Pub Date : 2025-06-18DOI: 10.1016/j.enzmictec.2025.110696
Dam-Seul Ko , Hyun-Mo Jeong , Yu-Jeong Shin, Da-Woon Jeong, Na-Ri Kim, Jae-Hoon Shim
In this study, three genes encoding novel Glycoside Hydrolase (GH) 97 enzymes were cloned from Bacteroides thetaiotaomicron and expressed in Escherichia coli. The recombinant enzymes (Bt_4581, Bt_0683, Bt_3163) were purified using Ni-NTA affinity chromatography and subsequently characterized. All three enzymes released glucose from the non-reducing ends of oligosaccharides and displayed metal ion dependency. Among them, Bt_4581 hydrolyzed a wide range of α-glycosidic linkages, while Bt_0683 and Bt_3163 showed narrower substrate specificity. Amino acid sequence analysis indicated that Bt_4581 and Bt_0683 belong to Group 1, whereas Bt_3163 is part of Group 3. Kinetic studies revealed that Bt_4581 preferred maltooligosaccharides with an odd number of glucosyl units. In contrast, Bt_3163 exhibited a preference for α-pNPG, confirming it as the first characterized α-glucosidase in Group 3 of the GH 97 family.
{"title":"Comparison of novel α-glucosidases in glycoside hydrolase family 97 isolated from Bacteroides thetaiotaomicron","authors":"Dam-Seul Ko , Hyun-Mo Jeong , Yu-Jeong Shin, Da-Woon Jeong, Na-Ri Kim, Jae-Hoon Shim","doi":"10.1016/j.enzmictec.2025.110696","DOIUrl":"10.1016/j.enzmictec.2025.110696","url":null,"abstract":"<div><div>In this study, three genes encoding novel Glycoside Hydrolase (GH) 97 enzymes were cloned from <em>Bacteroides thetaiotaomicron</em> and expressed in <em>Escherichia coli</em>. The recombinant enzymes (Bt_4581, Bt_0683, Bt_3163) were purified using Ni-NTA affinity chromatography and subsequently characterized. All three enzymes released glucose from the non-reducing ends of oligosaccharides and displayed metal ion dependency. Among them, Bt_4581 hydrolyzed a wide range of α-glycosidic linkages, while Bt_0683 and Bt_3163 showed narrower substrate specificity. Amino acid sequence analysis indicated that Bt_4581 and Bt_0683 belong to Group 1, whereas Bt_3163 is part of Group 3. Kinetic studies revealed that Bt_4581 preferred maltooligosaccharides with an odd number of glucosyl units. In contrast, Bt_3163 exhibited a preference for α-<em>p</em>NPG, confirming it as the first characterized α-glucosidase in Group 3 of the GH 97 family.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110696"},"PeriodicalIF":3.4,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144522232","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-18DOI: 10.1016/j.enzmictec.2025.110695
Jong-Hoon Kim , Hwa Lee , Kwang-Hee Son , Tae-Sook Jeong , Ho-Yong Park
Arazyme, an enzyme derived from Serratia proteamaculans, has demonstrated efficacy in enhancing skin barrier function in studies involving skin cell treatments and topical application on animal skin. The objective of this study was to assess the anti-wrinkle and anti-aging effects of Arazyme in skin keratinocytes and fibroblasts subjected to ultraviolet B (UVB) radiation and oxidative stress. Keratinocytes (HaCaT cells) and fibroblasts (CCD-986sk) were exposed to UVB (15 mJ/cm²) radiation or oxidative stress induced by 2 mM 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), followed by treatment with Arazyme (0.1–0.5 μM) for 24 h. The effects of Arazyme were compared to those of individual treatments with papain, trypsin, or retinol, which served as reference compounds. Key parameters examined included the expression of matrix metalloproteinases (MMP-1, MMP-3, and MMP-13), collagen synthesis, and cellular senescence markers (LMNB1, p16, p21, and p53). Additionally, the impact of Arazyme on cellular signaling pathways, including ERK, JNK, and NF-κB, was assessed. Arazyme significantly suppressed UVB-induced expression of MMP-1, MMP-3, and MMP-13 in a dose-dependent manner in HaCaT cells compared to other treatments. In UVB-exposed fibroblasts, Arazyme reduced both mRNA and protein levels of MMPs, while also enhancing procollagen concentration and collagen gene expression. Furthermore, Arazyme inhibited the activation of ERK, JNK, and NF-κB signaling pathways in keratinocytes. In AAPH-stimulated HaCaT cells, Arazyme significantly attenuated the expression of senescence-related markers, including LMNB1, p16, p21, and p53, and decreased the proportion of senescence-positive cells in fibroblasts. Our in vitro findings suggest that Arazyme may help attenuate UVB- and oxidative stress-induced markers of skin aging, indicating its potential as a candidate for further investigation in anti-aging skincare research.
{"title":"Arazyme prevents skin aging through regulation of matrix metalloproteinase and collagen synthesis","authors":"Jong-Hoon Kim , Hwa Lee , Kwang-Hee Son , Tae-Sook Jeong , Ho-Yong Park","doi":"10.1016/j.enzmictec.2025.110695","DOIUrl":"10.1016/j.enzmictec.2025.110695","url":null,"abstract":"<div><div>Arazyme, an enzyme derived from <em>Serratia proteamaculans</em>, has demonstrated efficacy in enhancing skin barrier function in studies involving skin cell treatments and topical application on animal skin. The objective of this study was to assess the anti-wrinkle and anti-aging effects of Arazyme in skin keratinocytes and fibroblasts subjected to ultraviolet B (UVB) radiation and oxidative stress. Keratinocytes (HaCaT cells) and fibroblasts (CCD-986sk) were exposed to UVB (15 mJ/cm²) radiation or oxidative stress induced by 2 mM 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), followed by treatment with Arazyme (0.1–0.5 μM) for 24 h. The effects of Arazyme were compared to those of individual treatments with papain, trypsin, or retinol, which served as reference compounds. Key parameters examined included the expression of matrix metalloproteinases (<em>MMP-1, MMP-3, and MMP-13</em>), collagen synthesis, and cellular senescence markers (<em>LMNB1, p16, p21</em>, and <em>p53</em>). Additionally, the impact of Arazyme on cellular signaling pathways, including ERK, JNK, and NF-κB, was assessed. Arazyme significantly suppressed UVB-induced expression of MMP-1, MMP-3, and MMP-13 in a dose-dependent manner in HaCaT cells compared to other treatments. In UVB-exposed fibroblasts, Arazyme reduced both mRNA and protein levels of MMPs, while also enhancing procollagen concentration and collagen gene expression. Furthermore, Arazyme inhibited the activation of ERK, JNK, and NF-κB signaling pathways in keratinocytes. In AAPH-stimulated HaCaT cells, Arazyme significantly attenuated the expression of senescence-related markers, including <em>LMNB1, p16, p21</em>, and <em>p53</em>, and decreased the proportion of senescence-positive cells in fibroblasts. Our in vitro findings suggest that Arazyme may help attenuate UVB- and oxidative stress-induced markers of skin aging, indicating its potential as a candidate for further investigation in anti-aging skincare research.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110695"},"PeriodicalIF":3.4,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144321341","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-11DOI: 10.1016/j.enzmictec.2025.110692
Zijuan Tao , Yusong Zhang , Yanmei Dai , Changshun Huang , Liangli Luo , Junhao Yue , Jing'e Yue , Hanbing Shi , Zhimin Ou
Steroid drugs play a pivotal role in clinical therapeutics, with androsta-1,4-diene-3,17-dione (ADD) serving as a critical intermediate whose efficient biosynthesis relies on the catalytic activity of 3-ketosteroid-Δ1-dehydrogenase (KstD). Building upon the previously engineered KstD2ep variant, this study employed semi-rational design strategies to enhance KstD2’s high-substrate-loading adaptability. Key residues (V332, L334, G534) were systematically identified through homology modeling and molecular docking, followed by constructing a combinatorial mutant library. Through alanine scanning and iterative screening of single/multiple-site mutations, the optimal mutant KstD2ep (V332E/L334T/G534V) demonstrated a 1.1-fold enhancement in catalytic efficiency compared to the KstD2ep. Molecular dynamics simulations confirmed significantly enhanced structural stability in the mutant. Whole-cell catalytic optimization revealed expanded operational tolerance to temperature fluctuations, pH variations, and co-solvent exposure. Implementing high-density fermentation coupled with fed-batch substrate supplementation, the process achieved a 1.5-fold increase in substrate conversion efficiency, yielding 117.75 g/L ADD. These advancements position the engineered variant as a high-potential candidate for scalable steroid biotransformation, addressing key barriers to enzymatic stability and process efficiency for enzyme-driven biosynthesis of steroidal pharmaceutical intermediates.
{"title":"Semi-rational engineering of 3-Ketosteroid-Δ1-dehydrogenase boosts catalytic efficiency and robustness for steroid bioconversion","authors":"Zijuan Tao , Yusong Zhang , Yanmei Dai , Changshun Huang , Liangli Luo , Junhao Yue , Jing'e Yue , Hanbing Shi , Zhimin Ou","doi":"10.1016/j.enzmictec.2025.110692","DOIUrl":"10.1016/j.enzmictec.2025.110692","url":null,"abstract":"<div><div>Steroid drugs play a pivotal role in clinical therapeutics, with androsta-1,4-diene-3,17-dione (ADD) serving as a critical intermediate whose efficient biosynthesis relies on the catalytic activity of 3-ketosteroid-Δ1-dehydrogenase (KstD). Building upon the previously engineered KstD<sub>2</sub><sup>ep</sup> variant, this study employed semi-rational design strategies to enhance KstD<sub>2</sub>’s high-substrate-loading adaptability. Key residues (V332, L334, G534) were systematically identified through homology modeling and molecular docking, followed by constructing a combinatorial mutant library. Through alanine scanning and iterative screening of single/multiple-site mutations, the optimal mutant KstD<sub>2</sub><sup>ep</sup> (V332E/L334T/G534V) demonstrated a 1.1-fold enhancement in catalytic efficiency compared to the KstD<sub>2</sub><sup>ep</sup>. Molecular dynamics simulations confirmed significantly enhanced structural stability in the mutant. Whole-cell catalytic optimization revealed expanded operational tolerance to temperature fluctuations, pH variations, and co-solvent exposure. Implementing high-density fermentation coupled with fed-batch substrate supplementation, the process achieved a 1.5-fold increase in substrate conversion efficiency, yielding 117.75 g/L ADD. These advancements position the engineered variant as a high-potential candidate for scalable steroid biotransformation, addressing key barriers to enzymatic stability and process efficiency for enzyme-driven biosynthesis of steroidal pharmaceutical intermediates.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110692"},"PeriodicalIF":3.4,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144280806","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-11DOI: 10.1016/j.enzmictec.2025.110694
Fuminori Kato
Fluorescent proteins (FPs) are widely used as molecular imaging tools for visualizing protein localization, gene expression, and bacterial labeling. However, most commercially available FPs are optimized for expression in human cells or Escherichia coli, and often exhibit poor expression in other bacterial species due to differences in codon usage preference, which critically affects translation efficiency. Staphylococcus aureus, a clinically important Gram-positive bacterium, presents additional challenges for heterologous protein expression due to codon usage bias and gene regulatory mechanisms. While our previous study has developed expression vectors that enable robust expression of GFP and its color variants in S. aureus, expression of red fluorescent proteins (RFPs) remain limited. In this study, I improved the expression of red fluorescent proteins (RFPs)—mCherry, mOrange2, E2-Crimson—and the photoconvertible protein Dendra2 in S. aureus. Codon optimization was performed based on S. aureus codon usage preferences, and RNA secondary structures at the 5′ region were minimized to enhance translation efficiency. The fully optimized mCherry(Sa2) exhibited strong fluorescence at both colony and single-cell levels. Similarly, mOrange2(Sa), E2-Crimson(Sa), and Dendra2(Sa) showed robust expression following host-adapted codon design, and Dendra2(Sa) retained its photoconvertible functionality. These results demonstrate that codon usage adjustment and RNA structure optimization are effective strategies for achieving high-level expression of diverse fluorescent proteins in S. aureus and provide valuable insights into optimizing heterologous protein expression in non-model bacterial systems.
{"title":"Expanding fluorescence imaging tools in Staphylococcus aureus: Optimized expression of red fluorescent proteins","authors":"Fuminori Kato","doi":"10.1016/j.enzmictec.2025.110694","DOIUrl":"10.1016/j.enzmictec.2025.110694","url":null,"abstract":"<div><div>Fluorescent proteins (FPs) are widely used as molecular imaging tools for visualizing protein localization, gene expression, and bacterial labeling. However, most commercially available FPs are optimized for expression in human cells or <em>Escherichia coli</em>, and often exhibit poor expression in other bacterial species due to differences in codon usage preference, which critically affects translation efficiency. <em>Staphylococcus aureus</em>, a clinically important Gram-positive bacterium, presents additional challenges for heterologous protein expression due to codon usage bias and gene regulatory mechanisms. While our previous study has developed expression vectors that enable robust expression of GFP and its color variants in <em>S. aureus</em>, expression of red fluorescent proteins (RFPs) remain limited. In this study, I improved the expression of red fluorescent proteins (RFPs)—mCherry, mOrange2, E2-Crimson—and the photoconvertible protein Dendra2 in <em>S. aureus</em>. Codon optimization was performed based on <em>S. aureus</em> codon usage preferences, and RNA secondary structures at the 5′ region were minimized to enhance translation efficiency. The fully optimized mCherry(Sa2) exhibited strong fluorescence at both colony and single-cell levels. Similarly, mOrange2(Sa), E2-Crimson(Sa), and Dendra2(Sa) showed robust expression following host-adapted codon design, and Dendra2(Sa) retained its photoconvertible functionality. These results demonstrate that codon usage adjustment and RNA structure optimization are effective strategies for achieving high-level expression of diverse fluorescent proteins in <em>S. aureus</em> and provide valuable insights into optimizing heterologous protein expression in non-model bacterial systems.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110694"},"PeriodicalIF":3.4,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144307024","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-07DOI: 10.1016/j.enzmictec.2025.110693
Rong-Huan Song , Dan Zhu , Zhong-qiao Yang , Jian-Ling Li , Zheng-Feng Yang , Zhi-Hua Lv , Kai-Qing Xie , Li-Quan Yang , A. Zhou-Cun , Peng Sang , Yi-Rui Yin
Xylanase is extensively employed in the food, feed, and paper sectors, with those derived from extreme environments offering distinct advantages. This study identified a novel xylanase gene (designated xynaes) through metagenomic analysis of samples from Aiting Lake, Xinjiang, China. Cloned and expressed in Escherichia coli after PCR amplification. The recombinant protein was purified using Ni-NTA affinity chromatography XynAES demonstrated optimal activity at pH 8.0 and 65 °C, its half-life (T1/2) was 120 min. XynAES preserved over 80 % residual activity after 12 h in pH 6.0–9.0 buffer. Its activity was enhanced to 132 % and 135 % in the presence of 1 mM Mg2+ and Zn2+, respectively. Additionally, XynAES maintained over 60 % relative activity in 0–3.0 M NaCl and its Km and Vmax of XynAES were determined to be 3.23 mg/mL and 72.46 μmol/min/mg, respectively. It is worth noting that the main products of XynAES enzymatic hydrolysis of xylan are xylose disaccharides and xylose tetrasaccharides, and XynAES shows obvious activity against the pre-treated wheat bran. In summary, XynAES is a thermophilic, alkali-tolerant, and salt-resistant xylanase, signifying its potential applications in the feed, food baking, paper manufacturing, and prebiotic production industries.
木聚糖酶广泛应用于食品、饲料和造纸行业，在极端环境中产生的木聚糖酶具有明显的优势。本研究通过对新疆艾亭湖样品的宏基因组分析，鉴定出一个新的木聚糖酶基因（称为xynaes）。经PCR扩增克隆并在大肠杆菌中表达。重组蛋白经Ni-NTA亲和层析纯化得到，XynAES在pH 8.0和65℃条件下活性最佳，半衰期（T1/2）为120 min。在pH 6.0-9.0的缓冲液中，xnaes在12 h后保留了80% %的残留活性。在1 mM Mg2+和Zn2+存在下，其活性分别提高到132 %和135 %。在0 ~ 3.0 M NaCl中，XynAES的相对活性保持在60% %以上，其Km和Vmax分别为3.23 mg/mL和72.46 μmol/min/mg。值得注意的是，XynAES酶解木聚糖的主要产物是木糖二糖和木糖四糖，并且XynAES对预处理后的麦麸具有明显的活性。综上所述，XynAES是一种耐热、耐碱、耐盐的木聚糖酶，在饲料、食品烘焙、造纸和益生元生产等行业具有潜在的应用前景。
{"title":"Characterization of a GH10 family thermophilic, alkali- and salt-tolerant xylanase from Xinjiang salt lake","authors":"Rong-Huan Song , Dan Zhu , Zhong-qiao Yang , Jian-Ling Li , Zheng-Feng Yang , Zhi-Hua Lv , Kai-Qing Xie , Li-Quan Yang , A. Zhou-Cun , Peng Sang , Yi-Rui Yin","doi":"10.1016/j.enzmictec.2025.110693","DOIUrl":"10.1016/j.enzmictec.2025.110693","url":null,"abstract":"<div><div>Xylanase is extensively employed in the food, feed, and paper sectors, with those derived from extreme environments offering distinct advantages. This study identified a novel xylanase gene (designated <em>xynaes</em>) through metagenomic analysis of samples from Aiting Lake, Xinjiang, China. Cloned and expressed in Escherichia coli after PCR amplification. The recombinant protein was purified using Ni-NTA affinity chromatography XynAES demonstrated optimal activity at pH 8.0 and 65 °C, its half-life (T<sub>1/2</sub>) was 120 min. XynAES preserved over 80 % residual activity after 12 h in pH 6.0–9.0 buffer. Its activity was enhanced to 132 % and 135 % in the presence of 1 mM Mg<sup>2</sup><sup>+</sup> and Zn<sup>2+</sup>, respectively. Additionally, XynAES maintained over 60 % relative activity in 0–3.0 M NaCl and its Km and Vmax of XynAES were determined to be 3.23 mg/mL and 72.46 μmol/min/mg, respectively. It is worth noting that the main products of XynAES enzymatic hydrolysis of xylan are xylose disaccharides and xylose tetrasaccharides, and XynAES shows obvious activity against the pre-treated wheat bran. In summary, XynAES is a thermophilic, alkali-tolerant, and salt-resistant xylanase, signifying its potential applications in the feed, food baking, paper manufacturing, and prebiotic production industries.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110693"},"PeriodicalIF":3.4,"publicationDate":"2025-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144262912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-31DOI: 10.1016/j.enzmictec.2025.110683
Weide Su , Huiying Luo , Xiaolian Chen , Gaoxiang Ai , Qipeng Wei , Zhiheng Zou , Chuanhui Xu , Jiang Chen , Pingwen Xiong , Wenjing Song , Qiongli Song
A xylanase derived from rumen ciliates designated as XynC, was successfully expressed and purified. The enzyme's catalytic properties, including its optimal pH (5.5) and temperature (40 °C), were determined, demonstrating high stability under mildly acidic and moderate thermal conditions. The substrate specificity of XynC was investigated by analyzing its hydrolysis pattern with beechwood xylan, where it preferentially degraded higher xylo-oligosaccharides (xylotetrose (X4), xylopentose (X5), xylohexose (X6)) while showing no activity on smaller oligosaccharides like xylobiose (X2) and xylotriose (X3). The in vivo effects of XynC on gut microbiota were evaluated by administering the enzyme to mice, followed by analysis of fecal 16S rRNA to assess shifts in gut bacterial populations. The results revealed dose-dependent modulation of gut microbiota composition, with a significant increase in Lactobacillus abundance observed in the medium- and high-dose groups, while no notable change was detected in the low-dose group. These dose-related microbial responses suggest that the efficacy of XynC in promoting probiotic growth is dependent on administration level. These findings suggest that XynC selectively promotes the growth of beneficial probiotics in a dosage-sensitive manner, supporting its potential as a functional feed additive for enhancing gut health and nutrient utilization in livestock.
{"title":"Influence of a rumen ciliate-derived xylanase on the gut microbiota composition: A potential enzyme for prebiotic applications","authors":"Weide Su , Huiying Luo , Xiaolian Chen , Gaoxiang Ai , Qipeng Wei , Zhiheng Zou , Chuanhui Xu , Jiang Chen , Pingwen Xiong , Wenjing Song , Qiongli Song","doi":"10.1016/j.enzmictec.2025.110683","DOIUrl":"10.1016/j.enzmictec.2025.110683","url":null,"abstract":"<div><div>A xylanase derived from rumen ciliates designated as XynC, was successfully expressed and purified. The enzyme's catalytic properties, including its optimal pH (5.5) and temperature (40 °C), were determined, demonstrating high stability under mildly acidic and moderate thermal conditions. The substrate specificity of XynC was investigated by analyzing its hydrolysis pattern with beechwood xylan, where it preferentially degraded higher xylo-oligosaccharides (xylotetrose (X4), xylopentose (X5), xylohexose (X6)) while showing no activity on smaller oligosaccharides like xylobiose (X2) and xylotriose (X3). The <em>in vivo</em> effects of XynC on gut microbiota were evaluated by administering the enzyme to mice, followed by analysis of fecal 16S rRNA to assess shifts in gut bacterial populations. The results revealed dose-dependent modulation of gut microbiota composition, with a significant increase in <em>Lactobacillus</em> abundance observed in the medium- and high-dose groups, while no notable change was detected in the low-dose group. These dose-related microbial responses suggest that the efficacy of XynC in promoting probiotic growth is dependent on administration level. These findings suggest that XynC selectively promotes the growth of beneficial probiotics in a dosage-sensitive manner, supporting its potential as a functional feed additive for enhancing gut health and nutrient utilization in livestock.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110683"},"PeriodicalIF":3.4,"publicationDate":"2025-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144204833","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-29DOI: 10.1016/j.enzmictec.2025.110671
Shitong Dong , Yuanwei Wang , Han Zhao , Yingshuai Wu , Ruiming Wang , Chunling Ma
D-allose is a rare hexose sugar with a variety of potential application in food, medicine and other fields. Ribose 5-phosphate isomerase（RPI） plays a pivotal role in the synthesis of D-allose. AtRpiB from Acetivibrio thermocellus can convert D-psicose to D-allose; however, improvements in its stability, optimal temperature, and conversion efficiency are necessary. This study aimed to investigate the catalytic properties, stability, and substrate-binding affinity of AtRpiB using D-psicose as the substrate. Using various strategies, 10 amino acid residues were selected to construct a library of 82 single mutants. Nine single mutants showed high conversion rates of D-allose. Furthermore, 27 double mutants were constructed by combining nine successful single mutation sites. Notably, the mutants R109W/R132Q, S39I/R109F, and S39V/R109F showed a 1.39-fold increase in enzyme activity at 40 ºC, 1.58-fold increase at 60 ºC, and 1.9-fold increase at 80 ºC, respectively. The S39V/R109F mutant converted 100 g/L D-psicose to 38.21 g/L D-allose after 18 hours at 40°C, the highest reported conversion rate for RpiB in D-allose production to date. Analysis of enzymatic characteristics, structure, and molecular dynamics simulations revealed that changes in the amino acid composition and conformational adjustments in loops 3, 9, and 10 of AtRpiB significantly affected the entry and exit of substrates and products into the active pocket, conversion efficiency, and enzyme stability.
d -醛糖是一种罕见的己糖，在食品、医药等领域具有多种潜在的应用前景。核糖5-磷酸异构酶（RPI）在D-allose的合成中起关键作用。来自热细胞活动弧菌的AtRpiB可以将D-psicose转化为D-allose；然而，它的稳定性，最佳温度和转换效率的改进是必要的。本研究旨在以D-psicose为底物考察AtRpiB的催化性能、稳定性和底物结合亲和力。采用不同的策略，选取10个氨基酸残基构建了82个单突变体文库。9个单突变体表现出较高的D-allose转化率。通过9个成功的单突变位点组合，构建了27个双突变体。值得注意的是，突变体R109W/R132Q、S39I/R109F和S39V/R109F在40℃、60℃和80℃下的酶活性分别提高了1.39倍、1.58倍和1.9倍。在40°C下，S39V/R109F突变体在18 小时后将100 g/L D-psicose转化为38.21 g/L D-allose，这是迄今为止报道的RpiB在D-allose生产中的最高转化率。酶学特性、结构分析和分子动力学模拟表明，AtRpiB的氨基酸组成和3、9、10环构象调整的变化显著影响底物和产物进入活性口袋的进出、转化效率和酶的稳定性。
{"title":"Mutational and structural analysis of ribose 5-phosphate isomerase B from Acetivibrio thermocellus: relationship between transformation efficiency and substrate binding pocket conformation","authors":"Shitong Dong , Yuanwei Wang , Han Zhao , Yingshuai Wu , Ruiming Wang , Chunling Ma","doi":"10.1016/j.enzmictec.2025.110671","DOIUrl":"10.1016/j.enzmictec.2025.110671","url":null,"abstract":"<div><div>D-allose is a rare hexose sugar with a variety of potential application in food, medicine and other fields. Ribose 5-phosphate isomerase（RPI） plays a pivotal role in the synthesis of D-allose. AtRpiB from <em>Acetivibrio thermocellus</em> can convert D-psicose to D-allose; however, improvements in its stability, optimal temperature, and conversion efficiency are necessary. This study aimed to investigate the catalytic properties, stability, and substrate-binding affinity of AtRpiB using D-psicose as the substrate. Using various strategies, 10 amino acid residues were selected to construct a library of 82 single mutants. Nine single mutants showed high conversion rates of D-allose. Furthermore, 27 double mutants were constructed by combining nine successful single mutation sites. Notably, the mutants R109W/R132Q, S39I/R109F, and S39V/R109F showed a 1.39-fold increase in enzyme activity at 40 ºC, 1.58-fold increase at 60 ºC, and 1.9-fold increase at 80 ºC, respectively. The S39V/R109F mutant converted 100 g/L D-psicose to 38.21 g/L D-allose after 18 hours at 40°C, the highest reported conversion rate for RpiB in D-allose production to date. Analysis of enzymatic characteristics, structure, and molecular dynamics simulations revealed that changes in the amino acid composition and conformational adjustments in loops 3, 9, and 10 of AtRpiB significantly affected the entry and exit of substrates and products into the active pocket, conversion efficiency, and enzyme stability.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110671"},"PeriodicalIF":3.4,"publicationDate":"2025-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144289008","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-27DOI: 10.1016/j.enzmictec.2025.110682
Sineenat Sripattanakul , Piyasiri Chueakwon , Le Thi Thuy Trinh , Rung-Yi Lai
Nicotinic acid, a bioactive form of niacin (vitamin B3), is an essential nutrient involved in various metabolic pathways. Its deficiency can lead to severe health issues, emphasizing the need for effective production methods. Traditional chemical methods for synthesizing nicotinic acid often require harsh reaction conditions and produce environmentally hazardous byproducts. This has prompted increasing interest in more sustainable biocatalytic alternatives. In this study, we investigated the bioconversion of 3-methylpyridine (3-MP) to nicotinic acid using resting cells of recombinant Escherichia coli expressing enzymes from Pseudomonas putida mt-2. Through systematic optimization, E. coli MG1655 RARE cells co-overexpressing xylene monooxygenase (XMO: XylM and its reductase XylA) and benzyl alcohol dehydrogenase (XylB) achieved efficient biotransformation of 9 mM 3-MP to 8.28 ± 0.35 mM nicotinic acid within 12 hours, corresponding to a yield of 92.0 % ± 3.9 %. This work presents a streamlined biocatalytic route for nicotinic acid synthesis and offers a foundation for further metabolic engineering of microbial production systems.
{"title":"Nicotinic acid production from 3-methylpyridine by E. coli whole-cell biocatalyst","authors":"Sineenat Sripattanakul , Piyasiri Chueakwon , Le Thi Thuy Trinh , Rung-Yi Lai","doi":"10.1016/j.enzmictec.2025.110682","DOIUrl":"10.1016/j.enzmictec.2025.110682","url":null,"abstract":"<div><div>Nicotinic acid, a bioactive form of niacin (vitamin B3), is an essential nutrient involved in various metabolic pathways. Its deficiency can lead to severe health issues, emphasizing the need for effective production methods. Traditional chemical methods for synthesizing nicotinic acid often require harsh reaction conditions and produce environmentally hazardous byproducts. This has prompted increasing interest in more sustainable biocatalytic alternatives. In this study, we investigated the bioconversion of 3-methylpyridine (3-MP) to nicotinic acid using resting cells of recombinant <em>Escherichia coli</em> expressing enzymes from <em>Pseudomonas putida</em> mt-2. Through systematic optimization, <em>E. coli</em> MG1655 RARE cells co-overexpressing xylene monooxygenase (XMO: XylM and its reductase XylA) and benzyl alcohol dehydrogenase (XylB) achieved efficient biotransformation of 9 mM 3-MP to 8.28 ± 0.35 mM nicotinic acid within 12 hours, corresponding to a yield of 92.0 % ± 3.9 %. This work presents a streamlined biocatalytic route for nicotinic acid synthesis and offers a foundation for further metabolic engineering of microbial production systems.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110682"},"PeriodicalIF":3.4,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144195821","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-26DOI: 10.1016/j.enzmictec.2025.110681
Pragya Gupta, Prakash C. Sahoo, Srikanth Sandipam, Manoj Kumar, Ravi Prakash Gupta, Rajesh Badhe, Umish Srivastva, Alok Sharma
This study investigates a bio-inorganic hybrid of selenium nanoparticles (SeNP@C. tropicalis) with an in-house developed mutant of Candida tropicalis (Castellani) Berkhout for enhanced bioconversion of crude dodecane (C₁₂H₂₆) into di-carboxylic acids (DCAs) under challenging conditions. The presence of SeNP@C. tropicalis improves NAD+ /NADH regeneration by enhancing enzymatic activity, thereby optimizing the metabolic transformation process. The activation of low-reactivity alkanes to DCAs, facilitated by cytochrome P450 monooxygenase, NADPH, and alcohol dehydrogenase (ADH), presents a sustainable approach for biorefineries when paired with effective downstream processing. At a 2 L scale, the SeNP@C. tropicalis hybrid achieved a 40.87 % increase in DCA production compared to the control. High-resolution transmission electron microscopy (HRTEM) confirmed the deposition of well-defined, spherical SeNP@C. tropicalis on the yeast surface, and X-ray diffraction (XRD) spectra validated the crystallinity of the nanoparticles, along with Confocal imaging. For downstream processing, four methods were evaluated, with vacuum distillation followed by crystallization achieving the highest DCA purity (>75 %) and recovery (>84 %). This research highlights the potential of SeNP@C. tropicalis as an effective catalyst for DCA production, offering new opportunities for advancing bioconversion technologies and enhancing the sustainability of biorefineries.
{"title":"Leveraging Se-nanoparticle@Candida tropicalis for efficient di-carboxylic acid production and methodological advances in selective product separation","authors":"Pragya Gupta, Prakash C. Sahoo, Srikanth Sandipam, Manoj Kumar, Ravi Prakash Gupta, Rajesh Badhe, Umish Srivastva, Alok Sharma","doi":"10.1016/j.enzmictec.2025.110681","DOIUrl":"10.1016/j.enzmictec.2025.110681","url":null,"abstract":"<div><div>This study investigates a bio-inorganic hybrid of selenium nanoparticles (SeNP@<em>C. tropicalis</em>) with an in-house developed mutant of <em>Candida tropicalis</em> (Castellani) Berkhout for enhanced bioconversion of crude dodecane (C₁₂H₂₆) into di-carboxylic acids (DCAs) under challenging conditions. The presence of <em>SeNP@C. tropicalis</em> improves NAD+ /NADH regeneration by enhancing enzymatic activity, thereby optimizing the metabolic transformation process. The activation of low-reactivity alkanes to DCAs, facilitated by cytochrome P450 monooxygenase, NADPH, and alcohol dehydrogenase (ADH), presents a sustainable approach for biorefineries when paired with effective downstream processing. At a 2 L scale, the SeNP@<em>C. tropicalis</em> hybrid achieved a 40.87 % increase in DCA production compared to the control. High-resolution transmission electron microscopy (HRTEM) confirmed the deposition of well-defined, spherical SeNP@<em>C. tropicalis</em> on the yeast surface, and X-ray diffraction (XRD) spectra validated the crystallinity of the nanoparticles, along with Confocal imaging. For downstream processing, four methods were evaluated, with vacuum distillation followed by crystallization achieving the highest DCA purity (>75 %) and recovery (>84 %). This research highlights the potential of SeNP@<em>C. tropicalis</em> as an effective catalyst for DCA production, offering new opportunities for advancing bioconversion technologies and enhancing the sustainability of biorefineries.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"189 ","pages":"Article 110681"},"PeriodicalIF":3.4,"publicationDate":"2025-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144154503","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-24DOI: 10.1016/j.enzmictec.2025.110677
Xin Kang , JiaQi Xu , ZiYuan Wang , Shenluan Yu , Long Qin , Baiqing Zhang , Shuguang Zhou , LiRong Yang
L-glufosinate (L-PPT) is a chiral herbicide with high herbicidal activity, environmental friendliness and significant market potential. The kinetic resolution of commercially available DL-PPT into optically pure L-PPT through four-enzyme one-pot biocatalysis presents a promising approach. D-amino acid oxidase (DAAO) catalyzes the conversion of D-PPT to 2-oxo-4-[(hydroxy)(methyl)phosphinyl] butyric acid (PPO), playing a critical role in the synthesis of L-PPT. However, the low solubility and enzyme activity of the wild-type DAAO limit its industrial applicability. In this study, we identified NcDAAO from Neurospora crassa OR74A, which exhibits high specific enzyme activity. First, the solubility of NcDAAO was significantly improved by combining N-terminal fusion tags with protein sequence truncation strategies, resulting in a 14.85-fold increase in enzyme activity. Subsequently, the modified variant was subjected to AlphaFold2 modeling, molecular docking and high-throughput screening. The V117N/Q325S variant demonstrated enhanced catalytic activity toward the substrate D-PPT. Finally, a one-pot biocatalysis was developed for the conversion of D-PPT to L-PPT, with the reaction completing within 3 hours and achieving an enantiomeric excess (ee) of > 99 %, highlighting the excellent catalytic performance of this variant in L-PPT synthesis.
{"title":"Soluble and pocket engineering of D-amino acid oxidase and its application in the biotransformation of L-glufosinate","authors":"Xin Kang , JiaQi Xu , ZiYuan Wang , Shenluan Yu , Long Qin , Baiqing Zhang , Shuguang Zhou , LiRong Yang","doi":"10.1016/j.enzmictec.2025.110677","DOIUrl":"10.1016/j.enzmictec.2025.110677","url":null,"abstract":"<div><div>L-glufosinate (L-PPT) is a chiral herbicide with high herbicidal activity, environmental friendliness and significant market potential. The kinetic resolution of commercially available DL-PPT into optically pure L-PPT through four-enzyme one-pot biocatalysis presents a promising approach. D-amino acid oxidase (DAAO) catalyzes the conversion of D-PPT to 2-oxo-4-[(hydroxy)(methyl)phosphinyl] butyric acid (PPO), playing a critical role in the synthesis of L-PPT. However, the low solubility and enzyme activity of the wild-type DAAO limit its industrial applicability. In this study, we identified NcDAAO from <em>Neurospora crassa</em> OR74A, which exhibits high specific enzyme activity. First, the solubility of NcDAAO was significantly improved by combining N-terminal fusion tags with protein sequence truncation strategies, resulting in a 14.85-fold increase in enzyme activity. Subsequently, the modified variant was subjected to AlphaFold2 modeling, molecular docking and high-throughput screening. The V117N/Q325S variant demonstrated enhanced catalytic activity toward the substrate D-PPT. Finally, a one-pot biocatalysis was developed for the conversion of D-PPT to L-PPT, with the reaction completing within 3 hours and achieving an enantiomeric excess (<em>ee</em>) of > 99 %, highlighting the excellent catalytic performance of this variant in L-PPT synthesis.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110677"},"PeriodicalIF":3.4,"publicationDate":"2025-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144204829","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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