Screening of Burkholderia sp. WGB31 producing anisic acid from anethole and optimization of fermentation conditions.

Anisic acid, the precursor of a variety of food flavors and industrial raw materials, can be bioconversed from anethole which extracted from star anise fruits. WGB31 strain with anisic acid molar production rate of 10.25% was isolated and identified as Burkholderia sp. Three significant influential factors, namely, glucose concentration, initial pH value, and medium volume were selected and their effects were evaluated by Box-Behnken Design (BBD). Regression analysis was performed to determine response surface methodology and the significance was tested to obtain the process model of optimal conditions for producing anisic acid. The fermentation conditions at the stable point of the model were obtained: glucose 6 g L(-1) , pH 6.2, culture medium volume 61 mL in a triangular flask with 250 ml volume. Verification test indicated that the production rate of anisic acid was 30.7%, which was three times of that before optimizing. The results provide a basis and reference for producing anisic acid by microbial transformation.

Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms.

BACKGROUND: Fumarase catalyzes the reversible hydration of fumarate to L-malate and is a key enzyme in the tricarboxylic acid (TCA) cycle and in amino acid metabolism. Fumarase is also used for the industrial production of L-malate from the substrate fumarate. Thermostable and high-activity fumarases from organisms that inhabit extreme environments may have great potential in industry, biotechnology, and basic research. The marine environment is highly complex and considered one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms are inaccessible in nature and are not easily cultivated in the laboratory. Metagenomic approaches provide a powerful tool to isolate and identify enzymes with novel biocatalytic activities for various biotechnological applications. RESULTS: A plasmid metagenomic library was constructed from uncultivated marine microorganisms within marine water samples. Through sequence-based screening of the DNA library, a gene encoding a novel fumarase (named FumF) was isolated. Amino acid sequence analysis revealed that the FumF protein shared the greatest homology with Class II fumarate hydratases from Bacteroides sp. 2_1_33B and Parabacteroides distasonis ATCC 8503 (26% identical and 43% similar). The putative fumarase gene was subcloned into pETBlue-2 vector and expressed in E. coli BL21(DE3)pLysS. The recombinant protein was purified to homogeneity. Functional characterization by high performance liquid chromatography confirmed that the recombinant FumF protein catalyzed the hydration of fumarate to form L-malate. The maximum activity for FumF protein occurred at pH 8.5 and 55°C in 5 mM Mg(2+). The enzyme showed higher affinity and catalytic efficiency under optimal reaction conditions: K(m) = 0.48 mM, V(max) = 827 µM/min/mg, and k(cat)/K(m) = 1900 mM/s. CONCLUSIONS: We isolated a novel fumarase gene, fumF, from a sequence-based screen of a plasmid metagenomic library from uncultivated marine microorganisms. The properties of FumF protein may be ideal for the industrial production of L-malate under higher temperature conditions. The identification of FumF underscores the potential of marine metagenome screening for novel biomolecules.

Changes in microbial community structure in two anaerobic systems to treat bagasse spraying wastewater with and without addition of molasses alcohol wastewater.

This study investigates the microbial community and structure in the internal circulation (IC) reactors that treat wastewater from bagasse spraying, with (reactor B) and without (reactor A) addition of molasses alcohol wastewater (MAW). The V3 regions in the 16S rRNA of bacteria were sequenced using illumina sequencing to characterize the microbial community structures. The results showed that there were approximately 34.8% more microorganisms were reduced, while the proportions of the three most predominant bacterial populations especially some sulfate-reducing bacteria increased in reactor B. The archaeal community composition was measured by PCR-DGGE (denaturing gradient gel electrophoresis) analysis and sequencing some clones from the 16S rRNA gene library. The results showed that numerous, mostly uncharacterized, archaeal genera are present in reactors A and B; the genus Methanomethylovorans was only detected in the samples that received MAW. This study demonstrated the significant effect of MAW on microbial communities in the wastewater treatment bioreactor.

Biochemical characterization of two novel ß-glucosidase genes by metagenome expression cloning.

Two novel ß-glucosidase genes designated as bgl1D and bgl1E, which encode 172- and 151-aa peptides, respectively, were cloned by function-based screening of a metagenomic library from uncultured soil microorganisms. Sequence analyses indicated that Bgl1D and Bgl1E exhibited lower similarities with some putative ß-glucosidases. Functional characterization through high-performance liquid chromatography demonstrated that purified recombinant Bgl1D and Bgl1E proteins hydrolyzed D-glucosyl-ß-(1-4)-D-glucose to glucose. Using p-nitrophenyl-ß-D-glucoside as substrate, K(m) was 0.54 and 2.11 mM, and k(cat)/K(m) was 1489 and 787 mM(-1) min(-1) for Bgl1D and Bgl1E, respectively. The optimum pH and temperature for Bgl1D was pH 10.0 and 30°C, while the optimum values for Bgl1E were pH 10.0 and 25°C. Bgl1D exhibited habitat-specific characteristics, including higher activity in lower temperature and at high concentrations of AlCl(3) and LiCl. Bgl1D also displayed remarkable activity across a broad pH range (5.5-10.5), making it a potential candidate for industrial applications.

[Cu and Fe bioleaching in low-grade chalcopyrite and bioleaching mechanisms using Penicillium janthinellum strain GXCR].

Bioleaching of Cu and Fe in low-grade chalcopyrite using Penicillium janthinellum strian GXCR was studied. As a result, shaking bioleaching was more efficient than submerged bioleaching; Cu bioleaching was much better than Fe bioleaching; under conditions of optimum carbon source (10% sucrose, W/V), optimum nitrogen source (1.5% NaNO3, W/V), shaking bioleaching and the optimum combination of conditions (initial pH 6.0 in leaching media, 5% (W/V) 200-mesh ore and initial inocula of 3.0x10(5) conidia/mL), Cu bioleaching efficiency reached 87.31% (W/W). One of the most important factors affecting Cu bioleaching in shaking bioleaching was the initial pH in leaching media (F > F0.05). The major organic acids for Cu and Fe bioleaching were citric and oxalic acids, respectively. Low bioleaching efficiency by submerged bioleaching was due to low production of citric and oxalic acids. The mechanisms employed by the GXCR in Cu bioleaching included biochemical functions of citric and oxalic acids as well as ore crack caused by mechanical power generated from mycelial growth.