Expression and Characterization of a GH16 Family beta-Agarase Derived from the Marine BacteriumMicrobulbifersp. BN3 and Its Efficient Hydrolysis of Agar Using Raw Agar-Producing Red SeaweedsGracilaria sjoestedtiiandGelidium amansiias Substrates

Agarases catalyze the hydrolysis of agarose to oligosaccharides which display an array of biological and physiological functions with important industrial applications in health-related fields. In this study, the gene encoding agarase (Aga-ms-R) was cloned fromMicrobulbifersp. BN3 strain. Sequence alignment indicated that Aga-ms-R belongs to the GH16 family and contains one active domain and two carbohydrate binding module (CBM) domains. The mature Aga-ms-R was expressed successfully by employing theBrevibacillussystem. Purified rAga-ms-R was obtained with a specific activity of 100.75 U/mg. rAga-ms-R showed optimal activity at 50 degrees C and pH 7.0, and the enzyme activity was stable at 50 degrees C and also over the pH range of 5.0-9.0. After exposure of rAga-ms-R to 70 degrees C for 30 min, only partial enzyme activity remained. Thin layer chromatographic analysis of the enzymatic hydrolysate of agar obtained using rAga-ms-R disclosed that the hydrolysate comprised, in a long intermediate-stage of the hydrolysis reaction, mainly neoagarotetraose (NA4) and neoagarohexaose (NA6) but ultimately, predominantly neoagarotetraose and trace amounts of neoagarobiose (NA2). Hydrolysates of the raw red seaweedsGracilaria sjoestedtiiandGelidium amansii, produced by incubation with rAga-ms-R, were mainly composed of neoagarotetraose. The results demonstrate the high efficiency of rAga-ms-R in producing neoagaraoligosaccharide under low-cost conditions.