Enzyme and Microbial Technology最新文献_第7页

Identification and characterization of a novel thermostable PL7 alginate lyase from a submarine volcanic metagenomic library 从海底火山元基因组文库中鉴定新型恒温 PL7 藻酸盐裂解酶并确定其特征

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-07-21 DOI: 10.1016/j.enzmictec.2024.110486

Vasileios Tsopanakis , Elena Anastasiadou , Maria D. Mikkelsen , Anne S. Meyer , Ioannis V. Pavlidis

{"title":"Identification and characterization of a novel thermostable PL7 alginate lyase from a submarine volcanic metagenomic library","authors":"Vasileios Tsopanakis , Elena Anastasiadou , Maria D. Mikkelsen , Anne S. Meyer , Ioannis V. Pavlidis","doi":"10.1016/j.enzmictec.2024.110486","DOIUrl":"10.1016/j.enzmictec.2024.110486","url":null,"abstract":"<div>Seaweed biomass is as an abundant and renewable source of complex polysaccharides, including alginate which has a variety of applications. A sustainable method for exploiting alginate towards the production of valuable oligosaccharides is through enzymatic processing, using alginate lyases. Industrial refinement methods demand robust enzymes. Metagenomic libraries from extreme environments are a new source of unique enzymes with great industrial potential. Herein we report the identification of a new thermostable alginate lyase with only 58 % identity to known sequences, identified by mining a metagenomic library obtained from the hydrothermal vents of the volcano Kolumbo in the Aegean Sea (Kolumbo Alginate Lyase, KAlLy). Sequence analysis and biochemical characterization of KAlLy showed that this new alginate lyase is a Polysaccharide Lyase of family 7 (PL7) enzyme with endo- and exo-action on alginate and poly-mannuronic acid, with high activity at 60°C (56 ± 8 U/mg) and high thermostability (half-life time of 30 h at 50°C). The response surface methodology analysis revealed that the reaction optimum conditions with poly-mannuronic acid as substrate are 44°C, pH of 5.5 with 440 mM NaCl. This novel alginate lyase is a valuable addition to the toolbox of alginate modifying enzymes, due to its diverse sequence and its good thermal stability.</div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"180 ","pages":"Article 110486"},"PeriodicalIF":3.4,"publicationDate":"2024-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141736457","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

High-efficient preparation of β-nicotinamide mononucleotides by crude enzymes cascade catalytic reaction 利用粗酶级联催化反应高效制备 β-烟酰胺单核苷酸。

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-07-20 DOI: 10.1016/j.enzmictec.2024.110482

Jiehu Liu , Runtian Huo , Huixian Fu , Shiheng Chen , Xueyi Qiao , Bo Xu , Zhaoyuan Zhang , Jing Wu , Lingqia Su

{"title":"High-efficient preparation of β-nicotinamide mononucleotides by crude enzymes cascade catalytic reaction","authors":"Jiehu Liu , Runtian Huo , Huixian Fu , Shiheng Chen , Xueyi Qiao , Bo Xu , Zhaoyuan Zhang , Jing Wu , Lingqia Su","doi":"10.1016/j.enzmictec.2024.110482","DOIUrl":"10.1016/j.enzmictec.2024.110482","url":null,"abstract":"<div>β-nicotinamide mononucleotide (β-NMN) is a key precursor of nicotinamide adenine dinucleotide, and becomes attractive in the nutrition and health care fields, but its enzymatic synthesis is expensive. In this study, a six-enzyme cascade catalytic system was constructed to produce β-NMN. Using D-ribose and nicotinamide as substrates, the β-NMN yield reached 97.5 % catalyzed by purified enzymes. Then, after knocking out the genes encoding proteins that consume β-NMN in E. coli BL21(DE3), the similar β-NMN yield, 97.2 %, using the crude enzymes could be also obtained. After that, β-NMN synthesis was performed under increased substrate concentration, and 'modular' crude enzymes cascade catalytic reaction system was proposed to reduce the inhibition of polyphosphate on ribose-phosphate diphosphokinase activity, and the β-NMN yield reached 78.4 % at 10 mM D-ribose, which is 1.82 times of that in 'one-pot' reaction and represents the highest β-NMN preparation level with phosphoribosylpyrophosphate as the core reported till now.</div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"180 ","pages":"Article 110482"},"PeriodicalIF":3.4,"publicationDate":"2024-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141765748","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

Efficiency enhancement in Aspergillus niger α-L-rhamnosidase reverse hydrolysis by using a tunnel site rational design strategy 利用隧道位点合理设计策略提高黑曲霉α-L-鼠李糖酶反向水解的效率

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-07-19 DOI: 10.1016/j.enzmictec.2024.110484

Yanling Lin , Yuchen Cai , Han Li , Lijun Li , Zedong Jiang , Hui Ni

{"title":"Efficiency enhancement in Aspergillus niger α-L-rhamnosidase reverse hydrolysis by using a tunnel site rational design strategy","authors":"Yanling Lin , Yuchen Cai , Han Li , Lijun Li , Zedong Jiang , Hui Ni","doi":"10.1016/j.enzmictec.2024.110484","DOIUrl":"10.1016/j.enzmictec.2024.110484","url":null,"abstract":"<div>There has been ongoing interest in improving the efficiency of glycoside hydrolase for synthesizing glycoside compounds through protein engineering, given the potential applications of glycoside compounds. In this study, a strategy of modifying the substrate access tunnel was proposed to enhance the efficiency of reverse hydrolysis catalyzed by Aspergillus niger α-L-rhamnosidase. Analysis of the tunnel dynamics identified Tyr299 as a key modifiable residue in the substrate access tunnel. The location of Tyr299 was near the enzyme surface and at the outermost end of the substrate access tunnel, suggested its role in substrate recognition and throughput. Based on the properties of side chains, six mutants were designed and expressed by Pichia pastoris. Compared to WT, the reverse hydrolysis efficiencies of mutants Y299P and Y299W were increased by 21.3 % and 11.1 %, respectively. The calculation results of binding free energy showed that the binding free energy was inversely proportional to the reverse hydrolysis efficiency. Further, when binding free energy levels were comparable, the mutants with shorter side chains displayed a higher reverse hydrolysis efficiency. These results proved that substrate access tunnel modification was an effective method to improve the reverse hydrolysis efficacy of α-L-rhamnosidase and also provided new insights for modifying other glycoside hydrolases.</div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"180 ","pages":"Article 110484"},"PeriodicalIF":3.4,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141852485","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

Biocatalytic approach for the synthesis of chiral alcohols for the development of pharmaceutical intermediates and other industrial applications: A review 生物催化方法合成手性醇，用于开发医药中间体和其他工业应用：综述

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-07-17 DOI: 10.1016/j.enzmictec.2024.110483

Mohd Naim , Mohd Fazli Mohammat , Putri Nur Arina Mohd Ariff , Mohamad Hekarl Uzir

{"title":"Biocatalytic approach for the synthesis of chiral alcohols for the development of pharmaceutical intermediates and other industrial applications: A review","authors":"Mohd Naim , Mohd Fazli Mohammat , Putri Nur Arina Mohd Ariff , Mohamad Hekarl Uzir","doi":"10.1016/j.enzmictec.2024.110483","DOIUrl":"10.1016/j.enzmictec.2024.110483","url":null,"abstract":"<div>Biocatalysis has emerged as a strong tool for the synthesis of active pharmaceutical ingredients (APIs). In the early twentieth century, whole cell biocatalysis was used to develop the first industrial biocatalytic processes, and the precise work of enzymes was unknown. Biocatalysis has evolved over the years into an essential tool for modern, cost-effective, and sustainable pharmaceutical manufacturing. Meanwhile, advances in directed evolution enable the rapid production of process-stable enzymes with broad substrate scope and high selectivity. Large-scale synthetic pathways incorporating biocatalytic critical steps towards >130 APIs of authorized pharmaceuticals and drug prospects are compared in terms of steps, reaction conditions, and scale with the corresponding chemical procedures. This review is designed on the functional group developed during the reaction forming alcohol functional groups. Some important biocatalyst sources, techniques, and challenges are described. A few APIs and their utilization in pharmaceutical drugs are explained here in this review. Biocatalysis has provided shorter, more efficient, and more sustainable alternative pathways toward existing small molecule APIs. Furthermore, non-pharmaceutical applications of biocatalysts are also mentioned and discussed. Finally, this review includes the future outlook and challenges of biocatalysis. In conclusion, Further research and development of promising enzymes are required before they can be used in industry.</div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"180 ","pages":"Article 110483"},"PeriodicalIF":3.4,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141732151","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

Enhanced L-theanine production through semi-rational design of γ-glutamylmethylamide synthetase from Methylovorus mays 通过对 Methylovorus mays 的 γ-谷氨酰甲酰胺合成酶进行半合理设计，提高 L-茶氨酸的产量。

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-07-17 DOI: 10.1016/j.enzmictec.2024.110481

Chao Fan , Jiakun Qi , Yunhan Cong , Chunzhi Zhang

引用次数: 0

Osmoregulation by choline-based deep eutectic solvent induces electroactivity in Bacillus subtilis biofilms 胆碱深共晶溶剂的渗透调节作用诱导枯草芽孢杆菌生物膜的电活性

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-07-16 DOI: 10.1016/j.enzmictec.2024.110485

Neda Eghtesadi , Kayode Olaifa , Tri T. Pham , Vito Capriati , Obinna M. Ajunwa , Enrico Marsili

{"title":"Osmoregulation by choline-based deep eutectic solvent induces electroactivity in Bacillus subtilis biofilms","authors":"Neda Eghtesadi , Kayode Olaifa , Tri T. Pham , Vito Capriati , Obinna M. Ajunwa , Enrico Marsili","doi":"10.1016/j.enzmictec.2024.110485","DOIUrl":"10.1016/j.enzmictec.2024.110485","url":null,"abstract":"<div>Gram-positive Bacillus subtilis is a model organism for the biotechnology industry and has recently been characterized as weakly electroactive in both planktonic cultures and biofilms. Increasing the extracellular electron transfer (EET) rate in B. subtilis biofilms will help to develop an efficient microbial electrochemical technology (MET) and improve the bioproduction of high-value metabolites under electrofermentative conditions. In our previous work, we have shown that the addition of compatible solute precursors such as choline chloride (ChCl) to the growth medium formulation increases current output and biofilm formation in B. subtilis. In this work, we utilized a low-carbon tryptone yeast extract medium with added salts to further expose B. subtilis to salt stress and observe the osmoregulatory and/or nutritional effects of a D-sorbitol/choline chloride (ChCl) (1:1 mol mol−1) deep eutectic solvents (DESs) on the electroactivity of the formed biofilm. The results show that ChCl and D-sorbitol alleviate the osmotic stress induced by the addition of NaH2PO4 and KH2PO4 salts and boost biofilm production. This is probably due to the osmoprotective effect of ChCl, a precursor of the osmoprotectant glycine betaine, and the induction of electroactive exopolymeric substances within the B. subtilis biofilm. Since high ionic strength media are commonly used in microbial biotechnology, the combination of ChCl-containing DESs and salt stress could enhance biofilm-based electrofermentation processes that bring significant benefits for biotechnological applications.</div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"180 ","pages":"Article 110485"},"PeriodicalIF":3.4,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0141022924000929/pdfft?md5=407aeace407d0e7ef745e4781151fc3c&pid=1-s2.0-S0141022924000929-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141709890","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Rational design of short-chain dehydrogenase DHDR for efficient synthesis of (S)-equol 合理设计短链脱氢酶 DHDR 以高效合成 (S)-equol

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-07-16 DOI: 10.1016/j.enzmictec.2024.110480

Weichuang Qin , Lujia Zhang , Yichen Yang , Wei Zhou , Shuting Hou , Jie Huang , Bei Gao

{"title":"Rational design of short-chain dehydrogenase DHDR for efficient synthesis of (S)-equol","authors":"Weichuang Qin , Lujia Zhang , Yichen Yang , Wei Zhou , Shuting Hou , Jie Huang , Bei Gao","doi":"10.1016/j.enzmictec.2024.110480","DOIUrl":"10.1016/j.enzmictec.2024.110480","url":null,"abstract":"<div>(S)-equol, the most influential metabolite of daidzein in vivo, has aroused great attention due to the excellent biological activities. Although existing studies have accomplished the construction of its heterologous synthetic pathway in the context of anaerobicity and inefficiency of natural strains, the low productivity of (S)-equol limits its industrial application. Here, rational design strategies based on decreasing the pocket steric hindrance and fine-tuning the pocket microenvironment to systematically redesign the binding pocket of enzyme were developed and processed to the rate-limiting enzyme dihydrodaidzein reductase in (S)-equol synthesis. After iterative combinatorial mutagenesis, an effective mutant S118G/T169A capable of significantly increasing (S)-equol yield was obtained. Computational analyses illustrated that the main reason of the increased activity relied on the decreased critical distance and more stable interacting conformation. Then, the reaction optimization was performed, and the recombinant Escherichia coli whole-cell biocatalyst harboring S118G/T169A enabled the efficient conversion of 2 mM daidzein to (S)-equol, achieving conversion rate of 84.5 %, which was 2.9 times higher than that of the parental strain expressing wide type dihydrodaidzein reductase. This study provides an effective idea and a feasible method for enzyme modification and whole-cell catalytic synthesis of (S)-equol, and will greatly accelerate the process of industrial production.</div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"180 ","pages":"Article 110480"},"PeriodicalIF":3.4,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141697498","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

Debridement efficacy of serine protease and formulated cream by In Vitro assessment against artificial wound eschar 通过体外评估丝氨酸蛋白酶和配制膏对人工伤口焦痂的清创功效

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-07-11 DOI: 10.1016/j.enzmictec.2024.110478

Julia Yunus, Haryati Jamaluddin, Wan Rosmiza Zana Wan Dagang

{"title":"Debridement efficacy of serine protease and formulated cream by In Vitro assessment against artificial wound eschar","authors":"Julia Yunus, Haryati Jamaluddin, Wan Rosmiza Zana Wan Dagang","doi":"10.1016/j.enzmictec.2024.110478","DOIUrl":"10.1016/j.enzmictec.2024.110478","url":null,"abstract":"<div>Chronic wounds typically comprise of necrotic tissue and dried secretions, often culminating in the formation of a thick and tough layer of dead skin known as eschar. Removal of eschar is imperative to facilitate wound healing. Conventional approach for eschar removal involves surgical excision and grafting, which can be traumatic and frequently leads to viable tissue damage. There has been growing interest in the use of enzymatic agents for a gentler approach to debridement, utilizing proteolytic enzymes. In this study, a purified intracellular recombinant serine protease from Bacillus sp. (SPB) and its cream formulation were employed to evaluate their ability to degrade artificial wound eschar; composed of collagen, fibrin, and elastin. Degradation was assessed based on percentage weight reduction of eschar biomass, analysis via sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and scanning electron microscopy (SEM). Both SPB and its cream formulation were able to degrade up to 50 % artificial wound eschar, with the SPB cream maintaining its degradation efficiency for up to 24 hours. Additionally, the SPB-based cream demonstrated the ability to hydrolyze proteinaceous components of eschars individually (fibrin and collagen) as determined through qualitative assessment. These findings suggest that SPB holds promise for the debridement of wound eschar.</div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"180 ","pages":"Article 110478"},"PeriodicalIF":3.4,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141695010","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

Control of Staphylococcus epidermidis biofilm by surfactins of an endophytic bacterium Bacillus sp. 15 F 内生细菌芽孢杆菌的表面活性剂对表皮葡萄球菌生物膜的控制 15 F

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-07-03 DOI: 10.1016/j.enzmictec.2024.110477

Marwa Jardak , Raphaël Lami , Oumaima Saadaoui , Hajer Jlidi , Didier Stien , Sami Aifa , Sami Mnif

{"title":"Control of Staphylococcus epidermidis biofilm by surfactins of an endophytic bacterium Bacillus sp. 15 F","authors":"Marwa Jardak , Raphaël Lami , Oumaima Saadaoui , Hajer Jlidi , Didier Stien , Sami Aifa , Sami Mnif","doi":"10.1016/j.enzmictec.2024.110477","DOIUrl":"https://doi.org/10.1016/j.enzmictec.2024.110477","url":null,"abstract":"<div>The present paper deals with the preparation and annotation of a surfactin(s) derived from a culture of the endophytic bacterium Bacillus 15 F. The LC-MS analysis of the acetonitrile fraction confirmed the presence of surfactins Leu/Ile7 C15, Leu/Ile7 C14 and Leu/Ile7 C13 with [M+H]+ at m/z 1036.6895, 1022.6741 and 1008.6581, respectively. Various concentrations of the surfactin(s) (hereafter referred to as surfactin-15 F) were used to reduce the adhesion of Staphylococcus epidermidis S61, which served as a model for studying antibiofilm activity on polystyrene surfaces. Incubation of Staphylococcus epidermidis S61 with 62.5 µg/ml of surfactin-15 F resulted in almost complete inhibition of biofilm formation (90.3 ± 3.33 %), and a significant reduction of cell viability (resazurin-based fluorescence was more than 200 times lower). The antiadhesive effect of surfactin-15 F was confirmed by scanning electron microscopy. Surfactin-15 F demonstrated an eradication effect against preformed biofilm, causing severe disruption of Staphylococcus epidermidis S61 biofilm structure and reducing viability. The results suggest that surfactins produced by endophytic bacteria could be an alternative to synthetic products. Surfactin-15 F, used in wound dressings, demonstrated an efficient treatment of the preformed Staphylococcus epidermidis S61 biofilm, and thus having a great potential in medical applications.</div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"180 ","pages":"Article 110477"},"PeriodicalIF":3.4,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141607715","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 an endonuclease and N6-adenine methyltransferase from Ureaplasma parvum SV3F4 strain 鉴定副脲原体 SV3F4 株的内切酶和 N6-腺嘌呤甲基转移酶。

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2024-06-26 DOI: 10.1016/j.enzmictec.2024.110471

Heng Ning Wu , Yuya Fujisawa , Zenzaburo Tozuka , Alexey Fomenkov , Yukiko Nakura , Shin-ichiro Kajiyama , Shinsuke Fujiwara , Kiyoshi Yasukawa , Richard J. Roberts , Itaru Yanagihara

{"title":"Identification of an endonuclease and N6-adenine methyltransferase from Ureaplasma parvum SV3F4 strain","authors":"Heng Ning Wu , Yuya Fujisawa , Zenzaburo Tozuka , Alexey Fomenkov , Yukiko Nakura , Shin-ichiro Kajiyama , Shinsuke Fujiwara , Kiyoshi Yasukawa , Richard J. Roberts , Itaru Yanagihara","doi":"10.1016/j.enzmictec.2024.110471","DOIUrl":"10.1016/j.enzmictec.2024.110471","url":null,"abstract":"<div>Here, we report a novel endonuclease and N6-adenine DNA methyltransferase (m6A methyltransferase) in the Ureaplasma parvum SV3F4 strain. Our previous study found that the SV3F4 strain carries 17 unique genes, which are not encoded in the two previously reported U. parvum serovar 3 strain, OMC-P162 and ATCC 700970. Of these 17 unique genes, UP3_c0261 and UP3_c0262, were originally annotated as encoding hypothetical proteins. Comparative genomics analyses more recently indicated they encode a Type II restriction endonuclease and an m6A methyltransferase, respectively. The UP3_c0261 and UP3_c0262 genes were individually expressed and purified in Escherichia coli. The UP3_c0261 recombinant protein showed endonuclease activity on the pT7Blue vector, recognizing and cleaving a GTNAC motif, resulting in a 5 base 5’ extension. The UP3_c0261 protein digested a polymerase chain reaction (PCR) product harboring the GTNAC motif. The endonuclease UP3_c0261 was designated as UpaF4I. Treatment of the PCR product with the recombinant protein UP3_c0262 completely blocked the restriction enzyme activity of UpaF4I. Analysis of the treated PCR product harboring a modified nucleotide by UP3_c0262 with HPLC-MS/MS and MS/MS showed that UP3_c0262 was an m6A methyltransferase containing a methylated A residue in both DNA strands of the GTNAC motif. Whole genome methylation analysis of SV3F4 showed that 99.9 % of the GTNAC motif was m6A modified. These results suggest the UP3_c0261 and UP3_c0262 genes may act as a novel Type II restriction-modification system in the Ureaplasma SV3F4 strain.</div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"180 ","pages":"Article 110471"},"PeriodicalIF":3.4,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0141022924000784/pdfft?md5=99c87b539386921e1e06916b966ff332&pid=1-s2.0-S0141022924000784-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141497491","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0