Extra carbohydrate binding module contributes to the processivity and catalytic activity of a non-modular hydrolase family 5 endoglucanase from Fomitiporia mediterranea MF3/22

Pan, Ronghua; Hu, Yimei; Long, Liangkun; Wang, Jing; Ding, Shaojun

doi:10.1016/j.enzmictec.2016.06.001

Cited by 21 publications

(10 citation statements)

References 39 publications

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“…5 , lines 7–9). Nonetheless, even with EG5C-1, a substantial fraction remained bound to Avicel, contrary to previous studies with other processive endoglucanases, where the catalytic domain alone had no binding affinity to Avicel [ 10 , 21 , 23 , 28 ]. The other known exception is the family 5 processive endoglucanase CHU_2103 from C. hutchinsonii [ 15 ].…”

Section: Resultscontrasting

confidence: 91%

“…A series of truncated derivatives of Cel9B was constructed and characterized, and the truncated forms devoid of CBM3c were completely inactivated after incubation for 30 min at 30 °C, while partially truncated forms that still contained CBM3c remained stable after 1 h at 50 °C [ 10 ]. Furthermore, Pan et al reported that fusion of an extra CBM1 domain with the non-modular family 5 endoglucanase FmEG∆24 had higher thermal stability [ 28 ]. To date only a few studies showed that deletion of a CBM domain could enhance the thermal stability of an intact endoglucanase.…”

Section: Resultsmentioning

confidence: 99%

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

Zheng

Pedroso

et al. 2018

Biotechnol Biofuels

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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.

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Section: Resultscontrasting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

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

Zheng

Pedroso

et al. 2018

Biotechnol Biofuels

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“…52 The same function of CBM1 was also found in the GH5 endoglucanase from Fomitiporia mediterranea. 51 For these endoglucanases, the function of CBM was to feed the cellulose chain from the end that was generated by the initial random endo-cleavage of the substrate into the active center, thus improving the processivity of the enzymes.…”

Section: Discussionmentioning

confidence: 99%

Starch-Binding Domain Modulates the Specificity of Maltopentaose Production at Moderate Temperatures

Ding

Zhao

Han

et al. 2022

J. Agric. Food Chem.

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Maltooligosaccharide-forming amylases (MFAs) hydrolyze starch into maltooligosaccharides with a defined degree of polymerization. However, the enzymatic mechanism underlying the product specificity remains partially understood. Here, we show that Saccharophagus degradans MFA (SdMFA) contains a noncatalytic starch-binding domain (SBD), which belongs to the carbohydrate-binding module family 20 and enables modulation of the product specificity. Removal of SBD from SdMFA resulted in a 3.5-fold lower production of the target maltopentaose. Conversely, appending SBD to another MFA from Bacillus megaterium improved the specificity for maltopentaose. SdMFA exhibited a higher level of exo-action and greater product specificity when reacting with amylopectin than with amylose. Our structural analysis and molecular dynamics simulation suggested that SBD could promote the recognition of nonreducing ends of substrates and delivery of the substrate chain to a groove end toward the active site in the catalytic domain. Furthermore, we demonstrate that a moderate temperature could mediate SBD to interact with the substrate with loose affinity, which facilitates the substrate to slide toward the active site. Together, our study reveals the structural and conditional bases for the specificity of MFAs, providing generalizable strategies to engineer MFAs and optimize the biosynthesis of maltooligosaccharides.

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“…Off-pathway non-productive binding for endocellulases collectively refers to those states where the CBM is bound to cellulose via planar aromatic residues while the CD is not catalytically engaged with substrate (see Figure 1). Although literature reports indicate that the attachment of CBMs to endocellulases leads to improve hydrolytic activity towards cellulose (Pan et al, 2016;Reyes-Ortiz et al, 2013), we hypothesized that tweaking the binding affinity of CBM through mutations can further improve endocellulase activity via reduced off-pathway nonproductive binding. In addition, although non-productive binding states cannot be directly characterized using simple biochemical methods, measurement of CBM adsorption and desorption constants ( and respectively) can lead to indirect insights into this phenomenon.…”

Section: Introductionmentioning

confidence: 99%

Reduced type‐A carbohydrate‐binding module interactions to cellulose I leads to improved endocellulase activity

Nemmaru¹,

Ramirez

Farino

et al. 2020

Biotech & Bioengineering

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Dissociation of non-productively bound cellulolytic enzymes from cellulose is hypothesized to be a key rate-limiting factor impeding cost-effective biomass conversion to fermentable sugars. However, the role of carbohydrate-binding modules (CBMs) in enabling non-productive enzyme binding is not well understood. Here, we examine the subtle interplay of CBM binding and cellulose hydrolysis activity for three model Type-A CBMs (families 1, 3a, and 64) tethered to a multifunctional endoglucanase (CelE) on two distinct cellulose allomorphs (i.e., cellulose I and III). We generated a small-library of mutant CBMs with varying cellulose affinity, as determined by equilibrium binding assays, followed by monitoring cellulose hydrolysis activity of CelE-CBM fusion constructs. Finally, kinetic binding assays using quartz crystal microbalance with dissipation (QCM-D) were employed to measure CBM adsorption and desorption rate constants and , respectively, towards nanocrystalline cellulose derived from both allomorphs. Overall, our results indicate that reduced CBM equilibrium binding affinity towards cellulose I alone, resulting from increased desorption rates () and reduced effective adsorption rates (), is correlated to overall improved endocellulase activity. Future studies could employ similar approaches to unravel the role of CBMs in non-productive enzyme binding and develop improved cellulolytic enzymes for industrial applications.

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Extra carbohydrate binding module contributes to the processivity and catalytic activity of a non-modular hydrolase family 5 endoglucanase from Fomitiporia mediterranea MF3/22

Cited by 21 publications

References 39 publications

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

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

Starch-Binding Domain Modulates the Specificity of Maltopentaose Production at Moderate Temperatures

Reduced type‐A carbohydrate‐binding module interactions to cellulose I leads to improved endocellulase activity

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