Abstract:

Through the separation and purification of the agar-degrading bacteria FG5 fermentation broth, agarase was obtained to study its enzymatic characterization, and whole genome sequencing was used to obtain high-potential agar-degrading engineering bacteria. The crude enzyme of FG5 agarase was obtained after centrifugation of the fermentation broth of FG5 strain followed by separation and purification via ultrafiltration concentration, dialysis, and dextran gel column chromatography. The DNS method was used to determine the reducing sugar content in the enzymatic hydrolysate, and then the enzyme activity was calculated. Then, a single factor experiment, and an orthogonal experiment design were utilized to optimize the enzymatic hydrolysis conditions for a single agarase component. A peak product with agarase activity is obtained after separation, purification, and UV detection. The molecular weight of the purified agarase protein is about 54 kD. The optimal conditions of FG5 agarase are as follows: pH 7.3, reaction temperature 51 ℃, and substrate concentration 0.351%. In this study, the FG5 crude enzyme is purified by 6.95 times using the dextran gel column, and the conditions for enzymatic hydrolysis of FG5 agarase are optimized. It provides a theoretical and practical foundation for the industrial production of high-purity agarase.

Key words: agar-degrading strain, agarase, dextran gel separation