurium and Escherichia coli. Unpublished report No. 20018049 from Novozymes A/S, Bagsvaerd, Denmark. Submitted to WHO by Novozymes A/S, Bagsvaerd, Denmark.

Whitwell, J. (2001) Alpha-Amylase. Induction of chromosome aberrations in cultured human peripheral blood lymphocytes. Unpublished report No. 1974/7-D6172 from Covance Labs. Ltd., Harrogate, England. Submitted to WHO by Novozymes A/S, Bagsvaerd, Denmark.



    See Also:
       Toxicological Abbreviations