Premium
1,4‐α‐Glucan phosphorylase from Klebsiella pneumoniae covalently coupled on porous glass
Author(s) -
Wengenmayer Friedrich,
Linder Dietmar,
Wallenfels Kurt
Publication year - 1977
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260190911
Subject(s) - glycogen phosphorylase , chemistry , glucan , carbodiimide , glutaraldehyde , covalent bond , enzyme , specific activity , yield (engineering) , enzyme assay , amylopectin , biochemistry , substrate (aquarium) , chromatography , organic chemistry , materials science , biology , amylose , ecology , starch , metallurgy
A simplified procedure for the preparation of 1,4‐α‐glucan phosphorylase from Klebsiella pneumoniae is described. An 80‐fold purification is achieved in two steps with an overall yield of about 50%. The specific activity of the homogeneous enzyme protein is 17.7 units/mg. Compared with glycogen phosphorylase from rabbit muscle the enzyme from K. pneumoniae shows a markedly higher stability against deforming and chaotropic agents. The 1,4‐α‐glucan phosphorylase was covalently bound to porous glass particles by three different methods. Coupling with glutaraldehyde gave the highest specific activity, i.e., 5.6 units/mg of bound protein or 133 units/g of glass with maltodextrin as substrate. This corresponds to about 30% of the specific activity of the soluble enzyme. With substrates of higher molecular weight, such as glycogen or amylopectin, lower relative activity was observed. The immobilized enzyme preparations showed pH activity profiles which were slightly displaced to higher values and exhibited an increased temperature stability.