Cloning, expression of a feruloyl esterase from Aspergillus usamii E001 and its applicability in generating ferulic acid from wheat bran.

A cDNA gene (AufaeA), which encodes a mature polypeptide of the type-A feruloyl esterase from Aspergillus usamii E001 (abbreviated to AuFaeA), was cloned and heterologously expressed in Pichia pastoris GS115. One transformant, labeled as P. pastoris GSFaeA4-8, expressing the highest recombinant AuFaeA (reAuFaeA) activity of 10.76 U/ml was selected by the flask expression test. The expressed reAuFaeA was purified to homogeneity with an apparent molecular weight of 36.0 kDa by SDS-PAGE analysis, and characterized using the model substrate of methyl ferulate (MFA). The purified reAuFaeA was optimally active at pH 5.0 and 45 °C, and highly stable at pH 4.0-6.5 and 45 °C or below. Its activity was not significantly affected by metal ions tested and EDTA. The K m and V max of reAuFaeA towards MFA were 4.64 mM and 115.5 U/mg, respectively. High-performance liquid chromatography analysis showed that only 9.7 % of total alkali-extractable ferulic acid (FA) was released from destarched wheat bran by reAuFaeA alone. The released FA increased to 36.5 % when reAuFaeA was used together with a recombinant Aspergillus usamii GH family 11 xylanase A, indicating a synergistic interaction between them.

Strong influence of surfactants on virgin hydrophobic microplastics adsorbing ionic organic pollutants.

Microplastic (MP) pollution has become an area of increasing concern because MPs accumulate various types of pollutants. Many previous studies have explored the interactions between MPs and hydrophobic pollutants. However, little research has been conducted on hydrophilic pollutants, which are of much higher concentration and ubiquitous in environment. Surfactants cause hydrophobic MPs to become hydrophilic, which may significantly enhance their capacities to adsorb hydrophilic pollutants. This study explored the influence of co-existing surfactants on the adsorption of ionic organic pollutants by MPs, and found that the presence of an ionic surfactant could significantly enhance the capacity of polyvinyl chloride (PVC, 0.2 mm) MPs to adsorb pollutants with opposite charges. The Langmuir methylene blue adsorption capacity of PVC could be increased from 172 to 4417 ppm in the presence of a sodium dodecyl benzene sulfonate surfactant. Nonionic surfactants impeded the adsorption of both cationic and anionic pollutants due to the steric resistance of the hydrophilic polyethelene glycol chains. The electrostatic interaction mechanism dominated the interfacial behaviors of ionic pollutants on surfactant-adsorbed MP interfaces. The effects of the surfactants were further verified using four different model pollutants and six surfactants. The adsorption capacities of real environmental MPs, including PVC, polyethylene (PE), polypropylene (PP), and polystyrene (PS), increased by three to twenty-six times. The adsorption properties of MPs may be determined by the presence of co-existing surfactants, rather than their polymer species or additives.

Surfactant stealth effect of microplastics in traditional coagulation process observed via 3-D fluorescence imaging.

Microplastics (MPs) have aroused rising social concerns. Although amounts of surfactants exist in wastewater and are expected to alter the surface properties of MPs significantly as they are designed to be adsorbed by hydrophobic particles. However, rare works have been done on the influence of surfactants on the coagulation removal process of MPs which was thought to be an effective way to remove MPs together with other natural particles, such as clay. We used 3-D fluorescence imaging to track the coagulation removal process of polystyrene MPs. Our results indicate that nonionic surfactant, tween 20 in ppm scale, could inhibit the coagulation removal of polystyrene MPs significantly. Residue MPs in the effluent is proportional with the surfactant concentration and increases up to tens of times, which will lead to a dramatic increase in their potential environmental risks. Apparent size effect exists in the coagulation in which smaller MPs can escape from the coagulation removal more easily. Mechanism study suggests that the steric resistance of the hydrophilic flexible polyethylene glycol (PEG) layer formed by tween 20 adsorbed on MP surface inhibits clay deposition and thus hinders subsequent agglomeration and precipitation. A surfactant stealth effect, which is used in the design of nanomedicine to avoid the human immune recognition and clearance of nano-drugs from blood circulation, also exists in the coagulation removal process of MPs. Our finding not only proves the strong influence of surfactants on MPs but also will stimulate related studies on other latent surfactant effects of MPs.

Cloning and expression of a family 10 xylanase gene (Aoxyn10) from Aspergillus oryzae in Pichia pastoris.

A full-length cDNA sequence of Aoxyn10, a gene encoding a glycoside hydrolase (GH) family 10 xylanase from Aspergillus oryzae, was amplified from the total RNA by 3' and 5' rapid amplification of cDNA ends. The cDNA sequence is 1,689 bp, containing 5', 3' untranslated regions and a 1,422 bp open reading frame (ORF) that encodes a 21-aa signal peptide and a 452-aa mature peptide (designated AoXyn10). Multi-alignment revealed that AoXyn10 contains two regions: a catalytic domain (CD) and a family 1 carbohydrate-binding module (CBM1). The three-dimensional (3-D) structure of the CD was predicted by multiple template-based homology modeling. A 2,308-bp complete DNA sequence of Aoxyn10 was obtained from the genomic DNA by both pUCm-T vector-mediated and conventional PCRs, harboring 5', 3' flanking regulatory regions, five exons and four introns. Moreover, Aoxyn10 was extracellularly expressed in Pichia pastoris. One transformant labeled as P. pastoris GS/Xyn4-11 was selected, expressing the highest recombinant AoXyn10 (named reAoXyn10) activity of 45.0 U/ml. SDS-PAGE analysis revealed that reAoXyn10, a glycoprotein with an apparent molecular weight (M.W.) of about 56.0 kDa, was secreted into the cultured medium. The purified reAoXyn10 displayed the maximum activity at pH 5.5 and 60°C. It was stable at a pH range of 4.0-7.0, and at 50°C or below. Its activity was not affected by an array of metal ions or EDTA, but was inhibited by Mn(2+) and Ba(2+). The Km and Vmax of reAoXyn10 were 1.7 mg/ml and 817 µmol/min/mg, respectively.

[Antigenic comparative analysis of Newcastle disease viruses with evolutional mutations in HN and F genes under antibody immune pressures].

In chicken fibroblast cell (CEF) cultures with antiserum against Newcastle disease virus (NDV) strain TZ060107, the virus was passed serially for 50 passages in 3 independent lineages. HN and F genes were amplified and sequenced every 10 passages. The derived virus A1-50 with most mutations among 3 lineages was further passed for another 50 passages in CEF with or without antiserum against A1-50, each in 3 independent lineages. Sequence comparisons for HN and F genes of 60, 70, 80, 90 and 100 passages indicated that the ratio of nonsynonymous mutations (NS) vs synonymous mutations (S) for HN genes in the lineages passed with antiserum against A1-50 was 5.25, which was obviously higher than 2. 375 of NS/ S in the lineages without the antiserum. The stable NS mutations occurred in the first 50 passages with the antiserum against the original TZ060107 were still maintained and one more new stable NS mutation appeared. For the F gene, 3 new stable NS mutations occurred during the second 50 passages in lineages with antiserum against A1-50 when the original NS mutations obtained in the first 50 passages with antiserum against TZ060107 still existed. Cross hemagglutination inhibition (HI) between original virus and its derivative viruses indicated that the more continuous passages in cell culture with antiserum passed, the bigger difference of antigenicity between the virus and the original virus had.

Influence of Landmarks on Spatial Memory in Short-nosed Fruit Bat, Cynopterus sphinx.

In order to study the relationship between landmarks and spatial memory in short-nosed fruit bat, Cynopterus sphinx (Megachiroptera, Pteropodidae), we simulated a foraging environment in the laboratory. Different landmarks were placed to gauge the spatial memory of C. sphinx. We changed the number of landmarks every day with 0 landmarks again on the fifth day (from 0, 2, 4, 8 to 0). Individuals from the control group were exposed to the identical artificial foraging environment, but without landmarks. The results indicated that there was significant correlation between the time of the first foraging and the experimental days in both groups (Pearson Correlation: experimental group: r=-0.593, P<0.01; control group: r=-0.581, P<0.01). There was no significant correlation between the success rates of foraging and the experimental days in experimental groups (Pearson Correlation: r=0.177, P>0.05), but there was significant correlation between the success rates of foraging and the experimental days in the control groups (Pearson Correlation: r=0.445, P<0.05). There was no significant difference for the first foraging time between experimental and control groups (GLM: F(0.05,1 )=4.703, P>0.05); also, there was no significant difference in success rates of foraging between these two groups (GLM: F(0.05,1 )=0.849, P>0.05). The results of our experiment suggest that spatial memory in C. sphinx was formed gradually and that the placed landmarks appeared to have no discernable effects on the memory of the foraging space.

[New record of a bat species from China, Megaderma spasma (Linnaeus, 1758)].

One male bat was collected in a cave in Cuibi Hill (N: 21 degree 53', E: 101 degree 18', H: 683 m a.s.l.), Menglun Town, Mengla County, Yunnan Province, on November 26, 2006. This bat is of medium body size, with 60.7 mm forearm and 21.4 g body mass. Its ears are large ovals and joined medially to the forehead at about 15% of the height of inner margin. The tragus of each ear is slender and distinctly bifid. The noseleaf is simple, and the posterior noseleaf is oval with obtuse tip and convex sides, a significant longitudinal ridge laying middle, which connects to intermediate noseleaf at the base. Intermediate noseleaf presents triangle with a W-shaped tip. Frontal noseleaf is horseshoe shape and attached directly to the muzzle. The tail is absent, and the second finger of each wing has only one phalanx. There are no upper incisors, Pm3 and Pm3. The upper canine has an anterior and a large posterior basal cusps. This bat is identified as lesser false vampire, Megaderma spasma, which is a new record of China. Its external and craniodental measurements were presented and compared with those of M. lyra. The specimen is preserved in Guangdong Entomological Institute.