Enzyme and Microbial Technology最新文献_第6页

Engineering glycolytic pathway for improved Lacto-N-neotetraose production in pichia pastoris 改良毕赤酵母生产乳酸-n -新四糖的工程糖酵解途径。

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-12-25 DOI: 10.1016/j.enzmictec.2024.110576

Jiao Yang , Nitesh Kumar Mund , Lirong Yang , Hao Fang

{"title":"Engineering glycolytic pathway for improved Lacto-N-neotetraose production in pichia pastoris","authors":"Jiao Yang , Nitesh Kumar Mund , Lirong Yang , Hao Fang","doi":"10.1016/j.enzmictec.2024.110576","DOIUrl":"10.1016/j.enzmictec.2024.110576","url":null,"abstract":"<div><div>Lacto-N-neotetraose (LNnT) is a primary solid component of human milk oligosaccharides (HMOs) with various promising health effects for infants. LNnT production by GRAS (generally recognized as safe) microorganisms has attracted considerable attention. However, few studies have emphasized <em>Pichia Pastoris</em> as a cell factory for LNnT’s production. Here, we have reported the first-ever synthesis of LNnT employing <em>P. pastoris</em> as the host. Initially, LNnT biosynthetic pathway genes β-1,3-N-acetylglucosaminyltransferase (<em>lgtA</em>) and β-1,4-galactostltransferase (<em>lgtB</em>) along with lactose permease (<em>lac12</em>) and galactose epimerase (<em>gal10</em>) were integrated into the genome of <em>P. pastoris</em>, but only 0.139 g/L LNnT was obtained. Second, the titer of LNnT was improved to 0.162 g/L via up-regulating genes to strengthen the supply of precursors, UDP-GlcNAc (Uridine diphosphate N-acetylglucosamine) and UDP-Gal (Uridine diphosphate galactose), for LNnT biosynthesis. Third, by knocking out critical mediator <em>pfk</em> (6-phosphofructokinase) genes in glycolysis, the major glucose metabolic flux was rewired to the LNnT biosynthesis pathway. As a result, the strain accumulated 0.867 g/L LNnT in YPG medium supplemented with glucose and lactose. Finally, LNnT production was increased to 1.24 g/L in a 3 L bioreactor. The work aimed to explore the potential of <em>P. pastoris</em> as a for LNnT production.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110576"},"PeriodicalIF":3.4,"publicationDate":"2024-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142913974","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}

引用次数: 0

Structures and properties of α-amylase and glucoamylase immobilized by ZIF-8 via one-pot preparation 用 ZIF-8 单锅制备固定α-淀粉酶和葡萄糖淀粉酶的结构和特性。

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-12-25 DOI: 10.1016/j.enzmictec.2024.110579

Yuxin Liu, Qinghua Pan, Zesheng Liang, Jingqiao Li, Rulong Wu

{"title":"Structures and properties of α-amylase and glucoamylase immobilized by ZIF-8 via one-pot preparation","authors":"Yuxin Liu, Qinghua Pan, Zesheng Liang, Jingqiao Li, Rulong Wu","doi":"10.1016/j.enzmictec.2024.110579","DOIUrl":"10.1016/j.enzmictec.2024.110579","url":null,"abstract":"<div><div>The immobilization of α-amylase and glucoamylase using a metal-organic framework (enzyme@ZIF-8) was prepared in situ through a one-pot method. The morphology, crystal structure, and molecular characteristics of the free enzyme and enzyme@ZIF-8 were characterized. The enzyme@ZIF-8 exhibited the rhombic dodecahedron morphology, with a decrease in particle size. Successful immobilization of α-amylase and glucoamylase within ZIF-8 was confirmed, with 30–40 % loading rate. The immobilization process did not significantly alter the crystal structure of ZIF-8. The changes in secondary structure of enzyme after immobilization resulted in modiﬁcation of catalytic activity of enzyme. The melting enthalpy of enzyme @ZIF-8 increased with the increase of enzyme content. The melting peak temperature of the enzyme immobilized by ZIF-8 increased. The activity of free and immobilized enzymes was influenced by the different time, pH and temperature. At pH 5–8 and temperature 60–80 °C, the activity of the immobilized enzyme was significantly greater than that of the free enzyme. The repeatability of enzyme@ZIF-8 was 61.52 % after three cycles. The kinetic parameters of Michaelis-Menten model for enzymatic reaction were determined by fitting the initial rate of reactions and initial substrate concentration data. The Michaelis-Menten constant (<em>K</em><sub><em>M</em></sub>) values of immobilized enzyme were lower than that of free enzyme, indicating the greater affinity between the enzyme and the substrate.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110579"},"PeriodicalIF":3.4,"publicationDate":"2024-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142926836","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}

引用次数: 0

Identification of a bacteria P450 enzyme from B. megaterium H-1 with vitamin D3 C-25 hydroxylation capabilities 具有维生素D3 C-25羟基化能力的巨型芽孢杆菌H-1细菌P450酶的鉴定。

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-12-24 DOI: 10.1016/j.enzmictec.2024.110578

Yulin He , Yina Hou , Hui Li , Fan He , Jingyi Zhou , Xiaomei Zhang , Jingsong Shi , Zhenghong Xu

{"title":"Identification of a bacteria P450 enzyme from B. megaterium H-1 with vitamin D3 C-25 hydroxylation capabilities","authors":"Yulin He , Yina Hou , Hui Li , Fan He , Jingyi Zhou , Xiaomei Zhang , Jingsong Shi , Zhenghong Xu","doi":"10.1016/j.enzmictec.2024.110578","DOIUrl":"10.1016/j.enzmictec.2024.110578","url":null,"abstract":"<div><div>Calcidiol (25(OH)VD<sub>3</sub>) and calcitriol (1<em>α</em>,25(OH)<sub>2</sub>VD<sub>3</sub>) are active vitamin D<sub>3</sub> with high medicinal value, which can maintain calcium and phosphorus balance and treat vitamin D deficiency. Microbial synthesis is an important method to produce high-value-added compounds. It can produce active vitamin D<sub>3</sub> through the hydroxylation reaction of P450, which can reduce the traditional chemical synthesis steps, and greatly improve the production efficiency and economic benefits. In this work, <em>Bacillus megaterium</em> H-1 was screened for its ability to produce 25(OH)VD<sub>3</sub> and 1<em>α</em>,25(OH)<sub>2</sub>VD<sub>3</sub> from vitamin D<sub>3</sub>. A new highly inducible vitamin D<sub>3</sub> hydroxylase CYP109E1-H was identified from <em>B. megaterium</em> H-1 through searching for transcripts with cytochrome P450 structural domains, combining the transcriptome sequencing with functional expression in <em>Bacillus subtilis</em> WB600. Biotransformation in recombinant <em>B. subtilis</em> confirmed that CYP109E1-H has C-25 hydroxylase activity towards vitamin D<sub>3</sub>. CYP109E1-H is a natural mutant of CYP109E1 with greater stereoselectivity and it is a new vitamin D<sub>3</sub> mono-hydroxylase. The cloning and characterization of the CYP109E1-H gene provide useful information on the structural basis for improving the regional and stereoselectivity of the CYP109E gene.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110578"},"PeriodicalIF":3.4,"publicationDate":"2024-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142892980","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}

引用次数: 0

Acidogenic fermentation of Ulva in a fed-batch reactor system: tubular versus foliose biomass 进料批式反应器系统中Ulva的产酸发酵：管状与卵泡生物量。

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-12-24 DOI: 10.1016/j.enzmictec.2024.110580

James Lawrence , Armando Oliva , Jerry D. Murphy , Piet N.L. Lens

{"title":"Acidogenic fermentation of Ulva in a fed-batch reactor system: tubular versus foliose biomass","authors":"James Lawrence , Armando Oliva , Jerry D. Murphy , Piet N.L. Lens","doi":"10.1016/j.enzmictec.2024.110580","DOIUrl":"10.1016/j.enzmictec.2024.110580","url":null,"abstract":"<div><div>The present study proposes a biorefinery of the macroalgae <em>Ulva,</em> focusing on evaluating two different morphologies of the species (foliose and tubular) during acidogenic fermentation in fed-batch reactors. Stage 1 of the study evaluates lyophilised foliose and tubular <em>Ulva</em>, whilst Stage 2 analyses the impact of ulvan extraction on volatile fatty acids yield and changes in carbohydrate availability. Acetic, propionic, and butyric acids were produced from each substrate, with peak concentrations of total VFAs recorded at 2179.5 mg HAc/L (foliose <em>Ulva</em>) and 2029.3 mg HAc/L (tubular <em>Ulva</em>) when ulvan was present. After ulvan extraction, the acidogenic fermentation of the foliose morphotype was negatively affected, reaching at most 315.3 mg HAc/L. In contrast, the extraction showed no influence on the tubular morphotype, peaking at 2165.0 mg HAc/L. Additional variations were noted in the availability of carbohydrates in each substrate during the acidogenic fermentation process. The ulvan-extracted tubular morphotype exhibited the highest peak in carbohydrate concentration (9.8 g glucose/L), whilst the ulvan-extracted foliose morphotype yielded up to 8.5 g glucose/L. This study highlights the biorefinery potential of <em>Ulva</em> biomass<em>,</em> proposing a multiple cascading approach linking multiple energy and biomolecule applications to maximise the valorisation of the biomass.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110580"},"PeriodicalIF":3.4,"publicationDate":"2024-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142946515","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}

引用次数: 0

Tailoring of levansucrase product size by a comparative molecular dynamics approach 用比较分子动力学方法裁剪左旋蔗糖酶产物大小。

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-12-23 DOI: 10.1016/j.enzmictec.2024.110577

Zhiwei Li, Tong Bao, Kaiwen Chen, Chao Hu, Xinyu Zhang, Xueqin Hu, Jingwen Yang, Hongbin Zhang

{"title":"Tailoring of levansucrase product size by a comparative molecular dynamics approach","authors":"Zhiwei Li, Tong Bao, Kaiwen Chen, Chao Hu, Xinyu Zhang, Xueqin Hu, Jingwen Yang, Hongbin Zhang","doi":"10.1016/j.enzmictec.2024.110577","DOIUrl":"10.1016/j.enzmictec.2024.110577","url":null,"abstract":"<div><div>Levan is widely used as food additives. Its utilization is significantly influenced by its molecular weight. <em>Bacillus subtilis</em> levansucrase (Bs-SacB) and <em>Priestia megaterium</em> levansucrase (Pm-SacB) yield levan of different weights. To delve deeper into the molecular underpinnings of the molecular weight disparity between the products of these two enzymes, we conducted a focused study on the eight loops surrounding the active sites of Bs-SacB and Pm-SacB and identified Loop3 and loop4 as critical determinants in changing the molecular weight of Bs-SacB 's products. Subsequently, leveraging mutation energy analysis and non-homologous substitution strategies, we crafted tailored modifications in loop3 and loop4, yielding a spectrum of mutant enzymes that exhibit diverse molecular weight profiles including F182Y (3698 Da), CYTI (3093 Da), 3-Pbl (2776 Da), 4-Bml (1845 Da), and F182K (1571 Da). This research provide a novel comparative molecular dynamics approach to change product molecular weight and it is successfully applied in the modification of levansucrase.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110577"},"PeriodicalIF":3.4,"publicationDate":"2024-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142892981","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}

引用次数: 0

Green and sustainable production of isofraxidin from Acanthopanax senticosus with cellulose-based immobilized probiotics 纤维素基固定化益生菌绿色可持续生产刺五加异黄酮。

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-12-23 DOI: 10.1016/j.enzmictec.2024.110574

Shuang Jin , Hongyao Cai , Cailiang Peng , Yupeng Cheng , Yubin Ren , Weili Liu , Yujie Fu , Chen Lv

{"title":"Green and sustainable production of isofraxidin from Acanthopanax senticosus with cellulose-based immobilized probiotics","authors":"Shuang Jin , Hongyao Cai , Cailiang Peng , Yupeng Cheng , Yubin Ren , Weili Liu , Yujie Fu , Chen Lv","doi":"10.1016/j.enzmictec.2024.110574","DOIUrl":"10.1016/j.enzmictec.2024.110574","url":null,"abstract":"<div><div>This study utilizes deep eutectic solvent (DES)-assisted enhancement of cellulose-based immobilized probiotics for efficient biotransformation of isofraxidin from <em>Acanthopanax senticosus</em>. Among seven probiotic strains tested, <em>Lactiplantibacillus plantarum</em> CICC 20767 exhibited the best catalytic activity. We explored the effects of 12 different DESs with varying hydrogen bond donors on the conversion capacity of the immobilized probiotics, with the highest efficiency observed using ChCl/EG (4.0 wt %). The optimized process, with a solid-to-liquid ratio of 1:5 (g/mL), a temperature of 35.6 °C, a reaction time of 4 d, and a pH of 6.9, resulted in a 5.53-folds increase in isofraxidin yield, reaching 0.4034 mg/g, compared to the untreated sample (0.0729 mg/g). The immobilized probiotics retained excellent catalytic activity after 12 cycles of use, demonstrating their stability and potential for large-scale, green production of isofraxidin. This study presents a valuable method for industrial isofraxidin production and highlights the broad potential of this environmentally friendly bioconversion process in the pharmaceutical industry.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110574"},"PeriodicalIF":3.4,"publicationDate":"2024-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142892979","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}

引用次数: 0

Cloning, purification and characterization of a novel thermostable recombinant tannase from Galactobacillus timonensis timmonensis半乳杆菌重组单宁酶的克隆、纯化及特性研究。

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-12-23 DOI: 10.1016/j.enzmictec.2024.110575

Jingya Wu , Huan Zeng , Xinyan Zhong , Xi Chen , Peng Zhang , Zeyuan Deng

{"title":"Cloning, purification and characterization of a novel thermostable recombinant tannase from Galactobacillus timonensis","authors":"Jingya Wu , Huan Zeng , Xinyan Zhong , Xi Chen , Peng Zhang , Zeyuan Deng","doi":"10.1016/j.enzmictec.2024.110575","DOIUrl":"10.1016/j.enzmictec.2024.110575","url":null,"abstract":"<div><div>The exorbitant production costs associated with natural tannases pose a significant challenge to their widespread industrial utilization. Microbial expression systems provide a cost-effective method for enzyme production. In this study, a putative gene encoding the subtype B tannase (Gt-Tan) was cloned from <em>Galactobacillus timonensis</em> and expressed heterologously in <em>Escherichia coli</em> BL21 (DE3) cells. The Gt-Tan was purified using metal affinity chromatography and exhibited a monomeric structure with a molecular weight of 55 kDa. Gt-Tan showed optimal activity at a temperature of 50 ℃ and a pH of 6.0. It was also quite thermostable, with approximately 68.3 % and 54.7 % of its maximal activity retained after incubation at 45 ℃ for 2 h and 40 ℃ for 48 h respectively. Addition of Mn<sup>2+</sup>, Zn<sup>2+</sup>, Al<sup>3+</sup>, urea, n-butanol, and dimethylsulfoxide at a low concentration slightly enhanced the activity of Gt-Tan, whereas Cu<sup>2+</sup>, Fe<sup>3+</sup>, Fe<sup>2+</sup>, Co<sup>2+</sup>, SDS, cetyltrimethylammonium bromide, DTT, Tween 80, and β-mercaptoethanol significantly inhibited its activity. <em>K</em><sub><em>m</em></sub> and <em>k</em><sub><em>cat</em></sub>/<em>K</em><sub><em>m</em></sub> values were estimated to be 0.83 mM and 19.7 s<sup><img>1</sup> mM<sup><img>1</sup> for methyl gallate, 0.67 mM and 65.4 s<sup><img>1</sup> mM<sup><img>1</sup> for propyl gallate, and 0.22 mM and 240.8 s<sup><img>1</sup> mM<sup><img>1</sup> for tannic acid. These results enhanced our understanding of tannase and provided potential sources for applications in the chemical, feed, and food industries.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110575"},"PeriodicalIF":3.4,"publicationDate":"2024-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142902714","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}

引用次数: 0

Structural and functional insights into recombinant β-glucosidase from Thermothelomyces thermophilus: Cello-oligosaccharide hydrolysis and thermostability 重组β-葡萄糖苷酶的结构和功能研究：纤维寡糖水解和热稳定性。

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-12-17 DOI: 10.1016/j.enzmictec.2024.110572

Ana Luiza da Rocha Fortes Saraiva , Gabriela Leila Berto , Bianca Oliva , Paula Macedo Cunha , Lucas Ramos , Leandro Cristante de Oliveira , Fernando Segato

{"title":"Structural and functional insights into recombinant β-glucosidase from Thermothelomyces thermophilus: Cello-oligosaccharide hydrolysis and thermostability","authors":"Ana Luiza da Rocha Fortes Saraiva , Gabriela Leila Berto , Bianca Oliva , Paula Macedo Cunha , Lucas Ramos , Leandro Cristante de Oliveira , Fernando Segato","doi":"10.1016/j.enzmictec.2024.110572","DOIUrl":"10.1016/j.enzmictec.2024.110572","url":null,"abstract":"<div><div>β-glucosidases (BGLs) are key enzymes in the depolymerization of cellulosic biomass, catalyzing the conversion of cello-oligosaccharides into glucose. This conversion is pivotal for enhancing the production of second-generation ethanol or other value-added products in biorefineries. However, the process is often cost-prohibitive due to the high enzyme loadings required. Therefore, the discovery of new highly efficient BGLs represents a significant advancement. In this study, a BGL from the glycoside hydrolase family 3 (GH3) of the thermophilic fungus <em>Thermothelomyces thermophilus</em> (<em>Tth</em>Bgl3A) was heterologously expressed in <em>Aspergillus nidulans</em>. The recombinant enzyme exhibited optimal activity at pH 5.0 and 55 °C, with noteworthy stability for up to 160 h. A distinctive, extensive loop within the catalytic cavity of <em>Tth</em>Bgl3A facilitates hydrophobic interactions that enhance the binding and hydrolysis of long cello-oligosaccharides. Consequently, <em>Tth</em>Bgl3A has proven to be an efficient enzyme for the hydrolysis lignocellulosic biomass. These findings are significant for expanding the repertoire of enzymes produced by <em>T. thermophilus</em> and provide new insights into the potential application of <em>Tth</em>Bgl3A in the degradation of cellulosic materials and the production of valuable compounds.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110572"},"PeriodicalIF":3.4,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142881501","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}

引用次数: 0

Potential of a newly isolated lytic bacteriophage to control Pseudomonas coronafaciens pv. garcae in coffee plants: Molecular characterization with in vitro and ex vivo experiments 一种新分离的噬菌体在控制咖啡植物中的冠突伪单胞杆菌（Pseudomonas coronafaciens pv. garcae）方面的潜力：体外和体内实验的分子特征。

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-12-15 DOI: 10.1016/j.enzmictec.2024.110573

Luan C. Mota , Erica C. Silva , Carlos A. Quinde , Basilio Cieza , Aakash Basu , Lucas M.R. Rodrigues , Marta M.D.C. Vila , Victor M. Balcão

{"title":"Potential of a newly isolated lytic bacteriophage to control Pseudomonas coronafaciens pv. garcae in coffee plants: Molecular characterization with in vitro and ex vivo experiments","authors":"Luan C. Mota , Erica C. Silva , Carlos A. Quinde , Basilio Cieza , Aakash Basu , Lucas M.R. Rodrigues , Marta M.D.C. Vila , Victor M. Balcão","doi":"10.1016/j.enzmictec.2024.110573","DOIUrl":"10.1016/j.enzmictec.2024.110573","url":null,"abstract":"<div><div>Traditionally, control of coffee plant bacterial halo blight (BHB) caused by the phytopathogen <em>Pseudomonas coronafaciens</em> pv. <em>garcae</em> (Pcg) involves frequent spraying of coffee plantations with non-environmentally friendly and potentially bacterial resistance-promoting copper products or with kasugamycin hydrochloride. In this study we report a leap forward in the quest for a new ecofriendly approach, characterizing (both physicochemically and biologically) and testing both <em>in vitro</em> and <em>ex vivo</em> a new lytic phage for Pcg. An in-depth molecular (genomic and DNA structural features) characterization of the phage was also undertaken. Phage PcgS01F belongs to the class Caudoviricetes, <em>Drexlerviridae</em> family and genus <em>Guelphvirus</em>, and presents a siphovirus-like morphotype. Phage PcgS01F showed a latency period of 40 min and a burst size of 46 PFU/host cell, allowing to conclude that it replicates well in Pcg IBSBF-158. At Multiplicity Of Infection (MOI, or the ratio of phage to bacteria) 1000, the performance of phage PcgS01F was much better than at MOI 10, promoting increasing bacterial reductions until the end of the <em>in vitro</em> inactivation assays, stabilizing at a significant 82 % bacterial load reduction. Phage PcgS01F infected and killed Pcg cells <em>ex vivo</em> in coffee plant leaves artificially contaminated, with a maximum of Pcg inactivation of 7.66 log CFU/mL at MOI 1000 after 36 h of incubation. This study provides evidence that the isolated phage is a promising candidate against the causative agent of BHB in coffee plants.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110573"},"PeriodicalIF":3.4,"publicationDate":"2024-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142863471","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}

引用次数: 0

Improving the anti-autolytic ability of alkaline protease from Bacillus alcalophilus by a rationally combined strategy 合理组合提高嗜钙芽孢杆菌碱性蛋白酶抗自溶能力。

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-12-07 DOI: 10.1016/j.enzmictec.2024.110561

Mian Wu, Lin Cao, Wei Tang, Zhemin Liu, Su Feng

{"title":"Improving the anti-autolytic ability of alkaline protease from Bacillus alcalophilus by a rationally combined strategy","authors":"Mian Wu, Lin Cao, Wei Tang, Zhemin Liu, Su Feng","doi":"10.1016/j.enzmictec.2024.110561","DOIUrl":"10.1016/j.enzmictec.2024.110561","url":null,"abstract":"<div><div>Detergent enzymes have been extensively developed as eco-friendly alternatives to harmful chemicals, with alkaline protease representing a significant portion of detergent enzyme sales. However, the self-cleavage function of alkaline protease impacts its activity and overall application. Therefore, a new rational combinatorial strategy is proposed based on self-molecular docking (Self-ZDOCK) and molecular dynamics (MD) simulations. Self-ZDOCK is a computational method for predicting the binding mode of proteins to themselves, which is crucial for understanding the self-cleavage mechanism of proteases. On the other hand, MD simulation is a powerful tool to gain insight into the dynamic behaviour of proteins over time, and thus to analyse the structural stability and flexibility of BpAP under various conditions. Experiments verified this strategy is an effective way to improve the anti-autolytic ability of BpAP. Among the 28 mutants of BpAP, 5 mutants showed increases in thermal stability, pH stability, and storage stability in detergent, indicating a significant enhancement in their anti-autolytic capacity. Structural analysis and MD simulations confirmed that the enhanced stability characteristic of BpAP is attributed to improved anti-autolytic ability rather than increased structural stability. The three points combined mutant (MT5) showed the best increases in autolytic ability, as well as advanced catalytic efficiency. The low rate of inactive mutants and the high rate of positive mutants indicated that newly introduced screening factors (distance from catalytic residues, Gibbs free energy term, molecular simulation, and visual inspections) greatly enhance the design of anti-autolytic alkaline protease. Additionally, these findings enhance the industrial use of alkaline protease in detergents and similar applications.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110561"},"PeriodicalIF":3.4,"publicationDate":"2024-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142817351","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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