Processivity and enzymatic mechanism of a multifunctional family 5 endoglucanase from Bacillus subtilis BS-5 with potential applications in the saccharification of cellulosic substrates

Abstract

BackgroundPresently, enzymes still constitute a major part of the cost of biofuel production from lignocellulosic biomass. Processive endoglucanases, which possess both endoglucanase and exoglucanase activity, have the potential to reduce the costs of biomass saccharification when used together with commercial cellulases. Therefore, the exploration of new processive endoglucanases has attracted much attention with a view to accelerating the industrialization of biofuels and biochemicals.ResultsThe endoglucanase EG5C and its truncated form EG5C-1 from Bacillus subtilis BS-5 were expressed and characterized. EG5C was a typical endoglucanase, comprised of a family 5 catalytic domain and family 3 carbohydrate-binding domain, and which had high activity toward soluble cellulosic substrates, but low activity toward insoluble cellulosic substrates. Importantly, the truncated form EG5C-1 was a processive endoglucanase that hydrolyzed not only carboxymethyl cellulose (CMC), but also insoluble cellulosic substrates. The hydrolytic activities of EG5C-1 towards CMC, phosphoric acid-swollen cellulose (PASC), p-nitrophenyl-β-d-cellobioside, filter paper and Avicel are 4170, 700, 2550, 405 and 320 U/μmol, respectively. These data demonstrated that EG5C-1 had higher activity ratio of exoglucanase to endoglucanase than other known processive endoglucanases. When PASC was degraded by EG5C-1, the ratio of soluble to insoluble reducing sugars was about 3.7 after 3 h of incubation with cellobiose and cellotriose as the main products. Importantly, EG5C-1 alone was able to hydrolyze filter paper and PASC. At 5% substrate concentration and 10 FPU/g PASC enzyme loading, the saccharification yield was 76.5% after 60 h of incubation. Replacement of a phenylalanine residue (F238) by an alanine at the entrance/exit of the substrate binding cleft significantly reduces the ability of EG5C-1 to degrade filter paper and Avicel, but this mutation has little impact on CMCase activity. The processivity of this mutant was also greatly reduced while its cellulose binding ability was markedly enhanced.ConclusionsThe processive endoglucanase EG5C-1 from B. subtilis BS-5 exhibits excellent properties that render it a suitable candidate for use in biofuel and biochemical production from lignocellulosic biomass. In addition, our studies also provide useful information for research on enzyme processivity at the molecular level.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1022-2) contains supplementary material, which is available to authorized users.

Resurrection of efficient Precambrian endoglucanases for lignocellulosic biomass hydrolysis

“…2e). The measured ratio for LFCA_EG is similar to that of other natural or designed EG with processive activity 26 . Although some hydrolases from family GH5 have been shown to be processive 27 , they have not been included in our phylogeny, which makes the processivity of the ancestral LFCA_EG a surprising feature.…”

“…Processivity assay. In order to determine the processivity 26 of the cellulases, there was a ratio of soluble to insoluble reducing sugar from PASC. The reaction was carried out at 45°C with 0.5% of PASC, and a sample was removed from the mixture at different time points.…”

Production of thermostable multiple enzymes from Bacillus amyloliquefaciens KUB29

“…Com exceção das celulasse produzidas por B. drentensis ME-2, as demais linhagens celulolíticas apresentaram elevada atividade enzimática em diversas temperaturas.Todos os isolados celulolíticos analisados exibiram máxima atividade enzimática a 60 °C.Muitas celulases são descritas na literatura com atividade ótima a 50-60 °C, como aquelas produzidas por B. amyloliquefaciens AK9 (Irfan M et al, 2017) e B. subtilis BS-5 (Wu B et al, 2018). Entretanto, temperatura ótima mais elevada já foi descrita, como 75 °C em celulases de Bacillus sp.…”

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

“…Some researches of hydrolytic B. subtilis have been focused on fermentation optimization to obtain higher yields, and increase recovery rates employing genetic engineering (Sarkar et al 2012). In this sense, there are some reports of characterization of complete genomes, genes and proteins and cloning to get more efficient enzymes (Yang et al 2009, Nurachman et al 2010, Vallenet et al, 2017, Chang et al 2018.…”