蜡样芽孢杆菌IndB1卤代酸脱卤酶基因的克隆与表达

Q4 Environmental Science

Indonesian Journal of Biotechnology Pub Date : 2018-02-13 DOI:10.22146/IJBIOTECH.27338

E. Ratnaningsih, I. Idris

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引用次数: 4

摘要

有机卤素化合物广泛用作农业中的杀虫剂和工业部门的溶剂，由于其毒性和持久性，会造成环境污染和健康问题。对有机卤素化合物的生物降解进行了大量研究，其中许多研究集中在细菌脱卤酶的使用上。卤代脂肪酸脱卤酶是一组切割卤代脂肪酸酯中碳卤键的酶。在先前的一项研究中，成功分离并鉴定了蜡样芽孢杆菌IndB1的bcfd1基因编码的卤酸脱卤酶。本研究旨在通过将bcfd1基因亚克隆到pET表达载体中，并在大肠杆菌BL21（DE3）中过表达该基因，从而建立该基因的表达系统。此外，对重组蛋白进行了表征，以更好地了解这种酶的催化作用。通过SDS-PAGE测定，使用0.01 mM IPTG在OD 550 0.8–1.0下诱导培养物，获得bcfd1的高表达。酶谱分析证明该重组蛋白具有脱卤酶活性。Bcfd1对一氯乙酸（MCA）的活性在30°C、pH 9时显示出37U/mg的比活性。据估计，Bcfd1的预测三级结构具有卤代酸脱卤酶超家族中保守的α/s水解酶折叠基序。Normal 0 false false IN X-NONE X-NONE/*样式定义*/table。MsoNormalTable｛mso style name:“Table Normal”；mso tstyle行带大小：0；mso ts style行带尺寸：0；mso style noshow:yes；mso style优先级：99；mso样式父级：“”；mso填充alt:0mm 5.4pt 0mm 5.4pt；mso对位边距顶部：0mm；mso段落右侧边距：0mm；mso段落边距底部：8.0pt；mso段落左边距：0mm；线路高度：107%；mso分页：寡妇孤儿；字体大小：11.0pt；font-family:“Calibri”，sans-serif；mso-ascii font-family:宋体；mso-ascii主题字体：小拉丁文；mso-hansi font-family:宋体；mso-hansi主题字体：小拉丁文；mso-bidi字体家族：“Times New Roman”；mso-bidi主题字体：minor bidi；mso-ansi语言：IN；｝

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Cloning and expression of haloacid dehalogenase gene from Bacillus cereus IndB1

Organohalogen compounds, widely used as pesticides in agriculture and solvents in the industrial sector, cause environmental pollution and health problems due to their toxicity and persistence. Numerous studies have been conducted on the biodegradation of organohalogen compounds, with many focusing on the use of dehalogenase from bacteria. Haloacid dehalogenase is a group of enzymes that cleaves the carbon-halogen bond in halogenated aliphatic acids. In a previous study, the bcfd1 gene encoded haloacid dehalogenase from Bacillus cereus IndB1 was successfully isolated and characterized. This research aimed to create an expression system of the bcfd1 gene by subcloning this gene into pET expression vector and to overexpress the gene in Escherichia coli BL21 (DE3). In addition, the recombinant protein was characterized to gain a better understanding of the catalytic action of this enzyme. A high expression of bcfd1 was obtained by inducing the culture at OD 550 0.8–1.0 using 0.01 mM IPTG as determined by SDS-PAGE. Zymogram analysis proved that the recombinant protein possessed dehalogenase activity. Bcfd1 activity toward monochloroacetic acid (MCA) showed specific activity of 37 U/mg at 30°C, pH 9. The predicted tertiary structure of Bcfd1 was estimated has conserved α/s hydrolase folding motif for haloacid dehalogenase superfamily. Normal 0 false false false IN X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0mm 5.4pt 0mm 5.4pt; mso-para-margin-top:0mm; mso-para-margin-right:0mm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0mm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-ansi-language:IN;}

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来源期刊

Indonesian Journal of Biotechnology Environmental Science-Environmental Science (miscellaneous)

CiteScore

1.00

自引率

0.00%

发文量

审稿时长

12 weeks