Purification and characterization of catechol 1,2-dioxygenase (EC 1.13.11.1; catechol-oxygen 1,2-oxidoreductase; C12O) using the local isolate of phenol-degrading Pseudomonas putida.

IF 2.4 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Folia microbiologica Pub Date : 2024-06-01 Epub Date: 2023-09-13 DOI:10.1007/s12223-023-01090-8
Huda Rasheed Tawfeeq, Sawsan Sajid Al-Jubori, Amel Hussaein Mussa
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Abstract

The purpose of the present study was to purify and characterize the catechol 1,2-dioxygenase (EC 1.13.11.1; catechol-oxygen 1,2-oxidoreductase; C12O) enzyme from the local isolate of Pseudomonas putida. This enzyme catalyzes the initial reaction in the ortho-pathway for phenol degradation in various gram-negative bacteria, including the genus of Pseudomonas. Pseudomonads are commonly used in the biodegradation of xenobiotics due to their versatility in degrading a wide range of chemical compounds. Eighty-nine soil samples were taken from the contaminated soil of the Midland Refineries Company (MRC) of Al-Daura refinery area at Baghdad from April to August 2021. The samples were grown in a mineral salt medium containing 250 mg per L of phenol to test their ability to biodegrade phenol. The pH was adjusted to 8.0 at 30 °C using a shaking incubator for 24-48 h. A number of 62 (69.6%) isolates of the total number were able to degrade phenol efficiently. The findings of the VITEK system and the housekeeping gene 16S rDNA confirmed that out of the positive isolates for phenol degradation, 36 from 62 (58.06%) were identified as Pseudomonas spp. isolates. Those isolates were distributed as P. aeruginosa 30 (83.3%) and P. putida 6 (16.6%). The enzyme production capabilities of the isolates were evaluated, and the highest activity was 2.39 U per mg for the isolate No. 15 which it was identified as P. putida. The previous isolate was selected for enzyme production, purification, and characterization. The enzyme was purified using ion exchange and gel filtration chromatography, with a combined yield of 36.12% and purification fold of 15.42 folds. Using a gel filtration column, the enzyme's molar mass was calculated to be 69 kDa after purification. The purified enzyme was stable at 35 °C and a pH of 6.0.

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利用本地分离的苯酚降解假单胞菌(Pseudomonas putida)纯化儿茶酚 1,2-二氧 化酶(EC 1.13.11.1;儿茶酚-氧 1,2-氧化还原酶;C12O)并确定其特性。
本研究的目的是从本地分离的假单胞菌(Pseudomonas putida)中纯化和鉴定儿茶酚 1,2-二氧 化酶(EC 1.13.11.1;儿茶酚-氧 1,2-氧化还原酶;C12O)。这种酶催化各种革兰氏阴性细菌(包括假单胞菌属)苯酚降解正交途径的初始反应。假单胞菌因其降解多种化合物的多功能性,常用于生物降解异种生物。2021 年 4 月至 8 月,研究人员从巴格达 Al-Daura 炼油厂地区米德兰炼油公司(MRC)受污染的土壤中采集了 89 个土壤样本。样本在每升含 250 毫克苯酚的矿物盐培养基中生长,以测试其生物降解苯酚的能力。将 pH 值调至 8.0,在 30 °C、振荡培养箱中培养 24-48 小时。在所有分离物中,有 62 个(69.6%)能够有效降解苯酚。VITEK 系统和管家基因 16S rDNA 的结果证实,在苯酚降解阳性分离物中,62 个分离物中有 36 个(58.06%)被鉴定为假单胞菌属分离物。这些分离物分布为铜绿假单胞菌 30 个(83.3%)和假单胞菌 6 个(16.6%)。对分离物的产酶能力进行了评估,其中 15 号分离物的活性最高,达到每毫克 2.39 U,被鉴定为普氏假单胞菌。前一种分离物被选中用于酶的生产、纯化和鉴定。使用离子交换和凝胶过滤色谱法纯化了酶,综合产率为 36.12%,纯化倍数为 15.42 倍。通过凝胶过滤柱,计算出纯化后酶的摩尔质量为 69 kDa。纯化后的酶在 35 °C 和 pH 值为 6.0 的条件下稳定。
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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.
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