BIOPROCESS AND BIOSYSTEMS ENGINEERING
·
2023
·
IF 3.8
Jia Dong-Xu,Yu Hai,Wang Fan,Jin Li-Qun,Liu Zhi-Qiang,Zheng Yu-Guo
Cellobiose 2-epimerase (CE) is ideally suited to synthesize lactulose from lactose, but the poor thermostability and catalytic efficiency restrict enzymatic application. Herein, a non-characterized CE originating from Caldicellulosiruptor morganii (CmCE) was discovered in the NCBI database. Then, a smart mutation library was constructed based on FoldX ΔΔG calculation and modeling structure analysis, from which a positive mutant D226G located within the α 8 /α 9 loop exhibited longer half-lives at 65–75 °C as well as lower K m and higher k cat / K m values compared with CmCE. Molecular modeling demonstrated that the improvement of D226G was largely attributed to the rigidification of the flexible loop, the compactness of the catalysis pocket and the increment of substrate-binding capability. Finally, the yield of synthesizing lactulose catalyzed by D226G reached 45.5%, higher than the 35.9% achieved with CmCE. The disclosed effect of the flexible loop on enzymatic stability and catalysis provides insight to redesign efficient CEs to biosynthesize lactulose. Graphical abstract
研究领域:
生物技术
·
酶学
·
分子生物学
·
化学工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIORESOURCE TECHNOLOGY
·
2026
·
IF 8.2
Wei Gao,Mengli Li,Ming Miao,Tao Zhang
A library of 37 Nicα GP mutants was built by computational engineering. • L226Y/V437L/F751H showed 2.10 fold higher activity than WT Nicα GP. • The triple mutant improved thermostability and kinetics. • Cascade productivity increased by 26.5 % at a cellobiose/G4 ratio of 1:2. • Structural analysis linked activity gains to regulatory layer optimization. Converting non-food cellulosic sugars into programmable amylose offers a route to higher-value carbohydrate materials. α -Glucan phosphorylase ( α GP) controls glucose-1-phosphate (G-1-P)-dependent chain elongation in cellobiose-to-amylose cascades. Here, Nicotiana attenuata α GP ( Nicα GP) was engineered by integrating AlphaFold3-based structural modeling, ProteinMPNN, EVcouplings, structural priors, and DynaMut2 filtering. Of 916 candidate substitutions, 37 were selected for experimental validation, and combinatorial screening identified L226Y/V437L/F751H as the optimal triple mutant, with 2.10-fold higher relative activity than the same-batch wild-type (WT) control and clear positive epistasis. The mutant further shifted the optimal temperature from 40 to 45 °C, increased thermal transition midpoint from 56.2 to 58.8 °C, and enhanced catalytic efficiency toward both maltotetraose and G-1-P. In the Clostridium thermocellum cellobiose phosphorylase ( Ct CBP)– Nicα GP cascade, L226Y/V437L/F751H reached 38.86 ± 2.06 % conversion at 72 h versus the previously reported WT value of 35.84 ± 1.04 % at 84 h, increasing apparent productivity by 26.5 %. Docking, CAVER, and molecular dynamics (MD) analyses suggested that these gains may be associated with optimization of structural regions surrounding the conserved catalytic core. This work provides an engineered α GP for amylose synthesis from cellobiose and supports peripheral regulatory engineering as a practical strategy for improving phosphorylase-based biocatalytic cascades. Download: Download high-res image (230KB) Download: Download full-size image
✓ 已验证
DOI
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Wei Gao,Ru Yang,Mengli Li,Ming Miao,Tao Zhang
The enzymatic synthesis of amylose is a sustainable solution for the valorisation of non-food cellulose. However, the limited thermostability and catalytic efficiency of existing α -glucan phosphorylases ( α GPs) hinders its potential for industrial scalability. In this study, we identified and characterised a novel α GP from Nicotiana attenuata ( Nicα GP) via genome mining. It exhibited superior catalytic performance, with a specific activity of 82.1 U/mg under optimised conditions (40 °C and pH 5.0), and notable temperature and pH stability. Supplementation of Mg 2+ further enhanced its activity by 5 %. Kinetic analysis revealed a catalytic efficiency ( k cat / K m ) of 27.46 mM −1 s −1 for maltotetraose (G4). Furthermore, a dual-enzyme cascade system integrating Nicα GP with Clostridium thermocellum cellobiose phosphorylase achieved up to 28.4 % cellulose conversion, synthesising amylose with tunable molecular weights (6602 ± 47.13–67,154 ± 47.28 g/mol) by modulating ratios of cellobiose/G4. Molecular docking identified the conserved residues (Gly196, Arg706, Lys711, and Lys821) critical for substrate binding and pyridoxal 5′-phosphate-mediated catalysis. Overall, this study provides a new enzyme component for the artificial synthesis of amylose from non-edible biomass, facilitating the widespread application of non-edible cellulosic resources for food conversion.
研究领域:
生物催化
·
工业酶学
·
生物质转化
·
植物生物技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2024
·
IF 7.7
Hu Xu,Mukan Ji,Dawei Xu,Yongqin Liu
Cellobiose 2-epimerase (CE) catalyzes the conversion of the lactose into its high-value derivatives, epilactose and lactulose, which has great prospects in food applications. In this study, CE sequences from the Qinghai-Tibet Plateau gene catalogue, we screened these for structural flexibility through molecular dynamics simulation to identify potential psychrophilic CE candidates. One such psychrophilic CE we termed psyCE demonstrated exceptional epimerization activity, achieving an optimum activity of 122.2 ± 1.6 U/mg. Its kinetic parameters (K cat and K m ) for epimerization activity were 219.9 ± 5.6 s −1 and 261.9 ± 18.1 mM, respectively, representing the highest K cat recorded among known cold-active CEs. Notably, this is the first report of a psychrophilic CE. The psyCE can effectively produce epilactose at 8 °C, converting 20.3 % of 200 mM lactose into epilactose within four hours. These findings suggest that psyCE is highly suitable for cryogenic food processing, and glaciers may serve as a valuable repository of psychrophilic enzymes.
研究领域:
生物化学
·
酶学
·
食品科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
·
2025
·Vol. 41
·期 11
·451
·
IF 4.2
Zhong Chenggang,Ding Weijian,Lin Haoran,Huang Jin
Cellobiose-2-epimerase is considered an ideal biocatalyst for the strictly thermodynamic synthesis of lactulose. However, its poor thermolability limits its industrial application. Herein, we successfully developed a novel thermostable cellobiose-2-epimerase for improving lactulose biosynthesis from lactose. The A242L mutant, derived from Dictyoglomus thermophilum (DiCE), was generated using semi-rational mutagenesis after computer-aided design via the FireProt web server. After systematic enzymatic characterization and optimization, A242L mutant exhibited a 123.5% T 1/2 in thermostability at 80 °C relative to the wild-type enzyme, and the E. coli BL21/pET28a(+)-A242L mutant harvested a maximal lactulose yield of 64.52%, representing a 51.38% improvement at a substrate concentration of 200 g/L for the whole-cell biotransformation reaction conducted at pH 7.0 and 80 °C for 4 h. Molecular docking and molecular dynamics (MD) simulation results indicated that the enhanced thermostability of the enzyme was closely related to the structure and rigidity of its flexible loops. The enhanced thermostability allows A242L to tolerate higher temperatures for extended periods, driving the reaction towards lactulose production, which is suitable for industrial lactulose production under harsh conditions.
研究领域:
酶工程
·
工业生物技术
·
生物化学
·
分子生物学
·
生物催化
✓ 已验证
产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
·
2025
·
IF 9.8
Zhou Chen,Yanyin Song,Yuhan Yan,Wei Chen,Tong Ren,Aijin Ma,Siting Li,Yingmin Jia
A novel cellobiose 2-epimerase gene ( CsCEase1 ) from Clostridium sp. TW13 was successfully overexpressed. Cs CEase1, with a molecular mass of 45.0 kDa, exhibited a specific activity of 254.75 U/mg and showed optimal activity at 40 °C and pH 7.5. Using Cs CEase1, 44.30 % of lactose was converted into epilactose, yielding a production level of 26.58 g/L. An efficient purification method was developed, incorporating crystallization and β-galactosidase treatment to remove the unconverted lactose, while Saccharomyces cerevisiae was used to consume the produced glucose. Final purification with activated carbon resulted in epilactose with 98 % purity and a recovery rate of 66.47 %. Furthermore, Cs CEase1 was incubated with whey power and expired milk, achieving epilactose conversion rates of 29.65 % and 32.69 %, respectively. These findings highlight the potential of Cs CEase1 for the cost-effective and environmentally sustainable production of epilactose from dairy waste.
研究领域:
生物化学
·
食品科学
·
环境科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
LANGMUIR
·
2024
·
IF 3.7
Lili Xu,Huanruo Liu,Xiaohan Wang,Qi Li,Suli Xu,Caizheng Sun,Hongbo Suo
β-Glucosidase (β-G) holds promising applications in various fields, such as biomass energy, food, pharmaceuticals, and environmental protection, yet its industrial application is still limited by issues of stability and recycling. Herein, we first immobilized β-G onto the surface of magnetic chitosan nanoparticles (MCS/β-G) through adsorption methods. Subsequently, utilizing the metal–organic framework (MOF), CaBDC, which possesses good stability under acidic conditions, we encapsulated MCS/β-G. The resulting biocatalyst (MCS/β-G@CaBDC) exhibited excellent activity and recyclability. MCS/β-G@CaBDC can convert 91.5% of cellobiose to glucose in 60 min and maintained 81.9% activity after 10 cycles. The apparent Km value of MCS/β-G@CaBDC was 0.148 mM, lower than free β-G (0.166 mM) and MCS/β-G (0.173 mM). The CaBDC layer increased the mass transfer resistance of the reaction but also triggered structural rearrangement of β-G during the encapsulation process. This resulted in the β-sheet content rising to 68.4%, which, in turn, contributed to enhancing the rigidity of β-G. Moreover, the saturated magnetic strength of this biocatalyst could reach 37.3 emu/g, facilitating its magnetic recovery. The biocatalyst prepared in this study exhibits promising application prospects, and the immobilization method can provide valuable insights into the field of enzyme immobilization.
研究领域:
生物化学
·
材料科学
·
酶工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ACS Sustainable Chemistry & Engineering
·
2024
·
IF 7.1
Xiaoyu Shi,Yang Yang,Xin Ye,Heng Zhong,Chunling Wang,Fangming Jin
Catalytic conversion of cellulosic biomass into polyols is an effective approach for biomass upgrading. However, the conversion involves cascade reactions which require various catalytic sites cooperation for activating specific steps, resulting in low efficiency. Here, we propose a bifunctional catalyst containing acid sites and hydrogenation capacity for polyol production with cellobiose as the model compound of cellulose. Phosphonic acids (PAs) containing either methyl (MPA) or aminomethyl (NH2MPA) tails were used to tune the Pd/Al2O3 catalyst to form monolayers, thereby modulating the surface physiochemical properties and introducing multiple catalytic sites. An increasing ratio of PdO/Pd and a strong metal–support interaction were observed after PA modification, which could boost the hydrogenation activity for cellobiose. Furthermore, Pd/Al2O3-MPA exhibited excellent hydrolytic hydrogenation activity due to the increase of Lewis acid sites with good durability under hydrothermal conditions by MPA modification, achieving a 78.5% yield of sorbitol at 180 °C for 5 h. A plausible mechanism for enhancing the synergistic effect of Pd and support over Pd/Al2O3-MPA was also proposed. This investigation provides a novel strategy for rationally designing hydrothermally stable bifunctional catalysts to utilize biomass in a more effective way.
研究领域:
催化化学
·
生物质能
✓ 已验证
产品关联
DOI
高影响因子
INDUSTRIAL & ENGINEERING CHEMISTRY RESEARCH
·
2016
·
IF 2.567
Guozhen Wang,Xuefeng Tan,Hui Lv,Mengmeng Zhao,Min Wu,Jinping Zhou,Xumu Zhang,Lina Zhang
The catalytic transformation of the most abundant cellulose to valuable platform chemicals is one of the significant issues to overcome the shortage of fossil fuels. Herein, we reported the first example of Ru-based homogeneous catalyst for the highly selective conversion of cellobiose and ball-milled cellulose to hexitols (including sorbitol, mannitol, and 1,4-sorbitan) under an acidic condition with the yield of 94.5% and 56.4%, respectively. The main features of this catalytic system were the high conversion efficiency of biomass, mild reaction condition (100 °C), and low catalyst loading, which was 1/20 of the related Ru/C heterogeneous catalyst. This work opened up a new avenue for the transformation of cellulose to hexitols under mild conditions.
研究领域:
化学工程
·
生物质能
✓ 已验证
产品关联
DOI
高影响因子
BIORESOURCE TECHNOLOGY
·
2026
·
IF 8.2
Hu Xu,Niannian Lv,Yongqin Liu
Psychrophilic cellobiose 2-epimerase (CE) is a promising biocatalyst for cold-process dairy manufacturing, yet its industrial application is hampered by the inherent trade-off between low-temperature activity and substrate affinity. To overcome this limitation, a semi-rational design strategy was implemented on a psychrophilic CE (psyCE). A GFP-based fluorescence assay was developed to normalize mutant expression, enabling precise identification of activity improvements. Structure analysis pinpointed six key residues for targeted mutagenesis, and a saturation mutagenesis library was constructed accordingly. Screening of the library yielded a robust double mutant (FR) with superior kinetics. The FR variant exhibited a record-breaking catalytic turnover (245.7 s −1 vs. 219.9 s −1 ) and an improved substrate affinity (208.9 mM vs. 261.9 mM), resulting in a 40% increase in overall catalytic efficiency. In a practical biocatalysis trial at 8 °C, FR achieved a 21% epilactose yield within 3 h, outperforming the wild type (20% within 4 h). Molecular dynamics simulations revealed the structural basis of this enhancement by showing the FR mutant evolves an enlarged binding pocket, while simultaneously establishing a strengthened hydrogen-bond network. This study not only provides a highly efficient cold-active biocatalyst but also establishes a screening platform for engineering psychrophilic enzymes.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Frontiers in Microbiology
·
2025
·
IF 4.5
Qiuqian Zeng,Xiaomei Lyu
Introduction: Cellobiose 2-epimerase (CE) catalyzes the interconversion of glucosyl and mannosyl groups at the reducing end of β-1,4-linked disaccharides. This enzyme is pivotal for converting lactose into prebiotics like epilactose, offering a potential solution for lactose-intolerant-friendly dairy products. However, current CEs are hindered by pH and thermal instability in milk processing, as their neutral-to-alkaline pH optima clash with milk’s mildly acidic conditions (pH 6.5–6.7), and their poor thermolability requires costly post-processing enzyme removal.Methods: We identified a novel CE from the acidophilic Acidobacteriota bacterium (Acba-CE) and characterized its properties. Its enzymatic activity was assessed under varying pH and temperature conditions, including milk-processing environments.Results: Acba-CE exhibits an acidic pH optimum (6.0), retaining 95% activity at milk pH (6.5). Notably, it undergoes rapid thermal inactivation at pasteurization temperatures, enabling complete enzyme deactivation during standard pasteurization without additional steps. In milk systems, Acba-CE achieves 28.5% lactose-to-epilactose conversion at refrigeration temperatures (10°C), demonstrating strong cold adaptability.Discussion: To our knowledge, this is the first reported CE from the Acidobacteriota phylum, combining acidophilic activity with low-temperature adaptability. Acba-CE represents a breakthrough for in situ dairy modification, eliminating key bottlenecks in milk processing and enabling next-generation functional milk production.
研究领域:
生物化学
·
食品科学
·
微生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ChemSusChem
·
2013
·Vol. 6
·期 8
·1545-1555
·
IF 7.475
Jinzhu Chen,Shengpei Wang,Jing Huang,Limin Chen,Longlong Ma,Xing Huang
Graphical Acid/Metal Balance : Bifunctional catalysts containing ruthenium and polyoxometalates as active species with a metal-organic framework as support and encapsulation matrix, respectively, are synthesized. Excellent yields in sorbitol are obtained in the conversion of cellobiose and ball-milled cellulose. The evaluation of the balance between the hydrogenation and hydrolysis functions of these bifucntional catalysts reveals that by carefully balancing the ratio of acid site density and the number of metal surface atoms a maximum conversion can be achieved. Cellulose and cellobiose were selectively converted into sorbitol over water-tolerant phosphotungstic acid (PTA)/metal– organic-framework-hybrid-supported ruthenium catalysts, Ru-PTA/MIL-100(Cr), under aqueous hydrogenation conditions. The goal was to investigate the relationship between the acid/metal balance of bifunctional catalysts Ru-PTA/MIL-100(Cr) and their performance in the catalytic conversion of cellulose and cellobiose into sugar alcohols. The control of the amount and strength of acid sites in the supported PTA/MIL-100(Cr) was achieved through the effective control of encapsulated-PTA loading in MIL-100(Cr). This design and preparation method led to an appropriately balanced Ru-PTA/MIL-100(Cr) in terms of Ru dispersion and hydrogenation capacity on the one hand, and acid site density of PTA/MIL-100(Cr) (responsible for acid-catalyzed hydrolysis) on the other hand. The ratio of acid site density to the number of Ru surface atoms ( n A / n Ru ) of Ru-PTA/MIL-100(Cr) was used to monitor the balance between hydrogenation and hydrolysis functions; the optimum balance between the two catalytic functions, that is, 8.84< n A / n Ru <12.90, achieves maximum conversion of cellulose and cellobiose into hexitols. Under the applied reaction conditions, optimal results (63.2 % yield in hexitols with a selectivity for sorbitol of 57.9 % at complete conversion of cellulose, and 97.1 % yield in hexitols with a selectivity for sorbitol of 95.1 % at complete conversion of cellobiose) were obtained using a Ru-PTA/MIL-100(Cr) catalyst with loadings of 3.2 wt % for Ru and 16.7 wt % for PTA. This research thus opens new perspectives for the rational design of acid/metal bifunctional catalysts for biomass conversion.
研究领域:
催化化学
·
生物质转化
·
材料科学
✓ 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）