Safety evaluation of pullulanase enzyme preparation derived from Bacillus licheniformis containing the pullulanase gene from Bacillus deramificans.

Pullulanase enzyme is an amylopectin debranching enzyme used in starch hydrolysis. This article describes studies conducted to investigate the safety of a pullulanase enzyme preparation produced by a strain of Bacillus licheniformis that has been transformed by introduction of genetic material from another Bacillus species, B. deramificans. A 4-week dietary toxicity study in rats was conducted in which test animals received pullulanase in the feed at concentrations of 0.2, 1.0, and 5.0%. No adverse treatment-related effects were observed. Lack of genetic toxicity potential was demonstrated by the results of a bacterial mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, in an in vitro histidine forward mutation study in mouse lymphoma cells, and in in vivo mouse bone marrow chromosome aberration and micronucleus assays. The enzyme preparation also has been shown to be a nonirritant in eye and primary dermal irritation tests in rabbits and is nontoxic by inhalation exposure. Finally, the genetically altered B. licheniformis has been demonstrated to be nonpathogenic upon single intraperitoneal injection to rats of both live and killed cells at doses up to 10(11) cells/kg. The results of these studies demonstrate that the enzyme preparation may be considered safe when employed in starch processing.