Asma Zafar, Ammara Masood, Ziaur Rahman, Attia Hamid, Mah Hoor Javed, Amna Zulfiqar, Samreen Fatima, Madood Makhdoom, Muhammad Nauman Aftab
{"title":"从蜡样芽孢杆菌(Bacillus cereusATCC 14579)中克隆、纯化和鉴定重组戊二酸酶的功能,以改善洗涤剂性能","authors":"Asma Zafar, Ammara Masood, Ziaur Rahman, Attia Hamid, Mah Hoor Javed, Amna Zulfiqar, Samreen Fatima, Madood Makhdoom, Muhammad Nauman Aftab","doi":"10.1002/jsde.12796","DOIUrl":null,"url":null,"abstract":"The present study outlines the approach that was employed for cloning, expression, and characterization of the recombinant pullulanase enzyme from <jats:italic>Bacillus cereus</jats:italic> ATCC 14579 into <jats:italic>Escherichia coli</jats:italic> BL21(DE3) using pET‐25b (+) expression vector. The recombinant pullulanase enzyme was purified using ammonium sulfate precipitation and immobilized metal ion affinity chromatography (IMAC). The molecular mass of the purified pullulanase enzyme was measured using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) as 95 kDa. The purified recombinant pullulanase enzyme demonstrated significant thermal stability, maintaining its structural integrity and functionality at temperatures as high as 90°C over a period of 4 h. The inclusion of divalent metal ions, specifically Ca<jats:sup>2+</jats:sup> and Mg<jats:sup>2+</jats:sup>, had a positive effect on the activity of the pullulanase enzyme. Conversely, the presence of Co<jats:sup>2+</jats:sup> and EDTA (Ethylene Diamine Tetra Acetic acid) resulted in suppression of the enzyme activity. The purified pullulanase enzyme demonstrated remarkable resistance when exposed to organic solvents. The enzyme activity was notably decreased in the presence of SDS (Sodium Dodecyl Sulfate) while β‐mercaptoethanol and tween‐60 did not substantially affect the enzyme activity and stability which suggest its potential applicability in the detergent sector. This discovery indicates a potential approach to improve the effectiveness of currently available detergents in the marketplace.","PeriodicalId":17083,"journal":{"name":"Journal of Surfactants and Detergents","volume":"60 1","pages":""},"PeriodicalIF":1.6000,"publicationDate":"2024-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Cloning, purification, and functional characterization of recombinant pullulanase from Bacillus cereusATCC 14579 for improved detergent performance\",\"authors\":\"Asma Zafar, Ammara Masood, Ziaur Rahman, Attia Hamid, Mah Hoor Javed, Amna Zulfiqar, Samreen Fatima, Madood Makhdoom, Muhammad Nauman Aftab\",\"doi\":\"10.1002/jsde.12796\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"The present study outlines the approach that was employed for cloning, expression, and characterization of the recombinant pullulanase enzyme from <jats:italic>Bacillus cereus</jats:italic> ATCC 14579 into <jats:italic>Escherichia coli</jats:italic> BL21(DE3) using pET‐25b (+) expression vector. The recombinant pullulanase enzyme was purified using ammonium sulfate precipitation and immobilized metal ion affinity chromatography (IMAC). The molecular mass of the purified pullulanase enzyme was measured using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) as 95 kDa. The purified recombinant pullulanase enzyme demonstrated significant thermal stability, maintaining its structural integrity and functionality at temperatures as high as 90°C over a period of 4 h. The inclusion of divalent metal ions, specifically Ca<jats:sup>2+</jats:sup> and Mg<jats:sup>2+</jats:sup>, had a positive effect on the activity of the pullulanase enzyme. Conversely, the presence of Co<jats:sup>2+</jats:sup> and EDTA (Ethylene Diamine Tetra Acetic acid) resulted in suppression of the enzyme activity. The purified pullulanase enzyme demonstrated remarkable resistance when exposed to organic solvents. The enzyme activity was notably decreased in the presence of SDS (Sodium Dodecyl Sulfate) while β‐mercaptoethanol and tween‐60 did not substantially affect the enzyme activity and stability which suggest its potential applicability in the detergent sector. This discovery indicates a potential approach to improve the effectiveness of currently available detergents in the marketplace.\",\"PeriodicalId\":17083,\"journal\":{\"name\":\"Journal of Surfactants and Detergents\",\"volume\":\"60 1\",\"pages\":\"\"},\"PeriodicalIF\":1.6000,\"publicationDate\":\"2024-08-29\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Surfactants and Detergents\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1002/jsde.12796\",\"RegionNum\":4,\"RegionCategory\":\"工程技术\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"CHEMISTRY, APPLIED\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Surfactants and Detergents","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1002/jsde.12796","RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"CHEMISTRY, APPLIED","Score":null,"Total":0}
Cloning, purification, and functional characterization of recombinant pullulanase from Bacillus cereusATCC 14579 for improved detergent performance
The present study outlines the approach that was employed for cloning, expression, and characterization of the recombinant pullulanase enzyme from Bacillus cereus ATCC 14579 into Escherichia coli BL21(DE3) using pET‐25b (+) expression vector. The recombinant pullulanase enzyme was purified using ammonium sulfate precipitation and immobilized metal ion affinity chromatography (IMAC). The molecular mass of the purified pullulanase enzyme was measured using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) as 95 kDa. The purified recombinant pullulanase enzyme demonstrated significant thermal stability, maintaining its structural integrity and functionality at temperatures as high as 90°C over a period of 4 h. The inclusion of divalent metal ions, specifically Ca2+ and Mg2+, had a positive effect on the activity of the pullulanase enzyme. Conversely, the presence of Co2+ and EDTA (Ethylene Diamine Tetra Acetic acid) resulted in suppression of the enzyme activity. The purified pullulanase enzyme demonstrated remarkable resistance when exposed to organic solvents. The enzyme activity was notably decreased in the presence of SDS (Sodium Dodecyl Sulfate) while β‐mercaptoethanol and tween‐60 did not substantially affect the enzyme activity and stability which suggest its potential applicability in the detergent sector. This discovery indicates a potential approach to improve the effectiveness of currently available detergents in the marketplace.
期刊介绍:
Journal of Surfactants and Detergents, a journal of the American Oil Chemists’ Society (AOCS) publishes scientific contributions in the surfactants and detergents area. This includes the basic and applied science of petrochemical and oleochemical surfactants, the development and performance of surfactants in all applications, as well as the development and manufacture of detergent ingredients and their formulation into finished products.