Extending the linker region increases the activity of the Bacillus subtilis cellulase CelI15

Li, Pingping; Zhou, Y.F.; Li, Qian; Zhang, Chong; Sun, Zhaowei; Tian, Liuyang; Weng, Haibo

doi:10.1007/s10529-016-2136-x

Cited by 3 publications

(1 citation statement)

References 14 publications

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“…Comparatively, the processivity may depend primarily on the length of the linker and less so on the specific amino acid composition, as a notable reduction of processivity was observed for EG1-Δ10 and EG1-Δ19, while other amino acid substitution variants had only moderate decreases in processivity. The importance of linker length for cellulase function is well documented [50, 51]. It has been proposed that the linker lengths between structured domains are optimized based on the GH domain and CBM type [31].…”

Section: Discussionmentioning

confidence: 99%

Altering the linker in processive GH5 endoglucanase 1 modulates lignin binding and catalytic properties

Wang

Zhang

Long

et al. 2018

Biotechnol Biofuels

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BackgroundThe non-productive adsorption of cellulases onto lignin in biomass is a key issue for the biofuel process economy. It would be helpful to reduce the inhibitory effect of lignin on enzymatic hydrolysis by engineering weak lignin-binding cellulases. Cellulase linkers are highly divergent in their lengths, compositions, and glycosylations. Numerous studies have revealed that linkers can facilitate optimal interactions between structured domains. Recently, efforts have focused on the contributions and mechanisms of carbohydrate-binding modules and catalytic domains that affect lignin affinity and processivity of cellulases, but our understanding of the effects of the linker regions on lignin adsorption and processivity of GH5 processive endoglucanases is still limited.ResultsEight GH5 endoglucanase 1 variants of varying length, flexibility, and sequence in the linker region were constructed. Their characteristics were then compared to the wild-type enzyme (EG1). Remarkably, significant differences in the lignin adsorption profiles and processivities were observed for EG1 and other variants. Our studies suggest that either the length or the specific amino acid composition of the linker has a prominent influence on the lignin-binding affinity of the enzymes. Comparatively, the processivity may depend primarily on the length of the linker and less so on the specific amino acid composition. EG1-ApCel5A, a variant with better performance in enzymatic hydrolysis in the presence of lignin, was obtained by replacing a longer, flexible linker. In total, up to between 28.2 and 30.1% more reducing sugars were generated from filter paper by EG1-ApCel5A in the presence of lignin compared to EG1.ConclusionsOur results highlight the relevance of the linker region in the lignin adsorption and processivity of a processive endoglucanase. Our findings suggest that the linker region may be used as a target for the design of more active and weaker lignin-binding cellulases.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1333-3) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Altering the linker in processive GH5 endoglucanase 1 modulates lignin binding and catalytic properties

Wang

Zhang

Long

et al. 2018

Biotechnol Biofuels

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Computational engineering of cellulase Cel9A-68 functional motions through mutations in its linker region

Costa

Silva

Batista

2018

Phys. Chem. Chem. Phys.

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Microbial cellulosic degradation by cellulases has become a complementary approach for biofuel production. However, its efficiency is hindered by the recalcitrance of cellulose fibres. In this context, computational protein design methods may offer an efficient way to obtain variants with improved enzymatic activity. Cel9A-68 is a cellulase from Thermobifida fusca that is still active at high temperatures. In a previous work, we described a collective bending motion, which governs the overall cellulase dynamics. This movement promotes the approximation of its CBM and CD structural domains (that are connected by a flexible linker). We have identified two residues (G460 and P461) located at the linker that act as a hinge point. Herein, we applied a new level of protein design, focusing on the modulation of this collective motion to obtain cellulase variants with enhanced functional dynamics. We probed whether specific linker mutations would affect Cel9A-68 dynamics through computational simulations. We assumed that P461G and G460+ (with an extra glycine) constructs would present enhanced interdomain motions, while the G460P mutant would be rigid. From our results, the P461G mutation resulted in a broader exploration of the conformational space, as confirmed by clustering and free energy analyses. The WT enzyme was the most rigid system. However, G460P and P460+ explored distinct conformational states described by opposite directions of low-frequency normal modes; they sampled preferentially closed and open conformations, respectively. Overall, we highlight two significant findings: (i) all mutants explored larger conformational spaces than the WT; (ii) the selection of distinct conformational populations was intimately associated with the mutation considered. Thus, the engineering of Cel9A-68 motions through linker mutations may constitute an efficient way to improve cellulase activity, facilitating the disruption of cellulose fibres.

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Enhancing the Catalytic Efficiency of Non-Modular Processive Endoglucanase Eg5c-1 by Incorporating an Extra Carbohydrate Binding Module

jingyi,

wenyu,

ziyu

et al. 2024

Preprint

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Extending the linker region increases the activity of the Bacillus subtilis cellulase CelI15

Abstract: The activity of CelI15 was increased by increasing the LR copy number, which could be a potential way to improve its enzymatic properties.

Cited by 3 publications

References 14 publications

Altering the linker in processive GH5 endoglucanase 1 modulates lignin binding and catalytic properties

Altering the linker in processive GH5 endoglucanase 1 modulates lignin binding and catalytic properties

Computational engineering of cellulase Cel9A-68 functional motions through mutations in its linker region

Enhancing the Catalytic Efficiency of Non-Modular Processive Endoglucanase Eg5c-1 by Incorporating an Extra Carbohydrate Binding Module

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