Characterization and application of a novel laccase derived from Bacillus amyloliquefaciens

Abstract

As the copper-containing enzymes, laccases demonstrate a promising potential in various environmental and industrial applications. In this study, a bacterial strain isolated from soil exhibited the laccase activity, which was subsequently characterized and named as Bacillus amyloliquefaciens TCCC 111018. The novel gene encoding CotA-laccase (lac) was amplified using the genome of B. amyloliquefaciens TCCC 111018 as the template and efficiently and actively expressed in Escherichia coli. The recombinant LAC (rLAC) exhibited its highest activity at 80 °C and pH 5.5 for 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) oxidization and 80 °C and pH 7.0 for 2,6-dimethoxyphenol (2,6-DMP) oxidization. rLAC was stable at up to 60 °C and within the pH ranging from 3.0 to 9.0 when using the substrate ABTS. Furthermore, rLAC demonstrated the relatively high tolerance to NaCl, SDS, and most metal ions. Moreover, rLAC was capable of decolorizing the structurally different azo, anthraquinone, and triphenylmethane with different mediator at 60 °C under pH 5.5, 7.0, and 9.0. Therefore, rLAC would be an ideal candidate for lots of biotechnological and industrial applications due to its stability in the extreme conditions, including but not limit to pH, high temperature, halides, heavy metals and detergents.

Introduction

As the multicopper-containing enzymes, laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) can catalyze the oxidation process of numerous substrates, including anilines, arylamines, ascorbic acid, phenols, and a few inorganic compounds [1,2]. As members of the multicopper oxidase family, laccases normally incorporate four copper atoms (a type I copper, a type II copper, and two type III copper sites [3]. They become promising biocatalysts for biobleaching, biosensors, textile dye decolorization, xenobiotics bioremediation, etc. in biotechnological and industrial applications due to their environmental friendliness and wide substrate spectrum [4].

Laccases are extensively existed in higher plants, insects, and microorganisms [5]. So far, almost all reported laccases are from fungi, which are the only laccases applied in the industries [2]. Nevertheless, lots of time is needed for most fungi to grow and thereafter produce laccases. Additionally, they become unstable under conditions of high temperatures, alkalinity stress, and high salt [6], leading to low efficiency and cost-prohibitive industrial processes [7]. In contrast, bacterial laccases are less frequently studied due to their lower redox potential than that of fungal laccases. But they demonstrated many advantages, including better stability under an extensive range of pH or high temperatures, less susceptible to inhibitory agents, and less dependent on metal ions [8]. Furthermore, bacterial laccases are facile to be heterologously expressed in Escherichia coli. In comparison to their fungal counterparts, it is easier to improve their catalytic properties, stability and expression level via directed evolution [9]. The first prokaryotic laccase was isolated from Azospirillum lipoferum [10], and then numerous laccases were gradually found in Pseudomonas sp. [11], E. coli [12], Streptomyces sp. [13], and Bacillus sp. [14]. As the most famous bacterial laccase yet, the spore coat protein A (CotA) of B. subtilis is the component of endospore coat and is of great help for Bacillus sp. to survive under harsh conditions [15]. Compared to other bacterial laccases, CotA was extensively studied by many researchers due to its excellent alkali-resistance and thermostability [9,[16], [17], [18]], demonstrating its great potential for industrial application. Up to now, the scientific teams have found and characterized various laccases in Bacillus species, including B. halodurans, B. amyloliquefaciens, B. licheniformis, B. pumilus, B. subtilis, B. thuringiensis, B. vallismortis, and Bacillus sp. [14]. However, little is known about the laccases derived from the other spore-forming Bacillus species.

Therefore, it is necessary to screen and identify new bacterial laccases with different catalytic properties to enlarge our knowledge on bacterial laccases and expand the available biocatalysts toolbox for the potential industrial applications. The aim of this work was to screen and characterize a new strain with laccase activity and heterologously express the corresponding gene in E. coli to characterize its biochemical and catalytic properties and thus analyze its ability in decolorizing synthetic dyes. This report would deepen our understanding of Bacillus laccases and facilitate their applications in biotechnology.