Preparation of carboxymethyl cellulose/chitosan-CuO giant vesicles for the adsorption and catalytic degradation of dyes.

To effectively remove the dyes from wastewater, novel carboxymethyl cellulose/chitosan-CuO giant vesicles with dual function of adsorption and catalytic degradation were prepared. The vesicles were facilely obtained via blending chitosan solution and carboxymethyl cellulose/CuO mixed solution with sequent fast and slow stirring. The removal ratios of methyl orange (MO) and acid black-172 (AB) can reach 86.3% and 88.6% with the catalytic oxidation system of ammonium persulfate and vesicles. Compared with the CuO catalysis without the vesicles, the degradation rates of MO and AB increased by 1.3 and 3.1 times, respectively. The enhanced dye removal is ascribed to the excellent dye adsorption capacity of giant vesicles. Furthermore, the giant vesicles worked well in wide ranges of environmental pH and temperature, and exhibited excellent stability and reusability. This study provides a facile method to load catalyst onto polymeric giant vesicle with outstanding performance for the adsorption and catalytic degradation of dyes.

Refolding and purification of rhNTA protein by chromatography.

RhNTA protein is a new thrombolytic agent which has potential medicinal and commercial value. Protein refolding is a bottleneck for large-scale production of valuable proteins expressed as inclusion bodies in Escherichia coli. The denatured rhNTA protein was refolded by an improved size-exclusion chromatography refolding process achieved by combining an increasing arginine gradient and a decreasing urea gradient (two gradients) with a size-exclusion chromatography refolding system. The refolding of denatured rhNTA protein showed that this method could significantly increase the activity recovery of protein at high protein concentration. The activity recovery of 37% was obtained from the initial rhNTA protein concentration up to 20 mg/mL. After refolding by two-gradient size-exclusion chromatography refolding processes, the refolded rhNTA was purified by ion-exchange and affinity chromatography. The purified rhNTA protein showed one band in SDS-PAGE and the specific activity of purified rhNTA protein was 110,000 U/mg.

Biodegradation of Structurally Diverse Phthalate Esters by a Newly Identified Esterase with Catalytic Activity toward Di(2-ethylhexyl) Phthalate.

Herein, we report a double enzyme system to degrade 12 phthalate esters (PAEs), particularly bulky PAEs, such as the widely used bis(2-ethylhexyl) phthalate (DEHP), in a one-pot cascade process. A PAE-degrading bacterium, Gordonia sp. strain 5F, was isolated from soil polluted with plastic waste. From this strain, a novel esterase (GoEst15) and a mono(2-ethylhexyl) phthalate hydrolase (GoEstM1) were identified by homology-based cloning. GoEst15 showed broad substrate specificity, hydrolyzing DEHP and 10 other PAEs to monoalkyl phthalates, which were further degraded by GoEstM1 to phthalic acid. GoEst15 and GoEstM1 were heterologously coexpressed in Escherichia coli BL21 (DE3), which could then completely degrade 12 PAEs (5 mM), within 1 and 24 h for small and bulky substrates, respectively. To our knowledge, GoEst15 is the first DEHP hydrolase with a known protein sequence, which will enable protein engineering to enhance its catalytic performance in the future.

Improving the refolding of NTA protein by urea gradient and arginine gradient size-exclusion chromatography.

Inclusion body refolding processes play a major role in the production of recombinant proteins. Improvement of the size-exclusion chromatography refolding process was achieved by combining a decreasing urea gradient with an increasing arginine gradient (two gradients) for the refolding of NTA protein (a new thrombolytic agent) in this paper. Different refolding methods and different operating conditions in two gradients gel filtration process were investigated with regard to increasing the NTA protein activity recovery and inhibition of aggregation. The refolding of denatured NTA protein showed this method could significantly increase the activity recovery of protein at high protein concentration. The activity recovery of 37% was obtained from the initial NTA protein concentration up to 20 mg/ml. The conclusions presented in this study could also be applied to the refolding of lysozyme.

[Bioassay-guided isolation of functional components from hot water extract of Chlorella pyrenoidosa].

The main functional ingredients of hot water extract of Chlorella pyrenoidosa (CPE) were investigated through a bioassay-guided fractionation based on free radical scavenging and macrophage proliferation effects. The main functional ingredients of CPE were polysaccharides (PS) that were isolated by high pressure extraction, Sevag method, ethanol precipitation and ultrafiltration separation. Crude polysaccharides were further separated and purified by ion exchange chromatography DEAE52 and size exclusion chromatography Sephadex G-100. The purified fractions were analyzed by gel permeation chromatography. Molecular weights of the purified fractions PS-1-4-2, PS-1-3-2 and PS-2-3-3 were 3.97×104, 2.28×104 and 4.1×10³ Da, respectively. Bioassay-guided fractionation results indicated that CPE could remove free radicals and promote Ana-1 cells proliferation, mainly due to its various components working together. The components of free radicals scavenging mainly concentrated in PS-1-3, PS-1-4, PS-2-3 and PS-2-4. The components of Ana-1 proliferation mainly concentrated in PS-1-3, PS-1-4 and PS-2-3. This study established the activity screening method of main functional component from CPE, and got three new functional ingredients. It can be used to guide the development of high value products, further promote the industrialization process of microalgae energy, and realize microalgae 'high value products, microalgae energy and microalgae carbon' integration of exemplary role.

Efficient Degradation of Malathion in the Presence of Detergents Using an Engineered Organophosphorus Hydrolase Highly Expressed by Pichia pastoris without Methanol Induction.

The biodegradation of pesticides by organophosphorus hydrolases (OPHs) requires an efficient enzyme production technology in industry. Herein, a Pichia pastoris strain was constructed for the extracellular expression of PoOPHM9, an engineered malathion-degrading enzyme. After optimization, the maximum titer and yield of fermentation reached 50.8 kU/L and 4.1 gprotein/L after 3 days, with the highest space-time yield (STY) reported so far, 640 U L-1 h-1. PoOPHM9 displayed its high activity and stability in the presence of 0.1% (w/w) plant-derived detergent. Only 0.04 mg/mL enzyme could completely remove 0.15 mM malathion in aqueous solution within 20 min. Furthermore, 12 µmol malathion on apples and cucumbers surfaces was completely removed by 0.05 mg/mL PoOPHM9 in tap water after 35 min washing. The efficient production of the highly active PoOPHM9 has cleared a major barrier to biodegradation of pesticide residues in food industry.

[Response of Soil Respiration and Organic Carbon to Returning of Different Agricultural Straws and Its Mechanism].

Soybean, maize and rice straws were selected as raw materials to study the response of the soil respiration (SR) and soil organic carbon (SOC) to returning of different straws in the Chongming Dongtan area. The results showed that all of SR, SOC and the plant biomass of the lands with returning of different straws were higher than those of the controls. The soil with soybean straw returning possessed the lowest SR and highest SOC among the three kinds of straws, meaning its higher soil organic carbon sequestration capability than corn and maize straws returning. Straw returning significantly enhanced soil dehydrogenase, ß-glycosidase activities and microbial biomass, and soil dehydrogenase activity was significantly correlated with soil respiration. The dehydrogenase activity of the soil with soybean straw returning was the lowest, thus, the lowest SR and highest SOC. Soybean straw had the highest cellulose and lignin contents and the lowest N content among the three kinds of straws, resulting in its lowest biodegradability. Therefore, when soybean straw was returned to soil, it was difficult to degrade completely by soil microorganisms, thus the lowest soil microbial activity, eventually leading to the lowest SR and highest SOC.

[The separation and identification of the flavonoids from the leaves of Bryophyllum pinnatum].

This study investigated the separation of the effective compositions containing quercetin derivatives from the leaves of Bryophyllum pinnatun by solvent extraction of methanol, polar adsorption by porous polymetric resins, mixed solvent extraction of methanol with chloroform and polyamide chromatography. The results indicated that the yield of product containing 51.23% quercetin in the form of quercetin derivatives was 0.2% (W/W). Through the NMR, Mass spectrum, Infrared spectrum, we try to identify the chemical structure of the flavonoids LEF. As a result we can conclude that the chemical structure of the flavonoids from the leaves of Bryophy Ilum pinnatum is C26H28O15 (quercetin3-alpha-L-rha-beta-D-xyl), molecular weight is 580.

[Potential Carbon Fixation Capability of Non-photosynthetic Microbial Community at Different Depth of the South China Sea and Its Response to Different Electron Donors].

The seawater samples collected from many different areas with different depth in the South China Sea were cultivated using different electron donors respectively. And the variation in the potential carbon fixation capability ( PCFC ) of non-photosynthetic microbial community (NPMC) in seawater with different depth was determined after a cycle of cultivation through the statistic analysis. In addition, the cause for the variation was clarified through analyzing key gene abundance regarding CO2 fixation and characteristics of seawater with different depth. The result showed that the PCFCs of NPMC in seawater with different depth were generally low and had no significant difference when using NaNO2 as the electron donor. The PCFC of NPMC in surface seawater was higher than that in deep seawater when using H2 as the electron donor, on the contrary, the PCFC of NPMC in deep seawater was higher than that in surface seawater when using Na2S2O3 as the electron donor. The abundance of the main CO2 fixation gene cbbL in surface seawater was higher than that in deep seawater while the cbbM gene abundance in deep seawater was higher than that in surface seawater. Most hydrogen-oxidizing bacteria had the cbbL gene, and most sulfur bacteria had the cbbM gene. The tendency of seawater cbbL/cbbM gene abundance with the change of depth revealed that there were different kinds of bacteria accounting for the majority in NPMC fixing CO2 at different depth of ocean, which led to different response of PCFC of NPMC at different depth of the sea to different electron donors. The distributions of dissolved oxygen and inorganic carbon concentration with the change of the depth of the sea might be an important reason leading to the difference of NPMC structure and even the difference of PCFC at different depth of the sea.

Similarity between segments in protein conformational epitopes and MHC II peptides.

In immune response, major histocompatibility complex class II (MHC II) molecules bind peptides derived from antigens and present them. The formation of MHC II-peptide complexes was a critical signal in inducing of antibodies production of B cells. In this research, we tried to investigate whether there was a similarity or relationship between the segments in B cell conformational epitopes and MHC II peptides. All the segments from the conformational epitopes and MHC II peptides were extracted and the relationship between them was analysed. From current results, it can be hinted that the epitope segments have a higher similarity to the known MHC II peptides. Furthermore, the majority of known conformational epitopes were found to contain at least one segment identical or similar to a MHC II peptide. Such results may not only facilitate the study of antigen information processing but also help to improve the conformational epitope prediction algorithm.

Purification and in situ immobilization of papain with aqueous two-phase system.

Papain was purified from spray-dried Carica papaya latex using aqueous two-phase system (ATPS). Then it was recovered from PEG phase by in situ immobilization or preparing cross-linked enzyme aggregates (CLEAs). The Plackett-Burman design and the central composite design (CCD) together with the response surface methodology (RSM) were used to optimize the APTS processes. The highly purified papain (96-100%) was achieved under the optimized conditions: 40% (w/w) 15 mg/ml enzyme solution, 14.33-17.65% (w/w) PEG 6000, 14.27-14.42% (w/w) NaH2PO4/K2HPO4 and pH 5.77-6.30 at 20°C. An in situ enzyme immobilization approach, carried out by directly dispersing aminated supports and chitosan beads into the PEG phase, was investigated to recover papain, in which a high immobilization yield (>90%) and activity recovery (>40%) was obtained. Moreover, CLEAs were successfully used in recovering papain from PEG phase with a hydrolytic activity hundreds times higher than the carrier-bound immobilized papain.

[Breeding, optimization and community structure analysis of non-photosynthetic CO2 assimilation microbial flora].

Isolation and screening from sea water and sediments, and the optimization of electron donor and inorganic carbon source structure were performed for obtaining microbial flora with high efficient inorganic carbon fixation without the light and hydrogen. In addition, the structure of the microbial flora was studied through 16S rDNA sequence analysis and contrast for providing theoretical basis to improve carbon fixation efficiency through optimizing microbial flora structure. The result showed that non-photosynthetic microbial flora with the capacity of inorganic carbon fixation under the general aerobic and anaerobic conditions could be obtained from the sea by long-term domestication and isolation. Inorganic carbon fixation efficiency of the microbial flora was enhanced significantly by adding of sodium thiosulfate, sodium sulfide and hydrogen as electron donor. Under the aerobic and anaerobic conditions with sodium thiosulfate as electron donor, the efficiency of inorganic carbon assimilation was 10.44 mg/L and 12.56 mg/L respectively. The assimilation efficiency of the microbial flora with mixed inorganic carbon source was higher than that with single carbon source. When CO2, sodium bicarbonate and sodium carbonate were added as carbon sources, carbon fixation efficiency of the microbial flora under the aerobic and anaerobic condition was 110 mg x (L x d)(-1) and 72 mg x (L x d)(-1) respectively which had been closed to the efficiency of hydrogen-oxidizing bacteria. The analysis results showed that the predominant species of the microbial flora varied significantly after the adding of different electron donor. And 11 species of the 16 predominant species in the microbial flora was uncultured. It means that the microbial flora could only exist in symbiotic manner. The inorganic carbon fixation effect of the microbial flora may be the results of co-function of multi-microbial species. Therefore, the optimization of microbial flora structure and proportion is benefit for the further improvement of carbon fixation efficiency.