The modular arabinanolytic enzyme Abf43A-Abf43B-Abf43C from Ruminiclostridium josui consists of three GH43 modules classified in different subfamilies

摘要

The abnA gene from Rwniniclostridium josui encodes the large modular arabinanolytic enzyme, Abf43A-Abf43B-Abf43C, consisting of an N-terminal signal peptide, a Laminin_G_3 module, a GH43_22 module, a Laminin_G_3 module, a Big_4 module, a GH43_26 module, a GH43_34 module and a dockerin module in order with a calculated molecular weight of 204,108. Three truncated enzymes were recombinantly produced in Escherichia coil and biochemically characterized, RjAbf43A consisting of the first Laminin_G_3 module and GH43_22 module, RjAbf43B consisting of the second Laminin_G_3 module, Big_4 module and GH43_26 module, and RjAbf43C consisting of the GH43_34 module. RjAbf43A showed a strong alpha-L-arabinofuranosidase activity toward sugar beet arabinan, highly branched arabinan but not linear arabinan, thus it acted in the removal of arabinose side chains from sugar beet arabinan. By contrast, RjAbf43B showed a strong exo-alpha-1,5-L-arabinofuranosidase activity toward linear arabinan and arabinooligosaccharides whereas RjAbf43C showed low activity toward these substrates. Although RjAbf43B was activated by the presence of some metal ions such as Zn2+, Mg2+ and Ni2+, RjAbf43A was inhibited by these ions. RjAbf43A and RjAbf43B attacked sugar beet arabinan in a synergistic manner. By comparison, RjAbf43A-Abf43B containing both GH43_22 and GH43_26 modules showed lower hydrolytic activity toward sugar beet arabinan but higher activity toward sugar beet fiber than the sum of the individual activities of RjAbf43A and RjAbf43B, suggesting that the coexistence of two distinct GH43 modules in a single polypeptide is important for the efficient hydrolysis of an insoluble and natural polysaccharide but not a soluble substrate.