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)