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.
Highlights
Enzymes still constitute a major part of the cost of biofuel production from lignocellulosic biomass
Purification and identification of enzymes EG5C and EG5C‐1 A purification strategy based on the high affinity of cellulolytic enzymes to cellulose was used to isolate proteins secreted by B. subtilis BS-5
We concluded that the 33 kDa enzyme observed on the SDS-PAGE gel represented a truncated form of the 46 kDa enzyme which might be produced by limited proteolysis with a protease from strain BS-5
Summary
Enzymes still constitute a major part of the cost of biofuel production from lignocellulosic biomass. An alternative mechanism for the bioconversion of cellulose was found in quite a few anaerobic microorganisms such as Clostridium thermocellum, which employs a large multiprotein complex (cellulosome) to hydrolyze cellulose [3] In both strategies, i.e. aerobic and anaerobic, the efficient hydrolysis of cellulose to glucose depends on the synchronized action of three classes of enzymes, endoglucanase, exoglucanase and β-glucosidase. Several endoglucanases that possess both endo and exoglucanase activity and appear to act in processive manner were found in these organisms, potentially presenting a novel strategy for the degradation of cellulose [5,6,7]. In contrast to other cellulolytic systems that dependent on both endo and exoglucanases, a processive endoglucanase coupled with a β-glucosidase may be sufficient for the degradation of cellulose and this enzyme has attracted considerable interest [6]
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