To plant crops (especially dry crops such as water spinach) with concomitant electricity recovery, a hanging-submerged-plant-pot system (HSPP) is developed. The HSPP consists of a soil pot (anodic) partially submerged under the water surface of a cathode tank. The microbial communities changed with conditions were also investigated. It was found that with chemical fertilizers the closed-circuit voltage (CCV, with 1 kΩ) was stable (approximately 250 mV) within 28 d; however, without fertilizer, the water spinach could adjust to the environment to obtain a better power output (approximately 3 mW m−2) at day 28. The microbial-community analyses revealed that the Pseudomonas sp. was the only exoeletrogens found in the anode pots. Using a secondary design of HSPP, for a better water-level adjustment, the maximum power output of each plant was found to be approximately 27.1 mW m−2. During operation, high temperature resulted in low oxygen solubility, and low CCV as well. At this time, it is yet to be concluded whether the submerged water level significantly affects electricity generation.
{"title":"Developing a plant microbial fuel cell by planting water spinach in a hanging-submerged plant pot system","authors":"Yi-Hsuan Chen, Shiue-Lin Li, Ching-Ya Hung, Pei-Ching Wu, Yue-Xiang Hong, Wen-Jing Chen, Shu-yi Chang, Yu-Ya Hsu, Wei-Yi Chao, Kai-Jhih Tsai, You-Chen Chen, Ji-Teng Chen, Chia-Le Hsu, Yun-Ju Lu, Li-Ming Fang, Ming-Han Yang, I-Ting Tan, Ying-Chuan Hsu, Hong-Yu Yang, Rui-Hong Jiang","doi":"10.1016/j.jbiosc.2024.08.007","DOIUrl":"10.1016/j.jbiosc.2024.08.007","url":null,"abstract":"<div><div>To plant crops (especially dry crops such as water spinach) with concomitant electricity recovery, a hanging-submerged-plant-pot system (HSPP) is developed. The HSPP consists of a soil pot (anodic) partially submerged under the water surface of a cathode tank. The microbial communities changed with conditions were also investigated. It was found that with chemical fertilizers the closed-circuit voltage (CCV, with 1 kΩ) was stable (approximately 250 mV) within 28 d; however, without fertilizer, the water spinach could adjust to the environment to obtain a better power output (approximately 3 mW m<sup>−2</sup>) at day 28. The microbial-community analyses revealed that the <em>Pseudomonas</em> sp. was the only exoeletrogens found in the anode pots. Using a secondary design of HSPP, for a better water-level adjustment, the maximum power output of each plant was found to be approximately 27.1 mW m<sup>−2</sup>. During operation, high temperature resulted in low oxygen solubility, and low CCV as well. At this time, it is yet to be concluded whether the submerged water level significantly affects electricity generation.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 6","pages":"Pages 533-540"},"PeriodicalIF":2.3,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142248091","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-08DOI: 10.1016/j.jbiosc.2024.08.008
Mikiko Nakamura , Rinji Akada
pET vectors allow inducible expression of recombinant proteins in Escherichia coli. In this system, isopropyl β-d-1-thiogalactopyranoside (IPTG) drives lacUV5 promoter to produce T7 RNA polymerase, simultaneously releasing the suppression of T7lac promoter. T7 RNA polymerase then strongly transcribes the target gene. A lac repressor encoded by lacI in the vector represses the promoters. Despite stringent repression and inducible expression achieved with the pET system, unexpected leaky expression can occur without IPTG induction. Here, by evaluating leaky expression in recombinant cells cultured in various Luria–Bertani (LB) media, prepared using yeast extract and peptone from different suppliers, as well as in five commercial premix-LB media, we confirmed the presence of unknown lac inducers in LB. To explore these inducers, we examined E. coli growth in media comprising yeast extract or peptone. At 4% concentration, five commercial yeast extract and six peptone samples individually allowed E. coli growth equivalent to that in LB medium. We determined the luciferase activity of the luxCDABE operon in the pET vector under these conditions. The presence of different concentrations of inducers was detected in both the yeast extract and peptone. Furthermore, we blended yeast extract and peptone with low or high concentrations of lac inducers. The low-expression blend, used as a basal medium before IPTG addition, allowed leak-free, tightly controlled expression. The high-expression blend was used for constitutive high-expression and pET induction with the basal medium, in lieu of IPTG. These blended media can be used for well-controlled inducible and constitutive expression using the pET system.
pET 载体可在大肠杆菌中诱导表达重组蛋白。在该系统中，异丙基β-d-1-硫代吡喃半乳糖苷（IPTG）驱动 lacUV5 启动子产生 T7 RNA 聚合酶，同时释放对 T7lac 启动子的抑制。然后，T7 RNA 聚合酶强烈转录目标基因。载体中由lacI编码的lac抑制因子抑制启动子。尽管 pET 系统实现了严格的抑制和诱导表达，但在没有 IPTG 诱导的情况下也会出现意外的泄漏表达。在这里，通过评估用不同供应商提供的酵母提取物和蛋白胨制备的各种 Luria-Bertani（LB）培养基以及五种商用预混合 LB 培养基培养的重组细胞的泄漏表达，我们证实了 LB 中存在未知的 lac 诱导因子。为了探究这些诱导剂，我们检测了大肠杆菌在含有酵母提取物或蛋白胨的培养基中的生长情况。在 4% 的浓度下，五种商用酵母提取物和六种蛋白胨样品分别允许大肠杆菌生长，与在 LB 培养基中的生长相当。在这些条件下，我们测定了 pET 载体中 luxCDABE 操作子的荧光素酶活性。在酵母提取物和蛋白胨中都检测到了不同浓度的诱导剂。此外，我们还将酵母提取物和蛋白胨与低浓度或高浓度的lac诱导剂混合。低表达混合培养基在添加 IPTG 前用作基础培养基，可实现无泄漏、严格控制的表达。高表达混合培养基用于组成型高表达，并用基础培养基诱导 pET，以代替 IPTG。这些混合培养基可用于使用 pET 系统进行良好的诱导型和组成型表达。
{"title":"Blending of selected yeast extract and peptone for inducible and constitutive protein production in Escherichia coli using the pET system","authors":"Mikiko Nakamura , Rinji Akada","doi":"10.1016/j.jbiosc.2024.08.008","DOIUrl":"10.1016/j.jbiosc.2024.08.008","url":null,"abstract":"<div><div>pET vectors allow inducible expression of recombinant proteins in <em>Escherichia coli</em>. In this system, isopropyl β-<span>d</span>-1-thiogalactopyranoside (IPTG) drives <em>lac</em>UV5 promoter to produce T7 RNA polymerase, simultaneously releasing the suppression of T7<em>lac</em> promoter. T7 RNA polymerase then strongly transcribes the target gene. A lac repressor encoded by <em>lacI</em> in the vector represses the promoters. Despite stringent repression and inducible expression achieved with the pET system, unexpected leaky expression can occur without IPTG induction. Here, by evaluating leaky expression in recombinant cells cultured in various Luria–Bertani (LB) media, prepared using yeast extract and peptone from different suppliers, as well as in five commercial premix-LB media, we confirmed the presence of unknown <em>lac</em> inducers in LB. To explore these inducers, we examined <em>E. coli</em> growth in media comprising yeast extract or peptone. At 4% concentration, five commercial yeast extract and six peptone samples individually allowed <em>E. coli</em> growth equivalent to that in LB medium. We determined the luciferase activity of the <em>luxCDABE</em> operon in the pET vector under these conditions. The presence of different concentrations of inducers was detected in both the yeast extract and peptone. Furthermore, we blended yeast extract and peptone with low or high concentrations of <em>lac</em> inducers. The low-expression blend, used as a basal medium before IPTG addition, allowed leak-free, tightly controlled expression. The high-expression blend was used for constitutive high-expression and pET induction with the basal medium, in lieu of IPTG. These blended media can be used for well-controlled inducible and constitutive expression using the pET system.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 6","pages":"Pages 548-556"},"PeriodicalIF":2.3,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142217177","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Gamma-aminobutyric acid (GABA), which is synthesized from l-glutamic acid via glutamate decarboxylase (Gad), is used as food, supplements, and biodegradable plastics. Our previous study demonstrated an Escherichia coli mutant (ΔΔ) strain, lacking type I NADH dehydrogenase (NDH-I) and cytochrome bo3 oxidase (Cytbo3), produced 7 g/L glutamic acid on MS1 glucose-minimal medium. In this study, the ΔΔ strain was used for improving GABA production. A plasmid (pMBL19-gadB′) expressing a mutated E. coli GadB (Glu89Gln/Δ452-466), retaining activity at neutral pH, was introduced into the ΔΔ strain and its parent strain (W1485). The ΔΔ strain carrying pMBL19-gadB′ exhibited a twofold increase in GABA production compared to the W1485 strain carrying pMBL19-gadB′. Deleting the C-terminal (Δ471–511) of GadC antiporter in the ΔΔ strain further improved GABA yield to 1.5 g/L when cultured in MS1 glucose-minimal medium. On the other hand, a large amount of glutamic acid produced by the ΔΔ strain was not fully converted to GABA, likely due to the inhibition of GadB activity by the accumulation of acetic acid. Although there is room for improvement, these results indicate the efficacy of the ΔNDH-IΔCytbo3 double mutation in augmenting GABA production.
{"title":"Improved fermentative gamma-aminobutyric acid production from glucose by the inactivation of respiratory chain components NDH-I and Cytbo₃ in Escherichia coli","authors":"Hiroki Wakahara , Takuya Mizokoshi , Kotaro Yamagami , Satoru Fukiya , Atsushi Yokota , Tomoya Maeda","doi":"10.1016/j.jbiosc.2024.08.004","DOIUrl":"10.1016/j.jbiosc.2024.08.004","url":null,"abstract":"<div><div>Gamma-aminobutyric acid (GABA), which is synthesized from <span>l</span>-glutamic acid via glutamate decarboxylase (Gad), is used as food, supplements, and biodegradable plastics. Our previous study demonstrated an <em>Escherichia coli</em> mutant (ΔΔ) strain, lacking type I NADH dehydrogenase (NDH-I) and cytochrome <em>bo</em><sub>3</sub> oxidase (Cyt<em>bo</em><sub>3</sub>), produced 7 g/L glutamic acid on MS1 glucose-minimal medium. In this study, the ΔΔ strain was used for improving GABA production. A plasmid (pMBL19-<em>gadB</em>′) expressing a mutated <em>E. coli</em> GadB (Glu89Gln/Δ452-466), retaining activity at neutral pH, was introduced into the ΔΔ strain and its parent strain (W1485). The ΔΔ strain carrying pMBL19-<em>gadB</em>′ exhibited a twofold increase in GABA production compared to the W1485 strain carrying pMBL19-<em>gadB</em>′. Deleting the C-terminal (Δ471–511) of GadC antiporter in the ΔΔ strain further improved GABA yield to 1.5 g/L when cultured in MS1 glucose-minimal medium. On the other hand, a large amount of glutamic acid produced by the ΔΔ strain was not fully converted to GABA, likely due to the inhibition of GadB activity by the accumulation of acetic acid. Although there is room for improvement, these results indicate the efficacy of the ΔNDH-IΔCyt<em>bo</em><sub>3</sub> double mutation in augmenting GABA production.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 6","pages":"Pages 501-506"},"PeriodicalIF":2.3,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142154190","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Plasmids are molecular genetic tools used for trans-complementation and gene expression in bacteria. Challenges faced by researchers include limited repertoire of antibiotic resistance of plasmids, issues related to plasmid compatibility and restricted or incompatible multiple cloning sites when needing to change plasmid copy number to tune production of their protein of interest. In this study, a series of plasmids were generated with compatible multiple cloning sites and homologous DNA regions to allow for modular cloning for rapid exchange of antibiotic resistance and plasmid origin. Plasmids generated in this series have options for high, mid, and low plasmid copy number, and have either an integrated FLAG epitope in the multiple cloning site or possess an uninterrupted multiple cloning site with the option of using the common LacZ-based blue/white screening method. Low copy plasmids also have one of five antibiotic selection markers. To demonstrate functionality of these plasmids, a representative FLAG tagged protein and mCherry were cloned into the low copy plasmids and expressed in various bacteria belonging to the Enterobacteriaceae family. In conclusion, by creating a new plasmid series, we have expanded the toolkit of available molecular biology tools for bacterial work.
质粒是用于细菌转基因和基因表达的分子遗传工具。研究人员面临的挑战包括：质粒的抗生素抗性种类有限、质粒兼容性相关问题，以及当需要改变质粒拷贝数以调整所需蛋白质的生产时，多重克隆位点受限或不兼容。在这项研究中，生成了一系列具有兼容的多重克隆位点和同源 DNA 区域的质粒，以便进行模块化克隆，快速交换抗生素抗性和质粒来源。这一系列质粒有高、中、低三种质粒拷贝数可供选择，在多重克隆位点上有一个整合的 FLAG 表位，或拥有一个不间断的多重克隆位点，可选择使用常见的基于 LacZ 的蓝/白筛选方法。低拷贝质粒还具有五种抗生素选择标记中的一种。为了证明这些质粒的功能，我们在低拷贝质粒中克隆了具有代表性的 FLAG 标记蛋白和 mCherry，并在属于肠杆菌科的各种细菌中进行了表达。总之，通过创建新的质粒系列，我们扩展了用于细菌工作的分子生物学工具包。
{"title":"Generation of a plasmid series for rapid sub-cloning and use in various Enterobacteriaceae","authors":"Hannah Gertrude Braun , Nabeela Kanwal , Luisa Fernanda Rivera Lopez , Jenny-Lee Thomassin","doi":"10.1016/j.jbiosc.2024.08.006","DOIUrl":"10.1016/j.jbiosc.2024.08.006","url":null,"abstract":"<div><div>Plasmids are molecular genetic tools used for trans-complementation and gene expression in bacteria. Challenges faced by researchers include limited repertoire of antibiotic resistance of plasmids, issues related to plasmid compatibility and restricted or incompatible multiple cloning sites when needing to change plasmid copy number to tune production of their protein of interest. In this study, a series of plasmids were generated with compatible multiple cloning sites and homologous DNA regions to allow for modular cloning for rapid exchange of antibiotic resistance and plasmid origin. Plasmids generated in this series have options for high, mid, and low plasmid copy number, and have either an integrated FLAG epitope in the multiple cloning site or possess an uninterrupted multiple cloning site with the option of using the common LacZ-based blue/white screening method. Low copy plasmids also have one of five antibiotic selection markers. To demonstrate functionality of these plasmids, a representative FLAG tagged protein and mCherry were cloned into the low copy plasmids and expressed in various bacteria belonging to the <em>Enterobacteriaceae</em> family. In conclusion, by creating a new plasmid series, we have expanded the toolkit of available molecular biology tools for bacterial work.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 6","pages":"Pages 478-487"},"PeriodicalIF":2.3,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142145722","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A bacterium that produces membrane vesicles (MVs), strain WSS15, was isolated from a traditional vinegar in Japan called Kurozu. A phylogenetic analysis of 16S rRNA gene sequences indicated that this bacterium belongs to the genus Acetobacter. MVs and peptidoglycan-associated lipoprotein (Pal) were detected in the MV fraction of strain WSS15. In the presence of the WSS15 MV fraction, murine macrophages produced the pro-inflammatory cytokine interleukin-6 (IL-6) via the recognition by superficial Toll-like receptor 2 (TLR2). WSS15 MVs adhered to the cell surface of macrophages. The macrophages secreted IL-6 through the TLR2 recognition of an acylated N-terminal peptide of Pal. We elucidated the mode of action of WSS15 MVs on immune cells and identified the Pal peptide from strain WSS15 as an agonist of TLR2.
{"title":"Characterization of the membrane vesicle fraction from Acetobacter sp. WSS15","authors":"Atsushi Kurata , Kota Aimatsu , Yuki Kimura , Hinako Hashiguchi , Asami Maeda , Tomoya Imai , Shino Yamasaki-Yashiki , Kensaku Hamada , Yuki Fujimoto , Akira Fujii , Koichi Uegaki","doi":"10.1016/j.jbiosc.2024.07.017","DOIUrl":"10.1016/j.jbiosc.2024.07.017","url":null,"abstract":"<div><div>A bacterium that produces membrane vesicles (MVs), strain WSS15, was isolated from a traditional vinegar in Japan called Kurozu. A phylogenetic analysis of 16S rRNA gene sequences indicated that this bacterium belongs to the genus <em>Acetobacter</em>. MVs and peptidoglycan-associated lipoprotein (Pal) were detected in the MV fraction of strain WSS15. In the presence of the WSS15 MV fraction, murine macrophages produced the pro-inflammatory cytokine interleukin-6 (IL-6) via the recognition by superficial Toll-like receptor 2 (TLR2). WSS15 MVs adhered to the cell surface of macrophages. The macrophages secreted IL-6 through the TLR2 recognition of an acylated N-terminal peptide of Pal. We elucidated the mode of action of WSS15 MVs on immune cells and identified the Pal peptide from strain WSS15 as an agonist of TLR2.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 6","pages":"Pages 495-500"},"PeriodicalIF":2.3,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142145721","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-03DOI: 10.1016/j.jbiosc.2024.07.019
Kazuhiro Kawamura , Eiichiro Fukusaki
Metabolomic research involves the comprehensive analysis of metabolites in biological samples and has many applications. Gas chromatography-mass spectrometry (GC-MS) is an established and widely used approach for metabolic profiling. However, sample preparation and metabolite derivatization are time-consuming, and derivatization options are limited. We propose gas–solid phase derivatization (GSPD) as a novel sampling and derivatization method that uses a silica monolith substrate and gaseous derivatization reagents for metabolomics using GC-MS. We developed a method to measure the organic acids and sugar phosphates responsible for glycolysis and the tricarboxylic acid (TCA) cycle. GSPD simplifies the sample preparation and can be applied to derivatization reactions that are difficult to perform in solution owing to solvent limitations. The developed method was applied to human plasma and tomato pulp and was shown to have a higher detection performance than the conventional method. This study provides a strategy to simplify sample preparation and expand derivatization options for GC-MS-based metabolomics.
{"title":"Novel sampling and gas-phase derivatization strategy: Proof-of-concept by profiling ionic polar metabolites using gas chromatography-mass spectrometry","authors":"Kazuhiro Kawamura , Eiichiro Fukusaki","doi":"10.1016/j.jbiosc.2024.07.019","DOIUrl":"10.1016/j.jbiosc.2024.07.019","url":null,"abstract":"<div><div>Metabolomic research involves the comprehensive analysis of metabolites in biological samples and has many applications. Gas chromatography-mass spectrometry (GC-MS) is an established and widely used approach for metabolic profiling. However, sample preparation and metabolite derivatization are time-consuming, and derivatization options are limited. We propose gas–solid phase derivatization (GSPD) as a novel sampling and derivatization method that uses a silica monolith substrate and gaseous derivatization reagents for metabolomics using GC-MS. We developed a method to measure the organic acids and sugar phosphates responsible for glycolysis and the tricarboxylic acid (TCA) cycle. GSPD simplifies the sample preparation and can be applied to derivatization reactions that are difficult to perform in solution owing to solvent limitations. The developed method was applied to human plasma and tomato pulp and was shown to have a higher detection performance than the conventional method. This study provides a strategy to simplify sample preparation and expand derivatization options for GC-MS-based metabolomics.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 5","pages":"Pages 462-468"},"PeriodicalIF":2.3,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142132788","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Koji mold (Aspergillus oryzae) is a key microorganism in brewing and fermentation in Japan. We isolated koji molds from the environment in Niigata Prefecture. Eighty-one environmental samples were placed on isolation medium made from steamed rice with wood ash and 36 Aspergillus section Flavi-like strains were obtained. Of those, 26 strains did not produce aflatoxin. We studied their morphology, sequence of ITS region, calmodulin gene, aflatoxin biosynthetic homologous gene cluster and α-amylase gene and fermentation-related enzyme activities. Furthermore, DNA-seq analysis of 14 strains from 26 non-aflatoxin producing strains were conducted and compared the three mycotoxin biosynthetic gene clusters (aflatoxin, cyclopiazonic acid, and aflatrem) and fermentation-related genes against those of reference strain A. oryzae RIB40. In some strains, gene sequences confirmed the absence of mycotoxin production, but differences in fermentation-related enzyme activities could not be explained well by amino acid substitutions. We classified the 26 isolates into 6 morphology types based on the appearance of colonies and mating types, and it was found that strains of the same morphology type had similar enzymatic profiles and gene sequences. Our results show that koji molds with various properties occur in the environment, and it will expand the possibilities of koji mold in industrial use.
{"title":"Isolation and characterization of koji mold (Aspergillus oryzae) from nature in Niigata","authors":"Kanae Sakai , Keigo Sato , Mitsuoki Kaneoke , Ken-Ichi Kusumoto","doi":"10.1016/j.jbiosc.2024.08.005","DOIUrl":"10.1016/j.jbiosc.2024.08.005","url":null,"abstract":"<div><div>Koji mold (<em>Aspergillus oryzae</em>) is a key microorganism in brewing and fermentation in Japan. We isolated koji molds from the environment in Niigata Prefecture. Eighty-one environmental samples were placed on isolation medium made from steamed rice with wood ash and 36 <em>Aspergillus</em> section <em>Flavi</em>-like strains were obtained. Of those, 26 strains did not produce aflatoxin. We studied their morphology, sequence of ITS region, calmodulin gene, aflatoxin biosynthetic homologous gene cluster and α-amylase gene and fermentation-related enzyme activities. Furthermore, DNA-seq analysis of 14 strains from 26 non-aflatoxin producing strains were conducted and compared the three mycotoxin biosynthetic gene clusters (aflatoxin, cyclopiazonic acid, and aflatrem) and fermentation-related genes against those of reference strain <em>A. oryzae</em> RIB40. In some strains, gene sequences confirmed the absence of mycotoxin production, but differences in fermentation-related enzyme activities could not be explained well by amino acid substitutions. We classified the 26 isolates into 6 morphology types based on the appearance of colonies and mating types, and it was found that strains of the same morphology type had similar enzymatic profiles and gene sequences. Our results show that koji molds with various properties occur in the environment, and it will expand the possibilities of koji mold in industrial use.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 5","pages":"Pages 415-422"},"PeriodicalIF":2.3,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142125825","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-03DOI: 10.1016/j.jbiosc.2024.08.001
Nattapong Thakham, Po-Hang Huang, Kai-Yuan Li, Sung-Chyr Lin
The effect of delignification on the adsorption capacity of loofah sponge-based immobilized metal affinity chromatography adsorbents was investigated with recombinant His-tagged trehalose synthase as the model protein. Pretreatments with [EMIM][Ac] ionic liquid at 80 °C for 5 h and with sodium chlorite/acetic acid at 80 °C for 2 h were found effective for the removal of lignin, leading to a loss in biomass of 15.7% and 25.2%, respectively. Upon delignification, the metal chelating capacities of the loofah sponge-based adsorbents prepared with 5-h ionic liquid pretreatment (712 ± 82 μmole Cu(II)/g) and with 2-h sodium chlorite/acetic acid pretreatment (1012 ± 18 μmole Cu(II)/g) were 38% and 97% higher than that of the control (514 ± 55 μmole Cu(II)/g), adsorbent prepared with untreated loofah sponge, respectively. Results of protein adsorption study indicated that the Co(II)-loaded adsorbent prepared with 2-h sodium chlorite/acetic acid pretreatment exhibited the highest adsorption capacity and selectivity for the recombinant His-tagged trehalose synthase, giving a purification product with a specific activity of 7.62 U/mg protein. The predicted maximum adsorption capacity of the delignified loofah sponge-based adsorbent, 2.04 ± 0.14 mg/g, was 73% higher than that of the control.
{"title":"Effect of delignification on the adsorption of loofah sponge-based immobilized metal affinity chromatography adsorbent for His-tagged trehalose synthase","authors":"Nattapong Thakham, Po-Hang Huang, Kai-Yuan Li, Sung-Chyr Lin","doi":"10.1016/j.jbiosc.2024.08.001","DOIUrl":"10.1016/j.jbiosc.2024.08.001","url":null,"abstract":"<div><div>The effect of delignification on the adsorption capacity of loofah sponge-based immobilized metal affinity chromatography adsorbents was investigated with recombinant His-tagged trehalose synthase as the model protein. Pretreatments with [EMIM][Ac] ionic liquid at 80 °C for 5 h and with sodium chlorite/acetic acid at 80 °C for 2 h were found effective for the removal of lignin, leading to a loss in biomass of 15.7% and 25.2%, respectively. Upon delignification, the metal chelating capacities of the loofah sponge-based adsorbents prepared with 5-h ionic liquid pretreatment (712 ± 82 μmole Cu(II)/g) and with 2-h sodium chlorite/acetic acid pretreatment (1012 ± 18 μmole Cu(II)/g) were 38% and 97% higher than that of the control (514 ± 55 μmole Cu(II)/g), adsorbent prepared with untreated loofah sponge, respectively. Results of protein adsorption study indicated that the Co(II)-loaded adsorbent prepared with 2-h sodium chlorite/acetic acid pretreatment exhibited the highest adsorption capacity and selectivity for the recombinant His-tagged trehalose synthase, giving a purification product with a specific activity of 7.62 U/mg protein. The predicted maximum adsorption capacity of the delignified loofah sponge-based adsorbent, 2.04 ± 0.14 mg/g, was 73% higher than that of the control.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 5","pages":"Pages 445-451"},"PeriodicalIF":2.3,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142125824","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Osteochondral tissue engineering using layered scaffolds is a promising approach for treating osteochondral defects as an alternative to microfracture procedure, autologous chondrocyte implantation, and cartilage-bone grafting. The team previously investigated the chondrogenesis of mesenchymal stem cells (MSCs) on a polycaprolactone (PCL)/acetylated hyaluronic acid scaffold. The present study first focused on fabricating a novel osteoconductive scaffold utilizing bismuth-nanohydroxyapatite/reduced graphene oxide (Bi-nHAp/rGO) nanocomposite and electrospun PCL. The osteoconductive ability of the scaffold was investigated by evaluating the alkaline phosphatase (ALP) activity and the osteogenic genes expression in the adipose-derived MSCs. The expression of Runx2, collagen I, ALP, and osteocalcin as well as the result of ALP activity indicated the osteoconductive potential of the Bi-nHA-rGO/PCL scaffold. In the next step, a bilayer scaffold containing Bi-nHAp/rGO/PCL as an osteogenic layer and acetylated hyaluronic acid/PCL as a chondrogenic layer was prepared by the electrospinning technique and transplanted into osteochondral defects of rats. The chondrogenic and osteogenic markers corresponding to the surrounding tissues of the transplanted scaffold were surveyed 60 days later by real-time polymerase chain reaction (PCR) and immunohistochemistry methods. The results showed increased chondrogenic (Sox9 and collagen II) and osteogenic (osteocalcin and ALP) gene expression and augmented secretion of collagens II and X after transplantation. The results strongly support the efficacy of this constructed cell-free bilayer scaffold to induce osteochondral defect regeneration.
使用分层支架进行骨软骨组织工程是治疗骨软骨缺损的一种很有前景的方法，可替代微骨折术、自体软骨细胞植入术和软骨-骨移植术。研究小组之前研究了间充质干细胞（MSCs）在聚己内酯（PCL）/乙酰化透明质酸支架上的软骨形成。本研究首先利用铋-纳米羟基磷灰石/还原氧化石墨烯（Bi-nHAp/rGO）纳米复合材料和电纺 PCL 制备了一种新型骨诱导支架。通过评估脂肪间充质干细胞的碱性磷酸酶（ALP）活性和成骨基因表达，研究了该支架的骨诱导能力。Runx2、胶原蛋白I、ALP和骨钙素的表达以及ALP活性的结果表明了Bi-nHA-rGO/PCL支架的成骨潜力。下一步，通过电纺丝技术制备了含有 Bi-nHAp/rGO/PCL 作为成骨层和乙酰化透明质酸/PCL 作为软骨层的双层支架，并将其移植到大鼠的骨软骨缺损处。60 天后，通过实时聚合酶链式反应（PCR）和免疫组化方法检测了移植支架周围组织相应的软骨和成骨标记物。结果显示，移植后软骨（Sox9 和胶原 II）和成骨（骨钙素和 ALP）基因表达增加，胶原 II 和 X 分泌增加。这些结果有力地证明了这种无细胞双层支架在诱导骨软骨缺损再生方面的功效。
{"title":"Cell-free bilayer functionalized scaffold for osteochondral tissue engineering","authors":"Seyedeh Mahsa Khatami , Hana Hanaee-Ahvaz , Kazem Parivar , Masoud Soleimani , Shabnam Abedin Dargoush , Alireza Naderi Sohi","doi":"10.1016/j.jbiosc.2024.07.018","DOIUrl":"10.1016/j.jbiosc.2024.07.018","url":null,"abstract":"<div><div>Osteochondral tissue engineering using layered scaffolds is a promising approach for treating osteochondral defects as an alternative to microfracture procedure, autologous chondrocyte implantation, and cartilage-bone grafting. The team previously investigated the chondrogenesis of mesenchymal stem cells (MSCs) on a polycaprolactone (PCL)/acetylated hyaluronic acid scaffold. The present study first focused on fabricating a novel osteoconductive scaffold utilizing bismuth-nanohydroxyapatite/reduced graphene oxide (Bi-nHAp/rGO) nanocomposite and electrospun PCL. The osteoconductive ability of the scaffold was investigated by evaluating the alkaline phosphatase (ALP) activity and the osteogenic genes expression in the adipose-derived MSCs. The expression of Runx2, collagen I, ALP, and osteocalcin as well as the result of ALP activity indicated the osteoconductive potential of the Bi-nHA-rGO/PCL scaffold. In the next step, a bilayer scaffold containing Bi-nHAp/rGO/PCL as an osteogenic layer and acetylated hyaluronic acid/PCL as a chondrogenic layer was prepared by the electrospinning technique and transplanted into osteochondral defects of rats. The chondrogenic and osteogenic markers corresponding to the surrounding tissues of the transplanted scaffold were surveyed 60 days later by real-time polymerase chain reaction (PCR) and immunohistochemistry methods. The results showed increased chondrogenic (Sox9 and collagen II) and osteogenic (osteocalcin and ALP) gene expression and augmented secretion of collagens II and X after transplantation. The results strongly support the efficacy of this constructed cell-free bilayer scaffold to induce osteochondral defect regeneration.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 5","pages":"Pages 452-461"},"PeriodicalIF":2.3,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142125823","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
In this study, we have demonstrated a complementary-determining region (CDR) grafting technology for the generation of rabbit scFvs with different antigen recognition and physicochemical properties. The antigen-binding affinity of the CDR-grafted anti-CRP scFv, C1R/B1R (V1), which was generated by the CDR/framework region (CDR/FR) definition based on the traditional numbering rule, was insufficient when compared to that of the original clone, C1R, suggesting that the amino acid residues outside the original CDRs might significantly contribute to antigen recognition in rabbit scFvs. We redefined new CDRs and FRs to maintain antigen-binding affinities through the extension of multiple amino acid residues for CDRH1 and CDRH2, based on the amino acid sequence alignments of rabbit scFvs isolated from phage libraries. The new version successfully maintained the antigen binding affinity. CDR-grafted scFvs possessing a common CDR sequence and different FR sequences were successfully generated based on this new CDR/FR definition, and their physicochemical properties were further investigated. The antigen-binding activities of rabbit scFvs on Maxisorp varied between the tested clones in sandwich ELISA, supporting the idea that the combination of CDR with different FRs might change the physicochemical properties of scFvs on a solid material. The CDR-grafted scFvs possessing a frame sequence of anti-CRP scFv C2R maintained the ability to bind to protein L and were successfully purified. Expression titers showed improved solubility by diminishing the amount of insoluble scFvs. Thus, the method developed in this study is promising for generating alternatives with strict antigen binding recognition and different physicochemical properties.
{"title":"Generation of rabbit single-chain variable fragments with different physicochemical and biological properties by complementary determining region-grafting technology","authors":"Ngoc Minh Nguyen , Kiichi Nakao , Ryo Kobayashi, Haruka Taniguchi, Fuki Yokoyama, Jun-ichi Horiuchi, Yoichi Kumada","doi":"10.1016/j.jbiosc.2024.07.009","DOIUrl":"10.1016/j.jbiosc.2024.07.009","url":null,"abstract":"<div><div>In this study, we have demonstrated a complementary-determining region (CDR) grafting technology for the generation of rabbit scFvs with different antigen recognition and physicochemical properties. The antigen-binding affinity of the CDR-grafted anti-CRP scFv, C1R/B1R (V1), which was generated by the CDR/framework region (CDR/FR) definition based on the traditional numbering rule, was insufficient when compared to that of the original clone, C1R, suggesting that the amino acid residues outside the original CDRs might significantly contribute to antigen recognition in rabbit scFvs. We redefined new CDRs and FRs to maintain antigen-binding affinities through the extension of multiple amino acid residues for CDRH1 and CDRH2, based on the amino acid sequence alignments of rabbit scFvs isolated from phage libraries. The new version successfully maintained the antigen binding affinity. CDR-grafted scFvs possessing a common CDR sequence and different FR sequences were successfully generated based on this new CDR/FR definition, and their physicochemical properties were further investigated. The antigen-binding activities of rabbit scFvs on Maxisorp varied between the tested clones in sandwich ELISA, supporting the idea that the combination of <span>CDR</span> with different FRs might change the physicochemical properties of scFvs on a solid material. The CDR-grafted scFvs possessing a frame sequence of anti-CRP scFv C2R maintained the ability to bind to protein L and were successfully purified. Expression titers showed improved solubility by diminishing the amount of insoluble scFvs. Thus, the method developed in this study is promising for generating alternatives with strict antigen binding recognition and different physicochemical properties.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 5","pages":"Pages 439-444"},"PeriodicalIF":2.3,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142093197","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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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