Nanomaterials
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2024
·Vol. 14
·期 4
·340
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IF 4.4
Xiaozhou Li,Jie Chen,Bin Wu,Zhen Gao,Bingfang He
Exploring an appropriate immobilization approach to enhance catalytic activity and reusability of cellulase is of great importance to reduce the price of enzymes and promote the industrialization of cellulose-derived biochemicals. In this study, Fe3O4magnetic nanoparticles (MNPs) were functionalized with meso-2,3-dimercaptosuccinic acid to introduce carboxyl groups on the surface (DMNPs). Then, melamine–glutaraldehyde dendrimer-like polymers were grafted on DMNPs to increase protein binding sites for the immobilization of processive endoglucanase EG5C-1. Moreover, this dendrimer-like structure was beneficial to protect the conformation of EG5C-1 and facilitate the interaction between substrate and active center. The loading capacity of the functionalized copolymers (MG-DMNPs) for EG5C-1 was about 195 mg/g, where more than 90% of the activity was recovered. Immobilized EG5C-1 exhibited improved thermal stability and increased tolerability over a broad pH range compared with the free one. Additionally, MG-DMNP/EG5C-1 biocomposite maintained approximately 80% of its initial hydrolysis productivity after five cycles of usage using filter paper as the substrate. Our results provided a promising approach for the functionalization of MNPs, enabling the immobilization of cellulases with a high loading capacity and excellent activity recovery.
研究领域:
生物化学
·
纳米生物学
·
酶工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Molecular Catalysis
·
2025
·
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
·
酶工程
✓ 已验证
产品关联
DOI
高影响因子
ACS Sustainable Chemistry & Engineering
·
2021
·
IF 8.198
Kemin Lv,Zhimeng Yu,Marcelo M. Pedroso,Bin Wu,Zhen Gao,Bingfang He,Gerhard Schenk
Establishing a suitable immobilization strategy to improve the accessibility of immobilized cellulase for insoluble cellulose and subsequently recover the enzyme from the remaining substrate is crucial to promote the industrialization of biofuels. Here, using iminodiacetic acid (IDA) and Ni2+ ions, a novel metal-chelated copolymer of methacrylic acid and methyl methacrylate was prepared for the immobilization of His-tagged processive endoglucanase EG5C-1 via affinity interaction between polyhistidine tag and Ni2+. The optimum loading capacity of the functionalized polymer (Eud-IDA-Ni2+) for EG5C-1 was about 280 mg/g, where more than 80% of the activity was recovered. Immobilized EG5C-1 exhibited improved thermal and pH stability and better reusability than the free one, and after five cycles of usage, the hydrolysis productivity remained above 70% of the initial value. The Eud-IDA-Ni2+/EG5C-1 biocomposite displayed reversibly soluble–insoluble characteristics with pH change, which was in the soluble state during the enzyme reaction process but could be recovered in an insoluble form by lowering the pH after the reaction. Thus, the yield obtained from the hydrolysis of an insoluble phosphoric acid-swollen cellulose substrate was similar for the free and immobilized form of EG5C-1. Our combined results suggested that a metal-ion-chelated, pH-responsive polymer had the potential for His-tagged cellulase immobilization to improve both the operational efficiency and economic benefit of the cellulosic biorefinery industry.
研究领域:
生物化学
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生物工程
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产品关联
DOI
高影响因子
ACS Synthetic Biology
·
2024
·
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
·
酶工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）