ACS Synthetic Biology
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2024
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IF 3.7
Jialing Wang,Jie Chen,Kemin Lv,Zhen Gao,Jiahuang Li,Bin Wu,Bingfang He,Gerhard Schenk
Processive endoglucanases have generated significant interest due to their bifunctionality in the degradation of cellulose and low product inhibition. However, enhancing their catalytic efficiency through engineering remains a formidable challenge. To address this bottleneck, our engineering efforts targeted loop regions located in the substrate channel of processive endoglucanase EG5C-1. Guided by a comparative analysis of characteristic structural features of the substrate channels in cellobiohydrolase, endoglucanase, and processive endoglucanase, a highly active triple mutant CM6 (N105H/T205S/D233L) was generated that had a 5.1- and 4.7-fold increase in catalytic efficiency toward soluble substrate carboxymethyl cellulose-Na and insoluble substrate phosphoric acid-swollen cellulose (PASC), compared with wild-type EG5C-1. Furthermore, this mutant exhibited greater processivity compared to EG5C-1. Molecular dynamics simulations unveiled that the mutations in the loop regions reshaped the substrate channel, leading to a deeper cleft, resembling the closed channel configuration of cellobiohydrolases. The increased compactness of the substrate channel induced changes in the substrate binding mode and substrate deformation, thereby enhancing both binding affinity and catalytic efficiency. Moreover, metadynamics simulations demonstrated that the processive velocity of cellulose chain through the binding channel in mutant CM6 surpassed that observed in EG5C-1.
研究领域:
生物化学
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酶工程
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PubMed
高影响因子
CARBOHYDRATE POLYMERS
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2024
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IF 10.7
Xueyan Wang,Le Liu,Ruiyu Shen,Qian Wang,Xiaoqi Xie,Weiming Liu,Zhimin Yu,Xianzhen Li,Xiaoyu Guo,Fan Yang
The inefficient decomposition of polysaccharides, particularly branched polysaccharides limits their large-scale industrial applications. Further understanding and modification of glycoside hydrolases (GHs) processivity is expected to overcome this limitation. Here, a novel xanthan-binding CBM ( Mi XBM), which was supposed to alter the processivity of GHs, was systematically characterized. Phylogeny and structure analyses indicated that Mi XBM is closely related to putative polysaccharide side chain-binding modules. Quantitative binding assays further revealed that Mi XBM probably has a high affinity for xanthan side chain via a variable loop site. Moreover, catalytic performance demonstrated that xanthanase chimeras containing Mi XBM promote highly efficient hydrolysis of xanthan because of improved substrate accessibility. Notably, Mi XBM was observed to enhance the processivity of xanthanase, owing to its high substrate affinity to the repeating unit xanthan. Furthermore, sequential hydrolysis of xanthan by xanthanases with varying processivity resulted in significantly increased hydrolytic efficiency and focused oligoxanthans array. These results expand understanding of CBM-substrate recognition and shed light on efficient degradation of other regularly branched polysaccharides using modified GHs.
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研究领域:
生物化学
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分子生物学
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PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
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IF 8.5
Wenming Shao,Junru Zhou,Yuzhen Huang,Ziyu Zhang,Yingwen Chen,Zhen Gao,Jiangfeng Ma,Bin Wu
The economic viability of cellulose biotransformation remains constrained by high enzyme costs, with processive endoglucanases emerging as promising candidates due to their dual-function hydrolysis mechanism. However, comprehensive kinetic and synergistic analyses of these enzymes are notably limited. This study investigates the kinetic properties of GH5 processive endoglucanase (M3-1) through various kinetic models. Inverse Michaelis-Menten analysis revealed M3-1's superior substrate recognition capacity, demonstrating 95.5 % productive binding site coverage compared to 48.8 % in non-processive endoglucanases. This enhanced efficiency is attributed to M3-1's distinctive structural features, particularly its open and deep cleft configuration. Pre-steady-state kinetics identified substrate association as the rate-limiting step, providing crucial direction for enzyme engineering efforts. Synergistic studies with cellobiohydrolase (CBH) demonstrated remarkable degradation synergy (DS value up to 8.2 on filter paper) and improved substrate resistance compared to traditional EG/CBH combinations. We propose a novel bidirectional degradation mechanism for the M3-1/CBH system, operating both inside-out and outside-in. The effectiveness of M3-1/CBH combination was further enhanced by up to 320 % through the addition of nonionic surfactants and expansin. These findings advance our understanding of processive endoglucanases and their potential applications in biomass conversion.
研究领域:
生物化学
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酶工程
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生物质转化
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PubMed
高影响因子
Molecular Catalysis
·
2025
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IF 4.9
Jingyi Chen,Wenyu Shao,Ziyu Zhang,Zhen Gao,Jiaxing Xu,Bin Wu
Processive endoglucanase with both endoglucanase and exoglucanase activity exhibits great potential in the degradation of cellulose. Carbohydrate Binding Modules (CBMs) can improve the catalytic efficiency by positioning the substrate closer to the catalytic domain and boosting the affinity between the enzyme and substrate. To investigate the impact of CBMs and linker peptides on the catalytic performance of the processive endoglucanase EG5C-1, various EG5C-1 derivatives with different CBMs and linker peptides were constructed. The results suggested the introduction of CBM1 from Volvariella volvacea and flexible peptide (GGGGS) 3 led to a notable 3.4-fold increase on the Avicel and a 3.7-fold increase on the overall enzymatic activity (FPase). In addition, the kinetic parameters of EG5C-1-F3- Vv CBM1 showed higher affinity ( K m ) and higher catalytic efficiency ( k cat / K m ) for insoluble substrates. Moreover, inverse kinetic parameters indicated that 99.5% of attack sites were occupied, surpassing the wild type. The processivity enhanced by 60% compared to EG5C-1. A series of cellooligosaccharides were hydrolyzed, with a pattern similar to EG5C-1. These results suggest that the fusing CBMs and linker peptides to the catalytic domain could effectively design a highly efficient processive endoglucanase.
研究领域:
生物化学
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酶工程
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高影响因子
JOURNAL OF APPLIED POLYMER SCIENCE
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2020
·Vol. 137
·期 43
·49319
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IF 2.52
Weihua Ye,Qinghua Chen,Zheng Chen,Zhi Wen,Zhengfu Liao
Uracil derivatives are potential nontoxic thermal stabilizers of poly(vinyl chloride) (PVC) and have a better stabilization effect. 5,6-diamino-1,3-dimethyluracil (DDU) was investigated as a thermal stabilizer for PVC. The stabilization effect of DDU was measured by thermogravimetric analysis, thermal aging test, and recording the time of the color change of the Congo red paper (Congo red test). Meanwhile, the processability of PVC stabilized by DDU was investigated through dynamic performance test. The results showed a better stabilizing effect compared with calcium stearate (CaSt 2 ) and zinc stearate (ZnSt 2 ). To explain the stabilization of DDU, the probable mechanism was suggested that DDU absorbed and chemically bonded with hydrogen chloride. Therefore, DDU could be used as a thermal stabilizer of PVC.
研究领域:
材料科学
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化学工程
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高影响因子
ACS Catalysis
·
2024
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IF 11.3
Kemin Lv,Xiaozhou Li,Kequan Chen,Bin Wu,Bingfang He,Gerhard Schenk
Processive endoglucanase is a typical bifunctional biocatalyst for cellulose degradation. A GH5 processive endoglucanase from Bacillus subtilis BS-5 was previously identified and shown to exhibit highly efficient catalytic performance. To further augment its catalytic efficiency, both consensus mutagenesis and loop engineering were applied. Compared to the wild-type enzyme, a variant (M3-1) with the four point mutations, i.e., K91I, A198T, Q237D, and V240P, exhibits an 8.5- and 4.8-fold increase in catalytic efficiency toward the soluble substrate carboxymethyl cellulose-Na (CMC) and the insoluble phosphoric acid-swollen cellulose (PASC), respectively. Molecular dynamics simulations were employed to elucidate the conformational changes that led to the enhanced catalytic efficiency. Structural superpositions suggest that the mutations cause a swing in loop 230–241, which in turn affects enzyme–substrate affinity and recognition. Residues K91 and A198 are located distal from the active site. The mutations K91I and A198T influence key amino acids within the active pocket through residue interaction networks in the protein. Furthermore, dynamic cross-correlation matrices (DCCMs) indicate that variant M3-1 possesses a conformation that is more favorable than that of the wild-type enzyme, promoting an increased frequency of interactions between active site residues and substrate molecules and thereby enhancing catalytic efficiency. The combined results provide valuable insights into the rational design and engineering of processive endoglucanases and other glycoside hydrolases, enabling the development of improved enzymatic catalysts for biotechnology applications.
研究领域:
生物催化
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酶工程
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生物化学
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分子生物学
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高影响因子
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