Inhibitors from lignocellulosic biomass have become the bottleneck of biorefinery development. Gluconobacter oxydans DSM2003 showed a high performance of inhibitors degradation, which had a short lag time in non-detoxified corn stover hydrolysate and could convert 90% of aldehyde inhibitors to weaker toxic acids. In this study, an aldehyde dehydrogenase gene W826-RS0111485, which plays an important function in the conversion of aldehyde inhibitors in Gluconobacter oxydans DSM2003, was identified. W826-RS0111485 was found by protein profiling, then a series of enzymatic properties were determined and were heterologously expressed in E. coli. The results indicated that NADP is the most suitable cofactor of the enzyme when aldehyde inhibitor is the substrate, and it had the highest oxidation activity to furfural among several aldehyde inhibitors. Under the optimal reaction conditions (50 °C, pH 7.5), the Km and Vmax of the enzyme under furfural stress were 2.45 and 80.97, respectively, and the Kcat was 232.22 min−1. The biodetoxification performance experiments showed that the recombinant E. coli containing the target gene completely converted 1 g/L furfural to furoic acid within 8 h, while the control E. coli only converted 18% furfural within 8 h. It was further demonstrated that W826-RS0111485 played an important role in the detoxification of furfural. The mining of this inhibitor degradation gene could provide a theoretical basis for rational modification of industrial strains to enhance its capacity of inhibitor degradation in the future.

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Identification of a Novel Dehydrogenase from Gluconobacter oxydans for Degradation of Inhibitors Derived from Lignocellulosic Biomass

Abstract Introduction Laccase is a copper-containing polyphenolic oxidase widely found in bacteria, archaea, fungi, animals, and plants. As a green biocatalyst with considerable potential for numerous environmental and industrial applications, the enzyme production efficiency of laccase in nature is low, and the cost is high. Purpose To examine the characterization and potential applications of laccase in this study, a novel laccase from Halomonas alkaliantartica (LacHa) was cloned and heterologously expressed it in Escherichia coli . Results To achieve heterologous and efficient laccase expression, a bacterial laccase gene designed as LacHa from Halomonas alkaliantartica of deep sea was cloned and expressed in E. coli . The results showed that the optimum temperature and pH of the enzyme reaction were 45 °C and 7.5. The 100 μM Cu 2+ and Fe 2+ ions had the strongest stimulatory effect on laccase activity, the surface-active agent SDS and organic solvent 5% ethanol had opposite effect. EDTA, and 5% DMSO have no effect on LacHa activity. The activity of LacHa was enhanced 1.5-fold by chloride at concentrations lower than 500 mM, and 57.6% of its initial activity remained in the reaction system containing 1000 mM. NaCl. Furthermore, LacHa showed decolorization rates ranging from 90.28 to 100% for indigo carmine and two azo dyes without mediators, with wide pH (5.0–9.0) and temperature (25–65 °C) ranges. Conclusions In this study, LacHa was expressed and showed unusual properties, indicating its great application potential in textile industries or environmental fields. 

Expression of a deep-sea bacterial laccase from Halomonas alkaliantartica and its application in dyes decolorization

The rapid growth of the human population in recent decades has resulted in the intensive development of various industries, the development of urban agglomerations and increased production of medicines for animals and humans, plant protection products and fertilizers on an unprecedented scale. Intensive agriculture, expanding urban areas and newly established industrial plants release huge amounts of pollutants into the environment, which, in nature, are very slowly degraded or not decomposed, which leads to their accumulation in water and terrestrial ecosystems. Researchers are scouring extremely contaminated environments to identify organisms that have the ability to degrade resistant xenobiotics, such as PAHs, some pharmaceuticals, plasticizers and dyes. These organisms are a potential source of enzymes that could be used in the bioremediation of industrial and municipal wastewater. Great hopes are pinned on oxidoreductases, including laccase, called by some a green biocatalyst because the end product of the oxidation of a wide range of substrates by this enzyme is water and other compounds, most often including dimers, trimers and polymers. Laccase immobilization techniques and their use in systems together with adsorption or separation have found application in the enzymatic bioremediation of wastewater.

https://www.mdpi.com/2076-3417/13/7/4394/pdf?version=1680259670

Biochemical Characteristics of Laccases and Their Practical Application in the Removal of Xenobiotics from Water

Abstract Introduction Laccase is a copper-containing polyphenolic oxidase widely found in bacteria, archaea, fungi, animals, and plants. As a green biocatalyst with considerable potential for numerous environmental and industrial applications, the enzyme production efficiency of laccase in nature is low, and the cost is high. Purpose To examine the characterization and potential applications of laccase in this study, a novel laccase from Halomonas alkaliantarctica (LacHa) was cloned and heterologously expressed in Escherichia coli . Results To achieve heterologous and efficient laccase expression, a bacterial laccase gene designed as LacHa from Halomonas alkaliantarctica of deep sea was cloned and expressed in E. coli . The results showed that the optimum temperature and pH of the enzyme reaction were 45°C and 7.5. The 100 μM Cu 2+ and Fe 2+ ions had the strongest stimulatory effect on laccase activity, the surface-active agent SDS and organic solvent 5% ethanol had opposite effect. EDTA, and 5% DMSO have no effect on LacHa activity. The activity of LacHa was enhanced 1.5 fold by chloride at concentrations lower than 500 mM, and 57.6% of its initial activity remained in the reaction system containing 1000 mM NaCl. Furthermore, LacHa showed decolorization rates ranging from 90.28% to 100% for indigo carmine and two azo dyes without mediators, with wide pH (5.0-9.0) and temperature (25-65°C) ranges. Conclusions In the present study, LacHa was expressed and showed unusual properties, suggesting its great application potential in the treatment of textile industries or environmental applications. 

/pdf/expression-of-a-deep-sea-bacterial-laccase-from-halomonas-27yxwswb.pdf

Expression of a deep-sea bacterial laccase from Halomonas alkaliantarctica and its application in dyes decolorization

Preparation and characterization of a laccase-like enzyme from Thermomicrobium roseum.

Laccases can catalyze the remediation of hazardous synthetic dyes in an eco-friendly manner, and thermostable laccases are advantageous to treat high-temperature dyeing wastewater. A novel laccase from Geothermobacter hydrogeniphilus (Ghlac) was cloned and expressed in Escherichia coli. Ghlac containing 263 residues was characterized as a functional laccase of the DUF152 family. By structural and biochemical analyses, the conserved residues H78, C119, and H136 were identified to bind with one copper atom to fulfill the laccase activity. In order to make it more suitable for industrial use, Ghlac variant Mut2 with enhanced thermostability was designed. The half-lives of Mut2 at 50 °C and 60 °C were 80.6 h and 9.8 h, respectively. Mut2 was stable at pH values ranging from 4.0 to 8.0 and showed a high tolerance for organic solvents such as ethanol, acetone, and dimethyl sulfoxide. In addition, Mut2 decolorized approximately 100% of 100 mg/L of malachite green dye in 3 h at 70 °C. Furthermore, Mut2 eliminated the toxicity of malachite green to bacteria and Zea mays. In summary, the thermostable laccase Ghlac Mut2 could effectively decolorize and detoxify malachite green at high temperatures, showing great potential to remediate the dyeing wastewater.

Kai Wang

Papers

An Engineered Thermostable Laccase with Great Ability to Decolorize and Detoxify Malachite Green