Inhibition of Microcystis aeruginosa by Bacillus subtilis fmb60 non-ribosome peptide metabolites / 生物工程学报

Microcystis aeruginosa, a type of algal bloom microalgae, is widely distributed in water, causing serious deteriorated effects on humans and the ecological environment. As a biocontrol microorganism, Bacillus subtilis can synthesize various bioactive substances through non-ribosomal peptide synthetase, to inhibit the growth of M. aeruginosa. Thus, it is imperative to investigate the non-ribosomal peptide (NRP) metabolites of B. subtilis fmb60. Three NRP metabolites from B. subtilis fmb60 including bacillibactin, surfactin and fengycin were extracted and identified by genome mining technology. The growth inhibition of M. aeruginosa was studied by adding various concentrations of NRP metabolites. The half-effect concentration value (EC50.4 d) of M. aeruginosa was 26.5 mg/L after incubation for 4 days. With the increasing concentration, the inhibitory effects of NRP metabolites of B. subtilis fmb60 on M. aeruginosa was enhanced significantly. Compared with the control group, with the addition of 50 mg/L NRP metabolites to the M. aeruginosa, the content of Fv/Fm, Fv/Fo and Yield parameter after cultured for 4 days were decreased by 2.8%, 1.7% and 2.0%, respectively. Those findings indicate that the NRP metabolites of B. subtilis fmb60 can significantly inhibit the photosynthesis and metabolism of M. aeruginosa, which provides a theoretical foundation for the development of biological algae inhibitor of B. subtilis.

Development and application of monoclonal antibodies anti-human lipoprotein-associated phospholipase A2 / 生物工程学报

The aim of this study is to prepare monoclonal anti-human Lp-PLA2 antibodies, and establish a rapid and accurate immunochromatographic Lp-PLA2 assay used in community medical institution. The gene sequence of human Lp-PLA2 was obtained from NCBI to construct the expression plasmid. Lp-PLA2 protein expressed in CHO-K1 cells was used to immune BALB/c mice. The monoclonal antibodies were produced in mouse ascites after hybridoma cells screening. Antibodies were evaluated by SDS-PAGE, ELISA and other methods. The Lp-PLA2 test strip was prepared based on sandwich method and evaluated with the portable detection instrument. The affinity of the paired antibodies, PLA1 and PLA5, both reached 1×10⁻⁸. The antibody subclass was IgG1. Both antibodies recognized the Lp-PLA2 protein in the blood specifically. The Lp-PLA2 test strip was prepared based on sandwich method, with linear range of 20-2000 ng/mL. The Lp-PLA2 test strip correlated well with the diaDexus ELISA test kit. In conclusion, the paired antibodies were successfully prepared with high affinity and specificity. The immunochromatographic test of Lp-PLA2 provided a fast and accurate method to detect the concentration of Lp-PLA2 in blood sample for clinical use in the community medical institution and could contribute to the management of cardiovascular diseases.

The progress of diagnosis and treatment of sepsis in traditional Chinese medicine / 国际中医中药杂志

Sepsis is a common complication in critically ill patients. The incidence rate was significantly higher in recent years, and it has been as the main cause of death in critically ill patients. The traditional treatment measures failed to significantly improve mortality. In recent years, the treatment of sepsis by traditional Chinese medicine are getting far more attention, the combination of Chinese traditional and western medicine treatment of sepsis bring out new treatment ideas and thoughts,and has achieved good results. This paper reviews the TCM recognition of sepsis from the etiology and pathogenesis, syndrome differentiation, treatments, signgle traditional Chinese medicine and compound preparations and so on,following a brief analysis of existing problems and solutions.

Breeding of high-producing LI-F lipopeptide Paenibacillus polymyxa by protoplast fusion and differential expression analysis of fusion strains / 生物工程学报

Auxotrophic strains of N1-37 (Phe-) and N2-27 (His-), screened from mutations of Paenibacillus polymyxa JSa-9 previously, were used as the parent strains to screen high-producing LI-F antibacterial lipopeptide fusion strain through protoplast fusion with polyethylene glycol as a promote agent. Fusion strain F5-15 was obtained. Then the product of LI-F antibacterial lipopeptide was quantified by HPLC, and the difference of expression of the key genes of lipopeptide synthase between wild strain JSa-9 and the fusion strain was analyzed by real-time PCR. LI-F antibacterial lipopeptide yield of the fusion strain F5-15 was 3.1-fold of the original strain JSa9's, and the expression levels of the target genes were 10.48, 2.48, 2.1 and 11.8 fold of the initial strain JSa-9, respectively.

Cloning and expression of lipoxygenase gene from Anabaena sp. PCC 7120 and purification, characterization of the recombinant enzyme / 生物工程学报

We cloned the lipoxygenase gene (ana-LOX) from Anabaena sp. PCC 7120 and expressed it in Escherichia coli BL21 (DE3) pLysS. We determined the active site of the recombinant ana-LOX through site-directed gene mutagenesis and obtained the shortest length of the functional gene. Meanwhile, we studied the properties of recombinant ana-LOX after purification. The C-terminal of the Aos (allene oxide synthase)-LOX fusion gene in Anabaena sp. PCC 7120 genome was found belonging to LOXs family by bioinformatics analysis. Further results of site-directed gene mutagenesis confirmed that the active sites of ana-LOX were His197, His202, His369, Asn373and Ile455. The shortest length of functional gene was identified to be 1 254 bp based on the strategy of shortening the gene length gradually. The highest activity of recombinant ana-LOX of 6 750 U/mL could be achieved when constructed to pET-32a vector and expressed at low temperature 16 degrees C. We purified the enzyme by Ni-NTA chelating affinity chromatography, with 60.89% yield and specific activity of 11.4 x 10(4) U/mg. The optimum reaction temperature and pH for ana-LOX were 45 degrees C and 6.0, respectively. Furthermore, the obtained ana-LOX was stable at room temperature. The effect of metal ions on ana-LOX was determined also. Fe2+, Mg2+ Ca2+ could markedly promote the activity of this enzyme whereas Fe3+ and Cu2+ had a strong inhibitory effect on it. Finally, the ana-LOX could improve the microscopical structure of dough. The results of this study will provide a basis for future improvements and food industrial applications of ana-LOX.

Fusion expression of fibrinolytic enzyme gene PPFE-I from endophytic Paenibacillus polymyxa in Escherichia coli and activity analysis / 生物工程学报

With the genomic DNA of strain EJS-3 as the template, we amplified the gene of fibrinolytic enzyme from Paenibacillus polymyxa (PPFE-I) by PCR. We purified the PCR product and ligated it into pMD19-T. After DNA sequencing, we cloned the PPFE-I gene into expression vector pET-DsbA and transformed it into Escherichia coli BL21(DE3). Upon induction of IPTG, we found that the activity of recombinant fibrinolytic enzyme fused with DsbA expressed in Escherichia coli was 228 IU/mL. SDS-PAGE analysis showed that the recombinant enzyme was soluble and accounted for about 18.4% of total cell protein. Western blotting demonstrated that the recombinant protein was DsbA-PPFE-I. We purified the recombinant enzyme by Ni affinity chromatography, thrombin digestion and sephadex G-100 gel-filtration, and identified the molecular weight of purified product to be 66.3 kDa with MALDI-TOF mass spectrometry. The purified enzyme exhibited distinct fibrinolytic activity on fibrin plate.

Cloning and expression of organic solvent tolerant lipase gene from Staphylococcus saprophyticus M36 / 生物工程学报

Lipases are important biocatalysts that are widely used in food processing and bio-diesel production. However, organic solvents could inactivate some lipases during applications. Therefore, the efficient cloning and expression of the organic solvent-tolerant lipase is important to its application. In this work, we first found out an organic solvent-tolerant lipase from Staphylococcus saprophyticus M36 and amplified the 741 bp Lipase gene lip3 (GenBank Accession No. FJ979867), by PCR, which encoded a 31.6 kD polypeptide of 247 amino acid residues. But the lipase shared 83% identity with tentative lip3 gene of Staphylococcus saprophyticus (GenBank Accession No. AP008934). We connected the gene with expression vector pET-DsbA, transformed it into Escherichia coli BL21 (DE3), and obtained the recombinant pET-DsbA-lip3. With the induction by 0.4 mmol/L of isopropyl beta-D-thiogalactopyranoside at pH 8.0, OD600 1.0, 25 degrees C for 12 h, the lipase activity reached up to 25.8 U/mL. The lipase expressed was stable in the presence of methanol, n-hexane, and isooctane, n-heptane.

Biodegradation of nicotine in tobacco extracts for making reconstituted tobacco by strain DN2 / 生物工程学报

The purpose of the study is to use O. intermedium DN2 to degrade nicotine in tobacco extracts for making reconstituted tobacco. Firstly, we studied the effects of various factors on degradation of nicotine in the extracts by strain DN2. When we added 0.1% yeast extract into the extracts, adjusted its pH value to 7.0 by ammonia solution, inoculated 15% cultures and maintained fermentation temperature of 30 degrees C, the degradation rate of nicotine by strain DN2 was the fastest. Furthmore, under these conditions, we studied the degradation rates of nicotine in three fed batches culture which carried out in a 30-L reactor, the result showed that the average degradation rate of nicotine by strain DN2 was 140.55 mg/L/h, which was much higher than that reported in other studies. These results indicated that strain DN2 may be useful for reducing nicotine content of reconstituted tobacco.

STUDY ON THE OPTIMUM FERMENTATION CONDITION FOR PRODUCTION OF PROTOPECTINASE BY ASPERGILLUS SP / 微生物学通报

Effects of carbon resource, nitrogen resource, metal irons and surface detergents on the production of pro topectinase by strain Aspergillus sp XZ 131 were studied The results showed that pectin substances were essential for the strain to produce protopectinase The enzyme activity reached to 300 U/mL, when(NH 4) 2SO 4 and(NH 4) 2HPO 4 were used as nitrogen resource Ca 2+ and Tween 20 were able to enhance the production of the enzyme The optimum composition of the medium was citrus peel powder 1g,(NH 4) 2SO 4 2g,CaCl 2 0 015g,Tween 20 0 2mL,KH 2PO 4 3 8g,K 2HPO 4?3H 2O 0 2g,H 2O 100mL,pH 6 5。

THE SCREENING OF THE CARBOXYPEPTIDASE PRODUCING STRAIN AND ITS PROPERTIES OF ENZYME PRODUCING / 微生物学通报

One carboxypeptidase producing strain was obtained from 28 proteinase producing strains, by analyzing the products of peptides hydrolyzed by the enzyme of the strains According to the characteristics of the morpha and the colonies, the screened strain belongs to aspergillus genera The activity of its carboxypeptidase reached maximum at the 84th hour after fermentation