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A non-blue laccase of Bacillus sp. GZB displays manganese-oxidase activity: A study of laccase characterization, Mn(II) oxidation and prediction of Mn(II) oxidation mechanism.
Chemosphere ( IF 8.1 ) Pub Date : 2020-03-28 , DOI: 10.1016/j.chemosphere.2020.126619
Ranjit Das 1 , Zhishu Liang 1 , Guiying Li 1 , Taicheng An 1
Affiliation  

Laccase, a unique class of multicopper oxidase, presents promising potential as a biocatalyst in many industrial and biotechnological applications. Recently, it has been significantly applied in many metal-polluted sites due to its Manganese (Mn)-oxidation ability. Here, we demonstrate the Mn(II)-oxidase activity of laccase obtained from Bacillus sp. GZB. The CotA gene of GZB was transformed in E. coli BL21 and overexpressed. The purified laccase (LACREC3-laccase) displayed the absence of a peak at 610 nm that is usually found in blue-laccase. Further, the LACREC3-laccase exhibited high activity and stability at different pH and temperatures with substrates 2, 2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonate) and syringaldazine, respectively. It also functioned in the presence of various metals and enzyme inhibitors. Most notably, LACREC3-laccase formed insoluble brown Mn(III)/Mn(IV)-oxide particles from Mn(II) mineral, exhibiting its Mn(II)-oxidase activity. In addition to native polyacrylamide gel electrophoresis and buffer test, we developed an 'agarose gel plate' assay to evaluate Mn(II) oxidation activity of laccase. Furthermore, using the leucoberbelin blue assay, a total of 44.45 ± 0.45% Mn(IV)-oxides were quantified, in which 5.87 ± 0.61% autoxidized after 24 h. The Mn(II) oxidation mechanisms were further predicted by trapping Mn(III) using pyrophosphate during Mn(II) to Mn(IV) conversion by LACREC3-laccase. Overall, the laccase of GZB has excellent activity and stability plus an ability to oxidize Mn(II). This study is the first report on a non-blue laccase, exhibiting Mn(II)-oxidase activity. Thus, it offers a novel finding of the Mn(II) oxidation processes that can be a valuable way of Mn(II)-mineralization in various metal-polluted environments.

中文翻译:

芽孢杆菌属的一种非蓝色漆酶。GZB显示锰氧化酶活性:漆酶表征,Mn(II)氧化和Mn(II)氧化机理预测的研究。

漆酶是一类独特的多铜氧化酶,在许多工业和生物技术应用中作为生物催化剂具有广阔的前景。最近,由于其锰(Mn)的氧化能力,它已被广泛应用于许多金属污染的场所。在这里,我们证明了从芽孢杆菌获得的漆酶的Mn(II)-氧化酶活性。GZB。GZB的CotA基因在大肠杆菌BL21中转化并过表达。纯化的漆酶(LACREC3-漆酶)显示出在610 nm处没有通常在蓝漆酶中发现的峰。此外,LACREC3-漆酶分别在不同的pH和温度下与底物2、2'-叠氮基双(3-乙基苯并噻唑啉-6-磺酸盐)和丁香醛嗪显示出高活性和稳定性。它在各种金属和酶抑制剂的存在下也起作用。最为显着地,LACREC3-漆酶由Mn(II)矿物形成不溶的棕色Mn(III)/ Mn(IV)-氧化物颗粒,表现出其Mn(II)-氧化酶活性。除了天然聚丙烯酰胺凝胶电泳和缓冲液测试外,我们还开发了“琼脂糖凝胶板”测定法来评估漆酶的Mn(II)氧化活性。此外,使用白细胞素蓝测定法,对总计44.45±0.45%的Mn(IV)-氧化物进行了定量,其中24 h后自动氧化为5.87±0.61%。通过LACREC3-漆酶将Mn(II)转化为Mn(IV)期间使用焦磷酸盐捕获Mn(III),可以进一步预测Mn(II)的氧化机理。总体而言,GZB的漆酶具有出色的活性和稳定性,并具有氧化Mn(II)的能力。这项研究是有关非蓝色漆酶的第一份报告,该漆酶具有Mn(II)-氧化酶活性。从而,
更新日期:2020-03-28
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