Initial studies on an amylopullulanase from Bacillus sp. DSM 405.

Pullulanase. a debranching enzyme that specifically cleaves a 1.6 linkages in amylopectin and pullulan, differs from a-amylase which hydrolyses the a 1,4 linkages of starch, amylose and amylopectin [ I ] . Pullulanases are inactive against linear a 1,4 linkages and a-amylases do not hydrolyse pullulan. Some novel amylolytic enzymes capable of hydrolysing both starch and pullulan have been discovered and, depending on the end-products formed on hydrolysis of pullulan, they can be classed into one of two groups [2] :1. Neopullulanases : These enzymes have activity on a 1,4 linkages in soluble starch and can also hydrolyse pullulan at a 1,4 linkages releasing the trisaccharide panose as the end-product [31 ; 2. Amylopullulanases : These enzymes hydrolyse the a I ,4 linkages in soluble starch but they can also hydrolyse the a 1,6 linkages in pullulan producing maltotriose as sole, and typically pullulanase type, end-product [4]. Both enzyme activities arise from the one protein but not necessarily from the one active site. Amylopullulanase is a novel type of enzyme which appears to be able to switch the type of bond it hydrolyses depending on the substrate presented to it. To date fourteen amylopullulanases have been described, and of these fourteen, five are produced by Bacillus spp. Bacillus sp. DSM 405 produces one such novel amylopullulanase enzyme. Buc.il1u.s sp. DSM 405 was grown at 6OoC and 200 rpm in a New Brunswick orbital shaker incubator in the following optimised medium conhining (g/L) : corn steep liquor. I .O; maltose, I .O; NH4N03, 1 .O; KH2P04, 0.5: NaCI. 0.25; MgS04.6H20, 0.25; FeS04, 0.05; CaCl2.2H.10, 0.05 and l m l L of IM NazSiOj, at an initial pH of 7.0. Along with the production of the extracellular a-amylase and pullulanase activities, low levels of maltase and a-glucosidase activities were detected, however these were produced intracellularly and were removed in the first step of purification. The amylopullulanase was purified via a series of steps including starch adsorption, ultrafiltration (UF), hydrophobic interaction chromatography (HIC) and gel filtration by FPLC (fast protein liquid chromatography) on a Superose 12 prep column. This protocol resulted in a 1,400 fold increase in the specific activity of the amylopullulanase (Table I). The two enzyme activities were associated throughout the purification protocol, with the ratio of the two activities remaining constant throughout the latter stages of the purification. The amylopullulanase appeared homogenous when analysed by SDS polyacrylamide gel electrophoresis. The amylopullulanase had pH optima of 6.5 and 6.0 on starch and pullulan, respectively, with a temperature Table 1 . Purification of the amylopullulanase of Bacillus sp. DSM 405.