Water → Agarose low melting and low gelling temperature

Participants

Reaction Steps

A solution containing agarose at a concentration of 0.2%(w/v) or 0.5%(w/v) in 20 mM Tris-HCl buffer (pH 7.2) containing 10 mM sodium chloride (in case of Agarase I) or 20 mM Tris-HCl buffer (pH 7.2) containing 10 mM calcium chloride and 10 mM sodium chloride (in case of Agarase II) was prepared. 180 μl of this solution as a substrate was mixed with 20 μl of an enzyme solution. The mixture was reacted at 50° C. for 5 to 30 minutes, preferably 10 minutes, and then heated in boiling water or at 75° C. for 1 minute to stop the reaction. 30 μl of the reaction mixture was subjected to a TSKgel α-2500 column (inner diameter: 7.8 mm; length: 300 mm; Tosoh). Elution was carried out using a 70% acetonitrile solution as an eluent at a flow rate of 0.8 ml/minute. Agarotetraose eluted at a retention time of about 25 minutes or neoagarotetraose eluted at a retention time of about 24 minutes, which was generated by the enzymatic reaction, was quantified. One unit (1 U) is defined as the amount of the enzyme that produces 1 μmol of agarotetraose or neoagarotetraose in 10 minutes.

Provenance & Evidence