Cloning and expression of pullulanase from Bacillus subtilis BK07 and PY22 in Pichia pastoris

In this study, pullulanase genes from a wild isolate B. subtilis BK07 and B. subtilis PY22 (mutant strain derived from B. subtilis 168) were transformed into P. pastoris KM71H. Extracellular recombinant protein production was achieved with methanol induction under the regulation of AOX1 promoter uti...

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Veröffentlicht in:Protein expression and purification 2019-10, Vol.162, p.83-88
Hauptverfasser: Erden-Karaoğlan, Fidan, Karakaş-Budak, Barçın, Karaoğlan, Mert, Inan, Mehmet
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Sprache:eng
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Zusammenfassung:In this study, pullulanase genes from a wild isolate B. subtilis BK07 and B. subtilis PY22 (mutant strain derived from B. subtilis 168) were transformed into P. pastoris KM71H. Extracellular recombinant protein production was achieved with methanol induction under the regulation of AOX1 promoter utilizing the Saccharomyces cerevisiae α-mating factor sequence for extracellular secretion. The molecular weight of the recombinant enzymes BK07pul and PY22pul were both approximately 90 kDa. Both enzymes showed highest activity at 40 °C, however PY22pul showed optimum activity at pH 6 whereas, BK07pul had highest activity at pH 8. BK07pul and PY22pul activities were determined as 8.46 U/mL and 15 U/mL. The enzyme stability of BK07pul was higher (89%) than PY22pul (68%) where relative activity was determined as activity remaining after 1 h at corresponding optimum conditions for each. Amino acid homology evaluation revealed the two enzymes had 80% identity in primary structure. The presence of conserved sequences consisting of 7 amino acids (YNWGYDP) in both enzymes confirmed these to be type I pullulanases, capable of hydrolyzing α-1,6 glucosidic bonds of pullulan resulting in maltotriose units. •Isolation of the pullulanase gene from a wild isolate Bacillus subtilis BK07.•Characterization of pullulanase enzymes from two different gene sources.•Effects of the buffer system and temperature on protein degradation problem.•The newly isolated BKpul sequence contained type I pullulanase motif (YNWGYDP).
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2019.05.008