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Isolation and Identification of a New Bacillus cereus Strain and Characterization of its Neopullulanase

Soheila Davaeifar, Parvin Shariati, Fatemeh Tabandeh, Bagher Yakhchali
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Abstract

Identification and use of more efficient enzymes in the food and pharmaceutical industries is the focus of many researchers. The aim of this study was to search for a new bacterial strain capable of producing high levels of pullulanase applicable to biotechnology, the starch bioprocessing and food industries. A new pullulan hydrolyzing Bacillus strain was isolated and designated SDK2. Morphological and biochemical tests identified the strain as a putative Bacillus cereus strain, which was further characterized and confirmed through 16s rRNA sequencing, and was submitted to GeneBank, under the accession number FR6864500. Quantitative analysis of the strain’s pullulanase activity was carried out by the Dintrosalicyclic (DNS) acid-based assay. Thin layer chromatography (TLC) of the culture supernatant, identified the extracellular pullulanase as neopullulanase. Effects of temperature and pH on pullulanase activity were also studied. The optimum conditions for enzyme activity, as represented by 60°C and a pH of 7, resulted in an activity of 13.43 U/ml, which is much higher than some of the previously reported activities. However, growth of B. cereus SDK2 was also observed at a pH range of 5 to 10, and temperatures of 30°C to 50°C. The effect of metal ions and reagents, such as Mg+2, Ca+2, Zn+2, Cu+2, Fe+2, Ni+2 on enzyme activity showed that Ca+2 ions increased pullulan activity, whereas the other ions and reagents inhibited pullulanase activity. The ability of B. cereus SDK2 to produce high levels of neopullulanase stable at 60°C that can generate panose from pullulan, make this newly isolated strain a valuable source of debranching enzyme for biotechnology, the starch bioprocess and medical industries.

Keywords

Bacillus cereus, DNS-based assay, Neopullulanse, 16s rRNA sequencing, Thin layer chromatography

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DOI: https://doi.org/10.22037/afb.v2i2.8151

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