Fangyuan Wang

Bio: Fangyuan Wang is an academic researcher from Henan Agricultural University. The author has contributed to research in topics: Malachite green & Laccase. The author has an hindex of 1, co-authored 2 publications receiving 1 citations.

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

Abstract: 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.

Stable and Functionally Diverse Versatile Peroxidases Designed Directly from Sequences

Abstract: White-rot fungi secrete a repertoire of high-redox potential oxidoreductases to efficiently decompose lignin. Of these enzymes, versatile peroxidases (VPs) are the most promiscuous biocatalysts. VPs are attractive enzymes for research and industrial use but their recombinant production is extremely challenging. To date, only a single VP has been structurally characterized and optimized for recombinant functional expression, stability, and activity. Computational enzyme optimization methods can be applied to many enzymes in parallel but they require accurate structures. Here, we demonstrate that model structures computed by deep-learning-based ab initio structure prediction methods are reliable starting points for one-shot PROSS stability-design calculations. Four designed VPs encoding as many as 43 mutations relative to the wildtype enzymes are functionally expressed in yeast, whereas their wildtype parents are not. Three of these designs exhibit substantial and useful diversity in their reactivity profiles and tolerance to environmental conditions. The reliability of the new generation of structure predictors and design methods increases the scale and scope of computational enzyme optimization, enabling efficient discovery and exploitation of the functional diversity in natural enzyme families directly from genomic databases.

Research Article: Determination of median lethal concentration (LC50) and histopathological effects of malachite green on Oncorhynchus mykiss

Abstract: Research Article: Determination of median lethal concentration (LC50) and histopathological effects of malachite green on Oncorhynchus mykiss

Stable and functionally diverse versatile peroxidases by computational design directly from sequence

Abstract: White-rot fungi secrete a repertoire of high-redox potential oxidoreductases to efficiently decompose lignin. Of these enzymes, versatile peroxidases (VPs) are the most promiscuous biocatalysts. VPs are attractive enzymes for research and industrial use, but their recombinant production is extremely challenging. To date, only a single VP has been structurally characterized and optimized for recombinant functional expression, stability and activity. Computational enzyme optimization methods can be applied to many enzymes in parallel, but they require accurate structures. Here, we demonstrate that model structures computed by deep-learning based ab initio structure prediction methods are reliable starting points for one-shot PROSS stability-design calculations. Four designed VPs encoding as many as 43 mutations relative to the wild type enzymes are functionally expressed in yeast whereas their wild type parents are not. Three of these designs exhibit substantial and useful diversity in reactivity profile and tolerance to environmental conditions. The reliability of the new generation of structure predictors and design methods increases the scale and scope of computational enzyme optimization, enabling efficient discovery and exploitation of the functional diversity in natural enzyme families.

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

Abstract: 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.

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

Abstract: 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.