Zemin Fang

Evidence for lignin oxidation by the giant panda fecal microbiome.

The digestion of lignin and lignin-related phenolic compounds from bamboo by giant pandas has puzzled scientists because of the lack of lignin-degrading genes in the genome of the bamboo-feeding animals. We constructed a 16S rRNA gene library from the microorganisms derived from the giant panda feces to identify the possibility for the presence of potential lignin-degrading bacteria. Phylogenetic analysis showed that the phylotypes of the intestinal bacteria were affiliated with the phyla Proteobacteria (53%) and Firmicutes (47%). Two phylotypes were affiliated with the known lignin-degrading bacterium Pseudomonas putida and the mangrove forest bacteria. To test the hypothesis that microbes in the giant panda gut help degrade lignin, a metagenomic library of the intestinal bacteria was constructed and screened for clones that contained genes encoding laccase, a lignin-degrading related enzyme. A multicopper oxidase gene, designated as lac51, was identified from a metagenomic clone. Sequence analysis and copper content determination indicated that Lac51 is a laccase rather than a metallo-oxidase and may work outside its original host cell because it has a TAT-type signal peptide and a transmembrane segment at its N-terminus. Lac51 oxidizes a variety of lignin-related phenolic compounds, including syringaldazine, 2,6-dimethoxyphenol, ferulic acid, veratryl alcohol, guaiacol, and sinapinic acid at conditions that simulate the physiologic environment in giant panda intestines. Furthermore, in the presence of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringic acid, or ferulic acid as mediators, the oxidative ability of Lac51 on lignin was promoted. The absorbance of lignin at 445 nm decreased to 36% for ABTS, 51% for syringic acid, and 51% for ferulic acid after incubation for 10 h. Our findings demonstrate that the intestinal bacteria of giant pandas may facilitate the oxidation of lignin moieties, thereby clarifying the digestion of bamboo lignin by the animal.

A mechanism of glucose tolerance and stimulation of GH1 β-glucosidases.

β-Glucosidases are enzymes that hydrolyze β-glycosidic bonds to release non-reducing terminal glucosyl residues from glycosides and oligosaccharides, and thus have significant application potential in industries. However, most β-glucosidases are feedback inhibited by the glucose product, which restricts their application. Remarkably, some β-glucosidases of the glycoside hydrolase (GH) 1 family are tolerant to or even stimulated by glucose. Elucidation of the mechanisms of glucose tolerance and stimulation of the GH1 β-glucosidases will be crucial to improve their application through enzyme engineering. In this study, by comparing the primary and tertiary structures of two GH1 β-glucosidases with distinct glucose dependence, some putative glucose-dependence relevant sites were mutated to investigate their exact roles. Both biochemical and structural characterization of the mutants suggested that some sites at the entrance and middle of the substrate channel regulate the effects of glucose, and the relative binding affinity/preference of these sites to glucose modulates the glucose dependence. A mechanism was therefore proposed to interpret the glucose dependence of GH1 β-glucosidases. This research provides fresh insight into our current understanding of the properties and mechanisms of GH1 β-glycosidases and related enzymes that modulate their activity via feedback control mechanism.

Structure-based rational design to enhance the solubility and thermostability of a bacterial laccase Lac15.

Bacterial laccases are ideal alternatives of fungal laccases for specific industrial applications due to specific characteristics such as alkalescence dependence and high chloride tolerance. However, some bacterial laccases presented as inclusion bodies when expressing in Escherichia coli and showed thermal instability. In this study, rational design was employed to enhance the solubility and the thermostablity of the bacterial laccase Lac15-His6 based on the crystal structure obtained previously. After deletion of His-tag and residues323–332, the obtained Lac15D was completely expressed in soluble form even at a higher temperature of 28°C, compared to only 50% of Lac15-His6 expressed solubly at 16°C. It showed a two-time higher activity at temperatures lower than 35°C and a half-life increasing from 72 min to 150 min at 45°C. When used in chromogenic reactions, Lac15D showed constant activity toward dye precursors and their combinations under alkaline conditions, demonstrating its application potential in hair coloring biotechnology.

Improve ethanol tolerance of β-glucosidase Bgl1A by semi-rational engineering for the hydrolysis of soybean isoflavone glycosides.

A β-glucosidase Bgl1A variant (A24S/F297Y) with improved ethanol tolerance was obtained by semi-rational engineering. At 30-40°C, IC50 values (the amount required for inhibiting 50% enzyme activity) of the variant for ethanol were 17-30% (v/v), 1.4- to 2.4-fold of Bgl1A. When incubating in 15% (v/v) ethanol at 30°C, the half-life of A24S/F297Y was 13min; whereas Bgl1A lost all enzyme activity within 5min. A24S/F297Y was more stable at pH 7.5 than at pH 6.5, and more than 50% of the original activity remained after incubation at 30°C for 10h. At 35°C and pH 7.5, the half-life of A24S/F297Y was 80min, 4.3 times longer than that of Bgl1A. When converting isoflavone glycosides to aglycones using A24S/F297Y as catalyst, the hydrolysis rates were 99% for daidzin and 98% for genistin. The concentrations of daidzein and genistein rapidly increased by 7.02mM and 4.35mM within 10min, respectively. These results showed that A24S/F297Y was a promising candidate for the enzymatic hydrolysis of soybean isoflavone glycosides.

Characterization of a novel β-glucosidase from Gongronella sp. W5 and its application in the hydrolysis of soybean isoflavone glycosides.

A novel β-glucosidase named BglW5 from Gongronella sp. was isolated, purified, and characterized for the first time. Under solid state fermentation, the yield of BglW5 was 49.9 U/g fermented medium. BglW5 was stable over a wide pH range of 3.0-8.5 and retained more than 50% of its maximal activity after incubation at 25 °C for 96 h. The half-lives of BglW5 were 20 h at 60 °C, and 1 h at 70 °C. The activity of BglW5 was stimulated by xylose and fructose at concentrations up to 500 mM, with maximal stimulatory effect of 1.6-fold and 2.2-fold, respectively. BglW5 converted isoflavone glycosides to aglycones, with a hydrolysis rate of 96.2% for daidzin and 96.7% for genistin. The productivities were 1.5 mmol L(-1) h(-1) for daidzein and 1.23 mmol L(-1) h(-1) for genistein, respectively. These features suggest that BglW5 has great application potential in the hydrolysis of soybean isoflavone glycosides.

Purification, crystallization and preliminary crystallographic analysis of recombinant Lac15 from a marine microbial metagenome

Laccases are members of the blue multi-copper oxidase family that can oxidize a wide range of aromatic compounds. A new bacterial laccase (Lac15) has recently been obtained from a marine microbial metagenome from the South China Sea and characterized. In this work, recombinant Lac15 was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.2 Å resolution. The crystal belonged to space group C121, with unit-cell parameters a = 123.41, b = 91.36, c = 86.157 Å, β = 112.10°.

Mechanism of salt-induced activity enhancement of a marine-derived laccase, Lac15

Laccase (benzenediol: oxygen oxidoreductases, EC1.10.3.2) is a multi-copper oxidase capable of oxidizing a variety of phenolic and other aromatic organic compounds. The catalytic power of laccase makes it an attractive candidate for potential applications in many areas of industry including biodegradation of organic pollutants and synthesis of novel drugs. Most laccases are vulnerable to high salt and have limited applications. However, some laccases are not only tolerant to but also activated by certain concentrations of salt and thus have great application potential. The mechanisms of salt-induced activity enhancement of laccases are unclear as yet. In this study, we used dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, intrinsic fluorescence emission, circular dichroism, ultraviolet–visible light absorption, and an enzymatic assay to investigate the potential correlation between the structure and activity of the marine-derived laccase, Lac15, whose activity is promoted by low concentrations of NaCl. The results showed that low concentrations of NaCl exert little influence on the protein structure, which was partially folded in the absence of the salt; moreover, the partially folded rather than the fully folded state seemed to be favorable for enzyme activity, and this partially folded state was distinctive from the so-called ‘molten globule’ occasionally observed in active enzymes. More data indicated that salt might promote laccase activity through mechanisms involving perturbation of specific local sites rather than a change in global structure. Potential binding sites for chloride ions and their roles in enzyme activity promotion are proposed.

Lutimaribacter marinistellae sp. nov., isolated from a starfish

A taxonomic study was carried out on a Gram-staining-negative bacterium, strain SF-12T, isolated from an unidentified starfish living in Sanya, PR China. Cells of SF-12T were non-spore-forming rods, 0.5-0.8 µm wide, 2.2-2.5 µm long and motile by means of flagella. SF-12T was facultatively anaerobic, heterotrophic, oxidase- and catalase-positive. Growth of SF-12T occurred at 15-38 °C (optimum, 30 °C), at pH 6.5-8.5 (optimum, pH 7.0), and in the presence of 2.0-7.0 % (w/v) NaCl (optimum, 3.0-4.0 %). The predominant fatty acids of SF-12T were C18 : 1ω7c and/or C18 : 1ω6c. Ubiquinone 10 was the sole respiratory quinone of SF-12T. The major polar lipids of SF-12T were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, three unknown aminolipids, and seven unknown phospholipids. The DNA G+C content was 61 mol%. SF-12T showed the highest 16S rRNA gene sequence similarity to Lutimaribacter pacificus W11-2BT (96.06 %), followed by Cribrihabitans neustonicus CC-AMHB-3T (96.02 %), Lutimaribacter saemankumensis SMK-117T (96.0 %), Cribrihabitans marinus CZ-AM5T (95.92 %), Lutimaribacter litoralis KU5D5T (95.92 %) and other species of the family Rhodobacteraceae(<95.9 %). However, phylogenetic trees based on 16S rRNA gene sequences showed that SF-12T formed a lineage with members of the genus Lutimaribacter in the trees. On the basis of phenotypic, chemotaxonomic and phylogenetic analyses, SF-12T is considered to represent a novel species of the genus Lutimaribacter, for which the name Lutimaribacter marinistellae sp. nov. is proposed. The type strain is SF-12T (=MCCC 1K01154T=KCTC 42911T).

Genome sequence of a laccase producing fungus Trametes sp. AH28-2.

Trametes sp. AH28-2 (CCTCC AF 2015027) is a white rot fungus isolated from rotting wood in China. Primary study indicated that this strain can be induced by kraft lignin to secrete high levels of extracellular laccase, and differentially express laccase genes upon addition of different phenolic compounds. Here we report the complete genome sequence of Trametes sp. AH28-2 and its genetic basis for lignin degradation and phenolic xenobiotics metabolism.

Complete genome of Gongronella sp. w5 provides insight into its relationship with plant

Gongronella sp. w5 (w5) is a soil fungus isolated from Anhui, China. Here we report the high-quality genome sequence of w5 and its phenotypic characteristics based on genomic information. The genome of w5 consists of 34,723,828 bp assembled into 149 scaffolds and 11,302 predicted protein-coding genes. Genome analysis suggested that w5 may possess host cell infection capacity and maybe a biotrophic fungus that relies on plant sucrose as carbon source. W5 shows the ability of rapid invasion into the plant root cells based on CAZymes analysis. Further results evidenced that w5 can use sucrose as the carbon source. Plant inoculation revealed that w5 penetrates the root cells of Actinidia chinensis with its hypha, and simultaneously promotes plant growth. It may promote plant growth by secreting organic acid and facilitating phosphate acquisition. The new genomic data and phenotype features will facilitate future applications of this strain in biotechnology.