Pub Date : 2025-11-20DOI: 10.1016/j.enzmictec.2025.110788
Huan Li, Shengjie Yuan, Ling Chen, Bing Wu
Thermoplastic polyurethane (TPU) with complex physical crosslinking is difficult to degrade under natural environmental conditions. Current degradation methods, particularly for aromatic TPU, suffer from poor degradation efficiency. This study investigated the degradation performance of lignin peroxidase (LiP) on aromatic TPU through protein engineering and multi-enzyme system construction. Results indicated that wild-type LiP (WT-LiP), expressed from the recombinant Pichia pastoris GS115, exhibited a certain degradation effect on aromatic TPU. By using molecular docking techniques to identify key mutation sites, three LiP mutants (F46W, H47W, and H175R) were successfully constructed. Under optimal conditions (30°C, pH 2.5, 1 mM H₂O₂, and 5 U/mg enzyme), the F46W mutant achieved a molecular weight degradation rate of 11.97 % after 3 days of degradation, which is 2.2 times higher than that of the WT-LiP with a weight loss of 2.22 %, and the degradation efficiency in 28 days was 26.59 %. Furthermore, the constructed multi-enzyme systems (LiP-manganese peroxidase-laccase and LiP-carboxylesterase) substantially improved the degradation efficiency of TPU. Specifically, the LiP-carboxylesterase system demonstrated superior performance, achieving molecular weight degradation rates of 29.20 % and weight loss of 5.07 % after 3 days of treatment. This study provides a green enzymatic approach for efficient aromatic TPU plastics degradation and offers more sustainable solutions for plastic waste management.
{"title":"Enhanced degradation of thermoplastic polyurethane plastics based on engineering lignin peroxidase","authors":"Huan Li, Shengjie Yuan, Ling Chen, Bing Wu","doi":"10.1016/j.enzmictec.2025.110788","DOIUrl":"10.1016/j.enzmictec.2025.110788","url":null,"abstract":"<div><div>Thermoplastic polyurethane (TPU) with complex physical crosslinking is difficult to degrade under natural environmental conditions. Current degradation methods, particularly for aromatic TPU, suffer from poor degradation efficiency. This study investigated the degradation performance of lignin peroxidase (LiP) on aromatic TPU through protein engineering and multi-enzyme system construction. Results indicated that wild-type LiP (WT-LiP), expressed from the recombinant <em>Pichia pastoris</em> GS115, exhibited a certain degradation effect on aromatic TPU. By using molecular docking techniques to identify key mutation sites, three LiP mutants (F46W, H47W, and H175R) were successfully constructed. Under optimal conditions (30°C, pH 2.5, 1 mM H₂O₂, and 5 U/mg enzyme), the F46W mutant achieved a molecular weight degradation rate of 11.97 % after 3 days of degradation, which is 2.2 times higher than that of the WT-LiP with a weight loss of 2.22 %, and the degradation efficiency in 28 days was 26.59 %. Furthermore, the constructed multi-enzyme systems (LiP-manganese peroxidase-laccase and LiP-carboxylesterase) substantially improved the degradation efficiency of TPU. Specifically, the LiP-carboxylesterase system demonstrated superior performance, achieving molecular weight degradation rates of 29.20 % and weight loss of 5.07 % after 3 days of treatment. This study provides a green enzymatic approach for efficient aromatic TPU plastics degradation and offers more sustainable solutions for plastic waste management.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"194 ","pages":"Article 110788"},"PeriodicalIF":3.7,"publicationDate":"2025-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145622365","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 : 2025-11-20DOI: 10.1016/j.enzmictec.2025.110783
Mohammed Alaa Kadhum, Mahmoud Hussein Hadwan
This study presents a novel fluorescence-based assay for quantifying Glyoxalase II (Glo II) enzymatic activity, using N-(9-Acridinyl)maleimide (NAM) as a fluorescent probe. The assay is designed to measure glutathione (GSH) production resulting from the hydrolysis of S-D-lactoylglutathione by Glo II, providing a sensitive and reliable method for assessing enzyme activity across various biological samples. The protocol involves incubating Glo II samples at 37 °C, then adding NAM, which reacts with thiol groups to form a fluorescent adduct. The fluorescence intensity is measured at excitation and emission wavelengths of 360 nm and 432 nm, respectively, allowing for precise quantification of Glo II activity. The NAM-Glo II method demonstrates exceptional sensitivity and specificity, with limits of detection (LOD) and quantification (LOQ) of 0.01 U/L and 0.033 U/L, respectively. This high sensitivity is crucial for accurately measuring Glo II activity in diverse bacterial strains, where enzyme levels may vary. Comparative studies with established methods reveal that the NAM-Glo II assay consistently yields results comparable to, and in some cases superior to, those obtained using UV-based techniques. Notably, the method effectively minimizes interference from common biomolecules, such as amino acids and carbohydrates, which can confound traditional assays. The NAM-Glo II method is a reliable, sensitive tool for quantifying Glo II activity, crucial for neurological and microbial studies. It enables accurate enzyme measurement, reveals higher activity in E. coli, aids bacterial metabolism research, and supports insights into detoxification, resistance, and targeted antimicrobial therapies.
{"title":"Sensitive and specific fluorometric assay for assessment of glyoxalase II enzymatic activity in microbial samples and biological tissue","authors":"Mohammed Alaa Kadhum, Mahmoud Hussein Hadwan","doi":"10.1016/j.enzmictec.2025.110783","DOIUrl":"10.1016/j.enzmictec.2025.110783","url":null,"abstract":"<div><div>This study presents a novel fluorescence-based assay for quantifying Glyoxalase II (Glo II) enzymatic activity, using N-(9-Acridinyl)maleimide (NAM) as a fluorescent probe. The assay is designed to measure glutathione (GSH) production resulting from the hydrolysis of S-<span>D</span>-lactoylglutathione by Glo II, providing a sensitive and reliable method for assessing enzyme activity across various biological samples. The protocol involves incubating Glo II samples at 37 °C, then adding NAM, which reacts with thiol groups to form a fluorescent adduct. The fluorescence intensity is measured at excitation and emission wavelengths of 360 nm and 432 nm, respectively, allowing for precise quantification of Glo II activity. The NAM-Glo II method demonstrates exceptional sensitivity and specificity, with limits of detection (LOD) and quantification (LOQ) of 0.01 U/L and 0.033 U/L, respectively. This high sensitivity is crucial for accurately measuring Glo II activity in diverse bacterial strains, where enzyme levels may vary. Comparative studies with established methods reveal that the NAM-Glo II assay consistently yields results comparable to, and in some cases superior to, those obtained using UV-based techniques. Notably, the method effectively minimizes interference from common biomolecules, such as amino acids and carbohydrates, which can confound traditional assays. The NAM-Glo II method is a reliable, sensitive tool for quantifying Glo II activity, crucial for neurological and microbial studies. It enables accurate enzyme measurement, reveals higher activity in <em>E. coli</em>, aids bacterial metabolism research, and supports insights into detoxification, resistance, and targeted antimicrobial therapies.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"194 ","pages":"Article 110783"},"PeriodicalIF":3.7,"publicationDate":"2025-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145578556","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 : 2025-11-20DOI: 10.1016/j.enzmictec.2025.110785
Haiying Lin , Man Zhang , Wenbin Kang , Jianru Pan
Nitrotryptophan and its derivatives are valuable building blocks for synthesizing bioactive compounds and functional materials. This study reports the development of an efficient and novel bio-catalytic bioreactor in Escherichia coli capable of the direct aromatic nitration of tryptophan, enabling the synthesis of nitrotryptophan isomers. The biosynthetic pathway incorporates a self-sufficient P450 enzyme (TB14, consisting of TxtE-linker14-BM3R) from Streptomyces for the direct insertion of a nitro group into the indole ring of L-tryptophan. This process is supported by a nitric oxide synthetase (BsNOS) from Bacillus subtilis or its chemical alternative, sodium nitroprusside (SNP), to produce nitric oxide (NO) from L-arginine, which facilitates the direct nitration. As both TB14 and BsNOS require the reductant NADPH for their respective biochemical reactions, a glucose dehydrogenase (GDH) from Bacillus subtilis was included in the experimental design to ensure NADPH regeneration within the system.The initial engineered strain produced 133.2 mg/L of nitrotryptophan in TB medium. Through systematic optimization, including pathway balancing, fermentation condition enhancement, and elimination of competing metabolic pathways, the final titer was successfully increased to 209.9 mg/L within 48 h. This work establishes a robust platform for the microbial production of valuable nitroaromatic compounds and provides key insights for future biocatalytic nitration strategies.
{"title":"Development and optimization of an engineered E. coli platform for nitrotryptophan biosynthesis","authors":"Haiying Lin , Man Zhang , Wenbin Kang , Jianru Pan","doi":"10.1016/j.enzmictec.2025.110785","DOIUrl":"10.1016/j.enzmictec.2025.110785","url":null,"abstract":"<div><div>Nitrotryptophan and its derivatives are valuable building blocks for synthesizing bioactive compounds and functional materials. This study reports the development of an efficient and novel bio-catalytic bioreactor in <em>Escherichia coli</em> capable of the direct aromatic nitration of tryptophan, enabling the synthesis of nitrotryptophan isomers. The biosynthetic pathway incorporates a self-sufficient P450 enzyme (TB14, consisting of TxtE-linker14-BM3R) from Streptomyces for the direct insertion of a nitro group into the indole ring of <span>L</span>-tryptophan. This process is supported by a nitric oxide synthetase (BsNOS) from <em>Bacillus subtilis</em> or its chemical alternative, sodium nitroprusside (SNP), to produce nitric oxide (NO) from <span>L</span>-arginine, which facilitates the direct nitration. As both TB14 and BsNOS require the reductant NADPH for their respective biochemical reactions, a glucose dehydrogenase (GDH) from Bacillus subtilis was included in the experimental design to ensure NADPH regeneration within the system.The initial engineered strain produced 133.2 mg/L of nitrotryptophan in TB medium. Through systematic optimization, including pathway balancing, fermentation condition enhancement, and elimination of competing metabolic pathways, the final titer was successfully increased to 209.9 mg/L within 48 h. This work establishes a robust platform for the microbial production of valuable nitroaromatic compounds and provides key insights for future biocatalytic nitration strategies.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"194 ","pages":"Article 110785"},"PeriodicalIF":3.7,"publicationDate":"2025-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145578555","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 : 2025-11-20DOI: 10.1016/j.enzmictec.2025.110786
Magno Sinval Pereira Ribeiro , Laura Cipolatto da Rosa , Giulia Bongiorni Galego , Mateus Dias de Oliveira , Carolina Wicteky Totti , Rogerio Margis , Geancarlo Zanatta , Tiana Tasca
Trichomonas vaginalis is the etiologic agent of trichomoniasis, the non-viral sexually transmitted infection most prevalent in world. It is important to investigate biochemical aspects of the parasite that contribute to our understanding of the biology and applications in the treatment and diagnosis of the infection. The nucleoside triphosphate diphosphohydrolase (NTPDase) is an enzyme that hydrolyses extracellular adenine and guanine nucleotides, forming nucleosides adenosine and guanosine. This is important for parasite survival through the purine salvage pathway, since adenosine is the precursor for the entire purine nucleotides pool in T. vaginalis. Herein we expressed TvNTPDase4 in the bacterial system Escherichia coli. Our data demonstrate that the enzyme is active, being able to hydrolyze ATP, ADP and AMP at a concentration of 10 μg of purified protein/reaction. The inhibitors gadolinium and adenosine 5′-[α,β-methylene]diphosphate (AMPCP) inhibited the hydrolysis of rTvNTPDase4. The inhibition of ATPase/ADPase activity was more effective with gadolinium, while the inhibition of AMPase activity was more effective with AMPCP. The enzyme rTvNTPDase4 was not cytotoxic to HMVII cells. In molecular dynamics, we observed that the ability of TvNTPDase4 to hydrolyze ATP, ADP, and AMP substrates occurs through direct interactions with the apyrase-conserved regions (ACR), especially ACR1 and ACR4. In this work, we did not find any candidate sequence for ecto-5′-nucleotidase (E-5′-N) in T. vaginalis, which leads us to believe that the parasite does not have this enzyme in its proteomic repertoire. Finally, we report that rTvNTPDase4 expressed and purified from a bacterial is active and has potential for biotechnological applications.
{"title":"Recombinant expression and nucleotide hydrolysis activity of NTPDase 4 from Trichomonas vaginalis","authors":"Magno Sinval Pereira Ribeiro , Laura Cipolatto da Rosa , Giulia Bongiorni Galego , Mateus Dias de Oliveira , Carolina Wicteky Totti , Rogerio Margis , Geancarlo Zanatta , Tiana Tasca","doi":"10.1016/j.enzmictec.2025.110786","DOIUrl":"10.1016/j.enzmictec.2025.110786","url":null,"abstract":"<div><div><em>Trichomonas vaginalis</em> is the etiologic agent of trichomoniasis, the non-viral sexually transmitted infection most prevalent in world. It is important to investigate biochemical aspects of the parasite that contribute to our understanding of the biology and applications in the treatment and diagnosis of the infection. The nucleoside triphosphate diphosphohydrolase (NTPDase) is an enzyme that hydrolyses extracellular adenine and guanine nucleotides, forming nucleosides adenosine and guanosine. This is important for parasite survival through the purine salvage pathway, since adenosine is the precursor for the entire purine nucleotides pool in <em>T. vaginalis</em>. Herein we expressed TvNTPDase4 in the bacterial system <em>Escherichia coli</em>. Our data demonstrate that the enzyme is active, being able to hydrolyze ATP, ADP and AMP at a concentration of 10 μg of purified protein/reaction. The inhibitors gadolinium and adenosine 5′-[α,β-methylene]diphosphate (AMPCP) inhibited the hydrolysis of rTvNTPDase4. The inhibition of ATPase/ADPase activity was more effective with gadolinium, while the inhibition of AMPase activity was more effective with AMPCP. The enzyme rTvNTPDase4 was not cytotoxic to HMVII cells. In molecular dynamics, we observed that the ability of TvNTPDase4 to hydrolyze ATP, ADP, and AMP substrates occurs through direct interactions with the apyrase-conserved regions (ACR), especially ACR1 and ACR4. In this work, we did not find any candidate sequence for ecto-5′-nucleotidase (E-5′-N) in <em>T. vaginalis</em>, which leads us to believe that the parasite does not have this enzyme in its proteomic repertoire. Finally, we report that rTvNTPDase4 expressed and purified from a bacterial is active and has potential for biotechnological applications.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"194 ","pages":"Article 110786"},"PeriodicalIF":3.7,"publicationDate":"2025-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145578557","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 : 2025-11-19DOI: 10.1016/j.enzmictec.2025.110780
Maitê Bernardo Correia dos Santos , Vitória Gonçalves Navarrete , Mohammed Anas Zaiter , Erike Jhonatan Pereira , Jorge Enrique Hernández González , Natália Ellen Castilho de Almeida , Roberto da Silva , Maurício Boscolo , Sérgio Goméz Alonso , Eleni Gomes
Several commercially important vinyl phenolic compounds can be produced by enzymatic decarboxylation of phenolic acids such as ferulic and p-coumaric acids which can be extracted from agro-industrial waste. Phenolic acid decarboxylase is an enzyme that acts in the decarboxylation of these acids to 4-vinylguaiacol and 4-vinylphenol, respectively. In this study, the gene encoding phenolic acid decarboxylase from Klebsiella pneumoniae TD 4.7 was isolated and identified as a 504 bp fragment, encoding a polypeptide of 167 amino acid residues. A 98 % predicted amino acid sequence identity between ferulic acid decarboxylase from other bacteria of the same genus was determined. The gene was successfully expressed in Escherichia coli BL21 (DE3), and the recombinant enzyme was purified as active in absence of cofactor. The protein had a mass of 22-kDa protein, with greater activity at pH 5.5 and 40 °C. The decarboxylase activity was inhibited by Hg2 + , Zn2+, Cu2+, and Cd2+ ions and increased by 20 % in the presence of Co2+. The Km and Vmax values for the recombinant enzyme were estimated at 2.95 mM and 102.10 µmol min−1 mg−1, respectively. The enzyme’s structure was modelled using the structural prediction programs AlphaFold Multimer and SWISS-MODEL, with an RMSD of just 0.7 Å, demonstrating the absence of cysteine and disulfide bonds in the homodimer, with the presence of a high number of lysine residues. The amino acids involved in the catalytic site were Tyr27, Glu134, and Asn23. The Enzyme activity on substrates ferulic and p-coumaric acids extracted from sugarcane bagasse, resulted in 4-vinylguaiacol and 4-vinylphenol, respectively, with conversion yields of 43 % for ferulic acid and 55 % for p-coumaric acid. These data are important in terms of obtaining an enzyme that decarboxylates ferulic and p-coumaric acids obtained from sugarcane bagasse hydrolyzed with similar efficiency, in a single step and without the need for a cofactor, making it an excellent option for bioprocesses using lignocellulosic biomass derivatives.
{"title":"Bioconversion of phenolic acids from hydrolyzed sugarcane bagasse into vinyl derivatives by recombinant phenolic acid decarboxylase","authors":"Maitê Bernardo Correia dos Santos , Vitória Gonçalves Navarrete , Mohammed Anas Zaiter , Erike Jhonatan Pereira , Jorge Enrique Hernández González , Natália Ellen Castilho de Almeida , Roberto da Silva , Maurício Boscolo , Sérgio Goméz Alonso , Eleni Gomes","doi":"10.1016/j.enzmictec.2025.110780","DOIUrl":"10.1016/j.enzmictec.2025.110780","url":null,"abstract":"<div><div>Several commercially important vinyl phenolic compounds can be produced by enzymatic decarboxylation of phenolic acids such as ferulic and <em>p</em>-coumaric acids which can be extracted from agro-industrial waste. Phenolic acid decarboxylase is an enzyme that acts in the decarboxylation of these acids to 4-vinylguaiacol and 4-vinylphenol, respectively. In this study, the gene encoding phenolic acid decarboxylase from <em>Klebsiella pneumoniae</em> TD 4.7 was isolated and identified as a 504 bp fragment, encoding a polypeptide of 167 amino acid residues. A 98 % predicted amino acid sequence identity between ferulic acid decarboxylase from other bacteria of the same genus was determined. The gene was successfully expressed in <em>Escherichia coli</em> BL21 (DE3), and the recombinant enzyme was purified as active in absence of cofactor. The protein had a mass of 22-kDa protein, with greater activity at pH 5.5 and 40 °C. The decarboxylase activity was inhibited by Hg<sup>2 +</sup> , Zn<sup>2+</sup>, Cu<sup>2+</sup>, and Cd<sup>2+</sup> ions and increased by 20 % in the presence of Co<sup>2+</sup>. The K<sub>m</sub> and V<sub>max</sub> values for the recombinant enzyme were estimated at 2.95 mM and 102.10 µmol min<sup>−1</sup> mg<sup>−1</sup>, respectively. The enzyme’s structure was modelled using the structural prediction programs AlphaFold Multimer and SWISS-MODEL, with an RMSD of just 0.7 Å, demonstrating the absence of cysteine and disulfide bonds in the homodimer, with the presence of a high number of lysine residues. The amino acids involved in the catalytic site were Tyr27, Glu134, and Asn23. The Enzyme activity on substrates ferulic and <em>p</em>-coumaric acids extracted from sugarcane bagasse, resulted in 4-vinylguaiacol and 4-vinylphenol, respectively, with conversion yields of 43 % for ferulic acid and 55 % for <em>p</em>-coumaric acid. These data are important in terms of obtaining an enzyme that decarboxylates ferulic and <em>p</em>-coumaric acids obtained from sugarcane bagasse hydrolyzed with similar efficiency, in a single step and without the need for a cofactor, making it an excellent option for bioprocesses using lignocellulosic biomass derivatives.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"193 ","pages":"Article 110780"},"PeriodicalIF":3.7,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145569257","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 : 2025-11-17DOI: 10.1016/j.enzmictec.2025.110782
Sonia Su, Amol D. Pagar, Julie M. Goddard
Environmental microplastic leads to bioaccumulation in humans, animals, and plants with potential toxicity. Polyethylene terephthalate (PET) degrading enzymes (PETases) present an opportunity to depolymerize PET in key intervention points, such as wastewater treatment. While PETase has been extensively studied since its discovery and modified for enhanced performance (especially thermostability), knowledge on immobilization for reusability remains limited. This study investigated the effect of linker peptides between functional, active, stable, and tolerant (FAST) PETase and a silica binding protein for immobilization onto silica and the reusability of the enzyme. Linker peptides and a silica binding protein were assembled onto FAST-PETase and expressed in Escherichia coli. The activity of the constructs was tested on PET before and after binding to silica. In the free system, repeating (GGGGS)3 flexible linker achieved the same activity as FAST-PETase parent enzyme after 48 h of degradation. Once immobilized to silica, repeating (GGGGS)3 flexible linker preserved 50 % of enzymatic activity, compared to free FAST-PETase, and 80 % compared to its free form. Silica-immobilized enzyme constructs all retain at least 15 % of relative activity compared to the first cycle of use after 5 reuse cycles. Integration of linker peptides between the enzyme and the silica binding peptide had a significant effect on the overall catalytic activity of FAST-PETase and advances our understanding of immobilized PETase for potential recovery and reuse in applications such as wastewater treatment.
Synopsis
Minimal research exists on the immobilization of polyethylene terephthalate degrading enzymes for reuse in environmental systems. This study reports the ability of silica immobilized enzyme, with aid of linker peptides, to minimize microplastic contamination from wastewater treatment plants.
{"title":"Silica immobilized PETase for microplastic bioremediation: Influence of linker peptides on activity","authors":"Sonia Su, Amol D. Pagar, Julie M. Goddard","doi":"10.1016/j.enzmictec.2025.110782","DOIUrl":"10.1016/j.enzmictec.2025.110782","url":null,"abstract":"<div><div>Environmental microplastic leads to bioaccumulation in humans, animals, and plants with potential toxicity. Polyethylene terephthalate (PET) degrading enzymes (PETases) present an opportunity to depolymerize PET in key intervention points, such as wastewater treatment. While PETase has been extensively studied since its discovery and modified for enhanced performance (especially thermostability), knowledge on immobilization for reusability remains limited. This study investigated the effect of linker peptides between functional, active, stable, and tolerant (FAST) PETase and a silica binding protein for immobilization onto silica and the reusability of the enzyme. Linker peptides and a silica binding protein were assembled onto FAST-PETase and expressed in <em>Escherichia coli</em>. The activity of the constructs was tested on PET before and after binding to silica. In the free system, repeating (GGGGS)<sub>3</sub> flexible linker achieved the same activity as FAST-PETase parent enzyme after 48 h of degradation. Once immobilized to silica, repeating (GGGGS)<sub>3</sub> flexible linker preserved 50 % of enzymatic activity, compared to free FAST-PETase, and 80 % compared to its free form. Silica-immobilized enzyme constructs all retain at least 15 % of relative activity compared to the first cycle of use after 5 reuse cycles. Integration of linker peptides between the enzyme and the silica binding peptide had a significant effect on the overall catalytic activity of FAST-PETase and advances our understanding of immobilized PETase for potential recovery and reuse in applications such as wastewater treatment.</div></div><div><h3>Synopsis</h3><div>Minimal research exists on the immobilization of polyethylene terephthalate degrading enzymes for reuse in environmental systems. This study reports the ability of silica immobilized enzyme, with aid of linker peptides, to minimize microplastic contamination from wastewater treatment plants.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"193 ","pages":"Article 110782"},"PeriodicalIF":3.7,"publicationDate":"2025-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145556535","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 : 2025-11-14DOI: 10.1016/j.enzmictec.2025.110781
Seung-Woo Yu , Hyeong-Jin Kim , Su-Yeon Song , Kee-Tae Kim , Dong Uk Ahn , Na-Kyoung Lee , Hyun-Dong Paik
The effects of ovomucoid (OVM), a by-product of egg white, and its hydrolysates on adipocyte differentiation and lipid accumulation were investigated. The OVM hydrolyzed using Alcalase® and pepsin was named AH and PH, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed significant changes in molecular weight of both hydrolysates, with AH showing a higher degree of hydrolysis. AH exhibited a more pronounced inhibitory effect on fat accumulation than PH. In in vitro experiments, AH and PH suppressed lipid accumulation during 3T3-L1 adipocyte differentiation, with AH inhibiting lipid accumulation most effectively. Oil red O staining and triglyceride measurements revealed lipid reduction in AH-treated cells, indicating that AH plays a major role in preventing lipid accumulation in adipocytes. In addition, AH inhibited the expression of lipid transcription factors (CCAAT/enhancer-binding protein alpha (C/EBP-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), and sterol regulatory element-binding proteins (SREBP-1c)), adipogenesis-related factors (fatty acid synthase (FAS) and ACC1), insulin-related factors (insulin receptor substrate (IRS2) and protein kinase B (AKT2)), and lipolysis-related factors (glycerol-3-phosphate acyltransferase (GPAT), CD36, and lipoprotein lipase (LPL)) in a concentration-dependent manner. Specifically, the effect of AH was most pronounced in the early stages of adipocyte differentiation, where it activated AMPK early to associate energy homeostasis and downregulate genes important for cell cycle and lipid formation. This study suggests that OVM hydrolysates prepared using Alcalase® may contribute to the development of new strategies for the obesity treatment market.
{"title":"Ovomucoid hydrolysates prepared using alcalase suppress adipogenesis at an early stage of 3T3-L1 cell differentiation","authors":"Seung-Woo Yu , Hyeong-Jin Kim , Su-Yeon Song , Kee-Tae Kim , Dong Uk Ahn , Na-Kyoung Lee , Hyun-Dong Paik","doi":"10.1016/j.enzmictec.2025.110781","DOIUrl":"10.1016/j.enzmictec.2025.110781","url":null,"abstract":"<div><div>The effects of ovomucoid (OVM), a by-product of egg white, and its hydrolysates on adipocyte differentiation and lipid accumulation were investigated. The OVM hydrolyzed using Alcalase® and pepsin was named AH and PH, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed significant changes in molecular weight of both hydrolysates, with AH showing a higher degree of hydrolysis. AH exhibited a more pronounced inhibitory effect on fat accumulation than PH. In <em>in vitro</em> experiments, AH and PH suppressed lipid accumulation during 3T3-L1 adipocyte differentiation, with AH inhibiting lipid accumulation most effectively. Oil red O staining and triglyceride measurements revealed lipid reduction in AH-treated cells, indicating that AH plays a major role in preventing lipid accumulation in adipocytes. In addition, AH inhibited the expression of lipid transcription factors (CCAAT/enhancer-binding protein alpha (C/EBP-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), and sterol regulatory element-binding proteins (SREBP-1c)), adipogenesis-related factors (fatty acid synthase (FAS) and ACC1), insulin-related factors (insulin receptor substrate (IRS2) and protein kinase B (AKT2)), and lipolysis-related factors (glycerol-3-phosphate acyltransferase (GPAT), CD36, and lipoprotein lipase (LPL)) in a concentration-dependent manner. Specifically, the effect of AH was most pronounced in the early stages of adipocyte differentiation, where it activated AMPK early to associate energy homeostasis and downregulate genes important for cell cycle and lipid formation. This study suggests that OVM hydrolysates prepared using Alcalase® may contribute to the development of new strategies for the obesity treatment market.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"193 ","pages":"Article 110781"},"PeriodicalIF":3.7,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145563223","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 : 2025-11-13DOI: 10.1016/j.enzmictec.2025.110779
Gyeongguk Park , Byung-Gee Kim , Joo-Hyun Seo
The site-specific methylation of (-)-epigallocatechin-3-O-gallate (EGCG), especially 3`` hydroxyl group in D-ring, significantly enhances its stability and bioavailability in human body. Therefore, the methylation is the way to make EGCG more orally active and potent nutraceutical agents. In this study, for the effective synthesis of the methylated EGCG, S-adenosyl-L-methionine dependent O-methyltransferase (OMT) was studied to synthesize the methylated EGCG. OMT from Bacillus licheniformis and B. megaterium were known to have methylation activity for EGCG. Because OMT from B. licheniformis (Bl_OMT) showed higher activity and regioselectivity between two OMTs, rational design was tried using Bl_OMT. F163W mutant of Bl_OMT showed 2-fold increased initial velocity for the methylation of EGCG than wild type Bl_OMT. This engineering result was further utilized as a basis for the identification of highly active OMT from sequence database. 100 homolog sequences of OMT of B. licheniformis and 100 homolog sequences of OMT of B. megaterium, were collected using BLAST. Multiple alignment of 202 sequences was used to generate subgroups. Four representative sequences from each subgroup were further studied. As a result, OMTs from Thermolongibacillus altinseunsis and B. subtilis, which were from homolog group of Bl_OMT, showed higher activity than Bl_OMT while showing the same high regioselectivity. OMTs from T. altinseunsis and B. subtilis showed kcat/KM of 17.4 M−1s−1 and 11.3 M−1s−1, respectively, while Bl_OMT showed 8.7 M−1s−1. Therefore, we could find that phenylalanine residue of the active site of OMT is very important to make strong binding of hydrophobic moiety of substrate and mutation to tryptophan is able to give higher binding strength.
EGCG(-)-表没食子儿茶素-3- o -没食子酸酯（epigallocatechin-3- o -gallate， EGCG）的位点特异性甲基化，尤其是d环上的3′′羟基甲基化，显著提高了EGCG在人体内的稳定性和生物利用度。因此，甲基化是使EGCG更具口服活性和有效的营养制剂的途径。在本研究中，为了有效合成甲基化EGCG，研究了s -腺苷- l-蛋氨酸依赖的o -甲基转移酶（OMT）来合成甲基化EGCG。已知地衣芽孢杆菌和巨芽孢杆菌的OMT对EGCG具有甲基化活性。由于地衣芽孢杆菌OMT （Bl_OMT）在两种OMT之间具有较高的活性和区域选择性，因此对Bl_OMT进行了合理设计。Bl_OMT突变体F163W的EGCG甲基化初始速度是野生型Bl_OMT的2倍。该工程结果进一步作为从序列数据库中识别高活性OMT的基础。利用BLAST采集了地衣芽孢杆菌OMT的100个同源序列和巨型芽孢杆菌OMT的100个同源序列。对202个序列进行多次比对，生成子群。对每个亚群的4个代表性序列进行进一步研究。结果表明，来自Bl_OMT同源群的高寒热隆杆菌和枯草芽孢杆菌的omt活性高于Bl_OMT，且具有相同的高区域选择性。T. altinseunsis和B. subtilis的omt的kcat/KM分别为17.4 M-1s-1和11.3 M-1s-1， Bl_OMT的kcat/KM分别为8.7 M-1s-1。因此，我们可以发现OMT活性位点的苯丙氨酸残基对底物疏水片段的强结合和突变与色氨酸的强结合非常重要，能够提供更高的结合强度。
{"title":"Mutational study-based identification of high activity O-methyltransferase for the regioselective methylation of epigallocatechin gallate","authors":"Gyeongguk Park , Byung-Gee Kim , Joo-Hyun Seo","doi":"10.1016/j.enzmictec.2025.110779","DOIUrl":"10.1016/j.enzmictec.2025.110779","url":null,"abstract":"<div><div>The site-specific methylation of (-)-epigallocatechin-3-<em>O</em>-gallate (EGCG), especially 3`` hydroxyl group in <span>D</span>-ring, significantly enhances its stability and bioavailability in human body. Therefore, the methylation is the way to make EGCG more orally active and potent nutraceutical agents. In this study, for the effective synthesis of the methylated EGCG, <em>S</em>-adenosyl-<span>L</span>-methionine dependent <em>O</em>-methyltransferase (OMT) was studied to synthesize the methylated EGCG. OMT from <em>Bacillus licheniformis</em> and <em>B. megaterium</em> were known to have methylation activity for EGCG. Because OMT from <em>B. licheniformis</em> (Bl_OMT) showed higher activity and regioselectivity between two OMTs, rational design was tried using Bl_OMT. F163W mutant of Bl_OMT showed 2-fold increased initial velocity for the methylation of EGCG than wild type Bl_OMT. This engineering result was further utilized as a basis for the identification of highly active OMT from sequence database. 100 homolog sequences of OMT of <em>B. licheniformis</em> and 100 homolog sequences of OMT of <em>B. megaterium</em>, were collected using BLAST. Multiple alignment of 202 sequences was used to generate subgroups. Four representative sequences from each subgroup were further studied. As a result, OMTs from <em>Thermolongibacillus altinseunsis</em> and <em>B. subtilis</em>, which were from homolog group of Bl_OMT, showed higher activity than Bl_OMT while showing the same high regioselectivity. OMTs from <em>T. altinseunsis</em> and <em>B. subtilis</em> showed <em>k</em><sub>cat</sub>/<em>K</em><sub>M</sub> of 17.4 M<sup>−1</sup>s<sup>−1</sup> and 11.3 M<sup>−1</sup>s<sup>−1</sup>, respectively, while Bl_OMT showed 8.7 M<sup>−1</sup>s<sup>−1</sup>. Therefore, we could find that phenylalanine residue of the active site of OMT is very important to make strong binding of hydrophobic moiety of substrate and mutation to tryptophan is able to give higher binding strength.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"193 ","pages":"Article 110779"},"PeriodicalIF":3.7,"publicationDate":"2025-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145548344","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 : 2025-11-08DOI: 10.1016/j.enzmictec.2025.110777
Arun Thapa, Ngangom Pravina Devi, Ashish Misra
Zymomonas mobilis has an extremely high specific glucose uptake rate and produces ethanol at high yields and productivities. For synthesizing products beyond ethanol in Z. mobilis, reducing ethanol yields is a major challenge. Previous efforts have sought to decrease PDC activity to minimize ethanol formation. Here, we sought to modulate ADH (encoded by adhA and adhB genes) activity to redirect flux away from ethanol. We found that deletion of adhB combined with co-expression of a heterologous NAD(P) regenerating enzyme (L-LDH) diverted more than 50 % of carbon flux away from ethanol formation. The sequential deletion of adhA in the adhB-deleted strain led to ∼90 % reduction in ADH activity. During batch growth, the strain showed ∼90 % reduction in ethanol titres (27 mM) compared to WT (209 mM) and significant flux redirection toward L-LA formation (169 mM). The results demonstrate that modulation of ADH activity by deletion of adhA and adhB genes is an effective strategy for rediverting flux toward products beyond ethanol in Z. mobilis.
{"title":"Modulation of ADH activity in Zymomonas mobilis provides significant flux redirection away from ethanol","authors":"Arun Thapa, Ngangom Pravina Devi, Ashish Misra","doi":"10.1016/j.enzmictec.2025.110777","DOIUrl":"10.1016/j.enzmictec.2025.110777","url":null,"abstract":"<div><div><em>Zymomonas mobilis</em> has an extremely high specific glucose uptake rate and produces ethanol at high yields and productivities. For synthesizing products beyond ethanol in <em>Z. mobilis,</em> reducing ethanol yields is a major challenge. Previous efforts have sought to decrease PDC activity to minimize ethanol formation. Here, we sought to modulate ADH (encoded by <em>adhA</em> and <em>adhB</em> genes) activity to redirect flux away from ethanol. We found that deletion of <em>adhB</em> combined with co-expression of a heterologous NAD(P) regenerating enzyme (<span>L</span>-LDH) diverted more than 50 % of carbon flux away from ethanol formation. The sequential deletion of <em>adhA</em> in the <em>adhB</em>-deleted strain led to ∼90 % reduction in ADH activity. During batch growth, the strain showed ∼90 % reduction in ethanol titres (27 mM) compared to WT (209 mM) and significant flux redirection toward <span>L</span>-LA formation (169 mM). The results demonstrate that modulation of ADH activity by deletion of <em>adhA</em> and <em>adhB</em> genes is an effective strategy for rediverting flux toward products beyond ethanol in <em>Z. mobilis.</em></div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"193 ","pages":"Article 110777"},"PeriodicalIF":3.7,"publicationDate":"2025-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145548408","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 : 2025-11-07DOI: 10.1016/j.enzmictec.2025.110776
Saloni Samant , Prashant S. Kharkar , Shreerang V. Joshi , Shamlan M.S. Reshamwala
Commercial production of 2-keto-L-gulonic acid (2-KGA), a crucial precursor in the synthesis of vitamin C, is carried out using the two-step fermentation process. This process uses a dual-strain system in the second step (conversion of L-sorbose to 2-KGA), necessitating complex process control. In this study, we report development of a recombinant Escherichia coli strain capable of producing 2-KGA from L-sorbose. L-Sorbose dehydrogenase (sdh) and L-sorbosone dehydrogenase (sndh) genes from Gluconobacter oxydans were expressed in E. coli as a synthetic operon in three different configurations under the control of the strong inducible T7 promoter. High-density whole-cell biotransformation was carried out to produce 2-KGA from L-sorbose, resulting in a yield of 0.69 g/g when the two genes were arranged in the non-native sdh-sndh configuration. Our design eliminates the need for multi-organism co-culture, intricate redox balancing, or nutrient-enriched media, representing a robust and scalable alternative to conventional production. This work demonstrates the feasibility of employing E. coli as a chassis strain for vitamin C precursor biosynthesis and offers a modular operon framework adaptable to other dehydrogenase-driven bioconversions. This approach enables efficient, scalable production of the vitamin C precursor in a genetically tractable host.
2-酮- l -谷醛酸（2-KGA）的商业化生产是合成维生素C的关键前体，采用两步发酵工艺进行。该工艺在第二步（L-sorbose到2-KGA的转换）中使用双应变系统，需要复杂的过程控制。在这项研究中，我们报道了一种重组大肠杆菌菌株的发展，该菌株能够从l -山梨糖中产生2-KGA。在强诱导型T7启动子的控制下，从氧葡萄糖杆菌中提取的l -山梨糖脱氢酶（sdh）和l -山梨糖脱氢酶（sndh）基因在大肠杆菌中以三种不同构型的合成操纵子表达。利用l -山梨糖进行高密度全细胞生物转化生产2-KGA，当这两个基因以非天然的sdh-sndh构型排列时，产量为0.69 g/g。我们的设计消除了对多生物共培养，复杂的氧化还原平衡或营养丰富的培养基的需求，代表了传统生产的强大和可扩展的替代方案。这项工作证明了利用大肠杆菌作为维生素C前体生物合成的基础菌株的可行性，并提供了一个适用于其他脱氢酶驱动的生物转化的模块化操纵子框架。这种方法使维生素C前体在遗传上易于处理的宿主中高效、可扩展地生产成为可能。
{"title":"Non-native genetic configuration of Gluconobacter oxydans dehydrogenases drives 2-keto-L-gulonic acid production in recombinant Escherichia coli","authors":"Saloni Samant , Prashant S. Kharkar , Shreerang V. Joshi , Shamlan M.S. Reshamwala","doi":"10.1016/j.enzmictec.2025.110776","DOIUrl":"10.1016/j.enzmictec.2025.110776","url":null,"abstract":"<div><div>Commercial production of 2-keto-L-gulonic acid (2-KGA), a crucial precursor in the synthesis of vitamin C, is carried out using the two-step fermentation process. This process uses a dual-strain system in the second step (conversion of L-sorbose to 2-KGA), necessitating complex process control. In this study, we report development of a recombinant <em>Escherichia coli</em> strain capable of producing 2-KGA from L-sorbose. L-Sorbose dehydrogenase (<em>sdh</em>) and L-sorbosone dehydrogenase (<em>sndh</em>) genes from <em>Gluconobacter oxydans</em> were expressed in <em>E. coli</em> as a synthetic operon in three different configurations under the control of the strong inducible T7 promoter. High-density whole-cell biotransformation was carried out to produce 2-KGA from L-sorbose, resulting in a yield of 0.69 g/g when the two genes were arranged in the non-native <em>sdh-sndh</em> configuration. Our design eliminates the need for multi-organism co-culture, intricate redox balancing, or nutrient-enriched media, representing a robust and scalable alternative to conventional production. This work demonstrates the feasibility of employing <em>E. coli</em> as a chassis strain for vitamin C precursor biosynthesis and offers a modular operon framework adaptable to other dehydrogenase-driven bioconversions. This approach enables efficient, scalable production of the vitamin C precursor in a genetically tractable host.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"193 ","pages":"Article 110776"},"PeriodicalIF":3.7,"publicationDate":"2025-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145494911","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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