构建带有合成脂肪酶基因盒的铜绿假单胞菌 SDK-6,并利用响应面方法优化不同参数以过度表达重组脂肪酶。

IF 2.4 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Folia microbiologica Pub Date : 2024-12-01 Epub Date: 2024-05-03 DOI:10.1007/s12223-024-01167-y
Damanjeet Kaur, Rupinder Pal Singh, Saurabh Gupta
{"title":"构建带有合成脂肪酶基因盒的铜绿假单胞菌 SDK-6,并利用响应面方法优化不同参数以过度表达重组脂肪酶。","authors":"Damanjeet Kaur, Rupinder Pal Singh, Saurabh Gupta","doi":"10.1007/s12223-024-01167-y","DOIUrl":null,"url":null,"abstract":"<p><p>Lipases are industrially important enzymes having vast applications in various fields. Cloning and expression of lipase enzyme-encoding genes in suitable host lead to their widespread use in different fields. The present study represents the first attempt towards the expression of the synthetic lipase gene in Pseudomonas aeruginosa. An alkalophilic lipase gene (GenBank accession number: NP_388152) from Bacillus subtilis was synthetically designed and introduced in the pJN105 vector and subsequently cloned in Pseudomonas aeruginosa SDK-6. Agarose gel electrophoresis confirmed the transformation of SDK-6, exhibiting a band difference of ~ 700 bp between native and recombinant pJN105. Further amplification of cloned lipase gene was confirmed using PCR amplification with Lip 1 and Lip 2 primers respectively, followed by restriction analysis. Approximately 15-fold increase in lipase production was observed in recombinant Pseudomonas as compared to the native strain. One factor at a time (OFAT) analysis revealed L-arabinose, inoculum size (0.5%; v/v), and agitation (120 rpm) as significant factors affecting the over-expression of lipase enzyme. Optimization of enzyme induction conditions by central composite design (CCD) led to 1.60-fold increase in the production of lipase at 0.65% (w/v) inducer concentration, OD<sub>600</sub>-1.075 before induction and 35 °C post induction temperature with overall lipase production of 50.50 IU/mL. Statistical validation of observed value via ANOVA showed an F-value of 138.70 at p < 0.01 with R<sup>2</sup> of 0.9921.</p>","PeriodicalId":12346,"journal":{"name":"Folia microbiologica","volume":" ","pages":"1279-1290"},"PeriodicalIF":2.4000,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Construction of Pseudomonas aeruginosa SDK-6 with synthetic lipase gene cassette and optimization of different parameters using response surface methodology for over-expression of recombinant lipase.\",\"authors\":\"Damanjeet Kaur, Rupinder Pal Singh, Saurabh Gupta\",\"doi\":\"10.1007/s12223-024-01167-y\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Lipases are industrially important enzymes having vast applications in various fields. Cloning and expression of lipase enzyme-encoding genes in suitable host lead to their widespread use in different fields. The present study represents the first attempt towards the expression of the synthetic lipase gene in Pseudomonas aeruginosa. An alkalophilic lipase gene (GenBank accession number: NP_388152) from Bacillus subtilis was synthetically designed and introduced in the pJN105 vector and subsequently cloned in Pseudomonas aeruginosa SDK-6. Agarose gel electrophoresis confirmed the transformation of SDK-6, exhibiting a band difference of ~ 700 bp between native and recombinant pJN105. Further amplification of cloned lipase gene was confirmed using PCR amplification with Lip 1 and Lip 2 primers respectively, followed by restriction analysis. Approximately 15-fold increase in lipase production was observed in recombinant Pseudomonas as compared to the native strain. One factor at a time (OFAT) analysis revealed L-arabinose, inoculum size (0.5%; v/v), and agitation (120 rpm) as significant factors affecting the over-expression of lipase enzyme. Optimization of enzyme induction conditions by central composite design (CCD) led to 1.60-fold increase in the production of lipase at 0.65% (w/v) inducer concentration, OD<sub>600</sub>-1.075 before induction and 35 °C post induction temperature with overall lipase production of 50.50 IU/mL. Statistical validation of observed value via ANOVA showed an F-value of 138.70 at p < 0.01 with R<sup>2</sup> of 0.9921.</p>\",\"PeriodicalId\":12346,\"journal\":{\"name\":\"Folia microbiologica\",\"volume\":\" \",\"pages\":\"1279-1290\"},\"PeriodicalIF\":2.4000,\"publicationDate\":\"2024-12-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Folia microbiologica\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1007/s12223-024-01167-y\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/5/3 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q3\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Folia microbiologica","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1007/s12223-024-01167-y","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/5/3 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0

摘要

脂肪酶是重要的工业酶,在各个领域都有广泛的应用。在合适的宿主中克隆和表达脂肪酶编码基因可使其在不同领域得到广泛应用。本研究首次尝试在铜绿假单胞菌中表达合成脂肪酶基因。我们合成了一种来自枯草芽孢杆菌的嗜碱性脂肪酶基因(GenBank登录号:NP_388152),并将其导入 pJN105 载体,随后克隆到铜绿假单胞菌 SDK-6 中。琼脂糖凝胶电泳证实了 SDK-6 的转化,在原生和重组 pJN105 之间显示出约 700 bp 的条带差异。分别用 Lip 1 和 Lip 2 引物进行 PCR 扩增,然后进行限制性分析,确认了克隆脂肪酶基因的进一步扩增。与原生菌株相比,重组假单胞菌的脂肪酶产量增加了约 15 倍。每次一个因素(OFAT)分析显示,L-阿拉伯糖、接种体大小(0.5%;v/v)和搅拌(120 转/分)是影响脂肪酶过度表达的重要因素。通过中央复合设计(CCD)优化酶诱导条件,在诱导剂浓度为 0.65%(w/v)、诱导前 OD600-1.075 和诱导后温度为 35 ℃ 时,脂肪酶产量增加了 1.60 倍,总脂肪酶产量为 50.50 IU/mL。通过方差分析对观察值进行的统计验证显示,F 值为 138.70,p 2 为 0.9921。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Construction of Pseudomonas aeruginosa SDK-6 with synthetic lipase gene cassette and optimization of different parameters using response surface methodology for over-expression of recombinant lipase.

Construction of Pseudomonas aeruginosa SDK-6 with synthetic lipase gene cassette and optimization of different parameters using response surface methodology for over-expression of recombinant lipase.

Lipases are industrially important enzymes having vast applications in various fields. Cloning and expression of lipase enzyme-encoding genes in suitable host lead to their widespread use in different fields. The present study represents the first attempt towards the expression of the synthetic lipase gene in Pseudomonas aeruginosa. An alkalophilic lipase gene (GenBank accession number: NP_388152) from Bacillus subtilis was synthetically designed and introduced in the pJN105 vector and subsequently cloned in Pseudomonas aeruginosa SDK-6. Agarose gel electrophoresis confirmed the transformation of SDK-6, exhibiting a band difference of ~ 700 bp between native and recombinant pJN105. Further amplification of cloned lipase gene was confirmed using PCR amplification with Lip 1 and Lip 2 primers respectively, followed by restriction analysis. Approximately 15-fold increase in lipase production was observed in recombinant Pseudomonas as compared to the native strain. One factor at a time (OFAT) analysis revealed L-arabinose, inoculum size (0.5%; v/v), and agitation (120 rpm) as significant factors affecting the over-expression of lipase enzyme. Optimization of enzyme induction conditions by central composite design (CCD) led to 1.60-fold increase in the production of lipase at 0.65% (w/v) inducer concentration, OD600-1.075 before induction and 35 °C post induction temperature with overall lipase production of 50.50 IU/mL. Statistical validation of observed value via ANOVA showed an F-value of 138.70 at p < 0.01 with R2 of 0.9921.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
Folia microbiologica
Folia microbiologica 工程技术-生物工程与应用微生物
CiteScore
5.80
自引率
0.00%
发文量
82
审稿时长
6-12 weeks
期刊介绍: Unlike journals which specialize ever more narrowly, Folia Microbiologica (FM) takes an open approach that spans general, soil, medical and industrial microbiology, plus some branches of immunology. This English-language journal publishes original papers, reviews and mini-reviews, short communications and book reviews. The coverage includes cutting-edge methods and promising new topics, as well as studies using established methods that exhibit promise in practical applications such as medicine, animal husbandry and more. The coverage of FM is expanding beyond Central and Eastern Europe, with a growing proportion of its contents contributed by international authors.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信