Open AccessPurification and biochemical characterization of pullulanase type I from Thermus caldophilus GK‐24
Author(s) -
Kim CheorlHo,
Nashiru Oyekanmi,
Ko Jeong Heon
Publication year - 1996
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1996.tb08148.x
Subject(s) - pullulan , pullulanase , maltotriose , amylopectin , chemistry , size exclusion chromatography , chromatography , enzyme , thermus , hydrolysis , enzyme assay , biochemistry , dextrin , maltose , starch , polysaccharide , thermophile , amylose
A thermostable pullulanase (pullulan 6‐glucanohydrolase, EC 3.2.1.41) has been purified to homogeneity from Thermus caldophilus GK‐24 by Chromatographic methods, including gel‐filtration and ion‐exchange chromatography. The specific activity of the enzyme was increased 431‐fold with a recovery of 13.2%. The purified enzyme was a monomer, M r = 65 kDa as estimated by SDS‐PAGE and gel filtration. The p I was 6.1. The enzyme was most active at pH 5.5. The activity was maximal at 75 °C and stable up to 95 °C for 30 min at pH 5.5. The enzyme was stable to incubation from pH 3.5 to pH 8.0 at 4 °C for 24 h. The activity of the enzyme was stimulated by Mn 2+ and Mg 2+ ions. Ni 2+ , Ca 2+ , Co 2+ ions and EDTA did not inhibit the enzyme activity. The enzyme hydrolyzed the α‐1,6 linkages of amylopectin, glycogens, α,β‐limited dextrin, and pullulan. The enzyme caused the complete hydrolysis of pullulan to maltotriose. The activity was inhibited by α‐, β‐, or γ‐cyclodextrins. The N‐terminal sequence [(Ala‐Pro‐Gln‐(Asp or Tyr)‐Asn‐Leu‐Leu‐Xaa‐ILe‐Gly‐Ala(Ser)] showed some similarity to those of bacterial pullulanases.