Pub Date : 2025-10-01Epub 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":"2025-10-01","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 : 2025-10-01Epub Date: 2025-07-02DOI: 10.1016/j.enzmictec.2025.110706
Lu Wang , Xiao He , Tian Tian , Jian Cheng , Ruolan Cao , Jie Hou , Hui Lin , Yonghao Li
The nuclear-localized β-glucosidase CEL3C in Trichoderma reesei plays a pivotal role in cellulase regulation, though its mechanism remains poorly understood. To address this, we disrupted CEL3C in the hypercellulolytic strain T. reesei Rut C30 via CRISPR-Cas9 and evaluated cellulase production under sophorose-rich MGD induction. Deletion of CEL3C significantly enhanced total cellulase activity by 31.28 % (p < 0.05), with β-glucosidase, endoglucanase, and cellobiohydrolase activities increasing by 94.97 %, 19.40 %, and 28.99 %, respectively. These improvements were driven by transcriptional upregulation of core cellulase genes (CEL7A: 2.01-fold; CEL6A: 1.5-fold; CEL12A: 2.0-fold; CEL5A: 1.32-fold) and β-glucosidases (CEL3A: 6.41-fold; CEL3B: 5.02-fold), confirming transcriptional-level control as the dominant regulatory mechanism. Transcriptomic profiling identified 688 differentially expressed genes (399 upregulated, 299 downregulated), with key changes including activation of transcriptional activators XYR1 (59.6 % increase), ACE3 (75.49 % increase), and RXE1 (161.56 % increase), suppression of repressors RCE1 (65.86 % decrease) and RCE2 (65.23 % decrease), and induction of sugar transporters (TrireC30_133589: 13.41-fold) and ER chaperones (BIP1: 1.26-fold; PDI1: 1.55-fold). These alterations collectively enhanced inducer uptake, enzyme maturation, and secretion while alleviating MAPK-mediated repression (TMK2: 110.54 % decrease). Intracellular sugar profiling revealed that gentiobiose and cellobiose were undetectable in the T. reesei ΔCEL3C, whereas glucose and sophorose levels increased by 31.71 % and 13.45 % (p < 0.05), respectively. These results suggest that CEL3C deletion enhances β-glucosidase-mediated hydrolysis of disaccharides into glucose and possibly promotes sophorose formation via transglycosylation. In parallel, the upregulation of disaccharide transporters may facilitate sophorose uptake. Together, these two mechanisms contributed to the intracellular enrichment of sophorose, thereby amplifying cellulase gene induction and enzyme production. Our findings establish CEL3C as a dual-function nuclear regulator that balances cellulase synthesis through transcriptional and enzymatic pathways, providing actionable targets for engineering T. reesei with optimized industrial cellulase yields.
{"title":"Functional characterization of CEL3C reveals its critical role in regulating cellulase gene expression in Trichoderma reesei Rut C30","authors":"Lu Wang , Xiao He , Tian Tian , Jian Cheng , Ruolan Cao , Jie Hou , Hui Lin , Yonghao Li","doi":"10.1016/j.enzmictec.2025.110706","DOIUrl":"10.1016/j.enzmictec.2025.110706","url":null,"abstract":"<div><div>The nuclear-localized β-glucosidase CEL3C in <em>Trichoderma reesei</em> plays a pivotal role in cellulase regulation, though its mechanism remains poorly understood. To address this, we disrupted CEL3C in the hypercellulolytic strain <em>T. reesei</em> Rut C30 via CRISPR-Cas9 and evaluated cellulase production under sophorose-rich MGD induction. Deletion of CEL3C significantly enhanced total cellulase activity by 31.28 % (<em>p</em> < 0.05), with β-glucosidase, endoglucanase, and cellobiohydrolase activities increasing by 94.97 %, 19.40 %, and 28.99 %, respectively. These improvements were driven by transcriptional upregulation of core cellulase genes (CEL7A: 2.01-fold; CEL6A: 1.5-fold; CEL12A: 2.0-fold; CEL5A: 1.32-fold) and β-glucosidases (CEL3A: 6.41-fold; CEL3B: 5.02-fold), confirming transcriptional-level control as the dominant regulatory mechanism. Transcriptomic profiling identified 688 differentially expressed genes (399 upregulated, 299 downregulated), with key changes including activation of transcriptional activators XYR1 (59.6 % increase), ACE3 (75.49 % increase), and RXE1 (161.56 % increase), suppression of repressors RCE1 (65.86 % decrease) and RCE2 (65.23 % decrease), and induction of sugar transporters (TrireC30_133589: 13.41-fold) and ER chaperones (BIP1: 1.26-fold; PDI1: 1.55-fold). These alterations collectively enhanced inducer uptake, enzyme maturation, and secretion while alleviating MAPK-mediated repression (TMK2: 110.54 % decrease). Intracellular sugar profiling revealed that gentiobiose and cellobiose were undetectable in the <em>T. reesei</em> ΔCEL3C, whereas glucose and sophorose levels increased by 31.71 % and 13.45 % (<em>p</em> < 0.05), respectively. These results suggest that CEL3C deletion enhances β-glucosidase-mediated hydrolysis of disaccharides into glucose and possibly promotes sophorose formation via transglycosylation. In parallel, the upregulation of disaccharide transporters may facilitate sophorose uptake. Together, these two mechanisms contributed to the intracellular enrichment of sophorose, thereby amplifying cellulase gene induction and enzyme production. Our findings establish CEL3C as a dual-function nuclear regulator that balances cellulase synthesis through transcriptional and enzymatic pathways, providing actionable targets for engineering <em>T. reesei</em> with optimized industrial cellulase yields.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110706"},"PeriodicalIF":3.4,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144549872","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-01Epub 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":"2025-10-01","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}
d-Amino acid oxidase from the thermophilic fungus Rasamsonia emersonii (ReDAAO) has garnered attention due to its high stability and broad substrate specificity, making it a promising candidate for various applications. In this study, we explored the structural factors underlying the unique substrate specificity of ReDAAO, particularly its broad substrate range and d-Glu oxidation ability. Comparing ReDAAO with TdDAAO—a homologous d-amino acid oxidase from the thermophilic fungus Thermomyces dupontii—revealed that ReDAAO lacks the YVLQG loop present in TdDAAO, which exhibited narrower substrate specificity. Inserting the YVLQG loop into ReDAAO narrowed its substrate specificity to match TdDAAO, while deleting the sequence from TdDAAO broadened its substrate specificity, resembling ReDAAO. A TdDAAO structural model suggests that the YVLQG loop could interact with a spatially adjacent region covering the active site, distinct from the canonical active-site lid in DAAOs, creating steric hindrance that limits access to the catalytic pocket. Additionally, the unexpected activity of ReDAAO toward d-Glu appears to depend on Arg97 and Ser231, which could interact with d-Glu side chain. Alanine substitutions at these residues significantly reduced d-Glu activity, revealing that Arg97 is essential for catalytic turnover while Ser231 is critical for substrate binding. Together, these results suggest that the YVLQG loop together with the spatially adjacent region acts as a steric gate that modulates access to the catalytic pocket, and Arg97/Ser231 plays an important role in d-Glu. These findings deepen our understanding of the structure–function relationship of DAAO and provide a foundation for developing improved DAAO variants for industrial applications.
{"title":"Structural determinants of unique substrate specificity of d-amino acid oxidase of the thermophilic fungus Rasamsonia emersonii","authors":"Yuya Shimekake, Takehiro Furuichi, Daiki Imanishi, Shouji Takahashi","doi":"10.1016/j.enzmictec.2025.110705","DOIUrl":"10.1016/j.enzmictec.2025.110705","url":null,"abstract":"<div><div><span>d</span>-Amino acid oxidase from the thermophilic fungus <em>Rasamsonia emersonii</em> (<em>Re</em>DAAO) has garnered attention due to its high stability and broad substrate specificity, making it a promising candidate for various applications. In this study, we explored the structural factors underlying the unique substrate specificity of <em>Re</em>DAAO, particularly its broad substrate range and <span>d</span>-Glu oxidation ability. Comparing <em>Re</em>DAAO with <em>Td</em>DAAO—a homologous <span>d</span>-amino acid oxidase from the thermophilic fungus <em>Thermomyces dupontii</em>—revealed that <em>Re</em>DAAO lacks the YVLQG loop present in <em>Td</em>DAAO, which exhibited narrower substrate specificity. Inserting the YVLQG loop into <em>Re</em>DAAO narrowed its substrate specificity to match <em>Td</em>DAAO, while deleting the sequence from <em>Td</em>DAAO broadened its substrate specificity, resembling <em>Re</em>DAAO. A <em>Td</em>DAAO structural model suggests that the YVLQG loop could interact with a spatially adjacent region covering the active site, distinct from the canonical active-site lid in DAAOs, creating steric hindrance that limits access to the catalytic pocket. Additionally, the unexpected activity of <em>Re</em>DAAO toward <span>d</span>-Glu appears to depend on Arg97 and Ser231, which could interact with <span>d</span>-Glu side chain. Alanine substitutions at these residues significantly reduced <span>d</span>-Glu activity, revealing that Arg97 is essential for catalytic turnover while Ser231 is critical for substrate binding. Together, these results suggest that the YVLQG loop together with the spatially adjacent region acts as a steric gate that modulates access to the catalytic pocket, and Arg97/Ser231 plays an important role in <span>d</span>-Glu. These findings deepen our understanding of the structure–function relationship of DAAO and provide a foundation for developing improved DAAO variants for industrial applications.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110705"},"PeriodicalIF":3.4,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144557435","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-01Epub Date: 2025-07-10DOI: 10.1016/j.enzmictec.2025.110711
Ahmed A. Allam , Hassan A. Rudayni , Noha A. Ahmed , Faris F. Aba Alkhayl , Al Mokhtar Lamsabhi , Emadeldin M. Kamel
UDP-glucuronosyltransferases (UGTs) are essential enzymes in the phase II metabolism of endogenous and exogenous compounds, playing a critical role in detoxification, drug metabolism, and clearance. Their function is crucial for the pharmacokinetics of numerous therapeutic agents, but UGT inhibition can result in altered drug metabolism, increased toxicity, or reduced efficacy. This review explores the mechanisms of UGT inhibition, its implications for drug metabolism and pharmacokinetics, and the clinical relevance of such inhibition in the context of drug-drug interactions (DDIs). We discuss the therapeutic strategies targeting UGTs, the impact of environmental and dietary factors on UGT activity, and the role of pharmacogenetics in modulating UGT function. Moreover, the review highlights the role of UGTs in xenobiotic detoxification and addresses the challenges in identifying and modulating UGT inhibition in drug development. Finally, we identify future research directions for understanding UGT inhibition and its clinical applications. By synthesizing recent advances in the field, this review provides a comprehensive overview of the dynamic role of UGTs in drug metabolism, offering insights for optimizing drug therapy and minimizing adverse interactions.
{"title":"Modulating UDP-glucuronosyltransferase activity: Mechanisms, clinical implications, therapeutic strategies, and future directions in drug development","authors":"Ahmed A. Allam , Hassan A. Rudayni , Noha A. Ahmed , Faris F. Aba Alkhayl , Al Mokhtar Lamsabhi , Emadeldin M. Kamel","doi":"10.1016/j.enzmictec.2025.110711","DOIUrl":"10.1016/j.enzmictec.2025.110711","url":null,"abstract":"<div><div>UDP-glucuronosyltransferases (UGTs) are essential enzymes in the phase II metabolism of endogenous and exogenous compounds, playing a critical role in detoxification, drug metabolism, and clearance. Their function is crucial for the pharmacokinetics of numerous therapeutic agents, but UGT inhibition can result in altered drug metabolism, increased toxicity, or reduced efficacy. This review explores the mechanisms of UGT inhibition, its implications for drug metabolism and pharmacokinetics, and the clinical relevance of such inhibition in the context of drug-drug interactions (DDIs). We discuss the therapeutic strategies targeting UGTs, the impact of environmental and dietary factors on UGT activity, and the role of pharmacogenetics in modulating UGT function. Moreover, the review highlights the role of UGTs in xenobiotic detoxification and addresses the challenges in identifying and modulating UGT inhibition in drug development. Finally, we identify future research directions for understanding UGT inhibition and its clinical applications. By synthesizing recent advances in the field, this review provides a comprehensive overview of the dynamic role of UGTs in drug metabolism, offering insights for optimizing drug therapy and minimizing adverse interactions.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110711"},"PeriodicalIF":3.4,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144595702","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-01Epub 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":"2025-10-01","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 : 2025-10-01Epub Date: 2025-06-23DOI: 10.1016/j.enzmictec.2025.110700
Jing Wu , Zhenggang Han , Pengrong Li , Jing Li , Yuanyuan Chen , Shangbo Ning , Hong-jun Chao , Xue-wang Gao , Dazhong Yan
Cyclohexylamine oxidase is a member of amine oxidases that catalyzes the conversion of cyclohexylamine to cyclohexanone. In our previous work, the enzymatic activity assay of cyclohexylamine oxidase CHAOYT-02 indicated that its specific activity towards cyclohexylamine of CHAOYT-02 was ten times higher than that of its homolog CHAOIH-35A. In this study, the crystal structure of CHAOYT-02 was determined by the molecular replacement method at a resolution of 1.49 Å. The atomic structure revealed that the amino acid residues Leu302, Trp70, Phe197, Phe349, and Tyr440 constitute the active center pocket of the enzyme. Amino acid residues Ile180, Leu181, and Trp332 separate the active center pocket and an intermediate pocket. Moreover, a molecular dynamics (MD) simulation and the calculation of the binding free energy were performed to predict substrate entry and product release from cyclohexylamine oxidases. Single-amino acid substitution mutants (W70A, I180A, L181A, I208A, F197A, L302A, W332A, F349A, and Y440A) of CHAOYT-02 were constructed to investigate the role of these amino acid residues in enzymatic properties and substrate specificity. The results indicated that both the amino acid residues in the active center pocket and gating the two pockets affected the activity or substrate specificity of CHAOYT-02. This study on the structure and catalytic mechanism of cyclohexylamine oxidase is beneficial to eliminating toxic amine compounds in the environment.
{"title":"Crystal structure of cyclohexylamine oxidase from Acinetobacter sp. YT−02 reveals key residues for catalytic activity and substrate specificity","authors":"Jing Wu , Zhenggang Han , Pengrong Li , Jing Li , Yuanyuan Chen , Shangbo Ning , Hong-jun Chao , Xue-wang Gao , Dazhong Yan","doi":"10.1016/j.enzmictec.2025.110700","DOIUrl":"10.1016/j.enzmictec.2025.110700","url":null,"abstract":"<div><div>Cyclohexylamine oxidase is a member of amine oxidases that catalyzes the conversion of cyclohexylamine to cyclohexanone. In our previous work, the enzymatic activity assay of cyclohexylamine oxidase CHAO<sub>YT-02</sub> indicated that its specific activity towards cyclohexylamine of CHAO<sub>YT-02</sub> was ten times higher than that of its homolog CHAO<sub>IH-35A</sub>. In this study, the crystal structure of CHAO<sub>YT-02</sub> was determined by the molecular replacement method at a resolution of 1.49 Å. The atomic structure revealed that the amino acid residues Leu302, Trp70, Phe197, Phe349, and Tyr440 constitute the active center pocket of the enzyme. Amino acid residues Ile180, Leu181, and Trp332 separate the active center pocket and an intermediate pocket. Moreover, a molecular dynamics (MD) simulation and the calculation of the binding free energy were performed to predict substrate entry and product release from cyclohexylamine oxidases. Single-amino acid substitution mutants (W70A, I180A, L181A, I208A, F197A, L302A, W332A, F349A, and Y440A) of CHAO<sub>YT-02</sub> were constructed to investigate the role of these amino acid residues in enzymatic properties and substrate specificity. The results indicated that both the amino acid residues in the active center pocket and gating the two pockets affected the activity or substrate specificity of CHAO<sub>YT-02</sub>. This study on the structure and catalytic mechanism of cyclohexylamine oxidase is beneficial to eliminating toxic amine compounds in the environment.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110700"},"PeriodicalIF":3.4,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144549870","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-01Epub 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":"2025-10-01","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}
Xylanase and cellulase have become increasingly significant due to their versatile applications in the food, paper, biofuel, and pharmaceutical industries. Nevertheless, the current production of these enzymes relies on costly substrates, with estimates indicating that over 30 % of the production expenses are attributed to these substrates. The objective of this study is to optimize the physicochemical parameters for obtaining the maximum production of xylanase & cellulase enzyme from Pantoea sp. (PQ584882). The production conditions were statistically optimized using Plackett-Burman design (PBD) and Central Composite design (CCD). The significant variables identified through PB design including temperature, substrate-to-moisture ratio, K2HPO4, peptone, surfactant, inoculum size, inoculum age, incubation time, and pH were further optimized using the CCD approach. This optimization process revealed the most influential factors affecting xylanase & cellulase production, with optimal conditions observed at a temperature of 40◦C, Moisture Proportion, 15 mL; K2HPO4 6 mM; peptone, 1.55 %; Castor oil 0.5 %; inoculum size, 1.55 % (v/w); inoculum age, 18 h; an incubation time, 87 h. The optimized CCD model displayed a 1.84-fold greater xylanase & cellulose production than the PB design approach. These findings suggest that wheat bran, a readily available agro-waste, could be a feasible alternative to the conventional substrate, beechwood xylan and CMC (Carboxy methyl cellulose) for the production of xylanase & cellulase enzymes with the possibility of achieving higher production levels optimized by using a statistical design approach.
{"title":"Optimization of physico-chemical parameters for synergistic production of xylano-cellulolytic enzymes by novel Pantoea sp. (PQ584882) under solid-state fermentation using statistical design approach","authors":"Neha Maurya , Harsh Sable , Jyoti Chauhan , Amit Kumar , Sharad Agrawal","doi":"10.1016/j.enzmictec.2025.110697","DOIUrl":"10.1016/j.enzmictec.2025.110697","url":null,"abstract":"<div><div>Xylanase and cellulase have become increasingly significant due to their versatile applications in the food, paper, biofuel, and pharmaceutical industries. Nevertheless, the current production of these enzymes relies on costly substrates, with estimates indicating that over 30 % of the production expenses are attributed to these substrates. The objective of this study is to optimize the physicochemical parameters for obtaining the maximum production of xylanase & cellulase enzyme from <em>Pantoea sp.</em> (PQ584882). The production conditions were statistically optimized using Plackett-Burman design (PBD) and Central Composite design (CCD). The significant variables identified through PB design including temperature, substrate-to-moisture ratio, K<sub>2</sub>HPO<sub>4</sub>, peptone, surfactant, inoculum size, inoculum age, incubation time, and pH were further optimized using the CCD approach. This optimization process revealed the most influential factors affecting xylanase & cellulase production, with optimal conditions observed at a temperature of 40◦C, Moisture Proportion, 15 mL; K<sub>2</sub>HPO<sub>4</sub> 6 mM; peptone, 1.55 %; Castor oil 0.5 %; inoculum size, 1.55 % (v/w); inoculum age, 18 h; an incubation time, 87 h. The optimized CCD model displayed a 1.84-fold greater xylanase & cellulose production than the PB design approach. These findings suggest that wheat bran, a readily available agro-waste, could be a feasible alternative to the conventional substrate, beechwood xylan and CMC (Carboxy methyl cellulose) for the production of xylanase & cellulase enzymes with the possibility of achieving higher production levels optimized by using a statistical design approach<strong>.</strong></div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110697"},"PeriodicalIF":3.4,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144489382","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}
This study investigates the dynamics of microbial communities and their functional characteristics during the natural fermentation of roses. Utilizing metagenomic sequencing and volatile compound analysis, the research elucidates the succession of microbial communities and their relationship with the flavor compound production. The findings indicate that Klebsiella and Pichia are predominant in the early stages of fermentation, while Acetobacter and Cyberlindnera become more abundant in the middle and later stages. The glycosyltransferase (GT) family is identified as the primary carbohydrate-active enzyme (CAZy) family involved in fermentation, with GT1 and GT2 exhibiting a higher gene abundance. Functional genes are predominantly associated with the carbohydrate and amino acid metabolism. Analysis of volatile compounds reveals that substances such as phenethyl acetate and (S,S)-2,3-Butanediol are closely related to the structure of the microbial community. These findings contribute to a deeper understanding of the mechanisms underlying rose fermentation and offer a theoretical foundation for technological advancements in the rose product industry.
{"title":"Microbial community dynamics and functional potential during the natural fermentation of rose: A metagenomic and volatile compound analysis","authors":"Zhiyuan Yin, Kangdi Cao, Ningfei Duan, Zhiguo Zhang","doi":"10.1016/j.enzmictec.2025.110703","DOIUrl":"10.1016/j.enzmictec.2025.110703","url":null,"abstract":"<div><div>This study investigates the dynamics of microbial communities and their functional characteristics during the natural fermentation of roses. Utilizing metagenomic sequencing and volatile compound analysis, the research elucidates the succession of microbial communities and their relationship with the flavor compound production. The findings indicate that <em>Klebsiella</em> and <em>Pichia</em> are predominant in the early stages of fermentation, while <em>Acetobacter</em> and <em>Cyberlindnera</em> become more abundant in the middle and later stages. The glycosyltransferase (GT) family is identified as the primary carbohydrate-active enzyme (CAZy) family involved in fermentation, with GT1 and GT2 exhibiting a higher gene abundance. Functional genes are predominantly associated with the carbohydrate and amino acid metabolism. Analysis of volatile compounds reveals that substances such as phenethyl acetate and (S,S)-2,3-Butanediol are closely related to the structure of the microbial community. These findings contribute to a deeper understanding of the mechanisms underlying rose fermentation and offer a theoretical foundation for technological advancements in the rose product industry.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110703"},"PeriodicalIF":3.4,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144481156","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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