Production of a high maltose-forming, hyperthermostable and Ca 2+ - independent amylopullulanase by an extreme thermophile Geobacillus thermoleovorans in submerged fermentation

Production of a hyperthermostable, high maltose-forming and Ca 2+ -independent amylopullulanase by an extreme thermophile Geobacillus thermoleovorans was studied in submerged fermentation. Parametric optimization led to secretion of amylopullulanase (2850 U L -1 of α-amylase, 840 U L -1 of pullulanase) in a starch-yeast extract medium (2% starch, 0.1% ammonium sulphate, 0.3% yeast extract, 0.1% K2HPO4, 0.1% NaCl, 0.01% MgSO4.7H2O and 0.1% Maltose) using 2% inoculum (12 h) at 70°C and 200 rpm in 20 h. The addition of trace elements, detergents, surfactants and additives did not stimulate growth or enzyme production. The hydrolysis of pullulan and starch yielded maltotriose, and maltose, maltotriose and maltotetraose as the major end products, respectively. The cultivation of the organism in a laboratory fermenter showed (i) abolition of the lag phase, (ii) a 2-fold increase in enzyme production (5880 U L -1 of α-amylase, 1900 U L -1 of pullulanase) and (iii) a reduction in fermentation time to 7-8 h.