Figure 2

BpGH16A and BpGH117 are the key enzymes for the accumulation of AgaDP3 from agarose in B. plebeius. (A) The PUL of B. plebeius. Numbers below gene arrows are locus tag numbers without their prefix, BACPLE. Based on the sequence analysis, three genes located in the PUL of B. plebeius—i.e., BACPLE_01670, BACPLE_01671, and BACPLE_01683—encoding BpGH16A, BpGH117, and BpGH50, respectively, are related to the agarolytic mechanism. Abbreviations: MFS, Major facilitator superfamily; HTCS, hybrid two-component system. (B) Purification of the agarose degradation pathway enzymes, BpGH16A, BpGH50, and BpGH117, originating from B. plebeius. (C) Enzymatic activities of purified recombinant BpGH16A, BpGH50, and BpGH117. The agarase activities of BpGH16A and BpGH50 were verified by the enzymatic reactions of each enzyme with agarose as the substrate. The NABH activity of BpGH117 was verified by the enzymatic reaction with NeoDP2 as the substrate. The reaction products were analyzed by TLC. (D) Simulation of the agarolytic mechanism of B. plebeius using the sequential and the simultaneous enzymatic reactions of BpGH16A and BpGH117 with agarose. Lanes 1–4, the standards of the agar-derived sugars; lane 5, the agarose substrate; lane 6, the reaction products of BpGH16A using agarose; lane 7, the reaction products of BpGH117 with the reaction products of BpGH16A; lane 8, simultaneous enzymatic reactions of BpGH16A and BpGH117 using agarose. (E) Effects of treatment of agarose with simulated gastric fluid on sequential or simultaneous hydrolysis of agarose using BpGH16A and BpGH117. The reaction products obtained from each step of agarose hydrolysis were analyzed by TLC. Lanes 1–3, the standards of agar-derived sugars, AHG, d-galactose (Gal), NeoDP2, AgaDP3, NeoDP4, and NeoDP6; lane 4, agarose substrate; lane 5, treatment of agarose using a simulated gastric fluid; lane 6, reaction products of BpGH16A with agarose treated using a simulated gastric fluid; lane 7, reaction products of BpGH117 with the reaction products of BpGH16A; lane 8, reaction products obtained from the simultaneous reaction of BpGH16A and BpGH117 with agarose treated using a simulated gastric fluid. (F) Proposed mechanism of agarose degradation by BpGH16A and BpGH117 based on the results of in vitro enzymatic reactions.