Purification of Glucosyltransferase from Streptococcus sobrinus Cell Culture Medium by Combined Use of Batch Extraction and Countercurrent Chromatography with a Polymer Phase System

Abstract

Glucosyltransferase (GTF), a cariogenic factor of Streptococcus sobrinus (SS), was extracted from a large quantity of cell culture medium by batch extraction with aqueous polymer two‐phase systems (APTPs). The small quantity of the extract was further purified by countercurrent chromatography (CCC) using polyethylene glycol (PEG) 8000‐dextran T500 system at pH 9.2, by eluting the impurities with the upper mobile phase at 1.0 mL/min. After the elution, the GTF still remaining in the dextran‐rich lower stationary phase was collected. The GTF fractions were subjected to hydroxyapatite chromatography to eliminate polymers, such as PEG 8000 and dextran T500. Purified fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and GTF enzyme activity, which rose over 270 fold from that in the original culture medium.

Keywords:

• Countercurrent chromatography (CCC)
• Hydroxyapatite chromatography
• Glucosyltransferase
• Aqueous polymer two‐phase systems
• Streptococcus sobrinus

Acknowledgment

The authors are grateful to Dr. I. Nasu for providing the bacterial strain used, and thank Dr. M. Tagashira of Asahi Breweries, Ltd., Ibaraki, Japan for useful suggestions for measurement of GTF activity. The authors are also indebted to Dr. Henry M. Fales of National Institutes of Health, Bethesda, Maryland for editing the manuscript with valuable suggestions.