Studies on cloning and expression of the gene encoding pullulanase from Bacillus sp.

This study focuses on isolation of the pullulanase gene from Bacillus sp. and recombinant production of the enzyme in Pichia pastoris. Bacillus strains isolated from ropey bread in a previous study were screened as sources of the gene and B. subtilis BK07 was used as the gene source. Pullulanase gene which was amplified with PCR to include a polyhistidine tag was transformed into P. pastoris KM71H yeast using the pPICZ?A vector and the enzyme production was achieved by methanol induced expression. In all steps, B. subtilis PY22 was used as positive control of the analysis.Optimum pH and temperature conditions for BK07 and PY22 pullulanase were investigated. Optimum pH and temperature was found as pH8-40°C and pH6-40°C, respectively. BK07 pullulanase activity was determined as 8.46 U/ml in supernatant and specific activity of the enzyme was calculated as 144 U/mg. PY22 pullulanase activity was determined as 2.95 U/ml in supernatant and specific activity of the enzyme was calculated as 51 U/mg. SDS-PAGE and Western blot analysis showed that the molecular weight of the recombinant enzyme BK07 pullulanase and PY22 pullulanase were determined to be about 81 kDa and 90 kDa, respectively.KEYWORDS: Pullulanase, Pichia pastoris, Bacillus sp., Recombinant protein production