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    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.


    See Also:
       Toxicological Abbreviations
       alpha-AMYLASE FROM BACILLUS STEAROTHERMOPHILUS EXPRESSED IN BACILLUS SUBTILIS (JECFA Evaluation)