ALPHA-AMYLASE FROM BACILLUS MEGATERIUM 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 B1-109 (ATCC 39,701) containing plasmid pCPC 800 [amylase
gene from B. megaterium (NCIB 11568)] and regulating sequences
from alpha-amylase of B. stearothermophilus (see Appendix 2) has
not been previously evaluated by the Committee. The Committee
reviewed the available data pertaining to the genetic modification
procedures employed, characterization of the producing organisms,
the fermentation process, and acute short-term 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
2.2.1.1 Rat
Groups of 6 male and 6 female rats (Fischer 344) were dosed by
gavage with the enzyme as an aqueous suspension at dose levels
ranging from 0 to 12 g/kg b.w. (alpha-amylase activity 5130 U/g).
There was no mortality and the acute LD50 was determined to be
greater than 12 g/kg b.w. (Weltman, 1986a).
2.2.2 Short term studies
2.2.2.1 Rat
Groups of 5 male and 5 female rat (Fischer 344) were exposed to
enzyme levels of either 0.0, 20.0, 60.0 or 100.0 U/g Bacillus
megaterium for 14 days (equivalent to 0, 1, 3 or 5% liquid use
product or approximately 0, 0.32, 0.99 or 1.57 g/kg b.w./day), in
the diet (alpha-amylase 6000 U/g). All animals were observed at
least twice daily and body weight and food consumption were recorded
periodically throughout the study. There were no significant
differences between treated control groups in body weight and a
slight lowering of food consumption in females of the low dose
group. The author concluded that there was no effect on
palatability (Weltman, 1986b).
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 either 0.0, 20.0, 60.0 or
100.0 U/g Bacillus megaterium for 15 days (approximately 0, 0.12,
0.29 or 0.74 g/kg b.w./day) in the diet (alpha-amylase 6000 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 (Weltman, 1986c).
Groups of 4 male and 4 female dogs (Beagle dogs, 6-7 months of
age) were exposed to enzyme at levels of 0, 20, 60 or 100 U/g
Bacillus megaterium amylase based on 5100 units of amylase/g of
test material for 13 weeks in the diet (approximately 0, 0.12, 0.32
or 0.57 g/kg b.w./day). All animals were observed at least twice
daily, body weight and food consumption were recorded periodically
throughout the study, 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, food consumption, clinical chemistry
and haematological parameters. There were no treatment-related
clinical observations, pathological changes or histopathological
observations. The authors determined a no effect level of 100 U/g
diet (Weltman, 1986d; MacKenzie et al., 1989).
2.2.3 Reproduction studies
2.2.3.1 Rat
Groups of 25 male and 25 female rats (Fischer 344,
approximately 6 weeks old) were exposed to alpha-amylase at levels
of 0, 20, 60 or 100 U/g Bacillus megaterium amylase based on 5100
units of amylase/g of test material (approximately 0, 0.27, 0.88 or
1.35 g/kg b.w./day) for 4 weeks and then allowed to mate. All
animals were observed at least twice daily, body weight and food
consumption recorded periodically throughout the study, and blood
samples were collected for clinical chemistry and haematology during
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 1
pup/sex/litter selected at random for continuation on 13 weeks of
exposure. There were no consistent treatment-related effects in the
F0 animals in body weight, or food consumption. There were no
treatment related reproductive effects. Kidney weights from the F0
females of the high dose group were significantly higher (but only
marginally) than controls. There was a higher incidence of
mineralization of kidney cortex in F0 females of the high dose
group compared to controls but mineralization in the 60 U/g group
was less than that of the controls. These changes were not
considered to be toxicologically significant. There were no other
treatment related effects in either F0 or F1 animals. There were
no significant treatment related effects on the F1 animals for body
weight food consumption, haematology, blood clinical chemistry,
pathology or histopathology. The authors concluded that the NOEL
for Bacillus megaterium amylase in rats exposed in utero and for
13 weeks after weaning is greater than 100 U/kg, which is equivalent
to approximately 1.35 g/kg b.w./day (Weltman, 1986e; MacKenzie et
al., 1989).
Appendix 2
Molecular Procedures used in cloning amylase from B. megaterium
to B. subtilis:
DNA from amylase-producing B. megaterium (NCIB 11568) and DNA
from phage lambda NM 590 were cleaved with the same restriction
enzyme, the DNA's mixed and ligated. The resulting chimaeric
lambda- B. megaterium amylase gene DNA was encapsidated in vitro
to produce biologically active phage particles. The phage particles
were used to infect E. coli HB101 and the transformed bacteria
were plated onto starch agar to screen for amylase activity (starch
digestion). The amylase gene was subcloned into plasmid pBR322
(conferring ampicillin resistance) by mixing amylase encoding lambda
DNA with pBR322 DNA followed by ligation. The resulting vector was
used to transform E. coli HB101 cells, and recombinant bacteria
selected for ampicillin resistance and amylase activity. DNA from
the amylase-bearing pBR322 plasmid and DNA from pUB110 were cleaved
with restriction endonucleases and ligated and used to transform
Bacillus subtilis. Transformants were identified by resistance to
kanamycin as well as by starch digestion activity. One of the
isolated transformed clones was designated pAMY100 (containing DNA
form pUB110, pBR322 and the amylase gene from B. megaterium ).
The B. stearothermophilus alpha-amylase gene, including its
regulatory sequences was inserted into the plasmid pUB110 to form
plasmid pCPC717. A fragment of the alpha-amylase gene was deleted
whereby no alpha-amylase was produced. The resulting plasmid was
pCPC 721. Plasmid pAMY100 was digested, mixed with pCPC71 and
ligated to form a chimaeric intermediate plasmid that contained
pUB110 from pAMY100, pUB100 from pCPC721, a 31 base pair fragment
from pBR322, amylase from B. megaterium and truncated alpha-
amylase from B. stearothermophilus. The promoter for B.
stearothermophilus lies ahead of the B. megaterium amylase gene
and the promoter from B. megaterium lies ahead of the B.
stearothermophilus alpha-amylase gene in the chimaeric plasmid.
The chimaeric plasmid was digested and religated to reverse the
orientation (from counterclockwise to clockwise) of a small
restriction fragment of pUB100 (derived from pAMY100). The
reoriented chimaeric plasmid was introduced into Bacillus subtilis
and by homologous recombination, repeated sequences of pUB100 from
pCPC720 and from pAMY100 were deleted. The resulting plasmid had
B. megaterium amylase, 31 bases from pBR322, the promoter region
of B. stearothermophilus, a truncated beta region and single
copies of alpha, kanamycin resistance, bleomycin resistance from
derived pUB100 from pCPC721 in addition to a beta region from pUB100
derived from pAMY100. This plasmid was designated pCPC801.
Finally, digestion of a part of the kanamycin and bleomycin genes
from pCPC801 was used to inactivate these antibiotic resistance
phenotypes. The final construct plasmid, pCPC800, had amylase from
B. megaterium, regulatory sequences from alpha-amylase of B.
stearothermophilus, 31 bases from pBR322 and pUB110 with kanamycin
or bleomycin sensitivity. This plasmid was introduced into the B.
subtilis asporogenic strain B1-109 and the host tested for amylase
activity and resistance to kanamycin and bleomycin.
Other information
Bacillus subtilis B1-109 and Bacillus subtilis B1-109 (ATCC
39,701) containing plasmid pCPC800 had the same susceptibility to
ampicillin and tetracycline.
Bacillus subtilis (B1-109 (ATCC 39,701) containing plasmid
pCPC800 exhibited no virulence potential when tested by
intraperitoneal injection and oral intubation in Balb/c mice.
Bacillus subtilis B1-109 (ATCC 39,701) containing plasmid
pCPC800 demonstrated no detectable cytotoxicity against vero cells
in 7 days of assays of shiga-like toxin. No viable cells and
transformable DNA of the production strain B. subtilis containing
plasmid pCPC800 were detected in the alpha-amylase product.
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 plasmid construct pCPC800,
containing the alpha-amylase gene, was introduced into B.
megaterium and the promoter region of the alpha-amylase gene from
B. stearothermophilus, was introduced into B. subtilis (ATCC 39
701) by standard transformation procedures. Data indicating the
absence of antibiotic resistance, production of "Shiga-like" toxin,
and infectivity potential of the alpha-amylase producing
microorganism were provided.
The B. subtilis was grown under properly controlled
conditions in media containing ingredients commonly used in the
production of food-grade substances by fermentation. Tfhe
fermentation broth was filtered and the filtrate lyophilized before
being mixed into the test diets. No viable cells or plasmid DNA
could be detected in the amylase product.
The lyophilized preparation produced no significant
toxicological effects in a 13-week study in dogs at levels of up to
0.57 g/kg b.w./day, nor in a one-generation (one-litter)
reproduction study in rats in which some of the offspring were
treated at levels up to 1.5 g/kg b.w./day for 13 weeks after
weaning.
4. EVALUATION
The Committee allocated an ADI "not specified" for this enzyme
preparation.
5. REFERENCES
BALBAS,P., SOBERON, X., MERINO, E., ZURITA, M., LOMELI, H., VALLE,
F., FLORES, N., & BOLIAR, F. (1987). Plasmid vector pBR322 and its
special-purpose derivatives - a review. Gene, 50, 3-40.
BAND, L., & HENNER, D.J. (1984). Bacillus subtilis requires a
stringent: Shine-Dalgarno region for gene expression. DNA, 3(1),
17-21.
MacKENIZE, K.M., PETSEL, R.W., WELTMAN, R.H., & ZEMAN, N.W. (1989).
Subchronic toxicity studies in dogs and in utero-exposed rats fed
diets containing Bacillus megaterium amylase derived from a
recombinant DNA organism. Fd. Chem. Toxicol., 27, 301-305.
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, NY.
WELTMAN, R.H. (1986a). Acute oral toxicity study in rats.
Unpublished report No. 6519-110 from Hazleton Laboratories America,
Inc. Madison, Wisconsin, USA. Submitted to WHO by CPC
International, Englewood Cliffs, NJ, USA.
WELTMAN, R.H. (1986b). Fourteen-day palatability study in rats.
Unpublished report No. 6159-106 from Hazleton Laboratories America,
Inc. Madison, Wisconsin, USA. Submitted to WHO by CPC
International, Englewood Cliffs, NJ, USA.
WELTMAN, R.H. (1986c). Fourteen-day palatability study in dogs.
Unpublished report No. 6159-108 from Hazleton Laboratories America,
Inc. Madison, Wisconsin, USA. Submitted to WHO by CPC
International, Englewood Cliffs, NJ, USA.
WELTMAN, R.H. (1986d). Subchronic toxicity study in dogs.
Unpublished report No. 6159-109 from Hazleton Laboratories America,
Inc. Madison, Wisconsin, USA. Submitted to WHO by CPC
International, Englewood Cliffs, NJ, USA.
WELTMAN, R.H. (1986e). Subchronic toxicity study in utero exposed
F1 rats. Unpublished report No. 6159-107 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 amylase of
Bacillus megaterium derived from Bacillus subtilis. Submitted
to WHO by Enzyme Bio-Systems Ltd. Arlington, Heights, IL, USA.
See Also:
Toxicological Abbreviations
alpha-AMYLASE FROM BACILLUS MEGATERIUM EXPRESSED IN BACILLUS SUBTILIS (JECFA Evaluation)