Improved purification procedure for the extracellular D-glucosyltransferase from Streptococcus mutans 6715

Area of Science:

• Microbiology
• Enzymology
• Biochemistry

Background:

• Streptococcus mutans produces exocellular enzymes crucial for dental plaque formation.
• D-glucosyltransferase (GTF) enzymes synthesize glucans from sucrose, contributing to biofilm structure.
• Efficient purification of GTF is essential for understanding its structure-function relationship and developing inhibitors.

Purpose of the Study:

• To highly purify exocellular D-glucosyltransferase from Streptococcus mutans 6715.
• To investigate and separate different D-glucosyltransferase activities present in the organism.
• To characterize the properties of the purified enzyme fractions, particularly in relation to dextran.

Main Methods:

• Culturing Streptococcus mutans 6715 in a specialized Trypticase soy broth supplemented with invertase and Tween 80.
• Ultrafiltration using a 10,000 molecular weight cut-off membrane to pre-purify the enzyme.
• Affinity chromatography employing insoluble streptococcal dextran as the stationary phase to isolate D-glucosyltransferase.

Main Results:

• High purification of exocellular D-glucosyltransferase was achieved with minimal loss of activity.
• Two distinct D-glucosyltransferase activities were identified: one that adsorbed to insoluble dextran and one that did not.
• The D-glucosyltransferase fraction that adsorbed to the insoluble dextran affinity support was significantly inhibited by the addition of insoluble dextran.

Conclusions:

• The purification protocol effectively isolated exocellular D-glucosyltransferase from Streptococcus mutans.
• The study successfully differentiated two GTF activities based on their interaction with insoluble dextran.
• The inhibitory effect of insoluble dextran on the adsorbed GTF fraction suggests a specific binding interaction relevant to enzyme function.