Pub Date : 2025-12-01Epub Date: 2025-07-08DOI: 10.1016/j.enzmictec.2025.110709
Ruth Chrisnasari, Roelant Hilgers, Guanna Li, Jean-Paul Vincken, Willem J H van Berkel, Marie Hennebelle, Tom A Ewing
Lipoxygenases (LOXs) are enzymes that catalyze the regioselective dioxygenation of polyunsaturated fatty acids (PUFAs), leading to the formation of fatty acid hydroperoxides (FAHPs). In addition to dioxygenase activity, some eukaryotic LOXs exhibit hydroperoxide isomerase (HPI) activity under specific conditions, resulting in the production of structurally diverse compounds such as epoxy alcohols and ketones. Until now, the presence of HPI activity in bacterial LOXs has not been documented. In this study, we investigated the HPI activity of LOX from Burkholderia thailandensis (Bt-LOX) and examined the effects of reaction conditions on its catalytic profile using three different C18 PUFA substrates. The results demonstrated that Bt-LOX exhibits significant HPI activity, especially at high enzyme concentrations, with ketone formation showing strong substrate dependence. Oxygen level was identified as a critical factor in directing the catalytic performance of Bt-LOX: HPI activity was inhibited under O₂-saturated conditions and enhanced under O₂-limited conditions. These findings establish Bt-LOX as the first bacterial LOX reported to exhibit pronounced HPI activity, and highlights its expanded potential for biocatalytic applications.
{"title":"Modulating dioxygenase and hydroperoxide isomerase activities in Burkholderia thailandensis lipoxygenase.","authors":"Ruth Chrisnasari, Roelant Hilgers, Guanna Li, Jean-Paul Vincken, Willem J H van Berkel, Marie Hennebelle, Tom A Ewing","doi":"10.1016/j.enzmictec.2025.110709","DOIUrl":"10.1016/j.enzmictec.2025.110709","url":null,"abstract":"<p><p>Lipoxygenases (LOXs) are enzymes that catalyze the regioselective dioxygenation of polyunsaturated fatty acids (PUFAs), leading to the formation of fatty acid hydroperoxides (FAHPs). In addition to dioxygenase activity, some eukaryotic LOXs exhibit hydroperoxide isomerase (HPI) activity under specific conditions, resulting in the production of structurally diverse compounds such as epoxy alcohols and ketones. Until now, the presence of HPI activity in bacterial LOXs has not been documented. In this study, we investigated the HPI activity of LOX from Burkholderia thailandensis (Bt-LOX) and examined the effects of reaction conditions on its catalytic profile using three different C18 PUFA substrates. The results demonstrated that Bt-LOX exhibits significant HPI activity, especially at high enzyme concentrations, with ketone formation showing strong substrate dependence. Oxygen level was identified as a critical factor in directing the catalytic performance of Bt-LOX: HPI activity was inhibited under O₂-saturated conditions and enhanced under O₂-limited conditions. These findings establish Bt-LOX as the first bacterial LOX reported to exhibit pronounced HPI activity, and highlights its expanded potential for biocatalytic applications.</p>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"191 ","pages":"110709"},"PeriodicalIF":3.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144811958","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}
Proteases have diverse industrial applications including keratinase, which degrade keratin-rich substrates, such as hair, nails, feathers, and skin. This study aimed to express a protease of Bacillus velezensis LPL061 (WP_003155195.1) in Escherichia coli BL21(DE3) and to evaluate its keratinolytic as a new source of keratinase. Two gene constructs were designed, one containing the propeptide domain (kerfull) and one without it (kerhalf). Expression was induced with 0.05 mM IPTG at 20°C for 20 h. KerFull was partially purified using sequential ultrafiltration. SDS-PAGE analysis showed that KerHalf as a 32.2 kDa protein, while KerFull appeared as a 38 kDa fusion and a 28.5 kDa mature form. QTOF-MS confirmed the amino acid sequence. Only KerFull exhibited proteolytic and keratinolytic, highlighting the importance of the I9 domain for proper folding and enzyme activation. The partially purified mature KerFull protein (P50 fraction) retained activity despite low yield. KerFull showed a broad pH stability (6−11) with optimum at pH 9 and active over a wide temperature range (37–70℃), with an optimum at 60℃. Protein remained stable at 20–40℃ and pH 8. Specific activity reached 16.67 U/mg in the crude and 34.14 U/mg of P50 fraction. When combined with DTT, KerFull effectively degraded chicken feather barbules within 4 h at 37 °C. These findings suggest that one of the proteases from B. velezensis LPL061 (WP_003155195.1) is a robust keratinase candidate, offering broad operational pH and temperature, and efficient keratin degradation even at low concentrations, making it a promising candidate for industrial keratinase applications.
{"title":"Overexpression of Keratinase Candidate from Bacillus velezensis LPL061 in Escherichia coli BL21(DE3)","authors":"Desi Sagita , Tirza Jeli Ping , Aluicia Anita Artarini , Rachmat Mauluddin , Edwin Setiawan , Anto Budiharjo , Catur Riani","doi":"10.1016/j.enzmictec.2025.110789","DOIUrl":"10.1016/j.enzmictec.2025.110789","url":null,"abstract":"<div><div>Proteases have diverse industrial applications including keratinase, which degrade keratin-rich substrates, such as hair, nails, feathers, and skin. This study aimed to express a protease of <em>Bacillus velezensis</em> LPL061 (WP_003155195.1) in <em>Escherichia coli</em> BL21(DE3) and to evaluate its keratinolytic as a new source of keratinase. Two gene constructs were designed, one containing the propeptide domain (<em>kerfull</em>) and one without it (<em>kerhalf</em>). Expression was induced with 0.05 mM IPTG at 20°C for 20 h. KerFull was partially purified using sequential ultrafiltration. SDS-PAGE analysis showed that KerHalf as a 32.2 kDa protein, while KerFull appeared as a 38 kDa fusion and a 28.5 kDa mature form. QTOF-MS confirmed the amino acid sequence. Only KerFull exhibited proteolytic and keratinolytic, highlighting the importance of the I9 domain for proper folding and enzyme activation. The partially purified mature KerFull protein (P50 fraction) retained activity despite low yield. KerFull showed a broad pH stability (6−11) with optimum at pH 9 and active over a wide temperature range (37–70℃), with an optimum at 60℃. Protein remained stable at 20–40℃ and pH 8. Specific activity reached 16.67 U/mg in the crude and 34.14 U/mg of P50 fraction. When combined with DTT, KerFull effectively degraded chicken feather barbules within 4 h at 37 °C. These findings suggest that one of the proteases from <em>B. velezensis</em> LPL061 (WP_003155195.1) is a robust keratinase candidate, offering broad operational pH and temperature, and efficient keratin degradation even at low concentrations, making it a promising candidate for industrial keratinase applications.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"194 ","pages":"Article 110789"},"PeriodicalIF":3.7,"publicationDate":"2025-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145622366","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}
Lignin is a major component of plant cell walls; however, its heterogeneous and complex structure has hindered its efficient utilization. Recently, strategies that combine chemical pretreatment with microbial conversion to produce valuable chemicals have attracted attention. To develop an ideal microbial platform for this purpose, it is essential to elucidate the complete bacterial catabolic system for lignin-derived aromatic compounds. Here, we identified an inner membrane transporter gene involved in the uptake of a metabolite of dehydrodiconiferyl alcohol (DCA), a lignin-derived β-5 dimer, in Sphingobium lignivorans SYK-6. SLG_12820 (phcK) was found to encode a major facilitator superfamily transporter belonging to the endosomal spinster family and is regulated by PhcR, a transcriptional regulator of DCA catabolism genes. Through mutant analysis and a specific uptake assay based on PhcR effector recognition, we demonstrated that PhcK functions as an inner membrane transporter that specifically imports the DCA metabolite, 3-(2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl)acrylic acid.
{"title":"PhcK mediates transport of a β-5-type lignin-derived dimer in Sphingobium lignivorans SYK-6","authors":"Mitsuru Kawazoe , Masaya Fujita , Shojiro Hishiyama , Naofumi Kamimura , Eiji Masai","doi":"10.1016/j.enzmictec.2025.110784","DOIUrl":"10.1016/j.enzmictec.2025.110784","url":null,"abstract":"<div><div>Lignin is a major component of plant cell walls; however, its heterogeneous and complex structure has hindered its efficient utilization. Recently, strategies that combine chemical pretreatment with microbial conversion to produce valuable chemicals have attracted attention. To develop an ideal microbial platform for this purpose, it is essential to elucidate the complete bacterial catabolic system for lignin-derived aromatic compounds. Here, we identified an inner membrane transporter gene involved in the uptake of a metabolite of dehydrodiconiferyl alcohol (DCA), a lignin-derived β-5 dimer, in <em>Sphingobium lignivorans</em> SYK-6. SLG_12820 (<em>phcK</em>) was found to encode a major facilitator superfamily transporter belonging to the endosomal spinster family and is regulated by PhcR, a transcriptional regulator of DCA catabolism genes. Through mutant analysis and a specific uptake assay based on PhcR effector recognition, we demonstrated that PhcK functions as an inner membrane transporter that specifically imports the DCA metabolite, 3-(2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl)acrylic acid.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"194 ","pages":"Article 110784"},"PeriodicalIF":3.7,"publicationDate":"2025-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145622367","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 : 2025-11-21DOI: 10.1016/j.enzmictec.2025.110787
Oksana V. Berezina , Alina I. Selimzyanova , Kirill V. Gordeev , Wolfgang H. Schwarz , Natalia A. Lunina
Carbohydrate-binding modules (CBMs) enhance enzymatic degradation of polysaccharides by improving substrate binding. CBM54, a family of carbohydrate-binding modules, is found in surface-displayed multimodular glycoside hydrolases (MGHs) of gram-positive bacteria. These modules bind insoluble polysaccharides, including chitosan, chitin, xylan, cellulose, and fungal cell wall β-glucans. In MGHs, the CBM54 module is invariably positioned downstream of three S-layer homology modules (SLHs), which anchor the enzyme to the bacterial cell surface. The SLHs-CBM54 tandem promotes bacterial adherence to substrates, concentrating hydrolytic enzymes at the interface and facilitating efficient uptake of soluble degradation products. CBM54 contains a cleavage site that divides it into two structurally distinct parts, which remain associated via hydrogen bonding. The processing of CBM54 promotes bacterial detachment from the substrate and their migration toward new nutrient sources. Beyond MGHs, the SLHs-CBM54 tandem occurs in many other bacterial proteins with uncharacterized functions. SLH modules may self-assemble into 2D arrays on synthetic supports, enabling their use as matrices for protein nucleation and crystal growth. The SLHs-CBM54 tandem fusion with functional modules offers an innovative approach to surface modification, with broad biomedical and biotechnological applications. Due to its versatile architecture, this system holds promise for antigen display, vaccine development, and enhanced polysaccharide degradation processes.
{"title":"Structure and functional role of the SLHs-CBM54 tandem in bacterial multimodular glycoside hydrolases","authors":"Oksana V. Berezina , Alina I. Selimzyanova , Kirill V. Gordeev , Wolfgang H. Schwarz , Natalia A. Lunina","doi":"10.1016/j.enzmictec.2025.110787","DOIUrl":"10.1016/j.enzmictec.2025.110787","url":null,"abstract":"<div><div>Carbohydrate-binding modules (CBMs) enhance enzymatic degradation of polysaccharides by improving substrate binding. CBM54, a family of carbohydrate-binding modules, is found in surface-displayed multimodular glycoside hydrolases (MGHs) of gram-positive bacteria. These modules bind insoluble polysaccharides, including chitosan, chitin, xylan, cellulose, and fungal cell wall β-glucans. In MGHs, the CBM54 module is invariably positioned downstream of three S-layer homology modules (SLHs), which anchor the enzyme to the bacterial cell surface. The SLHs-CBM54 tandem promotes bacterial adherence to substrates, concentrating hydrolytic enzymes at the interface and facilitating efficient uptake of soluble degradation products. CBM54 contains a cleavage site that divides it into two structurally distinct parts, which remain associated via hydrogen bonding. The processing of CBM54 promotes bacterial detachment from the substrate and their migration toward new nutrient sources. Beyond MGHs, the SLHs-CBM54 tandem occurs in many other bacterial proteins with uncharacterized functions. SLH modules may self-assemble into 2D arrays on synthetic supports, enabling their use as matrices for protein nucleation and crystal growth. The SLHs-CBM54 tandem fusion with functional modules offers an innovative approach to surface modification, with broad biomedical and biotechnological applications. Due to its versatile architecture, this system holds promise for antigen display, vaccine development, and enhanced polysaccharide degradation processes.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"194 ","pages":"Article 110787"},"PeriodicalIF":3.7,"publicationDate":"2025-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145603094","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.2025.110788
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}
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