•A novel gene ManB (GenBank: KJ806638) was synthesized following the amino acid sequence of mannanase 1BQC.•A multi-copy secretive expression vector pAOhr was constructed to introduce the gene ManB into Pichia pastoris GS115.•This recombinant alkaline thermostable mannanase ManB was characterized; Molecular docking showed that Tris molecule can bind to the enzyme active site.•The additive mannanase ManB was helpful to remove the gums when combined with Bacillus sp. HG-28 for ramie degumming.A codon optimized synthetic alkaline thermostable Thermobifida fusca β-mannanase ManB (KJ806638) was expressed in Pichia pastoris and used in ramie degumming. To improve the expression level, a multi-copy secretion expression vector pAOhr was constructed to introduce the ManB gene into Pichia pastoris GS115. The highest secretion yield was obtained from a transformant strain containing six copies of ManB gene. The size of ManB protein was 34 kDa in SDS-PAGE and the secreted protein was the main protein in the culture broth. The optimal activity region of ManB was at pH 7–9 and the enzyme was quite stable at pH 6–10. At pH 9, the specific activity of ManB was 493.8 IU/mg and the optimum temperature was 70–75 °C. ManB appeared to be inhibited by Tris buffer. Molecular docking showed that Tris molecule can bind to the enzyme active site. ManB exhibited high activity for locust bean gum, whereas it showed in practice no activity for CMC-Na. Ramie degumming was performed with combined treatment by ManB and Bacillus sp. HG-28 expressing pectinase and xylanase. The obtained results demonstrated that the combination treatment with additional mannanase enzyme was more efficient in removing the gums than the treatment merely by the bacterial strain.Download high-res image (233KB)Download full-size image

Methyl jasmonate (MeJA) is one of the most effective inducers of taxol biosynthetic genes, particularly the tasy gene. However, the mechanism underlying the regulation of tasy by MeJA is still unknown. In this study, a 550-bp 5′-flanking sequence was obtained and confirmed as the promoter of the tasy gene. Deletion analysis revealed that the fragment containing a GCC-box from −150 to −131 was the crucial jasmonate (JA)-responsive element, designated as JRE. Using JRE as bait, two binding proteins, namely TcERF12 and TcERF15, were discovered. Sequence alignment and phylogenetic analysis showed that TcERF12 was related to the repressor AtERF3, while TcERF15 was more related to the activator ORA59; these are typical GCC-box-binding ethylene-responsive factors. Both could significantly respond to MeJA for 10 and 4.5 times, respectively, in 0.5 h. When the two TcERFs were overexpressed in Taxus cells, tasy gene expression decreased by 2.1 times in TcERF12-overexpressing cells, but increased by 2.5 times in TcERF15-overexpressing cells. Results indicated that TcERF12 and TcERF15 were negative and positive regulators, respectively, in the JA signal transduction to the tasy gene by binding the GCC-box in the JRE of the tasy promoter. Our results promote further research on regulatory mechanisms of taxol biosynthesis.

The dynamics of calcium carbonate (CaCO3) precipitation induced by microbial intracellular or extracellular carbonic anhydrase (CA) at initial pH 6.0, 6.5, 7.0 and 8.0 were investigated through the gaseous diffusion method. The results indicated that both the intracellular and extracellular CA could promote CaCO3 precipitation. The Ca2+ ions in the enzymatic systems at initial pH 8.0 were completely deposited at 48 h, which were respectively 21 h, 15 h and 14 h earlier compared with that at initial pH 6.0, pH 6.5 and pH 7.0, indicating that higher pH favored CaCO3 precipitation in the experimental pH range, and was beneficial to the catalytic action of microbial CA on CaCO3 precipitation. In addition, XRD analysis indicated that the CaCO3 precipitates were mainly calcite crystals in the presence of microbial CA. With increasing deposition time, the crystals gradually changed from prism shape to pyramid-like or irregular polyhedral shape based on FESEM analysis.Graphical abstract.Highlights► Influence of initial pH on CaCO3 precipitation induced by microbial CA. ► Both the intracellular and extracellular CA promoted CaCO3 precipitation. ► The higher pH favored catalytic action of microbial CA on CaCO3 precipitation. ► The CaCO3 crystals were mainly calcite in the presence of microbial CA. ► The morphology of crystals induced by microbial CA changed with time.

In this paper, the interaction of human serum albumin (HSA) with phillygenin was investigated by fluorescence, circular dichroism (CD), UV–vis spectroscopic and molecular docking methods under physiological conditions. The Stern–Volmer analysis indicated that the fluorescence quenching of HSA by phillygenin resulted from static mechanism, and the binding constants were 1.71 × 105, 1.61 × 105 and 1.47 × 104 at 300, 305 and 310 K, respectively. The results of UV–vis spectra show that the secondary structure of the protein has been changed in the presence of phillygenin. The CD spectra showed that HSA conformation was altered by phillygenin with a major reduction of α-helix and an increase in β-sheet and random coil structures, indicating a partial protein unfolding. The distance between donor (HSA) and acceptor (phillygenin) was calculated to be 3.52 nm and the results of synchronous fluorescence spectra showed that binding of phillygenin to HSA can induce conformational changes in HSA. Molecular docking experiments found that phillygenin binds with HSA at IIIA domain of hydrophobic pocket with hydrogen bond interactions. The ionic bonds were formed with the O (4), O (5) and O (6) of phillygenin with nitrogen of ASN109, ARG186 and LEU115, respectively. The hydrogen bonds are formed between O (2) of phillygenin and SER419. In the presence of copper (II), iron (III) and alcohol, the apparent association constant KA and the number of binding sites of phillygenin on HSA were both decreased in the range of 88.84–91.97% and 16.09–18.85%, respectively. In view of the evidence presented, it is expected to enrich our knowledge of the interaction dynamics of phillygenin to the important plasma protein HSA, and it is also expected to provide important information of designs of new inspired drugs.Graphical abstractThe interaction between HSA and phillygenin at physiological conditions by fluorescence, circular dichroism (CD), UV–vis spectroscopy and molecular docking to enrich our knowledge of the interaction dynamics of phillygenin to the important plasma protein HSA, and it is also expected to provide important information of designs of new inspired drugs.Highlights► We investigate the interaction between HSA and phillygenin. ► Fluorescence, CD and UV spectra study shows complex formation between phillygenin and HSA. ► The fluorescence quenching of HSA by phillygenin was static mechanism. ► Van der Waals forces and hydrogen bonds play a role in stabilizing the complex. ► Metal ions and ethanol can increase the concentration of free phillygenin.

Gradual loss of secondary metabolite production is a common obstacle in the development of a large-scale plant cell production system. In this study, cell morphology, paclitaxel (Taxol®) biosynthetic ability, and genetic and epigenetic variations in the long-term culture of Taxus media cv Hicksii cells were assessed over a 5-year period to evaluate the mechanisms of the loss of secondary metabolites biosynthesis capacity in Taxus cell. The results revealed that morphological variations, gradual loss of paclitaxel yield and decreased transcriptional level of paclitaxel biosynthesis key genes occurred during long-term subculture. Genetic and epigenetic variations in these cultures were also studied at different times during culture using amplified fragment-length polymorphism (AFLP), methylation-sensitive amplified polymorphism (MSAP), and high-performance liquid chromatography (HPLC) analyses. A total of 32 primer combinations were used in AFLP amplification, and none of the AFLP loci were found to be polymorphic, thus no major genetic rearrangements were detected in any of the tested samples. However, results from both MSAP and HPLC indicated that there was a higher level of DNA methylation in the low-paclitaxel yielding cell line after long-term culture. Based on these results, we proposed that accumulation of paclitaxel in Taxus cell cultures might be regulated by DNA methylation. To our knowledge, this is the first report of increased methylation with the prolongation of culture time in Taxus cell culture. It provides substantial clues for exploring the gradual loss of the taxol biosynthesis capacity of Taxus cell lines during long-term subculture.Key message DNA methylation maybe involved in the regulation of paclitaxel biosynthesis in Taxus cell culture.

Plant secondary metabolites constitute are a wide range of compounds whose biosynthesis takes place in response to biotic and abiotic stresses. The phytohormone abscisic acid (ABA) acts as an important signaling molecule that regulates plant response to various stresses. Moreover, 9-cis-epoxycarotenoid dioxygenase (NCED) is one of the key enzymes in the ABA biosynthesis pathway in higher plants. In this study, a new NCED gene from Taxus chinensis, the TcNCED1, was overexpressed in transgenic T. chinensis cells, resulting in a maximum of 48 % more accumulation of ABA and a 2.7-fold increase of taxol production compared to the untransformed cells, respectively. These results indicate that overexpression of TcNCED1 can significantly increase the ABA and taxol level in T. chinensis cells, which probably provides an alternative approach in metabolic engineering to improve the yield of taxol in T. chinensis cells through genetic manipulation of the related genes in the ABA biosynthetic pathway.

Few environmentally friendly solvents are available to extract food-grade astaxanthin. In this paper, some environmentally friendly solvents, such as lactic acid, ethyl lactate, and ethanol, were employed in cell disruption and astaxanthin extraction from Xanthophyllomyces dendrorhous. The extraction procedure was optimized, validated and compared with other conventional extraction techniques. This method gave the best result due to the highest extraction efficiency within short extraction time. The optimum extraction conditions were as follows: the yeast cell wall was disrupted by lactic acid at 65° C for 1 h and then extracted with ethyl lactate:ethanol (1:1, v/v) for 0.5 h. It was proved that the extraction efficiency was enhanced by the addition of the natural antioxidant α-tocopherol. This new method showed low chemical toxicity and gave high extraction efficiency, which had good prospects for mass production at the industrial scale.

Taxus chinensis suspension cells were transformed by the Agrobacterium-mediated transformation method to overexpress the dbat gene coding for 10-deacetylbaccatin III-10 β-O-acetyltransferase, a key enzyme for taxol biosynthesis. A. tumefaciens strain LBA4404 harboring either pCAMBIA1303 or the recombinant plasmid p1303-SdbatN was used. Both plasmids harbored the hygromycin phosphotransferase gene (hptII) and gusA-mgfp5 gene as selectable markers, but the latter plasmid also harbored the dbat gene in the T-DNA region. The transgenic T. chinensis cells had been maintained in modified Gamborg’s B5 medium supplemented with hygromycin for more than 14 months and were subcultured at 4-week intervals. The selected transgenic cells were identified by PCR, Southern blot analysis, β-glucuronidase assay and western blot analysis, and the results showed that the transgenes were integrated in the chromosomal DNA of T. chinensis cells with single-copy style, and that the gusA-mgfp5 reporter gene was expressed in the transgenic cells. The dbat mRNA expression level in the p1303-SdbatN-transgenic T. chinensis cells tested by real-time quantitative PCR was 5.3 ± 0.6 times that of the non-transformed cells. Taxol yield of the p1303-SdbatN-transgenic T. chinensis cells was about 1.7 times that of the non-transformed cells. These results suggest that the overexpression of dbat gene in transgenic T. chinensis cells leads to increased taxol yield.

Genes encoding Δ6 desaturase, Δ6 fatty acid elongase, and Δ5 desaturase from the alga, Phaeodactylum tricornutum, were co-expressed in Pichia pastoris to produce arachidonic acid (ARA; 20:4 Δ5, 8, 11, 14) and eicosapentaenoic acid (EPA; 20:5 Δ5, 8, 11, 14, 17). A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro multimerization approach. ARA and EPA accumulated up to 0.3 and 0.1% of total fatty acids, respectively, in the recombinant P. pastoris carrying with double copies of these three heterologous genes, as compared to 0.1 and 0.05%, respectively, in the recombinant P. pastoris with single copy.

Microorganisms influence the dissolution of a number of minerals. Limestone is one of the most abundant rock types in karst areas, and is predominantly calcium carbonate. Two types of experimental systems were designed in this paper, to make comparisons of limestone dissolution rate among the acidic materials and extracellular carbonic anhydrase (CA) excreted by fungi and the enwrapping effect of fungal mycelia. One was the simulated experimental system containing microorganisms. Another was the simulated experimental system without microorganisms. Results of previous experiment indicated that the acidic materials and CA like enzymatic materials excreted by fungi and the enwrapping effect of fungal mycelia were important factors influencing limestone dissolution. In the three factors mentioned above, the dissolution effect was mycelia enwraping effect>acidic dissolution effect>CA enzymatic effect. The results of the second experiment demonstrated further that the limestone dissolution effect of the acidic materials excreted by fungi was stronger than that of CA excreted by fungi. Nevertheless, CA still played an important role in promoting the dissolution of limestone.

To explore the cause of plant vegetation distributed on the newly created wetland in estuary of modern Yellow River Delta, we took field-based observation of the wetland plant diversity of the Yellow River Delta, Shandong, China. Based on the results of field investigation, we studied the effects of salinity on germination characteristics of four dominant species in wetland of Yellow River Delta, i.e. Suaeda salsa, Apocynum venetum, Phragmites australis and Typha orientalis.The germination conditions of the four dominant emerging plants at 0, 50, 100, 150, 200, 250 and 300 mM NaCl were studied, the results indicated that all species had a trend of decreased germination percentage and rate of germination with the increase of salinity levels, but the inhibitory effect of salinity on the germination percentage of each was different. S. salsa and A. venetum had higher tolerance of salinity than P. australis and T. orientalis. Germination percentage and germination rate of seeds of the four species were significantly affected by salinity.In conclusion, the results of seeds germination of the four dominant species in the newly created wetland were accorded with the results of field investigation of plant community distribution along the gradients of environmental salinity, and salinity is one of the most pivotal factors limited the distribution of the plant vegetation in the newly created wetland of Yellow River Delta.

•Effect of bacterial CA on CO2 capture in CO2-H2O-carbonate system was explored.•Bacterial CA increased both rate and quantity of CO2 absorption in this system.•Overhigh CO2 concentration was not conducive to carbonate rock dissolution by CA.•The addition of bacterial CA could increase CO2 capture in the form of DIC.Atmospheric CO2 are closely connected to climate change and global carbon cycle. Karst processes in CO2-H2O-carbonate system can absorb atmospheric CO2. Carbonic anhydrase (CA) can efficiently catalyze the interconversion between CO2 and HCO3−, which may help accelerate karst processes. To explore the influence of bacterial CA on CO2 capture capacity in CO2-H2O-carbonate system, a flow dissolution experimental device was designed to simulate CO2 absorption by rainwater infiltration or surface water flushing. Results showed that, when bacterial CA was added into the experimental system, it increased not only the rate but also the quantity of CO2 absorption, and the CO2 captured by limestone or dolomite dissolution increased by at least 18.9% and 22.1%, respectively. However, excessively high concentration of CO2 was not conducive to carbonate rock dissolution by bacterial CA. In accordance with the fixed carbon caused by global carbonate dissolution, the contribution of soil CA to the CO2 sinks may reach 0.567–1.938 × 1014gC/a. In summary, the addition of microbial CA could increase the CO2 capture in the form of DIC in CO2-H2O-carbonate system, thereby increasing the carbon sink potential of karst systems. The results help establish a promising approach for reducing CO2 emission by microbial CA.