Pub Date : 2025-02-11DOI: 10.1016/j.btre.2025.e00878
Teresa Franchi-Mendes , Marília Silva , Maria Catarina Carreira , Ana Luísa Cartaxo , Paulo Vale , Panagiotis Karakaidos , Apostolos Klinakis , Ana Fernandes-Platzgummer , Cláudia L. da Silva
Introduction
Smooth muscle cells (SMC) play a crucial role in bladder tissue engineering strategies. Scalable, Good Manufacturing Practice (GMP)-compliant platforms are essential for producing clinically relevant cell numbers.
Materials & Methods
A gamma-irradiated human platelet lysate (HPL) supplement was used to develop a xeno(geneic)-free process for the isolation and scalable expansion of human bladder-derived SMC.
Results
Cells were isolated using an explant-based technique and expanded ex vivo, expressing typical SMC markers (α-SMA, desmin, caldesmon and SM22-α). Cell culture was successfully scaled-up using spinner flasks combined with plastic microcarriers, starting with a 2.8 × 103 cells/cm2 inoculum (i.e. 1 × 106 cells). Cell-microcarrier adhesion rates exceeded 80% within 24 hours with fold expansion ranging from 3.5 to 16.8 after 7 days, dependent on donor variability. Harvested cells retained their SMC phenotype.
Conclusions
This xeno-free, GMP compliant process enables scalable manufacturing of human bladder-derived SMC while preserving cell identity, potentially advancing clinical applications in bladder engineering.
{"title":"Xenogeneic-free platform for the isolation and scalable expansion of human bladder smooth muscle cells","authors":"Teresa Franchi-Mendes , Marília Silva , Maria Catarina Carreira , Ana Luísa Cartaxo , Paulo Vale , Panagiotis Karakaidos , Apostolos Klinakis , Ana Fernandes-Platzgummer , Cláudia L. da Silva","doi":"10.1016/j.btre.2025.e00878","DOIUrl":"10.1016/j.btre.2025.e00878","url":null,"abstract":"<div><h3>Introduction</h3><div>Smooth muscle cells (SMC) play a crucial role in bladder tissue engineering strategies. Scalable, Good Manufacturing Practice (GMP)-compliant platforms are essential for producing clinically relevant cell numbers.</div></div><div><h3>Materials & Methods</h3><div>A gamma-irradiated human platelet lysate (HPL) supplement was used to develop a xeno(geneic)-free process for the isolation and scalable expansion of human bladder-derived SMC.</div></div><div><h3>Results</h3><div>Cells were isolated using an explant-based technique and expanded ex vivo, expressing typical SMC markers (α-SMA, desmin, caldesmon and SM22-α). Cell culture was successfully scaled-up using spinner flasks combined with plastic microcarriers, starting with a 2.8 × 10<sup>3</sup> cells/cm<sup>2</sup> inoculum (i.e. 1 × 10<sup>6</sup> cells). Cell-microcarrier adhesion rates exceeded 80% within 24 hours with fold expansion ranging from 3.5 to 16.8 after 7 days, dependent on donor variability. Harvested cells retained their SMC phenotype.</div></div><div><h3>Conclusions</h3><div>This xeno-free, GMP compliant process enables scalable manufacturing of human bladder-derived SMC while preserving cell identity, potentially advancing clinical applications in bladder engineering.</div></div>","PeriodicalId":38117,"journal":{"name":"Biotechnology Reports","volume":"46 ","pages":"Article e00878"},"PeriodicalIF":0.0,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143488299","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-10DOI: 10.1016/j.btre.2025.e00879
Bhola Gautam , Sundar Tiwari , Min Raj Pokhrel , Jeffery K. Tomberlin , Prabhat Khanal
The growing global demand for food, particularly animal protein, is intensifying challenges related to food security and environmental sustainability. The increase in organic waste generation, coupled with inefficient waste management, is further deteriorating living conditions by negatively impacting the environment and public health, especially in developing nations. This study investigated the potential of black soldier fly larvae (BSFL) to recycle major daily organic waste fractions in Nepal. BSFL were exposed to seven different biowaste-based substrates locally sourced from fruit and vegetable markets, farms, and food industries. Additionally, the study evaluated rapeseed cake as a supplement to enhance BSFL growth and nutritional content. BSFL survival rates exceeded 80 % on food industry waste but dropped to 63 % on high-moisture substrates like vegetable waste. Mixed vegetable waste (14.7 mg/day) and bakery waste (11.5 mg/day) supported higher average daily weight gain, likely due to their better nutrient values (soluble carbohydrates and proteins). Although rapeseed cake alone hindered larval growth, its supplementation to biowastes improved growth, survival, and bioconversion rates, increased larval protein content up to 32 %, and reduced fat by 36 %. These findings indicate BSFL can effectively recycle diverse, locally available organic wastes in developing countries like Nepal, providing a sustainable source of domestic protein and contributing to feed security. As this is the first BSFL study in Nepal, further research is needed to elucidate the chemical and microbial safety of BSFL reared on biowastes and to develop technical solutions for commercial BSFL production in countries with a low-income economy.
{"title":"Expanding black soldier fly (BSF; Hermetia illucens; Diptera: Stratiomyidae) in the developing world: Use of BSF larvae as a biological tool to recycle various organic biowastes for alternative protein production in Nepal","authors":"Bhola Gautam , Sundar Tiwari , Min Raj Pokhrel , Jeffery K. Tomberlin , Prabhat Khanal","doi":"10.1016/j.btre.2025.e00879","DOIUrl":"10.1016/j.btre.2025.e00879","url":null,"abstract":"<div><div>The growing global demand for food, particularly animal protein, is intensifying challenges related to food security and environmental sustainability. The increase in organic waste generation, coupled with inefficient waste management, is further deteriorating living conditions by negatively impacting the environment and public health, especially in developing nations. This study investigated the potential of black soldier fly larvae (BSFL) to recycle major daily organic waste fractions in Nepal. BSFL were exposed to seven different biowaste-based substrates locally sourced from fruit and vegetable markets, farms, and food industries. Additionally, the study evaluated rapeseed cake as a supplement to enhance BSFL growth and nutritional content. BSFL survival rates exceeded 80 % on food industry waste but dropped to 63 % on high-moisture substrates like vegetable waste. Mixed vegetable waste (14.7 mg/day) and bakery waste (11.5 mg/day) supported higher average daily weight gain, likely due to their better nutrient values (soluble carbohydrates and proteins). Although rapeseed cake alone hindered larval growth, its supplementation to biowastes improved growth, survival, and bioconversion rates, increased larval protein content up to 32 %, and reduced fat by 36 %. These findings indicate BSFL can effectively recycle diverse, locally available organic wastes in developing countries like Nepal, providing a sustainable source of domestic protein and contributing to feed security. As this is the first BSFL study in Nepal, further research is needed to elucidate the chemical and microbial safety of BSFL reared on biowastes and to develop technical solutions for commercial BSFL production in countries with a low-income economy.</div></div>","PeriodicalId":38117,"journal":{"name":"Biotechnology Reports","volume":"45 ","pages":"Article e00879"},"PeriodicalIF":0.0,"publicationDate":"2025-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143429065","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-10DOI: 10.1016/j.btre.2025.e00883
Karla Flores-Zambrano , Wilson Tapia , Pablo Castillejo
Effluents generated by anthropogenic activities are a significant source of pollution and eutrophication in natural water bodies. In Ecuador, the increase in pig production has exacerbated this issue due to the untreated discharge of pig effluents. This study focused on the characterization of native microalgae present in pig effluents and the evaluation of their capacity to remove nitrogenous compounds under various conditions, with the aim of identifying efficient strains for phycoremediation. Four microalgal strains were isolated and molecularly identified as Radiococcus polycoccus, Chlorolobion braunii, Micractinium sp., and Desmodesmus multivariabilis. The cultures were exposed to initial concentrations of 100 mg L-1 N-NH₄ and 49.97 mg L-1 N-NO₃ for 12 days to assess their cellular growth and nutrient removal rates. Growth kinetics were analyzed under conditions of 2000 mg L-1 N-NH₄ and extreme pH levels of 3 and 10. Chlorolobion braunii demonstrated the highest productivity, achieving a removal of 67.73 % of N-NH₄ and 30.59 % of N-NO₃, and reached the highest cellular density under extreme ammonium conditions, being the only strain capable of growing at acidic pH. Conversely, Micractinium sp. exhibited the highest growth under alkaline conditions. These results highlight the promising potential of native microalgae from pig effluents for wastewater remediation and their adaptation to environmental conditions.
{"title":"Microalgae strains isolated from piggery wastewater in Ecuador: Effective nitrogen compound removal and growth potential in extremophile conditions","authors":"Karla Flores-Zambrano , Wilson Tapia , Pablo Castillejo","doi":"10.1016/j.btre.2025.e00883","DOIUrl":"10.1016/j.btre.2025.e00883","url":null,"abstract":"<div><div>Effluents generated by anthropogenic activities are a significant source of pollution and eutrophication in natural water bodies. In Ecuador, the increase in pig production has exacerbated this issue due to the untreated discharge of pig effluents. This study focused on the characterization of native microalgae present in pig effluents and the evaluation of their capacity to remove nitrogenous compounds under various conditions, with the aim of identifying efficient strains for phycoremediation. Four microalgal strains were isolated and molecularly identified as <em>Radiococcus polycoccus, Chlorolobion braunii, Micractinium</em> sp., and <em>Desmodesmus multivariabilis</em>. The cultures were exposed to initial concentrations of 100 mg L<sup>-1</sup> N-NH₄ and 49.97 mg L<sup>-1</sup> N-NO₃ for 12 days to assess their cellular growth and nutrient removal rates. Growth kinetics were analyzed under conditions of 2000 mg L<sup>-1</sup> N-NH₄ and extreme pH levels of 3 and 10. <em>Chlorolobion braunii</em> demonstrated the highest productivity, achieving a removal of 67.73 % of N-NH₄ and 30.59 % of N-NO₃, and reached the highest cellular density under extreme ammonium conditions, being the only strain capable of growing at acidic pH. Conversely, <em>Micractinium</em> sp. exhibited the highest growth under alkaline conditions. These results highlight the promising potential of native microalgae from pig effluents for wastewater remediation and their adaptation to environmental conditions.</div></div>","PeriodicalId":38117,"journal":{"name":"Biotechnology Reports","volume":"45 ","pages":"Article e00883"},"PeriodicalIF":0.0,"publicationDate":"2025-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143387611","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-09DOI: 10.1016/j.btre.2025.e00882
Araya Jivapetthai , Wanatchaporn Arunmanee , Natapol Pornputtapong
Quality by design (QbD) is applied to the upstream process to maximize the RBD-Fc fusion protein production in CHO cells. The three factors (culture duration, temperature, and polyethyleneimine to plasmid DNA (PEI-Max/pDNA) ratio) were identified as critical process attributes based on risk analysis (FMEA) and further optimized by response surface to maximize the protein yields. Using a Box-Behnken design, the optimal conditions for RBD-Fc production were determined to be a culture duration of 5 days, a culture temperature of 34.4 °C, and a PEI-Max/pDNA ratio of 4.2:1 (w/w) with a predictive value of 48 mg/L (desirability of 92.8 %). The PEI-Max/pDNA ratio and its interaction with culture duration to express the highest yield (47.78 ± 2.30 mg/l). In addition, the purified CHO-produced RBD-Fc fusion protein was highly pure and strongly bound to its receptor, ACE2. Our finding demonstrated that the QBD tools can identify the critical parameters to facilitate scaling-up production.
{"title":"Quality by design for transient RBD-Fc fusion protein production in Chinese hamster ovary cells","authors":"Araya Jivapetthai , Wanatchaporn Arunmanee , Natapol Pornputtapong","doi":"10.1016/j.btre.2025.e00882","DOIUrl":"10.1016/j.btre.2025.e00882","url":null,"abstract":"<div><div>Quality by design (QbD) is applied to the upstream process to maximize the RBD-Fc fusion protein production in CHO cells. The three factors (culture duration, temperature, and polyethyleneimine to plasmid DNA (PEI-Max/pDNA) ratio) were identified as critical process attributes based on risk analysis (FMEA) and further optimized by response surface to maximize the protein yields. Using a Box-Behnken design, the optimal conditions for RBD-Fc production were determined to be a culture duration of 5 days, a culture temperature of 34.4 °C, and a PEI-Max/pDNA ratio of 4.2:1 (w/w) with a predictive value of 48 mg/L (desirability of 92.8 %). The PEI-Max/pDNA ratio and its interaction with culture duration to express the highest yield (47.78 ± 2.30 mg/l). In addition, the purified CHO-produced RBD-Fc fusion protein was highly pure and strongly bound to its receptor, ACE2. Our finding demonstrated that the QBD tools can identify the critical parameters to facilitate scaling-up production.</div></div>","PeriodicalId":38117,"journal":{"name":"Biotechnology Reports","volume":"45 ","pages":"Article e00882"},"PeriodicalIF":0.0,"publicationDate":"2025-02-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143419172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-08DOI: 10.1016/j.btre.2025.e00881
Abdul Jalil Shah , Mohammad Younis Dar , Mohd Adnan , Tanmaykumar Varma , Dhairiya Agarwal , Prabha Garg , Reyaz Hassan Mir , Rampratap Meena , Mubashir Hussain Masoodi
Despite broad spectrum utility of Nardostachys jatamansi (D. Don) DC, little is known about the molecular processes that underlie its anti-Alzheimer action. To investigate the molecular targets and therapeutic potential of N. jatamansi for Alzheimer's disease (AD), we used Gas Chromatography-Mass Spectrometry (GC-MS), ADMET analysis, network pharmacology, differential gene expression analysis, molecular docking, and molecular dynamics (MD) simulations. The STITCH database was used for network creation and protein-protein interaction analysis, while Cytoscape was used for network visualization and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) for term enrichment. Additionally, to investigate the intermolecular interactions between the active chemicals and target proteins, molecular docking experiments were conducted using the Blind docking on the Achilles server. The stability of the PS1 gene complex with Spirojatamol, was further evaluated using MD simulations. With Spirojatamol showing the highest binding energy scores against PS1 (−6.9 kcal/mol), molecular docking confirmed the activity of this metabolite against AD targets PS1 and Spirojatamol formed a stable complex at 100 nanoseconds, according to additional investigation using MD simulations. Significant ligand-protein interactions were verified by binding free energy calculations using the MM/GBSA technique. The PS1-Spirojatamol complex had a binding energy of ΔG: −36.95 ± 5.00 kcal/mol. By focusing on several genes and pathways, involved in AD, this work reveals the molecular underpinnings behind N. jatamansi possible use in the treatment of AD.
尽管Nardostachys jatamansi (D. Don) DC具有广泛的用途，但人们对其抗阿尔茨海默病作用的分子过程知之甚少。为了研究jatamansi对阿尔茨海默病（AD）的分子靶点和治疗潜力，我们使用了气相色谱-质谱（GC-MS）、ADMET分析、网络药理学、差异基因表达分析、分子对接和分子动力学（MD）模拟。使用STITCH数据库进行网络创建和蛋白-蛋白相互作用分析，使用Cytoscape进行网络可视化，并使用京都基因与基因组百科全书（KEGG）途径富集和基因本体（GO）进行术语富集。此外，为了研究活性化学物质与靶蛋白之间的分子间相互作用，我们在Achilles服务器上进行了盲对接实验。利用MD模拟进一步评价PS1基因复合物与螺贾他莫的稳定性。根据MD模拟的额外研究，Spirojatamol对PS1的结合能得分最高（- 6.9 kcal/mol），分子对接证实了该代谢物对AD靶点PS1的活性，Spirojatamol在100纳秒内形成稳定的复合物。通过结合自由能计算，利用MM/GBSA技术验证了显著的配体-蛋白相互作用。ps1 -螺贾他莫配合物的结合能为ΔG:−36.95±5.00 kcal/mol。通过关注与阿尔茨海默病有关的几个基因和途径，这项工作揭示了N. jatamansi可能用于治疗阿尔茨海默病的分子基础。
{"title":"Integration of phytochemical profiling and computational approaches to evaluate the neuroprotective potential of Nardostachys jatamansi in Alzheimer's disease","authors":"Abdul Jalil Shah , Mohammad Younis Dar , Mohd Adnan , Tanmaykumar Varma , Dhairiya Agarwal , Prabha Garg , Reyaz Hassan Mir , Rampratap Meena , Mubashir Hussain Masoodi","doi":"10.1016/j.btre.2025.e00881","DOIUrl":"10.1016/j.btre.2025.e00881","url":null,"abstract":"<div><div>Despite broad spectrum utility of <em>Nardostachys jatamansi</em> (D. Don) DC, little is known about the molecular processes that underlie its anti-Alzheimer action. To investigate the molecular targets and therapeutic potential of <em>N. jatamansi</em> for Alzheimer's disease (AD), we used Gas Chromatography-Mass Spectrometry (GC-MS), ADMET analysis, network pharmacology, differential gene expression analysis, molecular docking, and molecular dynamics (MD) simulations. The STITCH database was used for network creation and protein-protein interaction analysis, while Cytoscape was used for network visualization and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) for term enrichment. Additionally, to investigate the intermolecular interactions between the active chemicals and target proteins, molecular docking experiments were conducted using the Blind docking on the Achilles server. The stability of the PS1 gene complex with Spirojatamol, was further evaluated using MD simulations. With Spirojatamol showing the highest binding energy scores against PS1 (−6.9 kcal/mol), molecular docking confirmed the activity of this metabolite against AD targets PS1 and Spirojatamol formed a stable complex at 100 nanoseconds, according to additional investigation using MD simulations. Significant ligand-protein interactions were verified by binding free energy calculations using the MM/GBSA technique. The PS1-Spirojatamol complex had a binding energy of ΔG: −36.95 ± 5.00 kcal/mol. By focusing on several genes and pathways, involved in AD, this work reveals the molecular underpinnings behind <em>N. jatamansi</em> possible use in the treatment of AD.</div></div>","PeriodicalId":38117,"journal":{"name":"Biotechnology Reports","volume":"45 ","pages":"Article e00881"},"PeriodicalIF":0.0,"publicationDate":"2025-02-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143419171","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The salinity susceptible CoC-671 and salinity tolerant sugarcane genotype CoM-265 were evaluated for Peroxidase (POX), Esterase (EST) and Alcohol Dehydrogenase (ADH) isozymes and soluble protein profiling by SDS and native-PAGE at salinity levels 0.41 dSm-1, 2.31 dSm-1, 4.21 dSm-1, and 8.01 dSm-1 maintained by NaCl solution. The plant height, number of leaves and seedling diameter got reduced in salinity susceptible sugarcane genotype CoC-671 as well as salinity tolerant sugarcane genotype CoM-265 with increase in salinity levels. However, reduction in plant height, number of leaves and seedling diameter was less in salinity tolerant sugarcane genotype CoM-265 as compared to salinity susceptible sugarcane genotype CoC-671. The POX isozyme profiling revealed that salinity susceptible CoC-671 and salinity tolerant sugarcane genotype CoM-265 had variation in soluble protein band intensity at different salinity levels with relative mobility (Rm) 0.137. The present study could be useful for genetic variability analysis in sugarcane genotypes differing in salinity stress tolerance capability.
{"title":"Salinity induced changes in esterase, peroxidase and alcohol dehydrogenase isozymes and leaf soluble proteins in salinity susceptible and salinity tolerant sugarcane genotypes","authors":"Manisha Rameshrao Patil , A.A. Kale , Ajay Kumar Singh , Priyanka Rameshrao Patil , Shaheen Badshah Inamdar , R.D. Satbhai","doi":"10.1016/j.btre.2025.e00880","DOIUrl":"10.1016/j.btre.2025.e00880","url":null,"abstract":"<div><div>The salinity susceptible CoC-671 and salinity tolerant sugarcane genotype CoM-265 were evaluated for Peroxidase (POX), Esterase (EST) and Alcohol Dehydrogenase (ADH) isozymes and soluble protein profiling by SDS and native-PAGE at salinity levels 0.41 dSm<sup>-1</sup>, 2.31 dSm<sup>-1</sup>, 4.21 dSm<sup>-1</sup>, and 8.01 dSm<sup>-1</sup> maintained by NaCl solution. The plant height, number of leaves and seedling diameter got reduced in salinity susceptible sugarcane genotype CoC-671 as well as salinity tolerant sugarcane genotype CoM-265 with increase in salinity levels. However, reduction in plant height, number of leaves and seedling diameter was less in salinity tolerant sugarcane genotype CoM-265 as compared to salinity susceptible sugarcane genotype CoC-671. The POX isozyme profiling revealed that salinity susceptible CoC-671 and salinity tolerant sugarcane genotype CoM-265 had variation in soluble protein band intensity at different salinity levels with relative mobility (Rm) 0.137. The present study could be useful for genetic variability analysis in sugarcane genotypes differing in salinity stress tolerance capability.</div></div>","PeriodicalId":38117,"journal":{"name":"Biotechnology Reports","volume":"45 ","pages":"Article e00880"},"PeriodicalIF":0.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143429064","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The four-way (Holliday) DNA junction is a key intermediate in homologous recombination, a ubiquitous process that is important in DNA repair and generation of genetic diversity. The final stages of recombination require resolution of the junction into nicked-duplex species by the action of a junction-resolving enzyme. The enzymes involved are nucleases that are highly selective for the structure of branched DNA. Here we present the isolation, expression and purification of a novel T7 endonuclease from the Kogelberg Biosphere Reserve (KBR), which possesses junction resolving capabilities. An initial approach was employed where the process was scaled up to 3 L with IPTG concentration of 0.1 mM at 30 °C and purified via immobilised metal affinity chromatography (IMAC). Expression titres of 20 ± 0.003 µg.L-1 culture were achieved with the amount of KBR-T7 endonuclease required per reaction ranging from as low as 10 to 100 nanograms. The solubility of the enzyme was relatively poor; however, enzyme activity was not affected. A derivative for improved solubility and efficacy was then designed from this original wild-type version, MBP-KBR-T7 and was expressed under similar conditions at 20 °C yielding 1.63 ± 0.154 mg.L-1 of formulated enzyme. This novel high value enzyme derivative is a valuable asset within the molecular reagent space as a tool for confirming both in vivo and in vitro genome editing; therefore, a means to produce it recombinantly in a scalable and technoeconomicaly viable process is highly desirable.
{"title":"Identification and purification of a novel bacteriophage T7 endonuclease from the Kogelberg Biosphere Reserve (KBR) biodiversity hotspot","authors":"Priyen Pillay , Maabo Moralo , Sibongile Mtimka , Taola Shai , Kirsty Botha , Lusisizwe Kwezi , Tsepo L. Tsekoa","doi":"10.1016/j.btre.2025.e00877","DOIUrl":"10.1016/j.btre.2025.e00877","url":null,"abstract":"<div><div>The four-way (Holliday) DNA junction is a key intermediate in homologous recombination, a ubiquitous process that is important in DNA repair and generation of genetic diversity. The final stages of recombination require resolution of the junction into nicked-duplex species by the action of a junction-resolving enzyme. The enzymes involved are nucleases that are highly selective for the structure of branched DNA. Here we present the isolation, expression and purification of a novel T7 endonuclease from the Kogelberg Biosphere Reserve (KBR), which possesses junction resolving capabilities. An initial approach was employed where the process was scaled up to 3 L with IPTG concentration of 0.1 mM at 30 °C and purified via immobilised metal affinity chromatography (IMAC). Expression titres of 20 ± 0.003 µg.L<sup>-1</sup> culture were achieved with the amount of KBR-T7 endonuclease required per reaction ranging from as low as 10 to 100 nanograms. The solubility of the enzyme was relatively poor; however, enzyme activity was not affected. A derivative for improved solubility and efficacy was then designed from this original wild-type version, MBP-KBR-T7 and was expressed under similar conditions at 20 °C yielding 1.63 ± 0.154 mg.L<sup>-1</sup> of formulated enzyme. This novel high value enzyme derivative is a valuable asset within the molecular reagent space as a tool for confirming both <em>in vivo</em> and <em>in vitro</em> genome editing; therefore, a means to produce it recombinantly in a scalable and technoeconomicaly viable process is highly desirable.</div></div>","PeriodicalId":38117,"journal":{"name":"Biotechnology Reports","volume":"45 ","pages":"Article e00877"},"PeriodicalIF":0.0,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143103879","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-14DOI: 10.1016/j.btre.2025.e00876
Thomas Loan , Avinash Karpe , Saeid Babaei , Stuart Denman , Chunghong Chen , Matthias Joust , Kristy Lam , Dawar Hussain , Sapna Pillai Vibhakaran , Matthew Callaghan , Abed Chaudhury , Karen Paco , Nigel Tomkins , Tristan Yang , Stephanie Payne , Michael Ayliffe , Ming Luo
The ruminant livestock industry is the largest agricultural emissions source of anthropogenic greenhouse gases, primarily from enteric methane. Several technologies, both natural and synthetic are being investigated to mitigate enteric methane emissions. Macroalgae derived feed ingredients that contain bromoform, a recognised inhibitor of methanogenesis, are an effective natural approach for methane mitigation. However, producing sufficient biomass economically to satisfy the livestock industry at a global scale is challenging. Here we demonstrate that a Curvularia soil fungi isolate can be cultured to produce bromoform and subsequently inhibit methanogenesis in pure cultures of Methanobrevibacter smithii and in mixed cultures of ovine rumen fluid. This highly culturable fungal species produces no known toxins and creates an exciting new, scalable and natural alternative for reducing ruminant livestock emissions.
{"title":"Biosynthesis of bromoform by Curvularia fungi provides a natural pathway to mitigate enteric methane emissions from ruminants","authors":"Thomas Loan , Avinash Karpe , Saeid Babaei , Stuart Denman , Chunghong Chen , Matthias Joust , Kristy Lam , Dawar Hussain , Sapna Pillai Vibhakaran , Matthew Callaghan , Abed Chaudhury , Karen Paco , Nigel Tomkins , Tristan Yang , Stephanie Payne , Michael Ayliffe , Ming Luo","doi":"10.1016/j.btre.2025.e00876","DOIUrl":"10.1016/j.btre.2025.e00876","url":null,"abstract":"<div><div>The ruminant livestock industry is the largest agricultural emissions source of anthropogenic greenhouse gases, primarily from enteric methane. Several technologies, both natural and synthetic are being investigated to mitigate enteric methane emissions. Macroalgae derived feed ingredients that contain bromoform, a recognised inhibitor of methanogenesis, are an effective natural approach for methane mitigation. However, producing sufficient biomass economically to satisfy the livestock industry at a global scale is challenging. Here we demonstrate that a <em>Curvularia</em> soil fungi isolate can be cultured to produce bromoform and subsequently inhibit methanogenesis in pure cultures of <em>Methanobrevibacter smithii</em> and in mixed cultures of ovine rumen fluid. This highly culturable fungal species produces no known toxins and creates an exciting new, scalable and natural alternative for reducing ruminant livestock emissions.</div></div>","PeriodicalId":38117,"journal":{"name":"Biotechnology Reports","volume":"45 ","pages":"Article e00876"},"PeriodicalIF":0.0,"publicationDate":"2025-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143103878","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-10DOI: 10.1016/j.btre.2025.e00875
Anna Civzele, Linda Mezule
The study investigates the application of white rot fungi for reactor–scale microalgae harvesting and explores the mechanisms underlying the algal–fungal interactions and their impact on biomass composition. Enzymatic analysis and microscopy revealed that the formation of algal-fungal complexes and successful harvesting are coupled with fungal cellulose-degrading enzyme production and hydrolytic processes of microalgae cells. Fluorescence intensity decreased by over 80 % in cells stained with Calcofluor-white after interaction with white rot fungi, indicating the reduction in cellulose content in microalgal cells caused by fungal enzymatic activity. These enzymes also caused significant cell damage and more than 50 % decrease in microalgae cell size. The presence of cellulolytic enzymes broadens the potential application of the resulting biomass in various biotechnological applications. Moreover, reactor-scale bioflocculation resulted in over 95 % T. obliquus and almost 85 % C. vulgaris harvesting efficiency from secondary wastewater within less than 24 h, demonstrating the method's scalability and industrial applicability.
{"title":"Fungal – assisted microalgae flocculation and simultaneous lignocellulolytic enzyme production in wastewater treatment systems","authors":"Anna Civzele, Linda Mezule","doi":"10.1016/j.btre.2025.e00875","DOIUrl":"10.1016/j.btre.2025.e00875","url":null,"abstract":"<div><div>The study investigates the application of white rot fungi for reactor–scale microalgae harvesting and explores the mechanisms underlying the algal–fungal interactions and their impact on biomass composition. Enzymatic analysis and microscopy revealed that the formation of algal-fungal complexes and successful harvesting are coupled with fungal cellulose-degrading enzyme production and hydrolytic processes of microalgae cells. Fluorescence intensity decreased by over 80 % in cells stained with Calcofluor-white after interaction with white rot fungi, indicating the reduction in cellulose content in microalgal cells caused by fungal enzymatic activity. These enzymes also caused significant cell damage and more than 50 % decrease in microalgae cell size. The presence of cellulolytic enzymes broadens the potential application of the resulting biomass in various biotechnological applications. Moreover, reactor-scale bioflocculation resulted in over 95 % <em>T. obliquus</em> and almost 85 % <em>C. vulgaris</em> harvesting efficiency from secondary wastewater within less than 24 h, demonstrating the method's scalability and industrial applicability.</div></div>","PeriodicalId":38117,"journal":{"name":"Biotechnology Reports","volume":"45 ","pages":"Article e00875"},"PeriodicalIF":0.0,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143103880","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A phosphorus (P)-accumulating mutant strain of Saccharomyces cerevisiae is used as a precursor for transition metal phosphides (TMPs) biochar. Tetrahydrofuran treatment of the P-accumulating yeast coupled with pyrolysis resulted in the formation of CoP-loaded biochar (CoP@P-yeast) unlike previously reported Co2P-loaded biochar using dry baker's yeast. The CoP@P-yeast exhibited the electrocatalytic activity for the hydrogen evolution with an overpotential of −192 mV at 10 mA cm−2. Furthermore, the CoP@P-yeast showed the highest ammonia production rate of 33 mg-NH3 h−1 mg-catalyst−1 in nitrate reduction reaction, as well as much higher than that with platinum on graphitized carbon. Scanning electron microscopy and transmission electron microscopy observations revealed that relatively large TMP crystals mainly located at the biochar surface, which may be beneficial to avoid catalytic deterioration during the nitrate reduction reaction. This study demonstrates that P-accumulating mutant strain of yeast is a suitable precursor to improve the activity of the resulting TMP biochar.
一种积累磷(P)的酿酒酵母突变株被用作过渡金属磷化物（TMPs）生物炭的前体。用四氢呋喃处理聚磷酵母，再加上热解，形成了负载二氧化碳的生物炭（CoP@P-yeast），这与之前报道的使用干面包酵母负载二氧化碳的生物炭不同。CoP@P-yeast在10 mA cm-2下的过电位为-192 mV，具有析氢电催化活性。在硝酸还原反应中，CoP@P-yeast的产氨率最高，为33 mg-NH3 h-1 mg-catalyst-1，远高于石墨化碳上铂的产氨率。扫描电镜和透射电镜观察发现，较大的TMP晶体主要位于生物炭表面，这可能有利于避免硝酸盐还原反应中的催化变质。本研究表明，积累磷的酵母菌突变株是提高TMP生物炭活性的合适前体。
{"title":"Cobalt phosphide-loaded biochar synthesis using phosphate-accumulating yeast and its application as an electrocatalyst","authors":"Yoshihiro Ojima , Riho Akiyoshi , Itto Tokiwa , Takashi Nakazono , Yusuke Yamada , Masayuki Azuma","doi":"10.1016/j.btre.2025.e00874","DOIUrl":"10.1016/j.btre.2025.e00874","url":null,"abstract":"<div><div>A phosphorus (P)-accumulating mutant strain of <em>Saccharomyces cerevisiae</em> is used as a precursor for transition metal phosphides (TMPs) biochar. Tetrahydrofuran treatment of the P-accumulating yeast coupled with pyrolysis resulted in the formation of CoP-loaded biochar (CoP@P-yeast) unlike previously reported Co<sub>2</sub>P-loaded biochar using dry baker's yeast. The CoP@P-yeast exhibited the electrocatalytic activity for the hydrogen evolution with an overpotential of −192 mV at 10 mA cm<sup>−2</sup>. Furthermore, the CoP@P-yeast showed the highest ammonia production rate of 33 mg-NH<sub>3</sub> h<sup>−1</sup> mg-catalyst<sup>−1</sup> in nitrate reduction reaction, as well as much higher than that with platinum on graphitized carbon. Scanning electron microscopy and transmission electron microscopy observations revealed that relatively large TMP crystals mainly located at the biochar surface, which may be beneficial to avoid catalytic deterioration during the nitrate reduction reaction. This study demonstrates that P-accumulating mutant strain of yeast is a suitable precursor to improve the activity of the resulting TMP biochar.</div></div>","PeriodicalId":38117,"journal":{"name":"Biotechnology Reports","volume":"45 ","pages":"Article e00874"},"PeriodicalIF":0.0,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11787416/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143081538","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
扫码关注我们
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号