Safary Azam, Moniri Rezvan, Hamzeh-Mivehroud Maryam, Dastmalchi Siavoush
Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran; Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran; Department of Microbiology and Immunology, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran.
J Biotechnol. 2016 Jun 10;227:56-63. doi: 10.1016/j.jbiotec.2016.04.006. Epub 2016 Apr 5.
An efficient method was introduced for soluble expression of recombinant laccase (rpCotA(SL-1)) from a newly isolated halo-thermotolerant Bacillus sp. SL-1 in modified Escherichia coli, trxB2/gor2 mutant (Origami™ B (DE3)). The yield of purified soluble laccase in Origami strain under micro-aerobic condition was ∼20mg/L of bacterial culture, showing significant improvement over the laccase produced in E.coli BL21 strain under aerobic condition. The specific activity of 13U/mg for purified laccase produced in micro-aerobic condition was higher than that of 1.07U/mg observed for the purified enzyme obtained in aerobic condition in Origami. The kinetic Km and kcat parameters for laccase-induced oxidation reactions were 46μM and 23s(-1) for ABTS (2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), and 19.6μM and 24s(-1) for SGZ (syringaldazine) substrates, respectively. The rpCotA(SL-1) displayed thermostability at 70°C and tolerance to specified concentrations of NaCl, NaN3, EDTA and SDS as inhibitors. The enzyme was relatively stable in the presence of different concentration of organic solvents, however the residual activity was adversely affected as the dipole moment of the solvents increase. Here we successfully report the production of soluble and functional laccase in Origami at the expression level suitable for industrial application.
一种高效方法被引入，用于在改良的大肠杆菌trxB2/gor2突变体（Origami™ B (DE3)）中可溶性表达来自新分离的嗜盐耐热芽孢杆菌SL-1的重组漆酶（rpCotA(SL-1)）。在微需氧条件下，Origami菌株中纯化的可溶性漆酶产量约为每升细菌培养物20毫克，与在有氧条件下大肠杆菌BL21菌株中产生的漆酶相比有显著提高。微需氧条件下产生的纯化漆酶的比活性为13U/mg，高于在Origami中需氧条件下获得的纯化酶所观察到的1.07U/mg。漆酶诱导氧化反应的动力学Km和kcat参数，对于ABTS（2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)）分别为46μM和23s(-1)，对于SGZ（丁香醛连氮）底物分别为19.6μM和24s(-1)。rpCotA(SL-1)在70°C显示热稳定性，并且对特定浓度的NaCl、NaN3、EDTA和SDS作为抑制剂具有耐受性。该酶在不同浓度的有机溶剂存在下相对稳定，然而随着溶剂偶极矩增加，残余活性受到不利影响。在此我们成功报道在Origami中以适合工业应用的表达水平生产出可溶性且有功能的漆酶。
