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

Abstract

An endo-β-1,4-glucanase gene, cel5L, was cloned using the shotgun 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 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°C, but had different half-lives of 4.0 and 22.8 min, respectively, at 70°C. The relative activities of Cel5L-p50 and Cel5L-p35 for barley β-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.

C-Terminal Bacterial Immunoglobulin-like Domain of κ-Carrageenase Serves as a Multifunctional Module to Promote κ-Carrageenan Hydrolysis

“…brachycerastes and a cellobiohydrolase from C. thermocellum (Xyl-ORF19). , These facts reinforce the role of the Big_2 domain in the enhancement of thermostability, which is consistent with the observations from some CBMs. , …”

“…17,24 These facts reinforce the role of the Big_2 domain in the enhancement of thermostability, which is consistent with the observations from some CBMs. 36,37 To date, CBMs have been extensively applied to construct functional fusion proteins with an enhanced catalytic activity, carbohydrate-binding activity, and stability. 38,39 In this study, similar to some unique properties of CBMs, PpBig_2 was demonstrated to play an essential role in the kinetic parameters (Table 1), κ-carrageenan-binding capability, mode of action, and thermostability of PpCgk.…”

Isolation of thermotolerant Bacillus subtilis DCH4 from Chancos hot spring (Carhuaz, Peru) with potential to degrade lignocellulosic agriculture wastes

“…In the same way, studies of the role of CMB3 of Bacillus sp. KD1014 suggest that it contribute in thermostability, and affinity and substrate specificity for small substrates (Lee et al 2018).…”