Abstract:[Objective] N-acetylneuraminic acid (Neu5Ac) has important medical value in the treatment of influenza, the neurological disease. The conventional methods of production have some shortcomings, such as high cost and low yield. So the available resource of Neu5Ac can not meet the large-scale demand of medical industry. [Methods] In earlier research, a metabolic engineering bacteria (Escherichia coli ΔnanTEK/pNA) has been constructed for the production of Neu5Ac, Whole-cell catalysis was used for the production of Neu5Ac with GlcNAc and pyruvate as the substrate. Culture condition (temperature and time) of E. coli ΔnanTEK/ pNA and subsequent biological transformation (temperature, time, Triton X-100 and so on) were studied by single factor experiment. [Results] After conditions optimization, the production of Neu5Ac was improved and 294.39 mmol/L of Neu5Ac was obtained. [Conclusion] The process can be used for industrial large-scale production of Neu5Ac in terms of convenience, efficiency and economy.

Abstract:[Objective] We optimized xylanase production from Malbranchea cinnamomea CAU521 by solid state fermentation (SSF) with agricultural wastes as carbon source. [Methods] single-factor experiment was used to optimize various factors affecting the enzyme production. There are six factors which include carbon source, nitrogen source, initial pH, initial moisture content, temperature and fermentation time were investigated. [Results] the optimum conditions for xylanase production were as follows: rice straw as carbon source, 2%?(w/w) yeast extract as nitrogen source, the ratio of solid substrate to mineral solution of 1:4 (W/W), the initial pH of 7.0, and incubation temperature of 45 °C. Under the optimized conditions, the highest xylanase production of 13 120 U/g dry carbon source was achieved after 6 days of cultivation. [Conclusion] the high-level xylanase production and low production cost may facilitate industrial scale enzyme application.

Abstract:[Objective] We isolated cadmium-resistant fungi from gangue pile of coal area and characterized their heavy-metal resistance. [Methods] The strain was identified by the morphological properties and 18S rDNA sequence homology analysis. Heavy-metal tolerance and the growth in acid leaching liquid of coal gangue were evaluated. The antioxidant enzyme activity of SOD (superoxide dismutase) and CAT (catalase) was assayed under mixed heavy-metals stress. [Results] The BJKD4 strain is the genus Penicillium sp. and could tolerate up to 29 mmol/L Cd2+, the optimum pH is 4.0?9.0. The order of heavy-metal toxicity to BJKD4 growth is: Cu2+>Ni2+>Cd2+>Pb2+ or Zn2+>Mn2+. Orthogonal experiment indicated that BJKD4 strain could survive in the medium containing different concentrations of a variety of heavy-metal ions, such as Cd2+, Zn2+, Ni2+ and Mn2+. SOD activity of BJKD4 is increased in response to heavy-metals, while the activity of CAT depended on the species and concentration of heavy-metal. Furthermore, BJKD4 could grow in medium containing an acidic leaching solution of coal gangue at alkali pH. [Conclusion] BJKD4 strain is tolerant to various heavy-metals and has the potential to prevent acidification of the gangue hill leachate, the antioxidant enzyme plays important role in alleviating the oxidative stress induced by heavy-metal ions.

Abstract:[Objective] We produced a novel manganese-dependent peroxidase (MnP) by Trametes sp. SQ01 and studied its resistance of MnP to hydrogen peroxide, substrate specificity and ability of decolorizing triphenylmethane dyes. [Methods] MnP was purified through acetone precipitation and DEAE-celluose 52 anion-exchange chromatography. The resistance of MnP against H2O2 and dye decolorization were measured with a UV-visible spectrophotometer. [Results] The homogenous MnP has been obtained through two-step purification. The optimum pH and temperature for the purified MnP were 4.5 and 70 °C, respectively. The enzyme was highly stable in the pH range 3.0?8.0. The enzyme oxidizes various substrates in the presence of manganese, such as 2,6-dimethoxyphenol, guaiacol, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and H2O2, simultaneously, the MnP also regards Mn2+ as its substrate. Among all the substrates tested, the optimum was H2O2 (Km, 3.7?μmmol/L). Interestingly, the enzyme was resistant to H2O2 bleaching. The enzyme retains 70% integrity of its heme after incubation with 2.5 mmol/L H2O2 for 60 min. All of the tested dyes could be discolored by MnP, among which Crystal violet was observed with the highest decolorization rate at 65.8%. The effects of Mn2+ and H2O2 on dye decolorization by MnP were also studied, which revealed that both H2O2 and Mn2+ had less influence on the decolorization of Remazol Brilliant Blue R (RBBR) than Malachite Green. [Conclusion] The resistance of MnP to hydrogen peroxide and its ability of decolorizing triphenylmethane dyes demonstrated its potential application on dye decolorization.

Abstract:[Objective] To study the classification of Aeromonas genus and its pathogenicity. [Methods] Various environmental samples were collected, Aeromonas strains were isolated and characterized, and multilocus sequence typing (MLST) method was adopted to classify the isolates. [Results] Among the isolates 7 Aeromonas strains were identified, belonging to 4 different species of the genus Aeromonas based on 16S rRNA sequence analysis. Furthermore, 5 major virulence genes including Aera, Hly, Aha1, GACT and Nuc were detected in the isolates by PCR and sequencing methods. All strains contained at least one virulence gene, and 3 of them contained 4 virulence genes, respectively. Antibiotic susceptibility test showed that 6 strains exhibited multiple drug resistances to at least 3 antibiotics. Finally, 6 housekeeping genes (gyrB, groL, gltA, metG, ppsA, and recA) were chosen for the MLST analysis by PCR and sequencing. Allele sequences were aligned by searching the MLST database. All 7 Aeromonas strains were assigned a new ST number. [Conclusion] High genetic diversity existed in Aeromonas strains.

Abstract:[Objective] We isolated and identified strains producing fibrinolytic enzyme. [Methods] A strain with fibrinolytic activity was isolated from the environment by using blood powder plates and agar-fibrin plates. We identified the strain by morphological, physiological and biochemical characterization, combined with 16S rRNA gene sequence and phylogenetic tree. [Results] A strain (named EF608) with fibrinolytic activity was obtained and identified as Enterococcus faecalis. Fermentation broth of strain EF608 had fibrinolytic activity. Its optimal reactive temperature and pH value were 35 °C and 7.5, respectively, and the molecular weight is 37 kD by SDS-PAGE and fibrin autography. EDTA completely inhibited the fibrinolytic activity, but PMSF exerted little effect on it. The fermentation broth dissolved thrombus in vitro, directly degrade the fibrin, and did not hydrolyze blood cells. [Conclusion] The strain might provide a new source for novel fibrinolytic enzymes.

Abstract:[Objective] The predation ability, morphological characteristics and 16S rDNA characteristics of Bdellovibrio-and-like organism (BALO) strain JU-PX1 will be studied. [Methods] The predation ability of JU-PX1 have been studied by double-layer agar and conical flask culture. The morphological characteristics of JU-PX1 were examined with optical microscope and transmission electron microscope. Using BALOs-specific primer and universal primer, 16S rDNA fragment of JU-PX1 were obtained by PCR amplification. [Results] Seven of the ten strains tested, especially Escherichia coli and Vibrio parahaemolyticus, could be hunted by JU-PX1. JU-PX1 exhibited typical BALOs pattern, showing vibrioid cell with a single polar flagellum. The cell size was (0.2?0.5) μm×(0.8?1.2) μm, and there was also 3.2?μm elongating cell in exponential phase. Two kinds of 16S rDNA fragment of JU-PX1 were obtained by PCR amplification, which lengths were 831 bp and 1 515 bp, respectively. The 16S rDNA was analysed on phylogenetic tree by NCBI BLAST and MEGA 5.10. [Conclusion] BALO JU-PX1 had the closest relationship with Bacteriovorax litoralis and the similarity was 93%.

Abstract:[Objective] The gene encoding carbonyl reductase was cloned and expressed for the synthesis of the chiral drug intermediates. [Methods] Based on the N-terminal amino acid sequence of carbonyl reductase from GenBank, cmcr was cloned from Kluyveromyce marxianus CGMCC 2.1977 and sequenced, an expression vector, pET28a-cMCR containing the full length of cmcr was constructed and introduced into Escherichia coli BL21(DE3) and Rosetta(DE3) to express the enzyme separately. [Results] This gene showing 100% similarity to reported mer contains an open reading frame of 1 038 bp encoding 345 amino acid residues. CMCR was overexpressed in Rosetta(DE3) with a protein molecular weight of 42 kD. The optimal temperature was 40 °C and pH 8. The enzyme has low thermal and pH stability. Ca2+ activates the enzyme activity, especially when its concentration is 0.5 mmol/L. The asymmetric reduction of 4-chloro-3-oxobutanoate ethyl ester to (S)-4-chloro-3-hydroxyl butanoate ethyl ester (CHBE) with Rosetta(pET28a-cMCR) cells, in which cmcr was expressed, as a catalyst was investigated (100% e.e., 81.0% yield). The application of the cells to the asymmetric reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-prapanamine(DKTP) to (S)-N,N-dimethyl-3- hydroxy-(2-thienyl)-1-propanamine [(S)-DHTP] was also attempted. [Conclusion] The gene encoding carbonyl reductase was cloned and expressed successfully in E. coli form Kluyveromyce marxianus CGMCC 2.1977 and can be applied to asymmetric reduction.

Abstract:[Objective] We cloned and expressed the pectate lyase gene (pel) from Dickeya sp. DCE-01, a strain for bast fiber bio-extracting, in E. coli BL21(DE3). Then we characterized the purified pectate lyase (Pel). [Methods] Primers were designed by the potential pel Q59419 annotated from the whole genome sequence of Dickeya sp. DCE-01. The pel was cloned, linked to pEASY-E1 and pACYCDuet-1, and expressed in E. coli BL21(DE3). After comparing the activities of extracellular Pel from the engineering strains, the Pel with highest activity was purified by the two-step process involving ultrafiltration and gel filtration (Sephadex G-100) and then characterized. [Results] The pel (GenBank: JX964997) was 1 128 bp and encoded 375 amino acids. The highest activity of the extracellular Pel from pACYCDuet-1-pel-BL strain was 298.8 IU/mL. The optimal condition for the purified enzyme was at 50 °C, pH 9.0 and polygalacturonic acid sodium as a substrate. The catalytic activity of the purified Pel was stable below 45 °C for 1 h at pH 9.0?10.0. It was dependent on Ca2+, while it was promoted by Zn2+ and NH4+ and inhibited seriously by Fe3+ and Pb2+. The maximal activity of the purified enzyme was obtained at Ca2+ concentration of 2 mmol/L. [Conclusion] An efficient alkaline pectate lyase gene has been excavated from strain DCE-01, and its expression product may be available for biorefinery.

Abstract:[Objective] In order to raising the antagonistic effect, the growth conditions of biocontrol strain CNY-04 was optimized. [Methods] The growth conditions of the biocontrol strain CNY-04 were optimized by response surface methodology basing on single factor tests. The growth curve was also measured. [Results] The optimal culture conditions of the biocontrol strain CNY-04 were determined for 0.5% glucose, 2.0% peptone, 0.1% yeast extract, 0.5% beef extract, temperature at 32 °C, pH 8.0, speeding at 150 r/min, the incubation time of 48 h, inoculation amount of 4%, liquid volume of 75 mL/250 mL. The OD600 of CNY-04 was 2.907 with the optimal growth conditions, consistenting with the predicted value. The antibacterial circle diameter was 44.5 mm, improving by 30.9%. [Conclusion] The optimal growth condition of the biocontrol strain CNY-04 was identified, providing a theoretical basis for enlargement of production.

Abstract:[Objective] Twelve ericoid mycorrhiza (ERM) fungal strain isolates were used to inoculate 2-year-old ornamental Rhododendron annae Franch. sterile seedlings, plant growth, chlorophyll, photosynthetic parameters and chlorophyll fluorescence parameters in plant leaves were investigated. [Methods] Greenhouse pot experiments were conducted in this study to test the effect of 12 ERM strains which were isolated from the roots of wild R. annae. [Results] The results revealed that the colonization ratio was notably higher. There were substantial differences on the aboveground, underground part and total biomass index of inoculated seedlings compared with those of uninoculated seedlings (P<0.01). Compared with the control treatment, the chlorophyll a and b, total chlorophyll content, leaf net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) were higher significantly, along with the increase of intercellular CO2 concentration (Ci). Practical quantum yield (ΦPSⅡ) of inoculated seedlings’ leaves were significantly higher than those of the uninoculated except strain TY19, TY24 and TY34; PSⅡ electron transfer rate (ETR), PSⅡmaximum photochemical quantum yield (Fv/Fm), potential activity (Fv/Fo) and photochemical quenching (qP) were significantly higher than those of the control plants. Non-photochemical quenching (NPQ) were higher as well, and the difference was very significant except strain TY29. The correlation of ΦPSⅡ and Pn, Gs were significantly greater than those of Fv/Fm, qP and NPQ. The correlation of ETR Fv/Fm, Fv/Fo, NPQ, Pn and Tr were greater than the qP and Gs. The correlation of Pn and Gs were greater than Tr, but it had significant negative correlation with Ci (P<0.01). [Conclusion] Through inoculation treatment, photosynthetic parameters and chlorophyll fluorescence parameters in plant leaves were improved, and meanwhile the effective use of light were enhanced. Thus, there were significant increases in seedling biomass. As for the comprehensive inoculation effects, TY18, TY29, TY35, TY02, TY07 and TY12 are the superior strains isolates to cultivate R. annae mycorrhizal seedlings.

Abstract:[Objective] Based on the bioinformations of the whole genomic sequences, we assessed biosynthetic pathway of mycotoxins and antibiotics in Rhizopus chinensis CCTCCM201021, an important fungus isolated from Daqu, a leaven used in the production of traditional Chinese liquor. [Methods] The sequencing was done using Solexa sequencing technology. The genome was assembled by using SOAP de novo software. Key synthetic pathways and genes of rhizoxin and rhizonin, as well as mycotoxins metabolism were analyzed, including PKS, NRPS and PKS-NRPS hybrid metabolic pathways, terpenoid metabolism and other metabolic pathways. [Results] The genome of R. chinensis has a total size of 45.70 Mb and a GC content of 36.99%. Compared with the genomes of the Zygomycetes, the sequence similarity is low, whereas the similarity of the homologous gene is about 60%. The genome differences between Rhizopus and the fungi belonging to the Zygomycete class were significant. By the analysis of typical mycotoxins synthesis pathways and genes based on the genome data, a few polyketide biosynthetic pathway metabolism and terpenoid metabolism genes were found in R. chinensis, but there were great quantity genes of xenobiotics biodegradation pathways. [Conclusion] R. chinensis cannot produce mycotoxins, and its products are generally considered as safe.

Abstract:[Objective] To establish the rapid screening method of mutant strain of Trichoderma reesei and to screen out mutants with high yield endoglucanase. [Methods] The screening medium of Trichoderma reesei T306 was optimized to establish the rapid screening method, and the mutant strain with high-production endoglucanase was screened through UV mutation. Furthermore, the fermentation medium of mutant strain was optimized. [Results] Addition of 0.1% (w/v) lactose, peptone and sodium deoxycholate was beneficial for the screening of mutants. The mutant strain 0516 was obtained, which had higher production of endoglucanase and obviously varied in colony morphology. The CMCase activity of the mutant was increased by 38.9% compared with original strain. The optimum carbon source and nitrogen source of culture medium were as follows: lactose 1.50%, ammonium sulfate 0.14%, urea 0.05%, and peptone 0.10%. Under these conditions, the CMCase activity of mutant 0516 reached 64.2 U/ mL, which was 2.3 times higher than before. [Conclusion] A method for rapidly screening mutant of Trichoderma reesei with high production endoglucanase was established, and mutant 0516 with higher production of endoglucanase and genetic stability was obtained after UV mutation.

Abstract:[Objective] This study was to detect the stability of biological characteristics of recombinant E. coli BL21/pET-22b(+)-rhTrx expressing human thioredoxin. [Methods] Recombinant E. coli BL21/pET-22b(+)-rhTrx was subcultured for 50 passages, and subjected to transmission electron microscopy, Gram staining and biochemical examination as well as test for expression level of target protein and property of plasmid every 10 passages. [Results] The results of restriction analysis and sequencing of various passages were in agreement. All the expression levels of target protein in recombinant E. coli BL21/pET-22b(+)-rhTrx of passages 0, 10, 20, 30, 40 and 50 passages were about 20%, which showed no significant difference. All the passages were negative in Gram staining and showed typical characteristics of E. coli under transmission electron microscope and in biochemical examination. [Conclusion] The recombinant E. coli BL21/pET-22b(+)-rhTrx showed stable biological characteristics and might be used as a bacterial seed for production.

Abstract:[Objective] Detect the effect of gene pfm on type Ⅲ secretion system effectors. [Methods] Complementary strain pfmC was constructed. Total RNA was extracted from the wild type strain PAO1, mutant strain Δpfm and complementary strain pfmC respectively and real-time PCR was performed to detect the transcription levels of exoS, exoT and exoY. Further more, both total intracellular proteins and secreted proteins of strains PAO1, Δpfm and pfmC were collected, and detected against the representative effector ExoS by western blot. [Results] The results showed that transcription levels of exoS, exoT and exoY were significantly decreased in Δpfm compared to PAO1, and recovered in pfmC. Western blot showed that both total intracellular and secreted ExoS of Δpfm were significantly lower than that of PAO1 and recovered in pfmC. [Conclusion] In conclusion, gene pfm of Pseudomonas aeruginosa can affect type Ⅲ secretion system effectors.

Abstract:As an emerging organism identification method, DNA barcoding has been widely used in plants, animals and microorganisms for its advantage of higher sensitivity, accuracy, and objectivity. Even the nuclear ribosomal internal transcribed spacer (ITS) is used as a standard barcode in fungal identification frequently, nowadays, there are more and more new barcodes, such as the microcoding, ND6 and EF3. In this article we summarized the generation and developing history of DNA barcoding, also we present the advantage, shortcomings and the development trend based on fungal barcoding case studies. We indicated that DNA barcoding technique will be a good supplementary to the traditional morphology-based taxonomy, and towards a combination of natural evolutional relationships and morphological taxonomy in fungi.

Abstract:Microbial enhanced oil recovery (MEOR) is a technology that utilizes bio-gas, bio-surfactants, bio-polymers and degradation produced by underground fermentation of bacteria for petroleum exploitation. In this study, it is introduced that progress in pilot tests of MEOR in Daqing Oilfield in recent decades, which analyzes suitable reservoir conditions and application characteristics. By the end of 2012, cumulative incremental oil production reached to 12×104 t, including 518 wells by single-well microbial huff-and-puff with cumulative incremental oil production of 6.3×104 t, and 10 projects (45 well patterns) by microbial flooding and profile modification with cumulative incremental oil of 5.7×104 t. The technology plays an important role in stabilizing production of Daqing oilfield.

Abstract:The pathogenic mechanism of Streptococcus suis serotype 2 is still unsolved. Pathogenicity island not only gives pathogen special ability of pathogenic but also plays an important role in bacterial evolution process. Thorough analysis of the functional genes in 89K pathogenicity island contributes to a more comprehensive grasp of the characteristics of Streptococcus suis serotype 2. This paper summarized the structure and evolution process of 89K pathogenicity island and the research progress of domestic and foreign, including in two-component signal transduction system, type IV secretion system, ATP-binding cassette transporter protein, toxin-antitoxin system and other important genes, trying in gene level to find a breakthrough in pathogenesis of Streptococcus suis serotype 2.

Abstract:Filamentous fungi are of great economic importance in industry, agriculture and medicine. On the other hand, some of them can infect human, animals and plants which caused huge economic losses. G protein signaling pathways are conserved in all eukaryotes for the transmembrane signaling transduction. To date, in fungi, G protein signaling and its regulation have been intensively studied. It has been demonstrated that G protein signaling pathways play an important role in sense and respond to various external signals which essential for the regulation of growth, development, reproduction, pathogenesis and secondary metabolite biosynthesis. This review summarized recent advances in the understanding of the basic components and biological functions of G protein signaling pathways in filamentous fungi.

Abstract:In the paper, “semi-self-help” teaching model was put forward. Proposition background, basic meaning, and the idea of the teaching model were introduced. Concrete implementing scheme and evaluation form were suggested. An example of applying in biotechnology was explained. Four steps were put forward to state the application of “semi-self-help” teaching model in biotechnology. That is, how to introduce the system’s content of the course by arranging engineering practice papers, how to organize the learning of basic knowledge, how to apply basic knowledge in specific production, and how to design the technology process of specific bioengineering products. The effect of “semi-self-help” teaching model was evaluated. The paper provides a new teaching model to improve students’ engineering practice ability.

Abstract:[Objective] To develop a new method based on PCR amplification and restriction endonuclease digestion of a 557 bp fragment of the 16S rRNA gene for identification of Legionella isolates. [Methods] The primers were designed according to sequences of 16S rRNA gene of Legionella. Pure culture microbial strains were used as templates. The reaction conditions were optimized and the specificity were verified by 16 L. pneumophila strains, 22 non-L. pneumophila strains and 12 other bacteria strains. One hundred sixty-nine isolates were identified by PCR-enzymatic digestion, and 16S rDNA and mip gene sequencing analysis. [Results] The results showed that 16 L. pneumophila strains, 22 non-L. pneumophila strains and 12 other bacteria strains could be correctly identified and differentiated by this scheme. 169 isolates examined by the scheme, 79 L. pneumophila strains and 81 non-L. pneumophila strains were identified by PCR-enzymatic digestion, and the results were consistent with 16S rDNA and mip gene sequencing analysis among them. [Conclusion] PCR combined with restriction endonuclease digestion technology is a simple and specific method for rapid identification and differentiation of L. pneumophila and non-L. pneumophila isolates.

Abstract:

Abstract:[Objective] In order to study the diversity of Dehalobacter spp. from six 1,1,1-trichloroethane (TCA) contaminated groundwater samples. [Methods] The concentration of 1,1,1-TCA, 1,1-dichloroethane (DCA) and chloroethane (CA) of each sample was determined using gas chromatography. Real-time qPCR was performed on each sample to estimate the ratio of Dehalobacter spp. (Dhb) to the total bacteria. PCR products amplified by combining 16S rRNA gene universal primer and Dehalobacter group-specific primer were used to construct six Dhb group-specific clone libraries, respectively. Phylogenetic tree was constructed based on the sequences from the clone libraries and their nearest neighbors from GenBank. [Results] The existence of 1,1-DCA and (or) CA in the 6 samples may associate with 1,1,1-TCA biodegradation. Real-time qPCR results demonstrated that the ratio of Dhb to the total bacteria varied in each sample. In total 41 sequences were obtained from these 6 clone libraries and BLAST showed all their nearest sequences were affiliated to Dhb. These sequences were clustered into 7 operational taxonomic units (OTUs) at a threshold of 99% similarity. OTU1 (24 sequences) has a similarity of 98% with the 1,1,1-TCA reductively dechlorinating strain Dehalobacter sp. str. TCA1. Three other OTUs only has 95%?96% similarity with their nearest 16S rRNA gene sequences in GenBank. [Conclusion] This study demonstrated the existence of Dhb in all 6 samples with a relatively high diversity and these Dhb bacteria might be responsible for the biodegradation of 1,1,1-TCA in the groundwater of sampling sites.

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