Purification and characterization of pullulanase from Lactococcus lactis

Prep Biochem Biotechnol. 2011;41(3):252-61. doi: 10.1080/10826068.2011.575316.


This paper describes a simple and efficient method of isolation of a plullulanase type I from amylolytic lactic acid bacteria (ALAB). Extracellular pullulanase type I was purified from a cell-free culture supernatant of Lactococcus lactis IBB 500 by using ammonium sulfate fractionation and dialysis (instead of ultrafiltration), and ion-exchange chromatography with CM Sepharose FF followed by gel filtration chromatography with Sephadex G-150 as the final step. A final purification factor of 14.36 was achieved. The molecular mass of the enzyme was estimated as 73.9 kD. The optimum temperature for the enzyme activity was 45°C and the optimum pH was 4.5. Pullulanase activity was increased by addition Co(2+) and completely inhibited by Hg(2+). The enzyme activity was specifically directed toward α-1,6 glycosidic linkages of pullulan giving maltotriose units. Enzymatic hydrolysis of starch and amylose produced a mixture of maltose and maltotriose.

MeSH terms

  • Bacterial Proteins
  • Chemical Fractionation / methods*
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange / methods*
  • Enzyme Stability
  • Glycoside Hydrolases / chemistry
  • Glycoside Hydrolases / isolation & purification*
  • Glycoside Hydrolases / metabolism*
  • Hydrogen-Ion Concentration
  • Lactococcus lactis / enzymology*
  • Lactococcus lactis / metabolism
  • Molecular Weight
  • Substrate Specificity
  • Temperature


  • Bacterial Proteins
  • Glycoside Hydrolases
  • pullulanase