Recombinant protein production in prokaryotic systems remains a major topic in biotechnology because of their rapid growth, cost-effectiveness, and ease of genetic manipulation. However, the production of functionally active proteins still faces significant challenges due to folding failures, insolubility, and the lack of the capability of most prokaryotes for complex post-translational processing. This review dwells into both traditional and emerging strategies for optimizing recombinant protein expression in various prokaryotic systems. It also highlights recent advances in genetic engineering and synthetic biology for expanding the toolkit available for protein production, which include refined expression vectors, engineered hosts with improved folding capabilities, and high-throughput screening platforms. Additionally, it provides a thorough discussion of how to optimize heterologous expression using fusion tag approaches, codon bias elimination, promoter and ribosome binding site (RBS) engineering, and chaperone-assisted folding. This review explores diverse prokaryotic expression systems that offer unique advantages for heterologous expression that extend far beyond the limitations of traditional hosts. Additionally, this review also emphasizes the need for the selection of the right expression system and optimizing conditions to fulfill the increasing demands for recombinant protein production in various fields.
{"title":"Revolutionizing recombinant protein production in prokaryotic platforms – Methodologies and advances","authors":"Shrinidhi Bhat, Senthamizh R, Mayur Mahindra Kedare, Sanjukta Patra","doi":"10.1016/j.enzmictec.2025.110778","DOIUrl":"10.1016/j.enzmictec.2025.110778","url":null,"abstract":"<div><div>Recombinant protein production in prokaryotic systems remains a major topic in biotechnology because of their rapid growth, cost-effectiveness, and ease of genetic manipulation. However, the production of functionally active proteins still faces significant challenges due to folding failures, insolubility, and the lack of the capability of most prokaryotes for complex post-translational processing. This review dwells into both traditional and emerging strategies for optimizing recombinant protein expression in various prokaryotic systems. It also highlights recent advances in genetic engineering and synthetic biology for expanding the toolkit available for protein production, which include refined expression vectors, engineered hosts with improved folding capabilities, and high-throughput screening platforms. Additionally, it provides a thorough discussion of how to optimize heterologous expression using fusion tag approaches, codon bias elimination, promoter and ribosome binding site (RBS) engineering, and chaperone-assisted folding. This review explores diverse prokaryotic expression systems that offer unique advantages for heterologous expression that extend far beyond the limitations of traditional hosts. Additionally, this review also emphasizes the need for the selection of the right expression system and optimizing conditions to fulfill the increasing demands for recombinant protein production in various fields.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"193 ","pages":"Article 110778"},"PeriodicalIF":3.7,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145463925","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}
Vibrio parahaemolyticus is a major seafood-associated foodborne pathogen whose lipopolysaccharide (LPS) plays an important role in virulence and antimicrobial resistance. The LPS of V. parahaemolyticus contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) sugar with phosphorylation. Previously, we have characterized the gene VP_RS01035 is responsible for the addition of Kdo; in this study, we characterized another gene VP_RS00960 which is responsible for the Kdo phosphorylation of V. parahaemolyticus LPS. To investigate its function, we first constructed an LPS-deficient Escherichia coli WH600 strain using CRISPR/Cas9. Heterologous expression of VP_RS01035 alone or in combination with VP_RS00960 yielded recombinant strains WH600/pB1–1 and WH600/pB2–12, respectively. Analysis of total lipids from the recombinant strains by thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry demonstrated that the VP_RS00960 gene encodes a Kdo kinase responsible for phosphorylating the 4-OH site of the Kdo sugar in V. parahaemolyticus. To further validate its role, we deleted VP_RS00960 gene in V. parahaemolyticus, resulting in mutant ΔRS00960. Notably, ΔRS00960 failed to produce polysaccharide-linked lipid A, although free lipid A synthesis remained unaffected. Furthermore, defective long-chain LPS assembly compromised outer membrane integrity, increasing permeability and hydrophobicity while reducing biofilm formation. Consequently, ΔRS00960 exhibited heightened susceptibility to membrane-targeting antibiotics, such as erythromycin and novobiocin. Macrophage infection assays using RAW264.7 cells revealed that VP_RS00960 deletion attenuated bacterial pathogenicity. These findings enhance the understanding of the pathogenicity and drug resistance of V. parahaemolyticus, and provide novel insights and strategies for addressing antibiotic resistance and food safety challenges posed by V. parahaemolyticus.
{"title":"Identification of a 3-deoxy-D-manno-octulosonic acid kinase of lipid A in Vibrio parahaemolyticus","authors":"Lingyan Chen , Danyang Huang , Xinrui Zhang , Hongchen Yin , Xiaoyuan Wang","doi":"10.1016/j.enzmictec.2025.110775","DOIUrl":"10.1016/j.enzmictec.2025.110775","url":null,"abstract":"<div><div><em>Vibrio parahaemolyticus</em> is a major seafood-associated foodborne pathogen whose lipopolysaccharide (LPS) plays an important role in virulence and antimicrobial resistance. The LPS of <em>V. parahaemolyticus</em> contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) sugar with phosphorylation. Previously, we have characterized the gene <em>VP_RS01035</em> is responsible for the addition of Kdo; in this study, we characterized another gene <em>VP_RS00960</em> which is responsible for the Kdo phosphorylation of <em>V. parahaemolyticus</em> LPS. To investigate its function, we first constructed an LPS-deficient <em>Escherichia coli</em> WH600 strain using CRISPR/Cas9. Heterologous expression of <em>VP_RS01035</em> alone or in combination with <em>VP_RS00960</em> yielded recombinant strains WH600/pB1–1 and WH600/pB2–12, respectively. Analysis of total lipids from the recombinant strains by thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry demonstrated that the <em>VP_RS00960</em> gene encodes a Kdo kinase responsible for phosphorylating the 4-OH site of the Kdo sugar in <em>V. parahaemolyticus</em>. To further validate its role, we deleted <em>VP_RS00960</em> gene in <em>V. parahaemolyticus,</em> resulting in mutant Δ<em>RS00960</em>. Notably, Δ<em>RS00960</em> failed to produce polysaccharide-linked lipid A, although free lipid A synthesis remained unaffected. Furthermore, defective long-chain LPS assembly compromised outer membrane integrity, increasing permeability and hydrophobicity while reducing biofilm formation. Consequently, Δ<em>RS00960</em> exhibited heightened susceptibility to membrane-targeting antibiotics, such as erythromycin and novobiocin. Macrophage infection assays using RAW264.7 cells revealed that <em>VP_RS00960</em> deletion attenuated bacterial pathogenicity. These findings enhance the understanding of the pathogenicity and drug resistance of <em>V. parahaemolyticus</em>, and provide novel insights and strategies for addressing antibiotic resistance and food safety challenges posed by <em>V. parahaemolyticus</em>.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"193 ","pages":"Article 110775"},"PeriodicalIF":3.7,"publicationDate":"2025-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145463985","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 : 2025-10-25DOI: 10.1016/j.enzmictec.2025.110767
H. Anjulal , Aritri Saha , Vitthal T. Barvkar , Kshitija Pawar , Manali Joshi , Smita S. Zinjarde
The ability of Nocardiopsis dassonvillei NCIM 5124 to synthesize polyhydroxybutyrate depolymerase (PHBD) was recently reported. In this investigation, in vitro codon optimized gene synthesis, overexpression, and biochemical characterization of this enzyme along with molecular docking studies are presented. The sequence of the PHBD was inserted in pET-28a(+) along with the PelB_Signal and His6 tag to generate the recombinant vector pET-Nd-pelB_PHBD. The transformed Escherichia coli BL21(DE3) could produce active PHBD. This enzyme was purified using Ni-NTA affinity chromatography, producing a product with a molecular weight of roughly 50 kDa. The optimum temperature and pH of the recombinant enzyme were 35°C and 8.0, respectively. Triton X100 and Tween 20 inhibited the enzyme activity by 90 %, indicating the role of hydrophobic residues in the active site of the enzyme, as also noted during docking studies. On the basis of the Michaelis-Menten equation, apparent Km and Vmax of recombinant PHBD were found to be 1.782 mg/mL and 4.79 U/mL/min, respectively. Molecular docking studies indicated that the hydrophobic amino acids Cys 39, Ala 40, Cys 77, Phe 158, Met 161, Val 201, Ala 272, and Tyr 273 present in the catalytic site were providing the necessary hydrophobic environment for binding of the ligand. The purified enzyme could also degrade films of PHB and poly (3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate (PHBVH). As far as we are aware, this is the first report on the overexpression of PHBD from Nocardiopsis sp.
{"title":"Overexpression, biochemical characterization, and structural modeling of polyhydroxybutyrate depolymerase from Nocardiopsis dassonvillei","authors":"H. Anjulal , Aritri Saha , Vitthal T. Barvkar , Kshitija Pawar , Manali Joshi , Smita S. Zinjarde","doi":"10.1016/j.enzmictec.2025.110767","DOIUrl":"10.1016/j.enzmictec.2025.110767","url":null,"abstract":"<div><div>The ability of <em>Nocardiopsis dassonvillei</em> NCIM 5124 to synthesize polyhydroxybutyrate depolymerase (PHBD) was recently reported. In this investigation, <em>in vitro</em> codon optimized gene synthesis, overexpression, and biochemical characterization of this enzyme along with molecular docking studies are presented. The sequence of the PHBD was inserted in pET-28a(+) along with the PelB_Signal and His<sub>6</sub> tag to generate the recombinant vector pET-Nd-pelB_PHBD. The transformed <em>Escherichia coli</em> BL21(DE3) could produce active PHBD. This enzyme was purified using Ni-NTA affinity chromatography, producing a product with a molecular weight of roughly 50 kDa. The optimum temperature and pH of the recombinant enzyme were 35°C and 8.0, respectively. Triton X100 and Tween 20 inhibited the enzyme activity by 90 %, indicating the role of hydrophobic residues in the active site of the enzyme, as also noted during docking studies. On the basis of the Michaelis-Menten equation, apparent K<sub>m</sub> and V<sub>max</sub> of recombinant PHBD were found to be 1.782 mg/mL and 4.79 U/mL/min, respectively. Molecular docking studies indicated that the hydrophobic amino acids Cys 39, Ala 40, Cys 77, Phe 158, Met 161, Val 201, Ala 272, and Tyr 273 present in the catalytic site were providing the necessary hydrophobic environment for binding of the ligand. The purified enzyme could also degrade films of PHB and poly (3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate (PHBVH). As far as we are aware, this is the first report on the overexpression of PHBD from <em>Nocardiopsis</em> sp.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"193 ","pages":"Article 110767"},"PeriodicalIF":3.7,"publicationDate":"2025-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145387386","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 : 2025-10-24DOI: 10.1016/j.enzmictec.2025.110763
Jie Wang , Linbo Gou , Di Liu , Shengfang Wu , Xiuwen Zhou , Tai-Ping Fan , Long Wang , Yujie Cai
Spermidine, a naturally occurring polyamine compound, has garnered significant attention due to its versatile physiological functions, including induction of cellular autophagy, antioxidant activity, and maintenance of mitochondrial homeostasis. In this study, we established a novel spermidine biosynthesis system in E. coli BL21 (DE3) by heterologously introducing the carboxyaminopropylagmatine (CAPA) pathway derived from cyanobacterium. To enhance precursor supply, we overexpressed key enzymes in the L-aspartate β-semialdehyde and agmatine branch metabolic pathways within the precursor metabolic module, while simultaneously knocking out competing metabolic pathways to redirect metabolic flux toward spermidine biosynthesis. To address the challenge of intracellular spermidine accumulation and its associated cytotoxicity, the high-efficiency spermidine efflux pump protein AmvA from Acinetobacter baumannii was heterologously expressed in E. coli BL21 (DE3). This engineering strategy enabled efficient extrusion of spermidine from the cells, alleviating the toxic effects of high intracellular spermidine concentrations on the host strain. Through these metabolic and efflux pump engineering modifications, the engineered strain SPD06-P5-P6 was constructed. Following 48 h of shake flask fermentation, SPD06-P5-P6 achieved a spermidine titer of 163.11 mg/L, which further increased to 1164.22 mg/L after 96 h of scale-up cultivation in a 5 L bioreactor.
{"title":"Highly efficient spermidine production system in Escherichia coli BL21 (DE3) based on precursor metabolic modules optimization and carboxyaminopropylagmatine pathway construction","authors":"Jie Wang , Linbo Gou , Di Liu , Shengfang Wu , Xiuwen Zhou , Tai-Ping Fan , Long Wang , Yujie Cai","doi":"10.1016/j.enzmictec.2025.110763","DOIUrl":"10.1016/j.enzmictec.2025.110763","url":null,"abstract":"<div><div>Spermidine, a naturally occurring polyamine compound, has garnered significant attention due to its versatile physiological functions, including induction of cellular autophagy, antioxidant activity, and maintenance of mitochondrial homeostasis. In this study, we established a novel spermidine biosynthesis system in <em>E. coli</em> BL21 (DE3) by heterologously introducing the carboxyaminopropylagmatine (CAPA) pathway derived from cyanobacterium. To enhance precursor supply, we overexpressed key enzymes in the L-aspartate β-semialdehyde and agmatine branch metabolic pathways within the precursor metabolic module, while simultaneously knocking out competing metabolic pathways to redirect metabolic flux toward spermidine biosynthesis. To address the challenge of intracellular spermidine accumulation and its associated cytotoxicity, the high-efficiency spermidine efflux pump protein AmvA from <em>Acinetobacter baumannii</em> was heterologously expressed in <em>E. coli</em> BL21 (DE3). This engineering strategy enabled efficient extrusion of spermidine from the cells, alleviating the toxic effects of high intracellular spermidine concentrations on the host strain. Through these metabolic and efflux pump engineering modifications, the engineered strain SPD06-P5-P6 was constructed. Following 48 h of shake flask fermentation, SPD06-P5-P6 achieved a spermidine titer of 163.11 mg/L, which further increased to 1164.22 mg/L after 96 h of scale-up cultivation in a 5 L bioreactor.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"193 ","pages":"Article 110763"},"PeriodicalIF":3.7,"publicationDate":"2025-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145361483","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 : 2025-10-24DOI: 10.1016/j.enzmictec.2025.110764
Mohammad J. Alsarraf , Aisha S.M. Al-Wahaibi , Steven L. Stephenson , Najla A. Alshaikh , Fuad Ameen
Forty-five fungal strains from decomposing wood, representing eight genera, were isolated. Among them, 27 cellulolytic strains were identified. The genera Trichoderma, Penicillium, and Phanerochaete (6, 2, and 4 isolates, respectively) demonstrated the highest cellulase production. The isolates TW-25, TW-28, and TW-33 exhibited superior enzyme activities, CMCase (5.4–6.5 U/mL), FPase (3.2–3.8 U/mL), pNPCase (2.6–3.0 U/mL), and pNPGase (3.5–4.0 U/mL) and were selected for further study. Internal transcribed spacer (ITS) region sequencing, combined with phenotypic characteristics, identified these strains as Trichoderma sp. TW-25, Trichoderma sp. TW-28, and Penicillium sp. TW-33. Maximum protoplast release was observed in Trichoderma sp. TW-25 (3.6 × 10⁶ protoplasts/mL), followed by Trichoderma sp. TW-28 (3.0 × 10⁶ protoplasts/mL) and Penicillium sp. TW-33 (2.8 × 10⁶ protoplasts/mL). Fusion frequencies were 2.8 × 10⁻³ for TW-25 × TW-28, 2.0 × 10⁻³ for TW-25 × TW-33, and 1.8 × 10⁻³ for TW-28 × TW-33. A total of 13 colonies obtained from TW-25 × TW-28, and 18 from intergeneric fusions (10 from TW-25 × TW-33 and 8 from TW-28 × TW-33). The cellulase activity of the fusants TWF1/1, TWF1/6, and TWF2/5 was the same as TW-25 and the fusants TWF1/3 and TWF3/2 the same as TW-28 while none of the fusants had the cellulase activity of TW-33. Fusants differed from their parental strains in their DNA content (3.25–3.65 µg/mg dry weight) and showed high cellulase activities in general. Among them, TWF1/10 demonstrated the highest enzymatic activity, producing CMCase, FPase, pNPCase, and pNPGase (10.5, 6.5, 5.8, and 7.5 U/mL), respectively, followed by TWF1/13, TWF2/8, TWF2/10, and TWF3/8. DNA banding patterns of TWF1/10, TWF1/13, TWF2/8, TWF2/10, and TWF3/8, analyzed using four RAPD and three ISSR primers, differed from their parental strains, except for ISSR-3 with fusants TWF1/10 and TWF1/13. These variations underscore the effectiveness of interspecific and intergeneric protoplast fusion. The supernatant of the hybrid strain TWF1/10 was concentrated and purified via ultrafiltration, and SDS-PAGE and zymogram assays confirmed its cellulase activity using CMC as the substrate.
{"title":"Development of high-efficiency hybrid strains for cellulolytic enzyme production via interspecific and intergeneric protoplast fusion of Trichoderma and Penicillium species","authors":"Mohammad J. Alsarraf , Aisha S.M. Al-Wahaibi , Steven L. Stephenson , Najla A. Alshaikh , Fuad Ameen","doi":"10.1016/j.enzmictec.2025.110764","DOIUrl":"10.1016/j.enzmictec.2025.110764","url":null,"abstract":"<div><div>Forty-five fungal strains from decomposing wood, representing eight genera, were isolated. Among them, 27 cellulolytic strains were identified. The genera <em>Trichoderma</em>, <em>Penicillium</em>, and <em>Phanerochaete</em> (6, 2, and 4 isolates, respectively) demonstrated the highest cellulase production. The isolates TW-25, TW-28, and TW-33 exhibited superior enzyme activities, CMCase (5.4–6.5 U/mL), FPase (3.2–3.8 U/mL), pNPCase (2.6–3.0 U/mL), and pNPGase (3.5–4.0 U/mL) and were selected for further study. Internal transcribed spacer (ITS) region sequencing, combined with phenotypic characteristics, identified these strains as <em>Trichoderma</em> sp. TW-25, <em>Trichoderma</em> sp. TW-28, and <em>Penicillium</em> sp. TW-33. Maximum protoplast release was observed in <em>Trichoderma</em> sp. TW-25 (3.6 × 10⁶ protoplasts/mL), followed by <em>Trichoderma</em> sp. TW-28 (3.0 × 10⁶ protoplasts/mL) and <em>Penicillium</em> sp. TW-33 (2.8 × 10⁶ protoplasts/mL). Fusion frequencies were 2.8 × 10⁻³ for TW-25 × TW-28, 2.0 × 10⁻³ for TW-25 × TW-33, and 1.8 × 10⁻³ for TW-28 × TW-33. A total of 13 colonies obtained from TW-25 × TW-28, and 18 from intergeneric fusions (10 from TW-25 × TW-33 and 8 from TW-28 × TW-33). The cellulase activity of the fusants TWF1/1, TWF1/6, and TWF2/5 was the same as TW-25 and the fusants TWF1/3 and TWF3/2 the same as TW-28 while none of the fusants had the cellulase activity of TW-33. Fusants differed from their parental strains in their DNA content (3.25–3.65 µg/mg dry weight) and showed high cellulase activities in general. Among them, TWF1/10 demonstrated the highest enzymatic activity, producing CMCase, FPase, pNPCase, and pNPGase (10.5, 6.5, 5.8, and 7.5 U/mL), respectively, followed by TWF1/13, TWF2/8, TWF2/10, and TWF3/8. DNA banding patterns of TWF1/10, TWF1/13, TWF2/8, TWF2/10, and TWF3/8, analyzed using four RAPD and three ISSR primers, differed from their parental strains, except for ISSR-3 with fusants TWF1/10 and TWF1/13. These variations underscore the effectiveness of interspecific and intergeneric protoplast fusion. The supernatant of the hybrid strain TWF1/10 was concentrated and purified via ultrafiltration, and SDS-PAGE and zymogram assays confirmed its cellulase activity using CMC as the substrate.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"193 ","pages":"Article 110764"},"PeriodicalIF":3.7,"publicationDate":"2025-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145376592","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}
To enhance the esterification and stability of lipase in organic solvents, the bio-imprinted Aspergillus niger lipase combined with cross-linked aggregate immobilization was investigated. The bio-imprinted lipase cross-linked aggregates were applied to the catalytic esterification for the synthesis of Vitamin E succinate in N, N-dimethylformamide (DMF) solution. Lauric acid, serving as a succinic acid analogue, was selected as the bio-imprinting molecule, 0.10 g lauric acid was added to 36 mL of 2.10 mg/mL lipase solution, imprinting 40 mins at pH 8.0, the immobilization yield achieved 91.5 % with cross-linked aggregates by glutaraldehyde. The catalytic activity of the bio-imprinted lipase cross-linked aggregates was significantly enhanced, achieving an esterification yield of 87.4 ± 0.43 % for Vitamin E succinate. Moreover, the bio-imprinted lipase cross-linked aggregates maintained their catalytic activity over five consecutive reaction cycles in DMF. Fluorescence spectroscopy analysis revealed that bio-imprinting promoted the exposure of the lipase active sites, which corresponded with the observed increase in esterification activity. In addition, the mechanism of the substrate analogue-imprinted lipase was characterized. This study provides a theoretical foundation for improving the catalytic esterification performance of lipase as well as a process basis for the enzymatic synthesis of Vitamin E succinate.
{"title":"Study on bio-imprinted Aspergillus niger lipase cross-linked aggregates and catalytic synthesis of Vitamin E succinate","authors":"Junqing Qian, Zhengze Yang, Aomei Huang, Qian Li, Hui Guo","doi":"10.1016/j.enzmictec.2025.110766","DOIUrl":"10.1016/j.enzmictec.2025.110766","url":null,"abstract":"<div><div>To enhance the esterification and stability of lipase in organic solvents, the bio-imprinted <em>Aspergillus niger</em> lipase combined with cross-linked aggregate immobilization was investigated. The bio-imprinted lipase cross-linked aggregates were applied to the catalytic esterification for the synthesis of Vitamin E succinate in <em>N, N</em>-dimethylformamide (DMF) solution. Lauric acid, serving as a succinic acid analogue, was selected as the bio-imprinting molecule, 0.10 g lauric acid was added to 36 mL of 2.10 mg/mL lipase solution, imprinting 40 mins at pH 8.0, the immobilization yield achieved 91.5 % with cross-linked aggregates by glutaraldehyde. The catalytic activity of the bio-imprinted lipase cross-linked aggregates was significantly enhanced, achieving an esterification yield of 87.4 ± 0.43 % for Vitamin E succinate. Moreover, the bio-imprinted lipase cross-linked aggregates maintained their catalytic activity over five consecutive reaction cycles in DMF. Fluorescence spectroscopy analysis revealed that bio-imprinting promoted the exposure of the lipase active sites, which corresponded with the observed increase in esterification activity. In addition, the mechanism of the substrate analogue-imprinted lipase was characterized. This study provides a theoretical foundation for improving the catalytic esterification performance of lipase as well as a process basis for the enzymatic synthesis of Vitamin E succinate.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"193 ","pages":"Article 110766"},"PeriodicalIF":3.7,"publicationDate":"2025-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145387388","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 : 2025-10-17DOI: 10.1016/j.enzmictec.2025.110765
Richa Raj , Pingping Shen , Xuewa Jiang , Jingling Zhang , Weihang Yao , Wei Wang , Jian Zhang
This study aims to diversify the structure of Soyasapogenol B (1) and generate novel metabolites through microbial-catalyzed biotransformation by two highly efficient microbial strains, Streptomyces griseus ATCC 13273 and Penicillium griseofulvum CICC 40293. Consequently, ten (2−11) unique bioactive metabolites were isolated. Their structures were determined using 1D/2D NMR and HR-ESI-MS data, revealing multiple tailoring reactions, including oxidation, C-C double bond rearrangement, hydroxylation, and dehydrogenation. This highlights the enzymatic ability of these strains to catalyze specific and diverse regioselective modifications on the Soyasapogenol B scaffold. Therefore, this study demonstrates that microbial-catalyzed biotransformation offers a promising approach to increase the chemical diversity of Soyasapogenol B (1), providing a sustainable alternative to chemical synthesis.
{"title":"Microbial-catalyzed biotransformation of Soyasapogenol B by Streptomyces griseus ATCC 13273, and Penicillium griseofulvum CICC 40293","authors":"Richa Raj , Pingping Shen , Xuewa Jiang , Jingling Zhang , Weihang Yao , Wei Wang , Jian Zhang","doi":"10.1016/j.enzmictec.2025.110765","DOIUrl":"10.1016/j.enzmictec.2025.110765","url":null,"abstract":"<div><div>This study aims to diversify the structure of Soyasapogenol B (1) and generate novel metabolites through microbial-catalyzed biotransformation by two highly efficient microbial strains, <em>Streptomyces griseus</em> ATCC 13273 and <em>Penicillium griseofulvum</em> CICC 40293. Consequently, ten (2−11) unique bioactive metabolites were isolated. Their structures were determined using 1D/2D NMR and HR-ESI-MS data, revealing multiple tailoring reactions, including oxidation, C-C double bond rearrangement, hydroxylation, and dehydrogenation. This highlights the enzymatic ability of these strains to catalyze specific and diverse regioselective modifications on the Soyasapogenol B scaffold. Therefore, this study demonstrates that microbial-catalyzed biotransformation offers a promising approach to increase the chemical diversity of Soyasapogenol B (1), providing a sustainable alternative to chemical synthesis.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"193 ","pages":"Article 110765"},"PeriodicalIF":3.7,"publicationDate":"2025-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145361482","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 : 2025-10-13DOI: 10.1016/j.enzmictec.2025.110762
Junjie Zhang , Guodong Zhang , Wenhu Zhu , Youzhi Li , Yutuo Wei , Xianwei Fan
p-Coumaric acid (p-CA) is widely utilized in the food, pharmaceutical and other industries, and has traditionally been derived from plant extraction or chemical synthesis. However, p-CA synthesized by the safe B. subtilis remains poorly explored. In this study, we first engineered a recombinant B. subtilis strain (PBK) capable of synthesizing p-Coumaric acid, achieving an initial yield of 3.81 mg L−1. A high-yielding strain PBnprE was then developed through promoter substitution, with a yield reaching 60.92 mg L−1, and the yield of PBnprE was further increased to 304.04 mg L−1 by optimizing fermentation conditions and substrates, showing an 80-fold increase over PBK. The optimized fermentation extract of PBnprE displayed increased antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, alongside enhanced DPPH and ABTS scavenging capabilities. Compared to PBK, the optimized extracts showed 4.81-fold higher in DPPH and 3.47-fold in ABTS scavenging, consistent with improved antioxidant properties driven by the increased presence of p-CA. This study first successfully constructed a high-yield p-CA producing engineered strain in B. subtilis, providing a valuable platform for synthesizing other secondary metabolites.
{"title":"Metabolic engineering of Bacillus subtilis for enhanced p-Coumaric acid production and antimicrobial applications","authors":"Junjie Zhang , Guodong Zhang , Wenhu Zhu , Youzhi Li , Yutuo Wei , Xianwei Fan","doi":"10.1016/j.enzmictec.2025.110762","DOIUrl":"10.1016/j.enzmictec.2025.110762","url":null,"abstract":"<div><div><em>p</em>-Coumaric acid (<em>p</em>-CA) is widely utilized in the food, pharmaceutical and other industries, and has traditionally been derived from plant extraction or chemical synthesis. However, <em>p</em>-CA synthesized by the safe <em>B. subtilis</em> remains poorly explored. In this study, we first engineered a recombinant <em>B. subtilis</em> strain (PBK) capable of synthesizing <em>p</em>-Coumaric acid, achieving an initial yield of 3.81 mg L<sup>−1</sup>. A high-yielding strain PBnprE was then developed through promoter substitution, with a yield reaching 60.92 mg L<sup>−1</sup>, and the yield of PBnprE was further increased to 304.04 mg L<sup>−1</sup> by optimizing fermentation conditions and substrates, showing an 80-fold increase over PBK. The optimized fermentation extract of PBnprE displayed increased antibacterial activity against <em>Staphylococcus aureus</em>, <em>Pseudomonas aeruginosa</em>, and <em>Escherichia coli</em>, alongside enhanced DPPH and ABTS scavenging capabilities. Compared to PBK, the optimized extracts showed 4.81-fold higher in DPPH and 3.47-fold in ABTS scavenging, consistent with improved antioxidant properties driven by the increased presence of <em>p</em>-CA. This study first successfully constructed a high-yield <em>p</em>-CA producing engineered strain in <em>B. subtilis</em>, providing a valuable platform for synthesizing other secondary metabolites.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"192 ","pages":"Article 110762"},"PeriodicalIF":3.7,"publicationDate":"2025-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145312635","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 : 2025-10-11DOI: 10.1016/j.enzmictec.2025.110761
Tatiana Leonidovna Gordeeva, Artur Aleksandrovich Tkachenko, Larisa Nikolaevna Borshchevskaya, Natalia Vladimirovna Bulushova, Egor Sergeevich Bobrov, Aleksandr Sergeevich Fedorov, Ekaterina Pavlovna Sakharova, Olga Evgenevna Melkina, Sergey Pavlovich Sineoky
The β-amylases AmyPf1 and AmyPf2 from two distinct Priestia flexa strains were successfully expressed for the first time in Komagataella phaffii. The main biochemical characteristics of the recombinant enzymes were determined. β-Amylases exhibited similar optimal temperature (55 °C) and pH (pH 8.0), as well as high stability at 55 °C and across a broad pH range (4.0–10.0). The thermal inactivation half-lives at 55 °C for both recombinant enzymes were approximately 4 h. The AmyPf2 demonstrated superior specific activity (3836 U•mg−1) and catalytic constant (6367 s−1) compared to AmyPf1 (3411 U•mg−1 and 4224 s−1, respectively). Comparison of the amino acid sequences of the two β-amylases revealed four residue differences. Site-directed mutagenesis introducing AmyPf2-specific substitutions into the AmyPf1 sequence showed that the V180A mutation significantly enhanced specific activity (4009 U•mg−1) and catalytic constant (6656 s−1) of the resulting r-V180A variant. In fed-batch fermentation, the activity of r-V180A reached 5130 U•mL−1. Hydrolysis of corn starch using r-V180A in combination with pullulanase (a starch-debranching enzyme) yielded 87.56 % maltose, indicating that a hydrolysate with high maltose content was obtained. These results highlight the potential of the recombinant r-V180A enzyme for industrial maltose production and underscore the utility of the K. phaffii expression system for efficient production of P. flexa β-amylases.
{"title":"Expression and biochemical characterization of wild-type and mutant β-amylases from Priestia flexa in Komagataella phaffii","authors":"Tatiana Leonidovna Gordeeva, Artur Aleksandrovich Tkachenko, Larisa Nikolaevna Borshchevskaya, Natalia Vladimirovna Bulushova, Egor Sergeevich Bobrov, Aleksandr Sergeevich Fedorov, Ekaterina Pavlovna Sakharova, Olga Evgenevna Melkina, Sergey Pavlovich Sineoky","doi":"10.1016/j.enzmictec.2025.110761","DOIUrl":"10.1016/j.enzmictec.2025.110761","url":null,"abstract":"<div><div>The β-amylases AmyPf1 and AmyPf2 from two distinct <em>Priestia flexa</em> strains were successfully expressed for the first time in <em>Komagataella phaffii</em>. The main biochemical characteristics of the recombinant enzymes were determined. β-Amylases exhibited similar optimal temperature (55 °C) and pH (pH 8.0), as well as high stability at 55 °C and across a broad pH range (4.0–10.0). The thermal inactivation half-lives at 55 °C for both recombinant enzymes were approximately 4 h. The AmyPf2 demonstrated superior specific activity (3836 U•mg<sup>−1</sup>) and catalytic constant (6367 s<sup>−1</sup>) compared to AmyPf1 (3411 U•mg<sup>−1</sup> and 4224 s<sup>−1</sup>, respectively). Comparison of the amino acid sequences of the two β-amylases revealed four residue differences. Site-directed mutagenesis introducing AmyPf2-specific substitutions into the AmyPf1 sequence showed that the V180A mutation significantly enhanced specific activity (4009 U•mg<sup>−1</sup>) and catalytic constant (6656 s<sup>−1</sup>) of the resulting r-V180A variant. In fed-batch fermentation, the activity of r-V180A reached 5130 U•mL<sup>−1</sup>. Hydrolysis of corn starch using r-V180A in combination with pullulanase (a starch-debranching enzyme) yielded 87.56 % maltose, indicating that a hydrolysate with high maltose content was obtained. These results highlight the potential of the recombinant r-V180A enzyme for industrial maltose production and underscore the utility of the <em>K. phaffii</em> expression system for efficient production of <em>P. flexa</em> β-amylases.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"192 ","pages":"Article 110761"},"PeriodicalIF":3.7,"publicationDate":"2025-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145291600","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 : 2025-10-09DOI: 10.1016/j.enzmictec.2025.110759
Meiyan Xin , Jitong Liu , Hongyu Zhou , Shengjun Bu , Zhuo Hao , He Sun , Jie Lu , Xiangru Feng , Xue Jiang , Qingshuang Wang , Jiayu Wan
The rapid and accurate detection of the H1N1 influenza virus is a key link in epidemic prevention and control. This study innovatively constructed a cascade signal amplification biosensor based on DNA polymerase activity regulation, aiming to achieve ultra-sensitive and highly specific detection of viral nucleic acids. This biosensor has the following significant advantages: (i) Molecular lock-key regulation mechanism: A functional DNA inhibitor is designed to form a complex with Taq DNA polymerase, and the target H1N1 RNA is specifically recognized to release enzyme activity inhibition, converting the target presence information into a PER reaction initiation signal. (ii) Cascade signal amplification system: The single-stranded DNA generated by PER activates Cas12a trans-cleavage activity, achieving a three-level signal amplification of enzyme activity activation → nucleic acid synthesis → CRISPR cleavage. The biosensor exhibits a linear detection range between 1 pM and 1 μM, with a detection limit of 25 fM. Moreover, the platform showed high versatility and could be readily adapted for the detection of other pathogens such as SARS-CoV-2 by simply modifying the nucleic acid sequences of the inhibitor and activator. This study not only provides a new tool for the screening of H1N1 influenza virus, but also offers a novel strategy for the development of next-generation molecular detection technologies suitable for point-of-care diagnostics, indicating considerable application potential.
{"title":"An H1N1 virus biosensor based on enzyme activity-gated PER-CRISPR/Cas12a cascade signal amplification","authors":"Meiyan Xin , Jitong Liu , Hongyu Zhou , Shengjun Bu , Zhuo Hao , He Sun , Jie Lu , Xiangru Feng , Xue Jiang , Qingshuang Wang , Jiayu Wan","doi":"10.1016/j.enzmictec.2025.110759","DOIUrl":"10.1016/j.enzmictec.2025.110759","url":null,"abstract":"<div><div>The rapid and accurate detection of the H1N1 influenza virus is a key link in epidemic prevention and control. This study innovatively constructed a cascade signal amplification biosensor based on DNA polymerase activity regulation, aiming to achieve ultra-sensitive and highly specific detection of viral nucleic acids. This biosensor has the following significant advantages: (i) Molecular lock-key regulation mechanism: A functional DNA inhibitor is designed to form a complex with Taq DNA polymerase, and the target H1N1 RNA is specifically recognized to release enzyme activity inhibition, converting the target presence information into a PER reaction initiation signal. (ii) Cascade signal amplification system: The single-stranded DNA generated by PER activates Cas12a trans-cleavage activity, achieving a three-level signal amplification of enzyme activity activation → nucleic acid synthesis → CRISPR cleavage. The biosensor exhibits a linear detection range between 1 pM and 1 μM, with a detection limit of 25 fM. Moreover, the platform showed high versatility and could be readily adapted for the detection of other pathogens such as SARS-CoV-2 by simply modifying the nucleic acid sequences of the inhibitor and activator. This study not only provides a new tool for the screening of H1N1 influenza virus, but also offers a novel strategy for the development of next-generation molecular detection technologies suitable for point-of-care diagnostics, indicating considerable application potential.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"192 ","pages":"Article 110759"},"PeriodicalIF":3.7,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145266795","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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