Effect of quality control on the proliferation of the extract from Taraxacum mongolicum Hand.-Mazz. in Lactobacillus plantarum.

In recent years, the fingerprint of high-performance liquid chromatography has been extensively applied in the identification and quality control of traditional Chinese medicine. It can be a potential protocol for assessing the authenticity, stability and consistency of traditional Chinese medicine and guaranteeing the expected biological activity. In this paper, a method using high-performance liquid chromatography to identify and control the quality of the extract of Taraxacum mongolicum Hand.-Mazz. (TME) was established. With this method, the correlation coefficients of the similarity of 10 batches were ≥0.994. The TME displayed a steady proliferative effect in Lactobacillus plantarum. In brief, this study successfully built a reliable, simple and efficient method to control and confirm the quality and the stability of biological activity of the TME.

Effect of quality control on the antiproliferative activity of the extract from Portulaca oleracea L. in Aspergillus flavus.

Similarity evaluation of complicated chromatographic profiles is a potential protocol for the identification and quality control of herbal medicinal products to ensure their biological activity. In this work, a high-performance liquid chromatography method was established for controlling the batch quality of the extract from Portulaca oleracea L. Using this method, the coefficients of correlation of the similarity of 10 batches extract of P. oleracea L. were ≥ 0.97. The 10 batch extracts from P. oleracea L. possessed stable antiproliferative activity in Aspergillus flavus. The antiproliferative activity stability is correlated with the stability quality of the of the extract from P. oleracea L. Therefore, the present study successfully set up a sensitive and efficient method which might be used to guarantee stable biological activity of the extract from P. oleracea L.

The Interaction between Oxidative Stress Biomarkers and Gut Microbiota in the Antioxidant Effects of Extracts from Sonchus brachyotus DC. in Oxazolone-Induced Intestinal Oxidative Stress in Adult Zebrafish.

Oxidative stress is a phenomenon caused by an imbalance between the production and accumulation of reactive oxygen species in cells and tissues that eventually leads to the production of various diseases. Here, we investigated the antioxidant effects of the extract from Sonchus brachyotus DC. (SBE) based on the 0.2% oxazolone-induced intestinal oxidative stress model of zebrafish. Compared to the model group, the treatment group alleviated oxazolone-induced intestinal tissue damage and reduced the contents of malondialdehyde, reactive oxygen species, IL-1ß, and TNF-α and then increased the contents of superoxide dismutase, glutathione peroxidase, and IL-10. The 16s rDNA gene sequencing findings demonstrated that SBE could increase the relative abundance of Fusobacteriota, Actinobacteriota, and Firmicutes and decrease the relative abundance of Proteobacteria. Based on the correlation analysis between the oxidative stress biomarkers and intestinal flora, we found that the trends of oxidative stress biomarkers were significantly correlated with intestinal microorganisms, especially at the genus level. The correlations of MDA, IL-1ß, and TNF-α were significantly negative with Shewanella, while SOD, GSH-Px, and IL-10 were significantly positive with Cetobacterium, Gemmobacter, and Flavobacterium. Consequently, we concluded that the antioxidant effect of SBE was realized through the interaction between oxidative stress biomarkers and gut microbiota.

Quercetin Inhibits the Proliferation and Aflatoxins Biosynthesis of Aspergillus flavus.

In this work of quercetin's anti-proliferation action on A. flavus, we revealed that quercetin can effectively hamper the proliferation of A. flavus in dose-effect and time-effect relationships. We tested whether quercetin induced apoptosis in A. flavus via various detection methods, such as phosphatidylserine externalization and Hoechst 33342 staining. The results showed that quercetin had no effect on phosphatidylserine externalization and cell nucleus in A. flavus. Simultaneously, quercetin reduced the levels of reactive oxygen species (ROS). For a better understanding of the molecular mechanism of the A. flavus response to quercetin, the RNA-Seq was used to explore the transcriptomic profiles of A. flavus. According to transcriptome sequencing data, quercetin inhibits the proliferation and aflatoxin biosynthesis by regulating the expression of development-related genes and aflatoxin production-related genes. These results will provide some theoretical basis for quercetin as an anti-mildew agent resource.

Eukaryotic expression and antimicrobial spectrum determination of the peptide tachyplesin II.

Ta0-a, the gene encoding the mature antimicrobial peptide tachyplesin II, was engineered to optimize the coding sequence according to codon usage bias in yeast. Ta0-a was efficiently expressed in the methylotrophic yeast Pichia pastoris strain SMD1168. The recombinant peptide Ta0 reached 150mg/L after methanol induction for 6 d. Ta0 was rapidly purified to homogeneity by a single step of size-exclusion chromatography. The minimal lethal concentrations of Ta0 to the Escherichia coli strain K12 was 30 microg/mL. Ta0 exhibited a wide range of antimicrobial activity: the growth of 26 bacterial and fungal strains, including some typical food/feed spoilage microorganisms, was all substantially inhibited. This result indicates the potential practical application of the recombinant peptide in various industrial products.

Induction of apoptosis in human cervical carcinoma HeLa cells by active compounds from Hypericum ascyron L.

Hypericum ascyron L. (Great St. Johnswort), which belongs to the Hypericaceae family, has been used for the treatment of hematemesis, metrorrhagia, rheumatism, swelling, stomach ache, abscesses, dysentery and irregular menstruation for >2,000 years in China. The aim of the present study was to clarify the anticancer activity compounds from H. ascyron L. and the underlying molecular mechanism. Anticancer activity of H. ascyron L. extract was evaluated using an MTT assay. To confirm the anticancer mechanism of activity compounds, Hoechst 33258, Annexin V-fluorescein isothiocyanate/propidium iodide, 2',7'-dichlorodihydrofluorescein diacetate, rhodamine 123 staining and caspase-3 activity analysis were performed. The results demonstrated that the anti-proliferative action of the mixture of kaempferol 3-O-ß-(2″-acetyl) galactopyranoside (K) and quercetin (Q) (molar ratio, 1:1) was significantly increased compared with either of these two compounds separately, and the active fraction of the H. ascyron L. extract |(HALE). HALE, indicating that the anti-proliferative function of H. ascyron L. may be a synergic effect of K and Q. Furthermore, the inhibitory effect of KQ on the growth of HeLa cells was mediated by the induction of apoptosis. To the best of our knowledge, the present study is the first to identify that KQ exhibits significant anti-proliferation activity on HeLa cells via the apoptotic pathway, and is also the first to evaluate the anticancer potential of H. ascyron L. The results of the present study may provide a rational base for the use of H. ascyron L. in the clinic, and shed light on the development of novel anticancer drugs.

Quercetin and aconitine synergistically induces the human cervical carcinoma HeLa cell apoptosis via endoplasmic reticulum (ER) stress pathway.

Up till now, studies have not been conducted on how the combination of Quercetin (Q), Aconitine (A) and apoptosis induction affects human cervical carcinoma HeLa cells. The result of our findings shows that the combination of Q and A (QA) is capable of synergistically inhibiting the proliferation of HeLa cells in a number of concentrations. QA synergistically inhibits the proliferation of MDR1 gene in the HeLa cells. It is concluded based on our result that QA induces apoptosis and ER stress just as QA-induced ER stress pathway may mediate apoptosis by upregulating mRNA expression levels of eIF2α, ATF4, IRE1, XBP1, ATF6, PERK and CHOP in the HeLa cells. The up-regulating of mRNA expression level of GRP78 and activation of UPR are a molecular basis of QA-induced ER stress.

[Overexpression of Citrobacter braakii phytase with high specific activity in Pichia pastoris].

Utilization of the phytase with high specific activity is an effective way to improve the fermentation potency of phytase in recombinant host and decrease the production cost. Up to now, the phytase APPA from Citrobacter braakii exhibits the highest specific activity in the all phytases recorded previously. The gene AppA encoding phytase was modified according to the bias in codon choice of the high expression gene in Pichia pastoris without changing the amino acid sequence and artificially synthesized. The modified gene, AppA ( m) , was inserted into the Pichia pastoris expression vector pPIC9 under the control of AOX1 promoter, and the resulted expression vector pPIC9-AppA ( m) was introduced into the host Pichia pastoris by electroporation. PCR analysis of the recombinant yeast indicated that AppA (m) gene was integrated into the chromosome of Pichia pastoris. The Pichia pastoris recombinants for phytase overexpression were screened by enzyme activity analysis and SDS-PAGE. The recombinant phytase APPA was purified by simple methods, such as dialysis, ultrafiltration and chromatography. After the simple purification, the purity of the recombinant phytase reached to electrophoresis purity, and the recombinant phytase was shown to be glycosylated by Endo-H treatment. The specific activity of the purified recombinant APPA was 3.5 x 10(6) IU/mg of protein. Recombinant phytase APPA showed activity at pH values from 2.0 through 7.0 with the optimum at 4.5. The temperature optimum was 55 degrees C at pH 4.5.The Km value for sodium phytate was 0.165mmol/L with a Vmax of 3.3 x 10(6)IU/mg min. In 5-liter fermentor in fed-batch fermentation, the expression level of phytase in recombinant Pichia pastoris was 3.2mg/mL and the fermentation potency exceeded 1.4 x 10(7) IU/mL, which is the highest level among all of the reported phytase recombinant strains at present.

Nesterenkonia alba sp. nov., an alkaliphilic actinobacterium isolated from the black liquor treatment system of a cotton pulp mill.

An alkaliphilic actinobacterium, designated strain CAAS 252(T), was isolated from the black liquor treatment system of a cotton pulp mill in Wuhan, China. Cells of strain CAAS 252(T) were Gram-positive, non-motile, non-endospore-forming, short rod-shaped, and grew optimally at 42 degrees C and pH 9-10 in the presence of 3 % (w/v) NaCl. Strain CAAS 252(T) contained MK-7, MK-8 and MK-9 as the major menaquinones and anteiso-C(17 : 0), anteiso-C(15 : 0) and C(16 : 0) as the predominant cellular fatty acids and had a peptidoglycan type of A4alpha, Lys-Gly-d-Asp. The DNA G+C content was 60.2 mol%. Based on analysis of 16S rRNA gene sequences (94.7-96.8 % similarity), DNA-DNA hybridization (<70 % relatedness) and chemotaxonomic characteristics, strain CAAS 252(T) belonged to the genus Nesterenkonia, but differed from all recognized species. Therefore, it is proposed that strain CAAS 252(T) represents a novel species of the genus Nesterenkonia, for which the name Nesterenkonia alba sp. nov. is proposed. The type strain is CAAS 252(T) (=CCTCC AB 207011(T)=DSM 19423(T)).

Nesterenkonia flava sp. nov., isolated from paper-mill effluent.

A Gram-positive, non-motile, rod-shaped, non-spore-forming bacterium, designated CAAS 251T, was isolated from paper-mill effluent in Wuhan, China. The organism grew optimally at 40-42 degrees C and at pH 9.0-10.0. The major menaquinones were MK-7, MK-8 and MK-9. The predominant cellular fatty acids were anteiso-C15:0 (34.78 %), anteiso-C17:0 (25.24 %) and C16:0 (13.37 %). The G+C content of the genomic DNA was 65.5 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain CAAS 251T belongs to the genus Nesterenkonia, having sequence identities ranging from 96.0 to 97.0 % with respect to eight recognized species of the genus Nesterenkonia. Data from DNA-DNA hybridization and physiological and biochemical tests indicated that strain CAAS 251T represents a novel species of the genus Nesterenkonia, for which the name Nesterenkonia flava sp. nov. is proposed. The type strain is CAAS 251T (=CCTCC AB 207010T=JCM 14814T).

[Gene cloning, expression and characterization of a novel phytase from Hafnia alvei].

A gene appA encoding a novel phytase was firstly cloned from Hafnia alvei by PCR and sequenced. The gene was consisted of 1335 bp, encoding 444 amino acids. The calculated molecular weight of the mature APPA was about 45.2 kD. The gene appA was expressed in E. coli BL21 (DE3). Recombinant APPA was purified and its enzymatic properties were determined. The optimum pH for the enzyme was 4.5 and the optimum temperature was 60 degrees C. The pH stability of r-APPA is good, the relative phytase activity was above 80% after treated in buffers of pH 2.0-10.0. The specific activity of r-APPA is 356.7 U/mg, and the Km value was 0.49 mmol/L and Vmax of 238 U/mg. The enzyme showed resistance to pepsin and trypsin treatment.

[Mutation research on Q23L and Q23LG272E in phytase derivated from Aspergillus fumigatus].

Aspergillus fumigatus wild-type phytase has many favorable properties, such as a good thermorstability and a broad pH optimum. However, the specific activity of the enzyme is relative low. A. fumigatus Q23L phytase resulted in a remarkable increase in specific activity around pH4.5 - 7.0, but the pH stability of Q23L was lower than A. fumigatus wild-type phytase. To increase the pH stability of Q23L, the mutant Q23LG272E was constructed by site-directed mutagenesis with PCR. The gene of A. fumigatus wild-type phytase and the mutant genes encoding the Q23LG272E and the Q23L were correctly expressed in Pichia pastoris GS115. Enzymes were purified and their enzymatic properties were determined. The results revealed that the specific activity of the Q23L improved remarkably, which increased from 51 u/mg of the wild type to 109 u/mg at pH5.5. Meanwhile, the pH stability of Q23L, decreased evidently, especially from pH3.0 to pH4.0.The pH stability of Q23LG272E in pH3.0 - 4.5 and pH6.5 - 7.0 has been improved compared with Q23L. The specific activity of Q23LG272E basically maintained at the level of Q23L. Analysis of 3-D structure and sequence similarity were used to reveal the presumable factors influencing the enzymatic properties of Q23LG272E, and discussion for the relationship between structure and function of phytase was given.

Functional expression of the keratinolytic serine protease gene sfp2 from Streptomyces fradiae var. k11 in Pichia pastoris.

We report the initial characterization and expression of sfp2, a gene encoding a keratinolytic serine protease from Streptomyces fradiae var. k11. Recombinant SFP2 was expressed in and secreted from the yeast Pichia pastoris with a final yield of 78 mg/L (136.2 U/mL caseinolytic activity) after 25 h of induction. The recombinant enzyme was purified using by ammonium sulfate precipitation and gel filtration chromatography to electrophoretic homogeneity, which was appropriately glycosylated and had a molecular mass of 26.0 kDa. The purified recombinant SFP2 was characterized. The optimal pHs and temperatures of SFP2 for proteolysis of casein and keratin azure were pH 10.0, 60 degrees C, and pH 9.0, 55 degrees C, respectively. SFP2 activity was stable from pH 3.0 to pH 11.0. The enzyme activity was inhibited by Co(2+) and Cr(3+) and enhanced by Ni(2+) and Cu(2+). The K(m) of 0.45 mmol/L and V(max) of 19.84 mmol/min mg were calculated using N-succinyl-Ala-Ala-Pro-Phe-pNA as a substrate. We tested the activity of SFP2 with soluble and insoluble substrates; SFP2 was more specific for keratinous substrates compared with proteinase K and other commercial proteases.

[Improving phytase expression by increasing the gene copy number of appA-m in Pichia pastoris].

In order to improve the fermentation potency of phytase in recombinant host and decrease the production cost, the pichia expression vector pGAPZalpha-A was modified by introduction of an AOX1 promoter from vector pPIC9 and the resulted vector pAOXZalpha is an methanol induced vector. After that, a phytase gene appA-m was cloned into pAOXZalpha to construct the recombinant vector pAOXZalpha-appA-m. The recombinant Pichia pastoris 74#, which already contains one copy of appA-m and its fermentation potency exceeded 7.5 x 10(6) IU/mL, was used as the host strain for the transformation of pAOXZalpha-appA-m. The Pichia pastoris transformants were gained by electroporation. PCR results indicated that the appA-m expression box has integrated into the genome of Pichia pastoris and the original construction of phytase gene has not changed. SDS-PAGE analysis revealed that phytase was overexpressed and secreted into the medium supernatant. Recombinants with high expression level were screened and used for fermentation. In 5L fermentor, the expression level of phytase protein achieved 4 mg/mL and the phytase activity (fermentation potency) exceeded 1.2 x 10(7) IU/mL, which was about 1.6-fold compared with that of the host strain 74#. Moreover, the improved recombinant Pichia pastoris is excellent at expression stability and heredity stability.