Pub Date : 2024-12-02DOI: 10.1016/j.enzmictec.2024.110562
Yidi Liu , Zhanzhi Liu , Xuehong Guo , Ke Tong , Yueheng Niu , Zhiyu Shen , Hanzhi Weng , Fengshan Zhang , Jing Wu
The substantial accumulation of polyethylene terephthalate (PET) plastic waste in the environment has exacerbated the issue of plastic pollution. The biodegradation of PET plastics using biological enzymes has garnered considerable attention due to its efficiency and environmentally friendly nature. Nevertheless, the low binding affinity of PET plastics presents a significant limitation to the application of biocatalysts in their degradation. This study endeavors to engineer a fusion protein comprising the anchor peptide LCI, derived from Bacillus subtilis A014, and a thermally stabilized variant of Thermobifida fusca cutinase, D204C/E253C (Tfuc2), with the objective of augmenting its polyethylene terephthalate (PET) degradation efficacy. The findings demonstrate that LCI exhibits a high binding affinity for PET, and the hydrolytic efficiency of the LCI-containing fusion protein is enhanced by a factor of 1.8–34.5 compared to the free Tfuc2 enzyme. The enzymatic characteristics and molecular dynamics simulation outcomes indicate that the improved hydrolytic efficiency of PET may originate from the flexible oscillatory behavior of LCI, which exhibits a high binding affinity for PET. This study presents a novel methodology for the enzymatic degradation of PET plastic waste.
{"title":"Enhanced degradation activity of PET plastics by fusion protein of anchor peptide LCI and Thermobifida fusca cutinase","authors":"Yidi Liu , Zhanzhi Liu , Xuehong Guo , Ke Tong , Yueheng Niu , Zhiyu Shen , Hanzhi Weng , Fengshan Zhang , Jing Wu","doi":"10.1016/j.enzmictec.2024.110562","DOIUrl":"10.1016/j.enzmictec.2024.110562","url":null,"abstract":"<div><div>The substantial accumulation of polyethylene terephthalate (PET) plastic waste in the environment has exacerbated the issue of plastic pollution. The biodegradation of PET plastics using biological enzymes has garnered considerable attention due to its efficiency and environmentally friendly nature. Nevertheless, the low binding affinity of PET plastics presents a significant limitation to the application of biocatalysts in their degradation. This study endeavors to engineer a fusion protein comprising the anchor peptide LCI, derived from <em>Bacillus subtilis</em> A014, and a thermally stabilized variant of <em>Thermobifida fusca</em> cutinase, D204C/E253C (Tfuc2), with the objective of augmenting its polyethylene terephthalate (PET) degradation efficacy. The findings demonstrate that LCI exhibits a high binding affinity for PET, and the hydrolytic efficiency of the LCI-containing fusion protein is enhanced by a factor of 1.8–34.5 compared to the free Tfuc2 enzyme. The enzymatic characteristics and molecular dynamics simulation outcomes indicate that the improved hydrolytic efficiency of PET may originate from the flexible oscillatory behavior of LCI, which exhibits a high binding affinity for PET. This study presents a novel methodology for the enzymatic degradation of PET plastic waste.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110562"},"PeriodicalIF":3.4,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142799958","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}
Glucosamine (GlcN), as one of the important derivatives of D-glucose, is formed by the substitution of the hydroxyl group at position 2 of glucose with an amino group. As a bioactive amino monosaccharide, GlcN is known for its various biological effects, including immune enhancement, antioxidant, anti-inflammatory, hepatoprotective, joint pain relief, and alleviation of osteoporosis. These properties highlight the broad applications of GlcN and its derivatives in pharmaceuticals, cosmetics, food production, and other fields, underscoring their promising prospects. Thus, the efficient industrial production of GlcN is gaining increasing attention as well. Here, we report a novel biosynthetic method for GlcN, utilizing engineered Escherichia coli expressing glucosamine-6-phosphate deaminase (GlmD) to directly convert D-fructose into GlcN. The best mutant screened using the Morgan-Elson colorimetric method is the triple mutant G42S/G43C/G136T (designated as GlmD-ZH11), which exhibits approximately 21 times higher catalytic activity towards D-fructose compared to the wild type. Using the purified enzyme of GlmD-ZH11 in shaken flask fermentation for six hours, we achieved a conversion rate of 72.11 % from D-fructose to GlcN. To further elucidate the mechanism behind the enhanced activity of the GlmD-ZH11 mutant, we conducted hydrogen bond network analysis to investigate the hydrogen bond interactions between the mutant and fructose. Additionally, we performed molecular dynamics simulations to study the RMSD and RMSF curves of the mutant. The results indicate that the protein structure of the mutant ZH11 is more stable and binds more tightly to the substrate. Calculations of the solvent-accessible surface area and binding free energy suggested that Thr41, Ser42, Asp72, Gly137, and Ala145 may be key amino acid residues in the catalytic process of ZH11. Finally, based on these findings and the catalytic mechanism of the wild type, we hypothesized a potential catalytic reaction mechanism for the ZH11 mutant.
{"title":"Semi-rational engineering of glucosamine-6-phosphate deaminase for catalytic synthesis of glucosamine from D-fructose","authors":"Zi-Hao Zhang, Yun-Xing Liao, Xue-Ting Deng, Zheng-Bing Guan","doi":"10.1016/j.enzmictec.2024.110552","DOIUrl":"10.1016/j.enzmictec.2024.110552","url":null,"abstract":"<div><div>Glucosamine (GlcN), as one of the important derivatives of D-glucose, is formed by the substitution of the hydroxyl group at position 2 of glucose with an amino group. As a bioactive amino monosaccharide, GlcN is known for its various biological effects, including immune enhancement, antioxidant, anti-inflammatory, hepatoprotective, joint pain relief, and alleviation of osteoporosis. These properties highlight the broad applications of GlcN and its derivatives in pharmaceuticals, cosmetics, food production, and other fields, underscoring their promising prospects. Thus, the efficient industrial production of GlcN is gaining increasing attention as well. Here, we report a novel biosynthetic method for GlcN, utilizing engineered <em>Escherichia coli</em> expressing glucosamine-6-phosphate deaminase (GlmD) to directly convert D-fructose into GlcN. The best mutant screened using the Morgan-Elson colorimetric method is the triple mutant G42S/G43C/G136T (designated as GlmD-ZH11), which exhibits approximately 21 times higher catalytic activity towards D-fructose compared to the wild type. Using the purified enzyme of GlmD-ZH11 in shaken flask fermentation for six hours, we achieved a conversion rate of 72.11 % from D-fructose to GlcN. To further elucidate the mechanism behind the enhanced activity of the GlmD-ZH11 mutant, we conducted hydrogen bond network analysis to investigate the hydrogen bond interactions between the mutant and fructose. Additionally, we performed molecular dynamics simulations to study the RMSD and RMSF curves of the mutant. The results indicate that the protein structure of the mutant ZH11 is more stable and binds more tightly to the substrate. Calculations of the solvent-accessible surface area and binding free energy suggested that Thr41, Ser42, Asp72, Gly137, and Ala145 may be key amino acid residues in the catalytic process of ZH11. Finally, based on these findings and the catalytic mechanism of the wild type, we hypothesized a potential catalytic reaction mechanism for the ZH11 mutant.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"183 ","pages":"Article 110552"},"PeriodicalIF":3.4,"publicationDate":"2024-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142746425","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 : 2024-11-23DOI: 10.1016/j.enzmictec.2024.110550
Astrid Müller , Jiali Meng , Robin Kuijpers , Miia R. Mäkelä , Ronald P. de Vries
Production of xylitol from agricultural by-products offers a promising approach for the circular bioeconomy. This study investigates the roles of transcription factors XlnR and CreA in xylitol production from wheat bran in Aspergillus niger by generating strains with a constitutively active XlnR (XlnRc, V756F mutation) and/or deletion of creA, in a previously generated xylitol-producing strain. The XlnRc mutation increased the initial rate of xylitol production but lowered the overall accumulation. Deletion of creA in this strain significantly improved both the onset and rate of xylitol production, indicating an inhibitory role of CreA in the PCP. These results demonstrate the complexity of metabolic engineering to generate fungal cell factories for valuable biochemicals, such as xylitol, as not only metabolic but also multiple gene regulation aspects need to be considered.
{"title":"Exploring the complexity of xylitol production in the fungal cell factory Aspergillus niger","authors":"Astrid Müller , Jiali Meng , Robin Kuijpers , Miia R. Mäkelä , Ronald P. de Vries","doi":"10.1016/j.enzmictec.2024.110550","DOIUrl":"10.1016/j.enzmictec.2024.110550","url":null,"abstract":"<div><div>Production of xylitol from agricultural by-products offers a promising approach for the circular bioeconomy. This study investigates the roles of transcription factors XlnR and CreA in xylitol production from wheat bran in <em>Aspergillus niger</em> by generating strains with a constitutively active XlnR (XlnR<sub>c</sub>, V756F mutation) and/or deletion of <em>creA</em>, in a previously generated xylitol-producing strain. The XlnR<sub>c</sub> mutation increased the initial rate of xylitol production but lowered the overall accumulation. Deletion of <em>creA</em> in this strain significantly improved both the onset and rate of xylitol production, indicating an inhibitory role of CreA in the PCP. These results demonstrate the complexity of metabolic engineering to generate fungal cell factories for valuable biochemicals, such as xylitol, as not only metabolic but also multiple gene regulation aspects need to be considered.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"183 ","pages":"Article 110550"},"PeriodicalIF":3.4,"publicationDate":"2024-11-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142699659","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-22DOI: 10.1016/j.enzmictec.2024.110547
Jiang Pan , Nan Yang , Yuan-Lin Lv , Zi-Yang Zhang , Chun-Xiu Li, Jian-He Xu
Lipase can mediate the chemo-enzymatic epoxidation of alkenes with the presence of free carboxylic acid and hydrogen peroxide. Four novel lipases with the abilities of chemo-enzymatic epoxidation were mined from the gene database. Lipase TiL originated from Tilletia indica was identified with significant activity on formation of methyl epoxystearate from methyl oleate. n-Heptanoic acid was determined as the optimal carboxylic acid substrate of TiL. Methyl oleate and α-pinene were efficiently converted to corresponding epoxy compound in micro-aqueous media and aqueous-organic biphase, respectively. A preparative scale chemo-enzymatic transformation of α-pinene was conduct using the optimized reaction condition, with 30 % yield of α-pinene oxide obtained.
脂肪酶可以在存在游离羧酸和过氧化氢的情况下介导烯烃的化学酶促环氧化反应。我们从基因数据库中挖掘出四种具有化学酶促环氧化能力的新型脂肪酶。确定了正庚酸为 TiL 的最佳羧酸底物。在微水介质和水有机双相中，油酸甲酯和 α-蒎烯分别被高效地转化为相应的环氧化合物。利用优化的反应条件对α-蒎烯进行了制备规模的化学酶转化，获得了 30% 的α-蒎烯氧化物收率。
{"title":"Screening of lipase TiL from Tilletia indica for chemo-enzymatic epoxidation of alkenes","authors":"Jiang Pan , Nan Yang , Yuan-Lin Lv , Zi-Yang Zhang , Chun-Xiu Li, Jian-He Xu","doi":"10.1016/j.enzmictec.2024.110547","DOIUrl":"10.1016/j.enzmictec.2024.110547","url":null,"abstract":"<div><div>Lipase can mediate the chemo-enzymatic epoxidation of alkenes with the presence of free carboxylic acid and hydrogen peroxide. Four novel lipases with the abilities of chemo-enzymatic epoxidation were mined from the gene database. Lipase <em>Ti</em>L originated from <em>Tilletia indica</em> was identified with significant activity on formation of methyl epoxystearate from methyl oleate. <em>n</em>-Heptanoic acid was determined as the optimal carboxylic acid substrate of <em>Ti</em>L. Methyl oleate and α-pinene were efficiently converted to corresponding epoxy compound in micro-aqueous media and aqueous-organic biphase, respectively. A preparative scale chemo-enzymatic transformation of α-pinene was conduct using the optimized reaction condition, with 30 % yield of α-pinene oxide obtained.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"183 ","pages":"Article 110547"},"PeriodicalIF":3.4,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142699660","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 : 2024-11-22DOI: 10.1016/j.enzmictec.2024.110551
Sota Kamba, Ryosuke Yamada , Takuya Matsumoto, Hiroyasu Ogino
The oleaginous yeast Lipomyces starkeyi is a promising triacylglycerol (TAG) producer for biodiesel fuel. However, it is necessary to further improve TAG productivity in L. starkeyi from a mixed sugar of glucose and xylose. This study aimed to construct an L. starkeyi mutant with increased TAG productivity from glucose/xylose mixed-sugar and to elucidate the causes underlying increased lipid productivity. Ultra-violet (UV) mutagenesis combined with enrichment culture with ethanol and H2O2 and selection of low-density cells was applied to L. starkeyi to obtain the L. starkeyi mutant strain UMP47, which exhibited higher TAG production from glucose/xylose. Transcriptome analysis revealed high expression of genes involved in transporter activity and carbohydrate metabolism, whereas genes involved in DNA replication exhibited lower expression in the mutant strain UMP47 than in the wild-type strain. Altogether, the lipid productivity of L. starkeyi was successfully improved by UV mutagenesis. Transcriptome analysis suggested the importance of previously unidentified genes in TAG production. This study provides information on potential target genes for improving TAG production through the genetic modification of oleaginous yeast.
含油酵母星形脂酵母是一种很有前途的生物柴油燃料三酰甘油（TAG）生产者。然而，有必要进一步提高星状酵母从葡萄糖和木糖混合糖中生产三酰甘油（TAG）的能力。本研究旨在构建一种可提高葡萄糖/木糖混合糖 TAG 生产率的星菌突变体，并阐明提高脂质生产率的原因。通过紫外线（UV）诱变、乙醇和 H2O2 富集培养以及低密度细胞的筛选，我们获得了星形菌突变株 UMP47，该突变株从葡萄糖/木糖中获得了更高的 TAG 产量。转录组分析显示，涉及转运体活性和碳水化合物代谢的基因表达量较高，而涉及 DNA 复制的基因在突变株 UMP47 中的表达量低于野生型菌株。总之，紫外诱变成功地提高了L. starkeyi的脂质生产率。转录组分析表明，以前未发现的基因在 TAG 生产中起着重要作用。这项研究为通过对含油酵母进行基因改造来提高 TAG 产量提供了潜在目标基因的信息。
{"title":"Improvement of lipid production from glucose/xylose mixed-sugar by the oleaginous yeast Lipomyces starkeyi through ultra-violet mutagenesis","authors":"Sota Kamba, Ryosuke Yamada , Takuya Matsumoto, Hiroyasu Ogino","doi":"10.1016/j.enzmictec.2024.110551","DOIUrl":"10.1016/j.enzmictec.2024.110551","url":null,"abstract":"<div><div>The oleaginous yeast <em>Lipomyces starkeyi</em> is a promising triacylglycerol (TAG) producer for biodiesel fuel. However, it is necessary to further improve TAG productivity in <em>L. starkeyi</em> from a mixed sugar of glucose and xylose. This study aimed to construct an <em>L. starkeyi</em> mutant with increased TAG productivity from glucose/xylose mixed-sugar and to elucidate the causes underlying increased lipid productivity. Ultra-violet (UV) mutagenesis combined with enrichment culture with ethanol and H<sub>2</sub>O<sub>2</sub> and selection of low-density cells was applied to <em>L. starkeyi</em> to obtain the <em>L. starkeyi</em> mutant strain UMP47, which exhibited higher TAG production from glucose/xylose. Transcriptome analysis revealed high expression of genes involved in transporter activity and carbohydrate metabolism, whereas genes involved in DNA replication exhibited lower expression in the mutant strain UMP47 than in the wild-type strain. Altogether, the lipid productivity of <em>L. starkeyi</em> was successfully improved by UV mutagenesis. Transcriptome analysis suggested the importance of previously unidentified genes in TAG production. This study provides information on potential target genes for improving TAG production through the genetic modification of oleaginous yeast.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"183 ","pages":"Article 110551"},"PeriodicalIF":3.4,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142699661","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 : 2024-11-21DOI: 10.1016/j.enzmictec.2024.110549
Hamza Moustakim, Aziz Amine, Hasna Mohammadi
The ongoing challenge of infectious pathogens highlights the need for accurate and accessible methods to discern their genetic signatures, especially in resource-limited settings. In response to this crucial requirement, we introduce an affordable large-scale screening platform for infectious pathogen detection, using Hepatitis B virus (HBV) as a fundamental model. This proposed biosensor integrates an exonuclease III-assisted target recycling amplification strategy within a high-throughput 96-well microplate format. The HBV DNA target binds to a capture probe DNA and exonuclease III digests the probe to release the target. This mechanism enables the target to engage in binding cycles with new probes, each digested in turn, increasing detection sensitivity for even small quantities of HBV DNA. The implemented approach incorporates a biotin-streptavidin interaction allowing the undigested capture probe DNA to bind to a 5′-biotin-modified detection probe for effective HBV DNA quantification. This interaction generates a signal that, following the enzyme-substrate reaction, can be detected on-site using a smartphone, offering either optical or electrochemical readouts. The developed biosensor was capable of detecting HBV DNA with a detection limit of 5.62 fM and provided a considerable linear range covering concentrations from 100 fM to 100 nM. The determination of HBV DNA quantities in spiked human serum was achieved with a recovery of 90.0 % – 107.4 % as well. The results suggest that the developed dual-mode biosensor offers an adaptable and cost-effective approach for detecting infectious diseases, with promising applications in medical diagnostics and environmental monitoring to support public health efforts.
传染病病原体带来的持续挑战凸显了对准确、易用的方法的需求，尤其是在资源有限的环境中。针对这一关键需求，我们以乙型肝炎病毒（HBV）为基本模型，推出了一种经济实惠的大规模传染病病原体检测筛查平台。这种拟议的生物传感器在高通量 96 孔微孔板格式中整合了外切酶 III 辅助的靶循环扩增策略。HBV DNA 靶标与捕获探针 DNA 结合，外切酶 III 消化探针以释放靶标。这种机制能使目标与新探针循环结合，每个探针依次被消化，从而提高对少量 HBV DNA 的检测灵敏度。这种方法结合了生物素-链霉亲和素的相互作用，使未消化的捕获探针 DNA 与 5′-生物素修饰的检测探针结合，从而有效地定量检测 HBV DNA。在酶-底物反应后，这种相互作用产生的信号可通过智能手机现场检测，提供光学或电化学读数。所开发的生物传感器能够检测出检测限为 5.62 fM 的 HBV DNA，其线性范围相当大，可覆盖 100 fM 到 100 nM 的浓度范围。在测定加标人血清中的 HBV DNA 数量时，回收率也达到了 90.0 % - 107.4 %。结果表明，所开发的双模式生物传感器为检测传染病提供了一种适应性强、成本效益高的方法，有望应用于医疗诊断和环境监测，为公共卫生工作提供支持。
{"title":"Affordable infectious pathogen detection using a dual-mode biosensor integrating exonuclease III-assisted target recycling amplification with high-throughput 96-well microplate format","authors":"Hamza Moustakim, Aziz Amine, Hasna Mohammadi","doi":"10.1016/j.enzmictec.2024.110549","DOIUrl":"10.1016/j.enzmictec.2024.110549","url":null,"abstract":"<div><div>The ongoing challenge of infectious pathogens highlights the need for accurate and accessible methods to discern their genetic signatures, especially in resource-limited settings. In response to this crucial requirement, we introduce an affordable large-scale screening platform for infectious pathogen detection, using Hepatitis B virus (HBV) as a fundamental model. This proposed biosensor integrates an exonuclease III-assisted target recycling amplification strategy within a high-throughput 96-well microplate format. The HBV DNA target binds to a capture probe DNA and exonuclease III digests the probe to release the target. This mechanism enables the target to engage in binding cycles with new probes, each digested in turn, increasing detection sensitivity for even small quantities of HBV DNA. The implemented approach incorporates a biotin-streptavidin interaction allowing the undigested capture probe DNA to bind to a 5′-biotin-modified detection probe for effective HBV DNA quantification. This interaction generates a signal that, following the enzyme-substrate reaction, can be detected on-site using a smartphone, offering either optical or electrochemical readouts. The developed biosensor was capable of detecting HBV DNA with a detection limit of 5.62 fM and provided a considerable linear range covering concentrations from 100 fM to 100 nM. The determination of HBV DNA quantities in spiked human serum was achieved with a recovery of 90.0 % – 107.4 % as well. The results suggest that the developed dual-mode biosensor offers an adaptable and cost-effective approach for detecting infectious diseases, with promising applications in medical diagnostics and environmental monitoring to support public health efforts.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"183 ","pages":"Article 110549"},"PeriodicalIF":3.4,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142699658","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 : 2024-11-19DOI: 10.1016/j.enzmictec.2024.110548
Larissa F. Santos , Denicezar Â. Baldo , José M. Oliveira Jr , Marta M.D.C. Vila , Victor M. Balcão
Contamination of water with mercury constitutes a serious public health problem, especially in locations where the use of Hg occurs improperly/illegally and negligently, as is the case in the Amazon region (Brazil). The riverside populations in the Amazon are frequently invaded by illegal mining, exposing these populations to significant risks, of which contamination by heavy metals such as mercury (Hg2+) has the potential to cause serious illnesses. Furthermore, exposure to this metal causes neurological, cardiovascular, immune and digestive system disorders, in addition to damaging the lungs, kidneys, skin and eyes. The aquatic biome is extremely important for the local economy and population, being drastically affected by Hg2+ contamination and its effects. Therefore, it is necessary to develop bioremediation/biomitigation methods that are effective and less harmful to the environment, aiming to remove Hg2+ from water. Hence, when we think about new methodologies that can lead to the reduction of mercury in water, the use of protein entities is a potential option and, for this reason, we can highlight the possibility of using bacteriophage virions to remove Hg2+ ions from water by biosorption using their negative Zeta Potential for this purpose. In this sense, the main goal of the research work undertaken was to test the possibility of mitigating the presence of mercury (II) ions in water through the immobilization of a bacteriophage isolated and already characterized by our research group (EcoM021, T4 myovirus of the Straboviridae family and genus Tequatrovirus), on a chitosan-coated Ca-alginate microparticle support, through which water contaminated with Hg2+ ions was percolated. The system developed in microparticle form integrating trapped phage virions showed to be very promising for retaining mercury ions through biosorption (electrostatic attraction), thus enabling the removal of ionic mercury from water.
{"title":"An environmental “fairytail”: Removal of mercury from water via phage virion-based biosorption","authors":"Larissa F. Santos , Denicezar Â. Baldo , José M. Oliveira Jr , Marta M.D.C. Vila , Victor M. Balcão","doi":"10.1016/j.enzmictec.2024.110548","DOIUrl":"10.1016/j.enzmictec.2024.110548","url":null,"abstract":"<div><div>Contamination of water with mercury constitutes a serious public health problem, especially in locations where the use of Hg occurs improperly/illegally and negligently, as is the case in the Amazon region (Brazil). The riverside populations in the Amazon are frequently invaded by illegal mining, exposing these populations to significant risks, of which contamination by heavy metals such as mercury (Hg<sup>2+</sup>) has the potential to cause serious illnesses. Furthermore, exposure to this metal causes neurological, cardiovascular, immune and digestive system disorders, in addition to damaging the lungs, kidneys, skin and eyes. The aquatic biome is extremely important for the local economy and population, being drastically affected by Hg<sup>2+</sup> contamination and its effects. Therefore, it is necessary to develop bioremediation/biomitigation methods that are effective and less harmful to the environment, aiming to remove Hg<sup>2+</sup> from water. Hence, when we think about new methodologies that can lead to the reduction of mercury in water, the use of protein entities is a potential option and, for this reason, we can highlight the possibility of using bacteriophage virions to remove Hg<sup>2+</sup> ions from water by biosorption using their negative Zeta Potential for this purpose. In this sense, the main goal of the research work undertaken was to test the possibility of mitigating the presence of mercury (II) ions in water through the immobilization of a bacteriophage isolated and already characterized by our research group (EcoM021, T4 myovirus of the <em>Straboviridae</em> family and genus <em>Tequatrovirus</em>), on a chitosan-coated Ca-alginate microparticle support, through which water contaminated with Hg<sup>2+</sup> ions was percolated. The system developed in microparticle form integrating trapped phage virions showed to be very promising for retaining mercury ions through biosorption (electrostatic attraction), thus enabling the removal of ionic mercury from water.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"183 ","pages":"Article 110548"},"PeriodicalIF":3.4,"publicationDate":"2024-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142692822","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}
Microalgae-based biofuel production is cost-effective only in a biorefinery, where valuable co-products offset high costs. Fatty acids produced by photosynthetic microalgae can serve as raw materials for bioenergy and pharmaceuticals. This study aims to understand the metabolic imprints of Scenedesmus sp. CABeR52, to decipher the physiological mechanisms behind lipid accumulation under nitrogen deprivation. Metabolomics profiles were generated using gas chromatography-mass spectrometry (GC–MS) of Scenedesmus sp. CABeR52 subjected to nutrient deprivation. Our initial data sets indicate that deprived cells have an increased accumulation of lipids (278.31 mg.g−1 dcw), 2.0 times higher than the control. The metabolomic profiling unveils a metabolic reprogramming, highlighting the upregulation of key metabolites involved in fatty acid biosynthesis, such as citric acid, succinic acid, and 2-ketoglutaric acid. The accumulation of trehalose, a stress-responsive metabolite, further underscores the microalga's adaptability. Interestingly, we found that a new fatty acid, nervonic acid, was identified in the complex, which has a significant role in brain development. These findings provide valuable insights into the metabolic pathways governing lipid accumulation in Scenedesmus sp., paving the way for its exploitation as a sustainable biofuel feedstock.
{"title":"Enhanced lipid accumulation in microalgae Scenedesmus sp. under nitrogen limitation","authors":"Getachew Tafere Abrha , Abdalah Makaranga , Pannaga Pavan Jutur","doi":"10.1016/j.enzmictec.2024.110546","DOIUrl":"10.1016/j.enzmictec.2024.110546","url":null,"abstract":"<div><div>Microalgae-based biofuel production is cost-effective only in a biorefinery, where valuable co-products offset high costs. Fatty acids produced by photosynthetic microalgae can serve as raw materials for bioenergy and pharmaceuticals. This study aims to understand the metabolic imprints of <em>Scenedesmus</em> sp. CABeR52, to decipher the physiological mechanisms behind lipid accumulation under nitrogen deprivation. Metabolomics profiles were generated using gas chromatography-mass spectrometry (GC–MS) of <em>Scenedesmus</em> sp. CABeR52 subjected to nutrient deprivation. Our initial data sets indicate that deprived cells have an increased accumulation of lipids (278.31 mg.g<sup>−1</sup> dcw), 2.0 times higher than the control. The metabolomic profiling unveils a metabolic reprogramming, highlighting the upregulation of key metabolites involved in fatty acid biosynthesis, such as citric acid, succinic acid, and 2-ketoglutaric acid. The accumulation of trehalose, a stress-responsive metabolite, further underscores the microalga's adaptability. Interestingly, we found that a new fatty acid, nervonic acid, was identified in the complex, which has a significant role in brain development. These findings provide valuable insights into the metabolic pathways governing lipid accumulation in <em>Scenedesmus</em> sp., paving the way for its exploitation as a sustainable biofuel feedstock.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"182 ","pages":"Article 110546"},"PeriodicalIF":3.4,"publicationDate":"2024-11-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142617167","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 : 2024-11-08DOI: 10.1016/j.enzmictec.2024.110544
Philippe de Castro Lins , Pedro Ricardo Vieira Hamann , Jônatas Cunha Barbosa Lima , João Alexandre Ribeiro Gonçalves Barbosa , João Lucas da Silva Correia , Ikaro Alves de Andrade , Débora Farage Knupp dos Santos , Betania Ferraz Quirino , Ricardo Henrique Krüger
Dioxygenases are enzymes involved in the conversion of polyconic aromatic hydroxycarbons (PAHs), attracting significant biotechnological interest for the conversion of recalcitrant organic compounds. Furthermore, few studies show that dioxygenases can take on the function of resistance genes in clones. This enzymatic versatility opens up new opportunities for elucidating the mechanisms of microbial resistance, as well as its biotechnological application. In this work, a Cerrado soil dioxygenase named CRB2(1) was biochemically characterized. The enzyme was shown to have optimal activity at pH 7; a temperature of 30 °C; and using iron ions as a cofactor for substrate cleavage. The kinetic catalytic parameters of CRB2(1) were Vmax = 0.02281 µM/min and KM = 97.6. Its predicted three-dimensional structure obtained using the Modeller software v9.22 based on the crystal structure of gentisate 1,2-dioxygenase from Silicibacter pomeroyi (GDOsp) (PDB ID 3BU7, resolution 2.80 Å, residues 17–374) revealed substrate binding to the cupin domain, where the active site is located. The analyzed substrates interact directly with the iron ion, coordinated by three histidine residues. Changing the iron ion charge modifies the binding between the active site and the substrates. Currently, there is a demand for enzymes that have biotechnological activities of interest. Metagenomics allows analyzing the biotechnological potential of several organisms at the same time, based on sequence and functional activity analyses.
{"title":"Biochemical characterization and structure prediction of the Cerrado soil CRB2(1) metagenomic dioxygenase","authors":"Philippe de Castro Lins , Pedro Ricardo Vieira Hamann , Jônatas Cunha Barbosa Lima , João Alexandre Ribeiro Gonçalves Barbosa , João Lucas da Silva Correia , Ikaro Alves de Andrade , Débora Farage Knupp dos Santos , Betania Ferraz Quirino , Ricardo Henrique Krüger","doi":"10.1016/j.enzmictec.2024.110544","DOIUrl":"10.1016/j.enzmictec.2024.110544","url":null,"abstract":"<div><div>Dioxygenases are enzymes involved in the conversion of polyconic aromatic hydroxycarbons (PAHs), attracting significant biotechnological interest for the conversion of recalcitrant organic compounds. Furthermore, few studies show that dioxygenases can take on the function of resistance genes in clones. This enzymatic versatility opens up new opportunities for elucidating the mechanisms of microbial resistance, as well as its biotechnological application. In this work, a <em>Cerrado</em> soil dioxygenase named CRB2(1) was biochemically characterized. The enzyme was shown to have optimal activity at pH 7; a temperature of 30 °C; and using iron ions as a cofactor for substrate cleavage. The kinetic catalytic parameters of CRB2(1) were <em>V</em><sub>max</sub> = 0.02281 µM/min and <em>K</em><sub>M</sub> = 97.6. Its predicted three-dimensional structure obtained using the Modeller software v9.22 based on the crystal structure of gentisate 1,2-dioxygenase from <em>Silicibacter pomeroyi</em> (GDOsp) (PDB ID <span><span>3BU7</span><svg><path></path></svg></span>, resolution 2.80 Å, residues 17–374) revealed substrate binding to the cupin domain, where the active site is located. The analyzed substrates interact directly with the iron ion, coordinated by three histidine residues. Changing the iron ion charge modifies the binding between the active site and the substrates. Currently, there is a demand for enzymes that have biotechnological activities of interest. Metagenomics allows analyzing the biotechnological potential of several organisms at the same time, based on sequence and functional activity analyses.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"182 ","pages":"Article 110544"},"PeriodicalIF":3.4,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142617164","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 : 2024-11-07DOI: 10.1016/j.enzmictec.2024.110545
Yusuf Alan , Ali-Osman Keskin , Mehmet Sönmez
Four Lactiplantibacillus plantarum strains newly isolated and identified from human breast milk in Türkiye, have probiotic, functional and proliferative inhibition potential of metabolites against colon cancer cell lines were evaluated. In simulated gastric and intestinal media, all strains exhibited strong probiotic character by showing resistance, although decreasing with time and concentration. The strains were sensitive to penicillin G, rifampin and chloramphenicol and showed antibacterial effect on all pathogenic bacteria. Citric acid, malic acid, tartaric acid, pyruvic acid and fumaric acid were not detected in the strains, while the highest amount of acetic acid was detected. The quantitative-qualitative analysis and structural characterization of exopolysaccharide (EPS) was confirmed and it was determined that the strains synthesized similar amounts. Compared to standard antioxidants, the strains showed less DPPH activity and similar ABTS activity. High amounts of metabolites of the strains showed good antiproliferative effect on Caco-2, while lower amounts showed good antiproliferative effect on the HT-29 cell line. When all the data were considered, it was determined that the strains were close to each other, but the YAAS 23 strain showed slightly better properties. In conclusion, breast milk is a unique environment harboring beneficial bacteria such as L. plantarum for human health.
{"title":"Probiotic and functional characterization of newly isolated Lactiplantibacillus plantarum strains from human breast milk and proliferative inhibition potential of metabolites","authors":"Yusuf Alan , Ali-Osman Keskin , Mehmet Sönmez","doi":"10.1016/j.enzmictec.2024.110545","DOIUrl":"10.1016/j.enzmictec.2024.110545","url":null,"abstract":"<div><div>Four <em>Lactiplantibacillus plantarum</em> strains newly isolated and identified from human breast milk in Türkiye, have probiotic, functional and proliferative inhibition potential of metabolites against colon cancer cell lines were evaluated. In simulated gastric and intestinal media, all strains exhibited strong probiotic character by showing resistance, although decreasing with time and concentration. The strains were sensitive to penicillin G, rifampin and chloramphenicol and showed antibacterial effect on all pathogenic bacteria. Citric acid, malic acid, tartaric acid, pyruvic acid and fumaric acid were not detected in the strains, while the highest amount of acetic acid was detected. The quantitative-qualitative analysis and structural characterization of exopolysaccharide (EPS) was confirmed and it was determined that the strains synthesized similar amounts. Compared to standard antioxidants, the strains showed less DPPH activity and similar ABTS activity. High amounts of metabolites of the strains showed good antiproliferative effect on Caco-2, while lower amounts showed good antiproliferative effect on the HT-29 cell line. When all the data were considered, it was determined that the strains were close to each other, but the YAAS 23 strain showed slightly better properties. In conclusion, breast milk is a unique environment harboring beneficial bacteria such as <em>L. plantarum</em> for human health.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"182 ","pages":"Article 110545"},"PeriodicalIF":3.4,"publicationDate":"2024-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142638571","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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