ALPHA-AMYLASE FROM BACILLUS STEAROTHERMOPHILUS
EXPRESSED IN BACILLUS SUBTILIS
1. EXPLANATION
Enzymes used for the hydrolysis of starch, generally called
amylases, have a long history of use by the food industry. The
amylase catalyzes the hydrolysis of 1,4 alpha-glucosidic linkages in
common polysaccharide. Bacterial (Bacillus subtilis) alpha-
amylase has been in common use to control the viscosity of chocolate
syrup since 1929 and in the brewing industry since 1936. The enzyme
preparation derived from these various Bacillus strains is usually
added directly to the food to be processed and then removed from the
final product by filtration. This alpha-amylase from Bacillus
subtilis ATCC 39,705 containing plasmid pCPC720 from Bacillus
stearothermophilus ATCC 39,709 has not been previously evaluated
by the Joint FAO/WHO Expert Committee on Food Additives. In its
meeting, the Committee reviewed the available data pertaining to the
genetic modification procedures employed, characterization of the
producing organisms, the fermentation process, and reproduction
studies with the lyophilized enzyme preparation.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
No information available.
2.2 Toxicological studies
2.2.1 Acute toxicity studies
2.2.1.2 Rat
Groups of 4 male and 4 female rats (Fischer 344) were dosed by
gavage with the enzyme as an aqueous suspension at dose levels
ranging from 0 to 6 g/kg b.w. (alpha-amylase activity 5800 U/g).
There was no mortality and the acute LD50 was determined to be
greater than 6 g/kg b.w. (Skalitzky, 1984).
2.2.2 Short term studies
2.2.2.1 Rat
Groups of 5 male and 5 female rats (Fischer 344, 28 days of
age) were exposed to enzyme levels of 0, 0.62 and 1.24% in the diet
for 2 weeks (alpha-amylase 5800 U/g). All animals were observed at
least twice daily and body weight and food consumption recorded
weekly throughout the study. There were no significant differences
between treated and control groups in body weight; there was a
slight lowering of food consumption in females of the low dose
group. The author concluded that there was no effect on
palatability (Weltman, 1984).
2.2.2.2 Dog
Groups of 1 male and 1 female dog (Beagle dogs, 6-7 months of
age) were exposed to enzyme levels of 0, 0.62 and 1.4% in the diet
for 2 weeks (alpha-amylase 5800 U/g). All animals were observed at
least twice daily and body weight and food consumption recorded
periodically throughout the study. There were no significant
differences between treated and control groups in body weight or
food consumption. The author concluded that there was no effect on
palatability (Dickie, 1984a).
Groups of 4 male and 4 female dogs (Beagle dogs, 6-7 months of
age) were exposed to enzyme at levels of 0, 0.36, and 0.72% in the
diet for 13 weeks (alpha-amylase activity 10000 U/g). All animals
were observed at least twice daily; body weight and food consumption
were recorded weekly throughout the study, ophthalmic examinations
were done prior to dosing and at termination, and blood samples were
collected for clinical chemistry and haematology prior to dosing and
at termination of the study. There were no significant differences
between treated and control groups in body weight, clinical
observations, and haematological parameters. Food consumption was
significantly increased during week 11 for males in the high dose
group, while there was significant decrease for females in the high
dose group during week 3 and in the low dose group for weeks 4, 5,
11 and 12. There was a significant reduction in serum calcium and
inorganic phosphorus concentrations for males in both dose groups
but changes were not considered to be toxicologically significant.
There were no treatment related clinical observations, gross
pathological changes or histopathological observations. The author
concluded that the changes observed were not clinically significant
and determined a NOEL of 0.72% in the diet (0.20 g/kg b.w./day)
(Dickie, 1984b).
2.2.3 Long-term/carcinogenicity studies
No information available.
2.2.4 Reproduction studies
2.2.4.1 Rat
Groups of 26 male and 26 female rats (Fischer 344 weanling
rats) were exposed to alpha-amylase at levels of 0, 0.36 and 0.72%
in the diet (alpha-amylase activity 10000 U/g) for 4 weeks and then
allowed to mate. All animals were observed at least twice daily;
body weight and food consumption were recorded periodically
throughout the study. Blood samples (10/sex/group) were collected
for clinical chemistry and haematology during the F1 component (45
days post weaning and at termination) of the study. Pups were
culled at random at day 4 to achieve a maximum litter size of 8.
Pups were weaned at 28 days of lactation and 2 pups/sex/litter were
selected at random for continuation for 14 weeks of exposure. There
were no consistent treatment-related effects in the F0 animals in
body weight, except for a reduction in food consumption for females
in the high dose group over days 14-20 of gestation. There were no
treatment-related reproductive effects. Pup weights in the high-
dose group were significantly lower than controls on days 14, 21,
and 28 and body weight gains were also reduced between days 4 and
14. There were no significant treatment related effects on the F1
animals for body weight, food consumption, haematology, blood
clinical chemistry, urinalysis, pathology or histopathology. The
author concluded that a NOEL of 0.72% in the diet (72 U/g or 0.50
g/kg b.w./day) could be established (Weltman, 1985).
APPENDIX 1
Molecular Procedures used in cloning alpha-amylase from B.
stearothermophilus to B. subtilis:
The alpha-amylase gene, located on a small plasmid from a non-
pathogenic strain of Bacillus stearothermophilus (ATCC 39,709),
was introduced into an Escherichia coli plasmid pBR327 (conferring
ampicillin-resistance) and used to transform the non-pathogenic,
non-toxigenic E. coli strain K12 (used as the intermediate host).
Plasmids containing a DNA insert from E. coli K12 that are
ampicillin-resistant and have alpha-amylase activity, when tested on
starch plates, were trimmed with restriction enzymes to obtain a
plasmid with the alpha-amylase gene (plasmid pCPC611). The plasmid
was digested with restriction enzymes and introduced into the B.
subtilis cloning vector pUB110 (conferring kanamycin-resistance).
The vector pUB110 has been used as a cloning vehicle for B.
subtilis for several years and is well characterized. Several
different sized vectors were obtained that contained alpha-amylase
activity. The plasmid with highest alpha-amylase activity, plasmid
pCPC704 had most of pBR327 DNA eliminated (thus lost ampicillin-
resistance), and contained all of pUB110 (conferred kanamycin-
resistance), and an active portion of the B. stearothermophilus
alpha-amylase gene (starch digesting). The pCPC704 was further
restricted to remove B. stearothermophilus DNA flanking the alpha-
amylase, and to remove part of the kanamycin-resistance gene as well
as other non-selected genes of pUB110 (such as bleomycin-
resistance). The final plasmid construct, pCPC720 contained a 2.4
kb portion of alpha-amylase gene with its own regulatory sequences
and a 2.6 kb portion of the pUB110 vector that lacks kanamycin-
resistance activity. pCPC720 was introduced into an asporogenic,
non-pathogenic and non-toxigenic strain of B. subtilis B1-109
(ATCC 39,701[Sac A321, AmyE]). The transformed B. subtilis cells
were shown to digest starch and were sensitive to kanamycin.
Other information
Concentrated cell supernatants from B. subtilis (ATCC 39,705)
did not contain any material that reacted with specific antibodies
raised against purified staphylococcal enterotoxins A, B, C or D.
Bacillus subtilis (ATCC 39,705) demonstrated no cytotoxicity
against Vero cells in the 4 days of assay for shiga-like toxin.
3. COMMENTS
The Committee noted that a well-documented non-pathogenic and
non-toxigenic strain of microorganisms had been employed in the
genetic modification procedures. The vector used, pUB110, is well-
characterized and has been used for several years as a cloning
vehicle for B. subtilis. The plasmid construct pCPC720,
containing the alpha-amylase gene, was introduced into B. subtilis
(ATCC 39 705) using standard transformation procedures. The
available data indicated the absence of antibiotic resistance, of
production of "Shiga-like" toxin and of production of enterotoxins
A, B, C and D by the alpha-amylase producing microorganism.
The B. subtilis was grown under properly controlled
conditions in media containing ingredients commonly used in the
production of food-grade substances by fermentation. The
fermentation broth was filtered and the filtrate lyophilized before
being mixed into the test diets.
When compared with alpha-amylase from B. stearothermophilus
(ATCC 39 709), the lyophilized preparation was shown to possess the
same enzyme-specific activity, relative molecular mass, peptide
maps, and reactivity towards antibody raised against alpha-amylase
from B. stearothermophilus (ATCC 39 709). This preparation
produced no significant toxicological effects in a 13-week feeding
study in dogs at levels of up to 0.20 g per kg of body weight per
day, nor in a one-generation (one-litter) reproduction study in rats
in which some of the offspring were treated at levels up to 0.50
g/kg b.w./day for 14 weeks after weaning.
4. EVALUATION
The Committee allocated an ADI "not specified" for this enzyme
preparation.
5. REFERENCES
DICKIE, B.C. (1984a). Fourteen-day palatability study in dogs.
Unpublished report No. 6159-103 from Hazleton Laboratories America,
Inc. Madison, Wisconsin, USA. Submitted to WHO by CPC
International, Englewood Cliffs, NJ, USA.
DICKIE, B.C. (1984b). Subchronic toxicity study in dogs.
Unpublished report No. 6159-100 from Hazleton Laboratories America,
Inc. Madison, Wisconsin, USA. Submitted to WHO by CPC
International, Englewood Cliffs, NJ, USA.
McKENZIE, T., HOSHINO, T., TANAKA, T., & SUEOKA, N. (1986). The
nucleotide sequence of pUB110: some salient features in relation to
replication and its regulation. Plasmid 15, 93-103.
McKENZIE, T., HOSHINO, T., TANAKA, T., & SUEOKA, N. (1987). A
revision of the nucleotide sequence and functional map of pUB110.
Plasmid, 17, 83.
OLD, R.W., & PRIMROSE, S.B. (1987). Principles of Gene
Manipulation, 3rd Edition, Blackwell Press, Oxford.
SAMBROOK, J., FRITSCH, E.F., & MANIATIS, T. (1989). Molecular
Cloning - A Laboratory Manual, 2nd Edition, CSHL Press, Cold Spring
Harbor, N.Y., USA
SKALITZKY, D.E. (1984). Acute oral toxicity study with Bacillus
subtilis alpha-amylase in rats. Unpublished report No. 6519-102
from Hazleton Laboratories America, Inc. Madison, Wisconsin, USA.
Submitted to WHO by CPC International, Englewood Cliffs, NJ, USA.
WELTMAN, R.H. (1984). Fourteen-day palatability study in rats.
Unpublished report No. 6159-104 from Hazleton Laboratories America,
Inc. Madison, Wisconsin, USA. Submitted to WHO by CPC
International, Englewood Cliffs, NJ, USA.
WELTMAN, R.H. (1985). Subchronic toxicity study in utero exposed
F1 rats. Unpublished report No. 6159-101 from Hazleton
Laboratories America, Inc. Madison, Wisconsin, USA. Submitted to
WHO by CPC International, Englewood Cliffs, NJ, USA.
ZEMAN, N.W. (1990). Additional safety information on the Alpha-
Amylase of Bacillus stearothermophilus derived from Bacillus
subtilis. Submitted to WHO by Enzyme Bio-Systems Ltd., Arlington
Heights, IL, USA.