Cloning and characterization of low-temperature adapted GH5-CBM3 endo-cellulase from Bacillus subtilis 1AJ3 and their application in the saccharification of switchgrass and coffee grounds

Abstract

Endocellulase is a key cellulase for cellulosic material pretreatment in the industry by hydrolyzing long cellulose chains into short chains. To investigate the endocellulase characteristics from Bacillus subtilis 1AJ3, and increase its production yield, this paper cloned an endocellulase gene denoted CEL-5A from strain 1AJ3 and expressed in E. coli BL21 (DE3). The CEL-5A gene was sequenced with a full-length of 1500 bp, encoding a totally of 500 amino acids, and containing two domains: the GH5 family catalytic domain (CD) and the CBM3 family cellulose-binding domain (CBD). Recombinant endocellulase Cel-5A with a His-tag was purified of the Ni-NTA column, and SDS-PAGE results demonstrated that Cel-5A exhibited a molecular weight of 56.4 kDa. The maximum enzyme activity of Cel-5A was observed at pH 4.5 and 50 °C. Moreover, it was active over the broad temperature region of 30-60 °C, and stable within the pH range of 4.5-10.0. In addition, Co 2+ was able to increase enzyme activity, while the majority of metal ions demonstrated stable enzyme activity under low-concentration. The substrate specificity of Cel-5A exhibited a high specific activity on the β-1,3-1,4 glucan linkage from barley. The Michaelis-Menten constant and the maximum velocity of the recombinant Cel-5A for CMC-Na were determined as 14.87 mg/mL and 19.19 μmol/min/mg, respectively. When Cel-5A was applied to the switchgrass and coffee grounds, its color became lighter and the biomass was observed to loosen following hydrolyzation. The saccharification rate reached 12% of the total weight of switchgrass in 20 h. These properties highlight the potential application of Cel-5A as an endocellulase in the pretreatment of biomass, for example, in the coffee grounds/waste, and related industries.

Biochemical characterization of a novel thermo‐halo‐tolerant GH5 endoglucanase from a thermal spring metagenome

“…Among the tested substrates, Cel M displayed the maximum specificity with β‐ d ‐glucan and the cellulose derivative, Na‐CMC (Table 1). This was in accordance with many previous reports on endoglucanases characterized from different sources (Guerrero et al, 2020; Ma et al, 2020; Wang et al, 2019; Yin et al, 2020). A limited activity was measured in the presence of galactomannan‐containing substrates, for example, Locust bean gum and Guar gum.…”

“…The kinetic parameter values, K m , k cat , and k cat /K m , of Cel M toward Na-CMC were computed as 19.87 mg mL −1 , 138 s −1 , 101.5 mg mL −1 s −1 , respectively. The kinetic analysis determined a fairly good substrate affinity of Cel M , which was comparable to that of the previously characterized GH5 endoglucanases from T. rubra YIM 77501 (Yin et al, 2020), B. subtilis 1AJ3 (Ma et al, 2020), and oil reservoir (Zhu et al, 2020). The turnover number and enzyme efficiency of Cel M were more or less similar to that of previously reported endoglucanases from termite microbiome (Guerrero et al, 2020) and cow rumen metagenome (Patel et al, 2020).…”

“…(d) Residual activity of Cel M after its exposure to salinity for 30 days. β-ME, β-mercaptoethanol; DMSO, dimethyl sulfoxide; SDS, sodium dodecyl sulfate catalytic activity of GH5 endoglucanases from T. rubra YIM 77501 (Yin et al, 2020) and B. subtilis 1AJ3 (Ma et al, 2020). The activity of a rhizosphere metagenomic GH5 endoglucanase had been shown to be severely affected by the presence of Zn in the catalytic reaction (Wierzbicka-Wos et al, 2019).…”

Diversity and Activity of Aquatic Cellulolytic Bacteria Isolated from Sedimentary Water in the Littoral Zone of Tonle Sap Lake, Cambodia

“…It was related to a GH family cellulase from B. velezensis by the BLASTx program. This putative endoglucanase was classified as an endoglucanase belonging to the GH5 family, which is the largest and most functionally diverse family of endoglucanases detected in numerous species [91][92][93]. The phylogenetic tree showed that this putative endoglucanase was clustered with the endoglucanases from B. amyloliquefaciens, B. siamensis, B. subtilis, B. thuringiensis, and B. velezensis.…”

Bioprospecção e caracterização de bactérias produtoras de celulase a partir do solo do Cerrado brasileiro

“…A estabilidade térmica em alta (60°C) e baixa (30-50°C) temperatura é um recurso desejável para a aplicação industrial de celulases, pois economiza energia da reação (Ma et al, 2020), e permite a interação com outras enzimas, tais como aquelas que degradam lignina (a 30°C) e xilanases (a 50-60°C) (Liu et al, 2019).…”