Pub Date : 2026-03-01Epub Date: 2025-12-06DOI: 10.1016/j.bej.2025.110042
Shimiao Chen , Yan Chen , Bin Shan , Yanyan Li , Fuhai Zheng , Yican Luo , Qinyu Lu
Recombinant papain production in Escherichia coli is limited by misfolding, aggregation, and host toxicity. Using a high-throughput microdroplet microbial culture (MMC) platform, we monitored chaperone-assisted folding and identified a transient “lysis window” during mid-log phase when activity peaked at 818.2 U/mg, doubling conventional yields. Co-expression with the Trigger Factor (TF) chaperone was most effective, whereas combining co-translational TF with the post-translational GroEL/ES system caused kinetic incompatibility, leading to massive aggregation and reduced activity (365.6 U/mg). Structural analyses revealed that optimal activity correlates with conformational flexibility rather than a static structure, a feature imparted by TF. Optimizing temporal dynamics and chaperone coordination is therefore essential for producing complex proteins. MMC provides a powerful platform for dissecting these folding pathways; in the redox-engineered E. coli SHuffle strain, this correct folding was synergistically enhanced, dramatically increasing soluble yields (to 450.2 mg/L) while maintaining high specific activity (857 U/mg) and confirming that an oxidizing cytoplasm is key for efficient production.
{"title":"High-throughput microdroplet screening reveals chaperone- and time-dependent enhancement of recombinant papain folding and activity in Escherichia coli","authors":"Shimiao Chen , Yan Chen , Bin Shan , Yanyan Li , Fuhai Zheng , Yican Luo , Qinyu Lu","doi":"10.1016/j.bej.2025.110042","DOIUrl":"10.1016/j.bej.2025.110042","url":null,"abstract":"<div><div>Recombinant papain production in <em>Escherichia coli</em> is limited by misfolding, aggregation, and host toxicity. Using a high-throughput microdroplet microbial culture (MMC) platform, we monitored chaperone-assisted folding and identified a transient “lysis window” during mid-log phase when activity peaked at 818.2 U/mg, doubling conventional yields. Co-expression with the Trigger Factor (TF) chaperone was most effective, whereas combining co-translational TF with the post-translational GroEL/ES system caused kinetic incompatibility, leading to massive aggregation and reduced activity (365.6 U/mg). Structural analyses revealed that optimal activity correlates with conformational flexibility rather than a static structure, a feature imparted by TF. Optimizing temporal dynamics and chaperone coordination is therefore essential for producing complex proteins. MMC provides a powerful platform for dissecting these folding pathways; in the redox-engineered <em>E. coli</em> SHuffle strain, this correct folding was synergistically enhanced, dramatically increasing soluble yields (to 450.2 mg/L) while maintaining high specific activity (857 U/mg) and confirming that an oxidizing cytoplasm is key for efficient production.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"227 ","pages":"Article 110042"},"PeriodicalIF":3.7,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145734005","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2025-12-14DOI: 10.1016/j.bej.2025.110050
Chenyang Huang , Wanning Gao , Mengting Chang , Xing Zhang , Junhua Tao , Yamei Lin , Lei Lin
Metal-organic frameworks (MOFs) have been widely explored as carriers for enzyme immobilization. However, conventional embedding and adsorption methods often suffer from limitations such as weak binding or enzyme leaching. In this study, we presented an enhanced immobilization strategy leveraging the chelation between histidine residues and nickel ions on Ni-based MOF, and we further validated the mechanism of enzyme immobilization through a histidine alkylation substitution strategy. This approach ensured strong enzyme anchoring on the MOF surface while providing superior protection to the enzyme, especially for the fragile glycoenzymes. Molecular dynamics (MD) simulations confirmed that not only the enzyme attachment was a rapid process, but also the structural integrity and catalytic activity of PmHS2 were preserved upon immobilization. The corresponding Enzyme-MOF system demonstrated remarkable stability, retaining 80.65 % activity after 7 recycling cycles and 80.95 % activity after 40 days of storage. These results confirmed that histidine-Ni²⁺ coordination is a highly effective strategy for improving enzyme immobilization performance.
金属有机骨架作为固定化酶的载体已被广泛探索。然而，传统的包埋和吸附方法往往受到弱结合或酶浸等限制。在本研究中，我们提出了一种利用组氨酸残基与镍离子在ni基MOF上螯合的强化固定化策略，并通过组氨酸烷基化取代策略进一步验证了酶固定化机制。这种方法确保了酶在MOF表面的强锚定，同时为酶提供了优越的保护，特别是对脆弱的糖酶。分子动力学（MD）模拟证实了PmHS2不仅是一个快速的酶附着过程，而且在固定后保持了PmHS2的结构完整性和催化活性。相应的酶- mof体系表现出显著的稳定性，在7次循环后保持80.65 %的活性，在储存40天后保持80.95 %的活性。这些结果证实了组氨酸- ni 2 +配合是提高酶固定化性能的一种非常有效的策略。
{"title":"Leveraging histidine-nickel coordination for stable enzyme immobilization on metal-organic frameworks","authors":"Chenyang Huang , Wanning Gao , Mengting Chang , Xing Zhang , Junhua Tao , Yamei Lin , Lei Lin","doi":"10.1016/j.bej.2025.110050","DOIUrl":"10.1016/j.bej.2025.110050","url":null,"abstract":"<div><div>Metal-organic frameworks (MOFs) have been widely explored as carriers for enzyme immobilization. However, conventional embedding and adsorption methods often suffer from limitations such as weak binding or enzyme leaching. In this study, we presented an enhanced immobilization strategy leveraging the chelation between histidine residues and nickel ions on Ni-based MOF, and we further validated the mechanism of enzyme immobilization through a histidine alkylation substitution strategy. This approach ensured strong enzyme anchoring on the MOF surface while providing superior protection to the enzyme, especially for the fragile glycoenzymes. Molecular dynamics (MD) simulations confirmed that not only the enzyme attachment was a rapid process, but also the structural integrity and catalytic activity of PmHS2 were preserved upon immobilization. The corresponding Enzyme-MOF system demonstrated remarkable stability, retaining 80.65 % activity after 7 recycling cycles and 80.95 % activity after 40 days of storage. These results confirmed that histidine-Ni²⁺ coordination is a highly effective strategy for improving enzyme immobilization performance.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"227 ","pages":"Article 110050"},"PeriodicalIF":3.7,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145786990","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2025-11-28DOI: 10.1016/j.bej.2025.110035
Lucky Caesar Direstiyani, Daisuke Inoue, Michihiko Ike
An electromethanogenesis (EM) system was successfully established using non-acclimated anaerobic digestion sludge as the inoculum. This study aimed to evaluate the EM performance by varying the electrolyte composition and the applied voltage. Alterations in the microbial community associated with CH4 generation and bioelectrochemchemical performance were also investigated. The findings indicated that the use of organic-rich electrolyte with a low applied voltage of 0.15 V showed a positive correlation with enhanced CH4 generation up to 59 % and a CH4 yield of 223.13 mmol day−1 m−2 which was ten times higher than the operation using the same electrolyte with an applied voltage of 0.35 V. Microbial community analysis revealed a shift of dominant methanogens from Methanosaeta to Methanosarcina and Methanoculleus at the cathodic biofilms when operated with organic-rich electrolyte at low voltage of 0.15 V. The presence of electroactive bacteria, such as DMER64 and JGI-0000079-D21, and syntrophic bacteria, including Desulfovibrio and Petrimonas, suggested the development of syntrophic interactions that strengthen biofilm resilience and the overall performance of the EM system. The microbial interaction network also emphasized the significance of electrolyte composition and adequate applied voltage in shaping microbial biofilms for efficient CH4 generation. The findings of this study accentuate the roles of sufficient electrolyte composition and low-voltage in enhancing the EM performance and corroborate the synergistic advantages of the EM system.
{"title":"Effects of electrolyte composition and applied voltage on methane generation and microbial community shifts in the electromethanogenesis system","authors":"Lucky Caesar Direstiyani, Daisuke Inoue, Michihiko Ike","doi":"10.1016/j.bej.2025.110035","DOIUrl":"10.1016/j.bej.2025.110035","url":null,"abstract":"<div><div>An electromethanogenesis (EM) system was successfully established using non-acclimated anaerobic digestion sludge as the inoculum. This study aimed to evaluate the EM performance by varying the electrolyte composition and the applied voltage. Alterations in the microbial community associated with CH<sub>4</sub> generation and bioelectrochemchemical performance were also investigated. The findings indicated that the use of organic-rich electrolyte with a low applied voltage of 0.15 V showed a positive correlation with enhanced CH<sub>4</sub> generation up to 59 % and a CH<sub>4</sub> yield of 223.13 mmol day<sup>−1</sup> m<sup>−2</sup> which was ten times higher than the operation using the same electrolyte with an applied voltage of 0.35 V. Microbial community analysis revealed a shift of dominant methanogens from <em>Methanosaeta</em> to <em>Methanosarcina</em> and <em>Methanoculleus</em> at the cathodic biofilms when operated with organic-rich electrolyte at low voltage of 0.15 V. The presence of electroactive bacteria, such as <em>DMER64</em> and <em>JGI-0000079-D21</em>, and syntrophic bacteria, including <em>Desulfovibrio</em> and <em>Petrimonas</em>, suggested the development of syntrophic interactions that strengthen biofilm resilience and the overall performance of the EM system. The microbial interaction network also emphasized the significance of electrolyte composition and adequate applied voltage in shaping microbial biofilms for efficient CH<sub>4</sub> generation. The findings of this study accentuate the roles of sufficient electrolyte composition and low-voltage in enhancing the EM performance and corroborate the synergistic advantages of the EM system.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"227 ","pages":"Article 110035"},"PeriodicalIF":3.7,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145682446","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2025-12-02DOI: 10.1016/j.bej.2025.110038
Xue-Chun Li , Hong-Jie Xie , Zheng-Xiong Zhou , Bin Sun , Jun-Jie Lin , Xiao-Dan Li , Ren Wang
1-aminocyclopropane-1-carboxylic acid (ACC), a crucial intermediate in the ethylene biosynthetic pathway, plays a decisive role in regulating plant developmental processes and stress responses. In heterologous biosynthesis systems, ACC biosynthesis is inhibited due to its antibacterial activity. Enhancing ACC export confers advantages to ACC-producing biotechnological platforms and microbial cell factories. While bacterial transporters for canonical amino acids in Corynebacterium glutamicum and Escherichia coli have been extensively studied, their roles in ACC transport remain unexplored. To identify the exporters mediating ACC transport in E. coli, relevant experiments were performed in this study. The results showed that the leucine-exporter LeuE acts as a high-efficiency ACC exporter in E. coli. Subsequently, structural modeling coupled with site-directed mutagenesis revealed that L160 is a critical residue in determining the ACC transport specificity of LeuE. The L160Y mutation enhanced E. coli tolerance to ACC, elevating the threshold from 50 mM to 100 mM. Structural modeling predicted that the L160Y mutation-induced compaction reduced the transporter channel diameter by 28 % (from 9.551 Å to 6.876 Å), a change that may enhance the binding affinity of ACC. Notably, a significant reduction in intracellular ACC levels was observed in both the LeuE-overexpressing and LeuE (L160Y)-overexpressing strains. These findings enable large-scale biosynthesis of ACC and its derivatives via engineered microbial systems, thereby facilitating cost-effective development of ACC-based pharmaceuticals and agrochemicals.
{"title":"An exporter of Leucine, LeuE functions as an exporter of 1-aminocyclopropane-1-carboxylic acid in Escherichia coli","authors":"Xue-Chun Li , Hong-Jie Xie , Zheng-Xiong Zhou , Bin Sun , Jun-Jie Lin , Xiao-Dan Li , Ren Wang","doi":"10.1016/j.bej.2025.110038","DOIUrl":"10.1016/j.bej.2025.110038","url":null,"abstract":"<div><div>1-aminocyclopropane-1-carboxylic acid (ACC), a crucial intermediate in the ethylene biosynthetic pathway, plays a decisive role in regulating plant developmental processes and stress responses. In heterologous biosynthesis systems, ACC biosynthesis is inhibited due to its antibacterial activity. Enhancing ACC export confers advantages to ACC-producing biotechnological platforms and microbial cell factories. While bacterial transporters for canonical amino acids in <em>Corynebacterium glutamicum</em> and <em>Escherichia coli</em> have been extensively studied, their roles in ACC transport remain unexplored. To identify the exporters mediating ACC transport in <em>E. coli</em>, relevant experiments were performed in this study. The results showed that the leucine-exporter LeuE acts as a high-efficiency ACC exporter in <em>E. coli</em>. Subsequently, structural modeling coupled with site-directed mutagenesis revealed that L160 is a critical residue in determining the ACC transport specificity of LeuE. The L160Y mutation enhanced <em>E. coli</em> tolerance to ACC, elevating the threshold from 50 mM to 100 mM. Structural modeling predicted that the L160Y mutation-induced compaction reduced the transporter channel diameter by 28 % (from 9.551 Å to 6.876 Å), a change that may enhance the binding affinity of ACC. Notably, a significant reduction in intracellular ACC levels was observed in both the LeuE-overexpressing and LeuE (L160Y)-overexpressing strains. These findings enable large-scale biosynthesis of ACC and its derivatives via engineered microbial systems, thereby facilitating cost-effective development of ACC-based pharmaceuticals and agrochemicals.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"227 ","pages":"Article 110038"},"PeriodicalIF":3.7,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145682449","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2025-12-11DOI: 10.1016/j.bej.2025.110046
Chenchen Yu, Yuyi Zheng, Weitie Lin, Jianfei Luo
Nitrifiers including ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) are well-known to release dissolved organic carbon (DOC) and synthesize extracellular polymeric substances (EPS), while the composition and difference of these products between them remain unclear. Moreover, the ecological role of this trait in nitrifier during their synergy with heterotroph is still largely unknown. In this study, both strains of AOB and NOB were found to produce significant amounts of DOC (a maximum of 5.22 and 8.91 mg/L) and EPS (a maximum of 11.39 and 4.63 mg/L). The DOC composition was similar between the tested strains of AOB and NOB. The EPS synthesis was found to occur throughout the whole growth phases, but their composition between AOB and NOB was different. The nitrifier released DOC supported the growth of heterotrophs (with the maximum cell number reached 5.0 × 107 CFU/mL), which in turn significantly promoted their growths (a maximum of 2.17 and 1.98 times higher), substrate oxidation activities and EPS syntheses. Co-cultivation with heterotroph significantly upregulated the functional genes of nitrifier that involving in substrate oxidation, CO2 fixation, and EPS synthesis. The nitrifier-heterotroph synergy enhanced the nitrifiers’ substrate oxidation, indicating its significant ecological role in promoting nutrient removal in wastewater treatment.
{"title":"Nitrifier released extracellular organics: Characterization and their ecological role in synergy with heterotrophs","authors":"Chenchen Yu, Yuyi Zheng, Weitie Lin, Jianfei Luo","doi":"10.1016/j.bej.2025.110046","DOIUrl":"10.1016/j.bej.2025.110046","url":null,"abstract":"<div><div>Nitrifiers including ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) are well-known to release dissolved organic carbon (DOC) and synthesize extracellular polymeric substances (EPS), while the composition and difference of these products between them remain unclear. Moreover, the ecological role of this trait in nitrifier during their synergy with heterotroph is still largely unknown. In this study, both strains of AOB and NOB were found to produce significant amounts of DOC (a maximum of 5.22 and 8.91 mg/L) and EPS (a maximum of 11.39 and 4.63 mg/L). The DOC composition was similar between the tested strains of AOB and NOB. The EPS synthesis was found to occur throughout the whole growth phases, but their composition between AOB and NOB was different. The nitrifier released DOC supported the growth of heterotrophs (with the maximum cell number reached 5.0 × 10<sup>7</sup> CFU/mL), which in turn significantly promoted their growths (a maximum of 2.17 and 1.98 times higher), substrate oxidation activities and EPS syntheses. Co-cultivation with heterotroph significantly upregulated the functional genes of nitrifier that involving in substrate oxidation, CO<sub>2</sub> fixation, and EPS synthesis. The nitrifier-heterotroph synergy enhanced the nitrifiers’ substrate oxidation, indicating its significant ecological role in promoting nutrient removal in wastewater treatment.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"227 ","pages":"Article 110046"},"PeriodicalIF":3.7,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145734003","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
L-Glutamine (L-Gln) was efficiently biosynthesized in Corynebacterium glutamicum using metabolic engineering strategies. Four C. glutamicum strains (ATCC 13032, ATCC 13809, s026, and s9114) were screened, and γ-glutamyl kinase was deleted using CRISPR/Cas12a system to redirect carbon flux, increasing L-Gln titer from 4.7 g/L to 5.3 g/L. Several glutamine synthetase (GS) were screened from different sources, and Saccharomyces cerevisiae-derived GS demonstrated to be with optimal activity. Combined with RBS optimization to enhance translational efficiency, L-Gln titer was increased to 14.62 g/L. Subsequently, a dual strategy of site-directed mutagenesis of GS and small RNA-mediated inhibition of adenylyltransferase were conducted to relieve the adenylylation of GS, increasing L-Gln titer to 19.06 g/L. A “pull-push” strategy was implemented by strengthening glutamate dehydrogenase to enhance L-glutamate precursor supply while blocking L-Gln catabolism via deletion of glutamate synthase and glutaminase, resulting in 28.51 g/L L-Gln. Self-regulatory metabolic control was achieved using a growth-responsive promoter Pcg2705 to downregulate α-ketoglutarate dehydrogenase during stationary phase, achieving a shake flask titer of 31.85 g/L. The engineered strain CG17 produced 58.96 g/L L-Gln in a 5 L fed-batch bioreactor, with a yield of 0.31 g/g glucose and productivity of 1.05 g/L/h. The work provides valuable insights for developing high-performance strains for amino acid biosynthesis.
{"title":"Rational engineering of Corynebacterium glutamicum for L-Glutamine biosynthesis","authors":"Ying-Tong Lin, Meng Chai, Qing-Hai Liu, Yu-Shu Ma, Xin-Yi Tao, Min Liu, Dong-Zhi Wei","doi":"10.1016/j.bej.2025.110011","DOIUrl":"10.1016/j.bej.2025.110011","url":null,"abstract":"<div><div><span><span>L</span></span>-Glutamine (<span>L</span>-Gln) was efficiently biosynthesized in <em>Corynebacterium glutamicum</em> using metabolic engineering strategies. Four <em>C. glutamicum</em> strains (ATCC 13032, ATCC 13809, s026, and s9114) were screened, and γ-glutamyl kinase was deleted using CRISPR/Cas12a system to redirect carbon flux, increasing <span>L</span>-Gln titer from 4.7 g/L to 5.3 g/L. Several glutamine synthetase (GS) were screened from different sources, and <em>Saccharomyces cerevisiae</em>-derived GS demonstrated to be with optimal activity. Combined with RBS optimization to enhance translational efficiency, <span>L</span>-Gln titer was increased to 14.62 g/L. Subsequently, a dual strategy of site-directed mutagenesis of GS and small RNA-mediated inhibition of adenylyltransferase were conducted to relieve the adenylylation of GS, increasing <span>L</span>-Gln titer to 19.06 g/L. A “pull-push” strategy was implemented by strengthening glutamate dehydrogenase to enhance <span>L</span>-glutamate precursor supply while blocking <span>L</span>-Gln catabolism via deletion of glutamate synthase and glutaminase, resulting in 28.51 g/L <span>L</span>-Gln. Self-regulatory metabolic control was achieved using a growth-responsive promoter Pcg2705 to downregulate α-ketoglutarate dehydrogenase during stationary phase, achieving a shake flask titer of 31.85 g/L. The engineered strain CG17 produced 58.96 g/L <span>L</span>-Gln in a 5 L fed-batch bioreactor, with a yield of 0.31 g/g glucose and productivity of 1.05 g/L/h. The work provides valuable insights for developing high-performance strains for amino acid biosynthesis.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"227 ","pages":"Article 110011"},"PeriodicalIF":3.7,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145617520","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2025-11-25DOI: 10.1016/j.bej.2025.110017
Pınar Kocabaş
Genome-scale metabolic models (GEMs) are powerful tools for exploring the metabolic state of cells and interpreting complex biological data. A key component of these models is the gene-protein-reaction (GPR) association, which links genes to enzymatic reactions via Boolean logic to account for isozymes and protein complexes. The accuracy of GPRs, alongside the stoichiometric matrix, critically determines the predictive performance of GEMs. GPRs play a central role in constructing condition-specific, cell line, and disease models, and are widely used in gene essentiality analysis, expression profiling, and strain design. This review presents a historical overview of GPR construction in mammalian and yeast GEMs, summarizes the main tools, databases, and methods used for their generation and curation, and identifies current challenges and limitations. Finally, potential improvements in GPR generation frameworks to enhance their utility in systems biology and metabolic engineering applications are discussed.
{"title":"Generation of gene-protein-reaction association rules in genome scale metabolic models: Chronology, challenges, and future perspectives","authors":"Pınar Kocabaş","doi":"10.1016/j.bej.2025.110017","DOIUrl":"10.1016/j.bej.2025.110017","url":null,"abstract":"<div><div>Genome-scale metabolic models (GEMs) are powerful tools for exploring the metabolic state of cells and interpreting complex biological data. A key component of these models is the gene-protein-reaction (GPR) association, which links genes to enzymatic reactions via Boolean logic to account for isozymes and protein complexes. The accuracy of GPRs, alongside the stoichiometric matrix, critically determines the predictive performance of GEMs. GPRs play a central role in constructing condition-specific, cell line, and disease models, and are widely used in gene essentiality analysis, expression profiling, and strain design. This review presents a historical overview of GPR construction in mammalian and yeast GEMs, summarizes the main tools, databases, and methods used for their generation and curation, and identifies current challenges and limitations. Finally, potential improvements in GPR generation frameworks to enhance their utility in systems biology and metabolic engineering applications are discussed.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"227 ","pages":"Article 110017"},"PeriodicalIF":3.7,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145617518","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2025-11-27DOI: 10.1016/j.bej.2025.110029
Shuai Huang , Haoyuan Tan , Luxuan Sun , Meng Wang , Biqiang Chen
This study demonstrated the advantages of the rotating bed reactor (RBR) over the conventional turbine stirred tank reactor (TSTR) for enzymatically catalyzed biodiesel synthesis using D311-resin immobilized lipase. Integrating simulation and experimental analyses, the work revealed that the rotating bed generated significantly lower shear forces compared to the turbine stirred paddles, thereby preserving lipase integrity and enhancing reusability. Simulations identified tangential velocity—modulated by rotational speed and bed porosity—as the dominant factor governing hydrodynamic velocity and liquid-solid mass transfer coefficients. Experimental validation confirmed these findings: Under optimized conditions, the yield of the fatty acid methyl esters (FAMEs) decreased from 87.49 % to 60.33 % after continuous use of immobilized lipase for 48 cycles in the RBR. In contrast, TSTR systems exhibited accelerated activity loss (≤69.9 % retention after 9 cycles) and yield deterioration (60.3 %). By mitigating shear-induced lipase deactivation and optimizing mass transfer, RBR technology paired with D311-resin immobilized lipase offers a scalable, cost-effective strategy for industrial biodiesel production.
{"title":"Hydrodynamic velocity and interfacial mass transfer dynamics in the rotating bed reactor: Application to enzymatically catalyzed biodiesel production","authors":"Shuai Huang , Haoyuan Tan , Luxuan Sun , Meng Wang , Biqiang Chen","doi":"10.1016/j.bej.2025.110029","DOIUrl":"10.1016/j.bej.2025.110029","url":null,"abstract":"<div><div>This study demonstrated the advantages of the rotating bed reactor (RBR) over the conventional turbine stirred tank reactor (TSTR) for enzymatically catalyzed biodiesel synthesis using D311-resin immobilized lipase. Integrating simulation and experimental analyses, the work revealed that the rotating bed generated significantly lower shear forces compared to the turbine stirred paddles, thereby preserving lipase integrity and enhancing reusability. Simulations identified tangential velocity—modulated by rotational speed and bed porosity—as the dominant factor governing hydrodynamic velocity and liquid-solid mass transfer coefficients. Experimental validation confirmed these findings: Under optimized conditions, the yield of the fatty acid methyl esters (FAMEs) decreased from 87.49 % to 60.33 % after continuous use of immobilized lipase for 48 cycles in the RBR. In contrast, TSTR systems exhibited accelerated activity loss (≤69.9 % retention after 9 cycles) and yield deterioration (60.3 %). By mitigating shear-induced lipase deactivation and optimizing mass transfer, RBR technology paired with D311-resin immobilized lipase offers a scalable, cost-effective strategy for industrial biodiesel production.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"227 ","pages":"Article 110029"},"PeriodicalIF":3.7,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145617578","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Vanillin is one of the monomers originated from lignin. We have synthesized divanillin from vanillin as a precipitate using the enzymatic reaction of horseradish peroxidase (HRP), to examine the effects of the concentration of HRP and of the reaction medium on the reactivity of the substrate and the morphology of the product, divanillin. Mathematical modeling was used to analyze the effects of solvent-induced enzyme inactivation and substrate reactivity on substrate concentration. The amount of HRP on the reaction influenced the inactivation due to the reaction media. It was quantitatively shown that changing the concentration of methanol in the reaction medium increased the solvation to the substrate vanillin and increased the contact efficiency with HRP. SEM observation of the precipitates revealed that they were spherical and needle-like and varied with the reaction conditions. Divanillin is a substance with potential for subsequent effective utilization of biomass, and this research will lead to future applications of novel materials.
{"title":"Divanillin synthesis from vanillin by horseradish peroxidase in consideration of mathematical model and morphology of product","authors":"Ikuya Teranishi, Shusuke Ito, Shintaro Morisada, Keisuke Ohto, Hidetaka Kawakita","doi":"10.1016/j.bej.2025.110016","DOIUrl":"10.1016/j.bej.2025.110016","url":null,"abstract":"<div><div>Vanillin is one of the monomers originated from lignin. We have synthesized divanillin from vanillin as a precipitate using the enzymatic reaction of horseradish peroxidase (HRP), to examine the effects of the concentration of HRP and of the reaction medium on the reactivity of the substrate and the morphology of the product, divanillin. Mathematical modeling was used to analyze the effects of solvent-induced enzyme inactivation and substrate reactivity on substrate concentration. The amount of HRP on the reaction influenced the inactivation due to the reaction media. It was quantitatively shown that changing the concentration of methanol in the reaction medium increased the solvation to the substrate vanillin and increased the contact efficiency with HRP. SEM observation of the precipitates revealed that they were spherical and needle-like and varied with the reaction conditions. Divanillin is a substance with potential for subsequent effective utilization of biomass, and this research will lead to future applications of novel materials.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"227 ","pages":"Article 110016"},"PeriodicalIF":3.7,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145594584","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-03-01Epub Date: 2025-11-27DOI: 10.1016/j.bej.2025.110030
Yanling Wang , Lei Ma , Wilburt Tam, Weibin Zheng, Dana Lee, Qinhong Yu, Lixin Feng
AntibodyPlus denotes engineered antibodies that incorporate an added functional module, such as an enzyme, cytokine, peptide, or small-molecule payload, to expand or enhance their biological activity beyond that of a conventional antibody. Among its formats, enzyme-fusion antibodies, coupling binding modules to enzymes, enable precise functions for enzyme replacement, prodrug activation, diagnostics, and cancer therapy. However, as a hard-to-express protein, challenges remain in maintaining enzyme integrity, activity, and proper bispecific assembly during its manufacture. Thus, for enzyme-fusion antibodies, optimizing transfection and clone selection strategies that consider both titer and enzymatic activity is key for manufacturing and clinical translation. In this study, a Leap-In transposon site-specific integration strategy was first introduced to the enzyme-fusion antibody CHO transfection and expression system, providing sufficient, high-quality clones for screening. Sequentially, a novel tailored multidimensional single-clone screening method was originally established and introduced for the expression of asymmetric enzyme-fused antibodies, achieving both high titer and high enzyme activity systematically. It enabled efficient identification of high-performing clones, achieving titers above 6 g/L and enzymatic specific activities over 1200 mU/mg through vector chain balancing, optimized screening strategies, and analytical validation such as CE-SDS, SEC-HPLC, and ELISA. This work establishes a robust and resource-efficient CLD workflow for enzyme-fusion antibodies, offering a significant advancement for the expression and development of complex, hard-to-express biologics.
{"title":"AntibodyPlus enzyme fusion protein cell line development using a novel multidimensional screening of productivity, purity, and specific activity","authors":"Yanling Wang , Lei Ma , Wilburt Tam, Weibin Zheng, Dana Lee, Qinhong Yu, Lixin Feng","doi":"10.1016/j.bej.2025.110030","DOIUrl":"10.1016/j.bej.2025.110030","url":null,"abstract":"<div><div>AntibodyPlus denotes engineered antibodies that incorporate an added functional module, such as an enzyme, cytokine, peptide, or small-molecule payload, to expand or enhance their biological activity beyond that of a conventional antibody. Among its formats, enzyme-fusion antibodies, coupling binding modules to enzymes, enable precise functions for enzyme replacement, prodrug activation, diagnostics, and cancer therapy. However, as a hard-to-express protein, challenges remain in maintaining enzyme integrity, activity, and proper bispecific assembly during its manufacture. Thus, for enzyme-fusion antibodies, optimizing transfection and clone selection strategies that consider both titer and enzymatic activity is key for manufacturing and clinical translation. In this study, a Leap-In transposon site-specific integration strategy was first introduced to the enzyme-fusion antibody CHO transfection and expression system, providing sufficient, high-quality clones for screening. Sequentially, a novel tailored multidimensional single-clone screening method was originally established and introduced for the expression of asymmetric enzyme-fused antibodies, achieving both high titer and high enzyme activity systematically. It enabled efficient identification of high-performing clones, achieving titers above 6 g/L and enzymatic specific activities over 1200 mU/mg through vector chain balancing, optimized screening strategies, and analytical validation such as CE-SDS, SEC-HPLC, and ELISA. This work establishes a robust and resource-efficient CLD workflow for enzyme-fusion antibodies, offering a significant advancement for the expression and development of complex, hard-to-express biologics.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"227 ","pages":"Article 110030"},"PeriodicalIF":3.7,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145617579","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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