Spin Labeling and Kinetic Studies of a Membrane‐Immobilized Proteolytic Enzyme

Ping Zhuangt, D. Allan Butterfield

Research output: Contribution to journalArticlepeer-review

24 Scopus citations


Membrane‐based bioreactors can greatly influence the rate and extent of chemical reactions and consequently lower the costs associated with the corresponding engineering processes. However, in order to progress in this area, greater understanding of the relationship of the structure and function of bioreactor systems is required. In this study, a proteolytic enzyme, papain (EC, was covalently coupled onto the surface of a vinyl alcohol/vinyl butyral copolymer (PVB) membrane employing either glutaraldehyde (GA) or l,l'‐carbonyldiimidazole (CDI). Various kinetic and performance properties of the immobilized papain were studied. It was found that these characteristics of the membrane‐bound papain depended on the immobilization method. The CDI‐immobilized papain bioreactor was used, although the apparent Michaelis constant, Km, of the CDI‐immobilized papain was larger than that of the GA‐immobilized enzyme. In separate experiments, a six‐carbon spacer was also used between the membrane support and the covalently‐linked enzyme. It was found that the insertion of the spacer reduced the disturbance of the enzyme system, resulting in a decreased Km, which was now closer to the value for the free enzyme. Electron paramagnetic resonance (EPR) techniques of spin labeling were used for the first time to examine the conformational change and the active site structure of an enzyme covalently immobilized to a membrane. The structural changes of the active site of papain upon immobilization with and without a spacer were in agreement with the functional properties of the enzyme.

Original languageEnglish
Pages (from-to)204-210
Number of pages7
JournalBiotechnology Progress
Issue number3
StatePublished - 1992

ASJC Scopus subject areas

  • Biotechnology


Dive into the research topics of 'Spin Labeling and Kinetic Studies of a Membrane‐Immobilized Proteolytic Enzyme'. Together they form a unique fingerprint.

Cite this