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Roles of Carbohydrate-Binding Module (CBM) of an Endo-β-1,4-Glucanase (Cel5L) from Bacillus sp. KD1014 in Thermostability and Small-Substrate Hydrolyzing Activity

  • Lee, Jae Pil (Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University) ;
  • Shin, Eun-Sun (Department of Agricultural Chemistry, Sunchon National University) ;
  • Cho, Min Yeol (Department of Agricultural Chemistry, Sunchon National University) ;
  • Lee, Kyung-Dong (Department of Oriental Medicine Materials, Dongshin University) ;
  • Kim, Hoon (Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University)
  • Received : 2018.10.02
  • Accepted : 2018.10.19
  • Published : 2018.12.28

Abstract

An endo-${\beta}$-1,4-glucanase gene, cel5L, was cloned using the shot-gun method from Bacillus sp.. The gene, which contained a predicted signal peptide, encoded a protein of 496 amino acid residues, and the molecular mass of the mature Cel5L was estimated to be 51.8 kDa. Cel5L contained a catalytic domain of glycoside hydrolase (GH) family 5 and a carbohydrate-binding module family 3 (CBM_3). Chromatography using HiTrap Q and CHT-II resulted in the isolation of two truncated forms corresponding to 50 (Cel5L-p50) and 35 kDa (Cel5L-p35, CBM_3-deleted form). Both enzymes were optimally active at pH 4.5 and $55^{\circ}C$, but had different half-lives of 4.0 and 22.8 min, respectively, at $70^{\circ}C$. The relative activities of Cel5L-p50 and Cel5L-p35 for barley ${\beta}$-glucan were 377.0 and 246.7%, respectively, compared to those for carboxymethyl-cellulose. The affinity and hydrolysis rate of pNPC by Cel5L-p35 were 1.7 and 3.3 times higher, respectively, than those by Cel5L-p50. Additions of each to a commercial enzyme set increased saccharification of pretreated rice straw powder by 17.5 and 21.0%, respectively. These results suggest CBM_3 is significantly contributing to thermostability, and to affinity and substrate specificity for small substrates, and that these two enzymes could be used as additives to enhance enzymatic saccharification.

Keywords

References

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