Characterization of two novel highly active glycoside hydrolase family 53 endo-1,4-ß-galactanases and their synergism with other carbohydrases in plant polysaccharide decomposition.

Two novel Bacillus glycoside hydrolase family 53 (GH53) endo-1,4-ß-galactanases (Bs936 and Bs4828) were identified. The recombinant Bs936 and Bs4828 displayed maximal activities at pH 5.5/55 °C and pH 6.5/65 °C, respectively. Stability analyses revealed that the enzymes were stable between pH 4.5-10, retaining over 80% activities after 12 h incubation at 25 °C. Moreover, Bs936 maintained about 75% activity after being treated at 45 °C for 2 h, while Bs4828 kept full activity after 4 h of incubation at 50 °C. Importantly, Bs936 and Bs4828 exhibited good activity towards ß-1,4-galactan, showing specific activities of 1859.46- and 3110.79 U/mg towards potato galactan, respectively. The corresponding Km and kcat/Km were 4.93 mg/mL and 296.35 mL/mg/s for Bs936, and 6.60 mg/mL and 707.12 mL/mg/s for Bs4828. Extensive synergy assays revealed that each endo-1,4-ß-galactanase acted synergistically with the other ß-galactosidase (Bs937 or Bs4829), endo-1,5-α-L-arabinosidase (BsAbn2) and an endoxylanase/endoglucanase+cellulase mixture (BaXynA + LsCel) towards the hydrolysis of galactan, debranched arabinan/arabinan, and pretreated corn stover, respectively. The corresponding degrees of synergy were 1.28, 1.17/1.09, and 1.15 for Bs486 and 1.55, 1.04/1.08, and 1.11 for Bs4828, respectively. This study expands the repertoire of GH53 enzymes, and demonstrates the role of two highly active endo-1,4-ß-galactanases in polysaccharides hydrolysis.