Secretory Expression of a Maltooligosaccharide-forming Amylase in Bacillus subtilis and Its Enzymatic Properties
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Abstract:
A secretory expression system for the maltooligosaccharide-forming amylase from B. stearothermophilus STB04 was constructed in Bacillus subtilis and the fermentation conditions of B. subtilis WB600 containing the expression vectors for the production of the recombinant enzyme were optimized. The results showed that the secretory expression of recombinant enzyme was optimal in terrific broth (TB) medium as the fermentation medium and at a fermentation temperature of 30 ℃. The recombinant amylase was purified through a combination of hydrophobic interaction and strong anion exchange chromatography to yield electrophoretically pure enzyme. The purified enzyme was characterized with respect to its properties and the biochemical reaction it catalyzes. The results showed that this enzyme was a monomer in solution with an optimum reaction temperature of 45 ℃, and a half-life of approximately 30 min at this temperature. The enzyme functioned optimally at pH 6.5 with high pH stability. The activity of this enzyme was dependent on metal ions to a certain extent and was dramatically inhibited by some heavy metal ions, especially Fe3+ and Sn2+. The kinetics of the amylase-catalyzed reaction could be fairly well described by the Michaelis-Menten equation, and the maltodextrin hydrolysis rate was the highest compared with other substrates. The main products of the hydrolysis of native starches from different sources and maltodextrin with this recombinant enzyme were maltotriose, maltotetrose, and maltopentose. Maltopentose was predominant among the hydrolysis products.
