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Abstract:

Abstract:[Objective] A microorganism from the samples of industrial spoilage with high-yield bacterium biofilm was isolated and identified. Meanwhile studies on characterization of biofilm formation were also carried out. [Methods] First of all, crystal violet stain was used to evaluate the ability of the biofilm-forming of screened strains in 96-cell microplates. And then, colonial morphology, physio-biochemical tests and 16S rRNA sequence analysis were adopted to determine the phylogenetic position of the isolated strains. Finally, the effects of attachment materials and culture temperature on the characterization of biofilm formation were also researched using scanning electron microscope (SEM) and crystal violet stain respectively. [Results] The best biofilm-forming strain was identified and clarified into Citrobacter werkmanii. And biofilm could be formed on the surfaces of glass, stainless steel and polyvinylchloride (PVC) respectively. Moreover the characterization of biofilm could be significantly influenced by temperature and the best biofilm was found on the PVC at 30 °C no matter which materials. [Conclusion] In industrial spoilage, the biofilm-forming strains could be found and its ability to produce biofilm could also be affected by materials and temperature.

Abstract:[Objective] To investigate the characters of hydroxymandelate synthase (HmaS) gene in Escherichia coli, the activities of HmaS encoded by hmas isolated from Amycolatopsis orientalis and Streptomyces coelicolor were compared. [Methods] The coding sequences of hmas were respectively amplified from the genomic DNAs of A. orientalis and S. coelicolor and were heterologous expressed in E. coli. The expressing proteins were isolated and purified by anion exchange chromatography and gel filtration chromatography, then the enzymatic activity and catalytic properties of HmaS were evaluated. [Results] The activity of HmaSSC2 from S. coelicolor was almost 3.6 times as of A. orientalis. The optimum reaction temperature of HmaSAO is 28 °C with high storage stability in weak alkaline condition. HmaSSC2 has the optimum reaction temperature of 35 °C and maintains high activity between 28 °C and 45 °C. HmaSSC2 functions well under neutral conditions. [Conclusion] The characters of HmaSSC2 from S. coelicolor is more suitable for metabolic engineering of E. coli to produce mandelic acid.

Abstract:[Objective] A raw starch-digesting glucoamylase was got, and its characterizations were studied. [Methods] A raw starch-digesting glucoamylase was purified from the fermentation broth of Aspergillus sp. RSD using the combination of ammonium sulfate fractionation, HiPrep DEAE FF16/10 anion exchange, Gel filtration chromatography and Hiprep 16/10 source 30S cation exchange. [Results] The crude enzyme was purified 12.65 times with 9.02% recovery of enzyme activity. The molecular weigh of the purified raw starch glucoamylase was about 82 kD identified by SDS-PAGE. The enzyme properties showed that it’s optimal reaction temperature was 50 °C, and the enzyme had a good stability under 50 °C as well as unstable under high temperature; the optimal reaction pH of this enzyme was 4.5, and there was a good stability between pH 3.5 and pH 7.0. At the condition of 40 °C and pH 4.6, its Km and Vmax for soluble starch were 7.44 g/L and 1.45 g/(L·min). The effects of different metal ions on enzyme activity showed that the enzyme was strongly activated by Fe2+, however, EDTA, Cu2+ and K+ inhibited the enzyme activity at various extents. The substrate specificity of raw starch-digesting glucoamylase showed that the enzyme had higher enzyme activity on malt dextrin. [Conclusion] Compared with commercially available glucoamylase and raw starch-digesting glucoamylase, this enzyme has higher degradation ability on raw materials, and has a good application prospect in industry.

Abstract:[Objective] To examine the effects of light on stability of carotenoids (Cars) and bacteriochlorophyll (BChl) a of anoxygenic phototrophic bacteria. [Methods] Using Rhodopesudomonas palustris CQV97 as a reference, we used silica gel column chromatography, HPLC and absorption spectrophotometry for the separation, composition analysis and light stability of Cars and BChl. [Results] The recovery of Cars was higher than that of BChl a whereas the recovery rate of BChl a showed light-dependent fluctuation. Six Cars and trace amount of bacteriopheophytin a (<0.25%) were detected in Car separation fraction. Four intermediates of BChl a including BChl aGG, BChl aDHGG, BChl aTHGG and BChl aP were detected in BChl a fraction, and BChl aP was predominated (76.73%). In the dark, both Cars and BChl a were very stable. Under different light irradiation of 2 000 lx, Cars kept stable within 70 minutes, but it was sensitive to UV radiation and its half-life was about 11.15 min. Under incandescent lamp, fluorescent lamp, natural light and UV radiation, the photodegradation rate constants (min–1) of BChl a were 0.169 8, 0.028 9, 0.213 9 and 0.026 4, corresponding half-lives were 4.47 min, 29.68 min, 4.20 min and 26.19 min, respectively. [Conclusion] BChl a and Cars could be seperated simultaneously by one-step gel column chromatography. Cars were stable toward light irradiation, but sensitive to UV radiation. BChl a was unstable toward light irradiation, the short-term light irradiation resulted in the fluctuation of BChl a recovery rate, a stable intermediate was found in the process of BChl a photolysis. The results will be helpful for refining and functional analyzing of the photosynthetic pigments of the anoxygenic phototrophic bacteria.

Abstract:[Objective] Analysis of the denitrifying bacteria composition in a long-term amended soil by pyrosequencing and isolating functional bacteria. [Methods] Microbial community structure was analyzed by 454 pyrosequencing of 16S rRNA gene, Giltay medium was used to cultivate and screen denitrifying bacteria from the isolates obtained from the soil, 16S rRNA genes of the detected denitrifying bacteria were also identified. [Results] Proteobacteria, Acidobacteria, Bacteroidetes, Chloroflexi were dominant in the soil sample and nearly 70% sequences were unclassified in genus level. Among 1 344 isolates, 12 isolates were found that could efficiently remove nitrate under anaerobic condition and were affiliated to Pseudomonas, Aeromonas, Serratia and Acinetobacter. [Conclusion] The results showed a high microbial diversity in the soil and most of them were unclassified bacteria. Eleven strains of denitrifying bacteria were identified via nitrate removal and gas formation. These strains could be used for studying the denitrification property.

Abstract:[Objective] Influence on bacterial communities of different ore composition. [Methods] The change of acidic microbial community dominance before and after leaching ore were investigated by using 16S rRNA-RFLP methods. [Results] AMD of three sampling points are the independent Acidithiobacillus bacteria genera of microbes, which accounts for about half the number of tested strains, three sample points between ore leaching microbial species and quantity larger similarity. Meantime, we use the AMD to bioleaching ore, the result of microbial community of four ore samples indicated that H71 and F11 community structure difference are bigger, J72 and S71 are almost the same. [Conclusion] The test sequences can be divided into two branches: Acidithiobacillus bacteria genera and relatively independent bacteria genera.

Abstract:[Objective] MilA, a cytidine 5′-monophosphate (CMP) hydroxymethylase which can convert CMP to 5-hydroxymethylcytidine-5′-monophosphate (HmCMP), was discovered and characterized in the mildiomycin biosynthetic pathway. MilA belongs to the superfamily of thymidylate synthase (TS) and cytidylate hydroxymethylase (CH), and was the only enzyme to date that was able to efficiently convert CMP other than dCMP to HmCMP. Three-dimensional structure prediction of MilA revealed high similarity to that of CH and TS. Ser94 of CH was proved to be a key residue mediating hydroxylation of the 5′-methyl cytosine of dCMP, corresponding residue is Thr102 in MilA and Pro92 in TS, respectively. In order to study the role of Thr102 in MilA, we constructed mutants MilA T102V and MilA T102L. [Methods] Based on above analysis, Thr102 of MilA was site-mutated into valine (Val, V) and leucine (Leu, L) to assay their catalytic efficiency. [Results] Compared to the wild type, activity of MilA T102V for CMP was significantly reduced by 87.1%, while that for dCMP was maintained at a similar level. By contrast, MilA T102L could not use CMP and dCMP any more. As for MilA T102L, the side chain of Leu102 was one carbon atom longer than both of Val102 and Thr102, implying that steric hindrance effect was involved in the substrate binding by MilA T102L. [Conclusion] The results demonstrated that Thr102 in MilA was essential for HmCMP formation. The function of MilAT102 may have reached optimization after naturally evolutionary fine-tuning.

Abstract:[Objective] In the salinomycin biosynthetic gene cluster, slnTI and slnTII encode the ABC transporter ATP-binding subunit and trans-membrane subunit, respectively. We speculate that slnTI and slnTII are most likely related to salinomycin export. Here, the role of slnTI and slnTII on salinomycin biosynthesis and resistance was investigated by gene replacement and overexpression. [Methods] Using REDIRECT? technology, the slnTI mutant LJ01 and the slnTII mutant LJ02 were constructed, and further verified by trans-complementation with cloned corresponding genes. SlnTI and SlnTII were overexpressed in the wild-type Streptomyces albus XM211. Also, slnTI and slnTII were introduced into S. lividans 1326, and the resistance to salinomycin of the resulted strains was tested. [Results] Compared with the wild-type strain, salinomycin production in LJ01 and LJ02 decreased by 27.2% and 45.4%, respectively. The transcription of structure gene slnA3 and regulatory gene slnR were both reduced in LJ01 and LJ02. The overexpression of slnTI and slnTII in the wild-type XM211 resulted in a 14.6% enhanced salinomycin production. Correspondingly, the transcription of slnA3 and slnR was also increased. Additionally, heterologous expression of slnTI and slnTII in S. lividans 1326 slightly increased its resistance to salinomycin. [Conclusion] slnTI and slnTII are identified to be related to the production of salinomycin, but not to be the main resistant gene for salinomycin in the producer.

Abstract:[Objective] Express and purify the Pyrococcus furiosus (P. furiosus) 8-oxoguanine DNA glycosylase; characterize the enzymatic properties of the recombinant P. furiosus 8-oxoguanine DNA glycosylase. [Methods] First, pfogg gene was cloned into an expression vector. Second, the expression of the recombinant protein was induced in Escherichia coli Rosetta (DE3) by IPTG. Third, the recombinant protein was purified through Ni2+ affinity chromatography. Finally, the enzymatic reaction of PfOgg was biochemically characterized using different oligonucleotides as substrates. [Results] The recombinant P. furiosus 8-oxoguanine DNA glycosylase was successfully expressed in E. coli and the purity was up to 95%. The enzymatic characterizations of recombinant P. furiosus 8-oxoguanine DNA glycosylase were assayed in vitro. The results showed that (1) the recombinant P. furiosus 8-oxoguanine DNA glycosylase could efficiently remove the 8-oxoguanine damage from both single-stranded and double-stranded DNA and the excision efficiency of 8-oxoguanine from various DNA substrates was the following order: GO/C≈GO/G≈GO/T≈GO/A>GO/?; (2) the optimal pH value and temperature for the reaction were pH 8.5 and 55 °C, respectively; (3) Zn2+ clearly inhibited the removal of 8-oxo-G by PfOgg, and the other divalent ions, such as Mn2+, Mg2+, Ca2+, Ni2+, Co2+ and Cu2+ had no significant effect on the enzymatic reaction; (4) the removal of 8-oxo-G was highly efficient with the ionic strength ranging from 50 to 100 mmol/L, but was clearly inhibited by higher ionic strength (above 100 mmol/L). (5) the PfOgg is highly heat-resistant, with a half lifetime of 5 min at 100 °C. [Conclusion] P. furiosus 8-oxoguanine DNA glycosylase was successfully expressed in E. coli, and had the typical enzymatic activities of removing the 8-oxoguanine damaged base from single-stranded and double-stranded DNA.

Abstract:[Objective] In order to improve the production of Iturin A from Bacillus amyloliquefaciens CC09. [Methods] We first investigate the alterative constitutes and their concentrations (various carbon and nitrogen sources), NaCl concentration and pH based on LB medium, and the cultural conditions in the flask including temperature, shaking speed, medium volume using single factor experiments. Based on the results obtained from the single factor experiments, four key factors, including nitrogen concentration, pH, temperature, and medium volume were further optimized using orthogonal experiment. [Results] Through the optimization of medium and cultural condition, the growth speed and Iturin A production of strain CC09 could be improved. Soluble starch and a suitable ratio of tryptone to yeast powder were respectively good carbon and nitrogen sources for strain CC09 to synthesize Iturin A. Moreover, cultural conditions including temperature, medium volume and pH had significant influences on the Iturin A production of strain CC09. The optimal fermentation condition is as follow, medium composition is 5 g/L starch, 15 g/L tryptone and yeast powder mix (3:1, V/V), 1 g/L NaCl; cultural condition is pH 6.0, shaking speed 120 r/min, medium volume 20%, temperature 28 °C. [Conclusion] Under the optimal fermentation condition, the production of Iturin A of strain CC09 was up to 690 mg/L, 4 times higher than when it was cultured in LB medium (138 mg/L).

Abstract:[Objective] This study aimed to purify and characterize proteases from Aspergillus oryzae. [Methods] Ammonium sulfate precipitation, DEAE-Sepharose FF anion exchange chromatography, Phenyl-Sepharose HP hydrophobic chromatography and Superdex-G75/200 gel filtration chromatography were used to purify the proteases from A. oryzae. The molecular weight and purity were determined by SDS-PAGE, and the enzymatic hydrolyzates of soy protein isolated were assayed by HPLC. [Results] Two proteases (P1 and P2) were obtained from Aspergillus oryzae and their molecular weights were approximately 37 kD and 45 kD, respectively. Using casein as a substrate, the optimum conditions of P1 and P2 were pH 8.0, 45 °C and pH 7.0, 45 °C, respectively. The Km of P1 and P2 were 8.36 g/L and 4.11 g/L, respectively. Moreover, the Vm of P1 and P2 were 12.95 μg/(mL·min) and 4.86 μg/(mL·min), respectively. Both P1 and P2 had the highest hydrolytic activity to casein while lowest activity to BSA (Bull Serum Albumin). Meanwhile, the molecular weight of peptides in soy protein isolated hydrolyzates had different distributions. [Conclusion] Purified P1 and P2 have some differences in enzymatic properties. And they both have strong cutting capacity towards the hydrophobic peptide bond, but there are also differences in the specificity of bond groups. These results have a great significance for the application of proteases from Aspergillus oryzae in the food industry.

Abstract:[Objective] To study the relationship between S-adenosylmethionine synthetase (SAMs) activity and the biosynthesis of epothilone. [Methods] We analyzed the relationship between the activities of SAMs and the yields of epothilone by the addition of inhibitor and accelerator. [Results] SAMs showed high activities in the period of the biosynthesis of epothilone. SAMs activities and the yields of epothilones both reduced with the indole-3-acetic acid addition and increased with the sodium p-toluenesulfonate addition. The SAMs activities were positive correlated with the yields of epothilone when the inhibitor or accelerator was added. [Conclusion] SAMs play an important role in the biosynthesis of epothilone in Sorangium cellulosums.

Abstract:[Objective] Lipoglycans were purified and characterized from mycobacteria, and the structure of lipoarabinomannan (LAM), lipomannan (LM) from diverse mycobacterial strains were compared, the effects of lipoglycan trigged cyclooxygenase-2 (COX-2) protein expression in murine macrophages were studied. [Methods] Lipoglycans were extracted using the Triton X-114 phase partition, and then purified by electroelution. MALDI-TOF/TOF-MS was used to analyze the molecular weight of lipoglycans. We analyzed the structure of lipoglycans from different strains by concanavalin A (ConA), which specifically reacted with the α-D-mannosyl domain at the non-reducing end of the molecule. Western blot was used to determine the expression of COX-2 in RAW 264.7 macrophages. [Results] Lipoglycans were successfully obtained from M. sinov JDM601, M. tuberculosis H37Rv and M. smegmatis mc2155 by using electroelution. Furthermore, using MALDI-TOF/TOF-MS, we found the ranking of molecular mass from small to large are M. sinov JDM601, M. smegmatis mc2155, M. tuberculosis H37Rv, respectively. Con A interacted with LAM from M. tuberculosis H37Rv but not with those of M. sinov JDM601 and M. smegmatis mc2155; LM from M. sinov JDM601 reacted strongly with Con A compared to LM from the other strains. In addition, all of the lipoglycans enhanced COX-2 expression in RAW 264.7 macrophages. [Conclusion] This is the first study to purify the lipoglycans from Chinese clinical mycobacterial isolate, and the result showed that the lipoglycans have different structures in comparison with the standard stains and indicated that LAM and LM can enhance COX-2 expression in macrophages. The study also laid the foundation for further researches on their virulence and immune mechanism on hosts.

Abstract:Aspergillus nidulans is a filamentous fungus used as a model system for asexual developmental mechanisms study. Taking research of the FluG-BrlA pathway involved in Aspergillus nidulans asexual developmental as the starting point, we summarized current understanding of the asexual developmental including the central regulatory pathway genes (brlA, abaA and wetA), the central regulatory pathway modifiers (stuA and medA) and the central regulatory pathway activators (fluG, flbA?E), as well as the genetic model for related genes of Aspergillus nidulans conidiation. This review can be used as references for researching asexual developmental mechanisms of other filamentous fungi.

Abstract:Diketopiperazines (DKPs) are derivatives of cyclodipeptides resulted from the condensation of two amino acids. The conformationally constrained six-membered ring makes DKP an attractive pharmacophore in medicinal chemistry. Recently, there has been increasing interest in the natural DKPs due to their diverse bioactivities and pharmacological activities. Advances in modern biotechnologies and high throughput sequencing technologies have promoted our understanding of DKPs biosynthesis both on enzymatic and genetic levels. Biosynthesis of DKPs can be achieved either by non-ribosomal peptide synthases (NRPSs) or cycliodipeptide synthases (CDPSs). This paper provides a brief overview of recent progresses on DKPs biosynthesis.

Abstract:Lactobacilli, one of the most common members of intestinal microbiota, have been applied and studied considerably. Because it can regulate the host intestinal microbiota balance and improve the immunity and resistance of host. Lactobacillus surface layer proteins (S-layer proteins) have been a research hotspot for playing an important role in the adhesion to the host. Here we summarize the advances in researches about the structure, the method of separation and purification, the situation of gene cloning and the probiotic functions of S-layer protein.

Abstract:Microbial diversity of hot springs includes species diversity, genetic diversity, physiological diversity and ecological diversity. It is influenced by geographical position, temperature, pH value, chemical composition (sulfur, boron, iron, arsenic ions and compounds), oxygen and light. Many thermophilic enzymes have been isolated from thermophilic microorganisms in hot springs. This paper summarizes the microbial diversity of hot springs and its influencing factors, discusses the widely used microbial enzymes produced by microorganisms from hot springs. This would promote the research of hot springs’ microbial diversity, and benefit for the exploitation of hot springs’ microbial resources.

Abstract:For health and disease, the gut microbial community of humans has received significant attention from researchers. The application of 16S rRNA-based molecular technology enables researchers to study the microbial composition in the gut ecosystem. Developments in “omics” approaches have made it possible to reveal the function of microbes in the gastrointestinal tract. In this paper, combined with our own findings, we summarized advances in molecular ecology applied to studying gastrointestinal tract microorganism.

Abstract:Butanol, as a new generation of biofuels, has become the hot spot of the biofuel reseach in the world. Production of butanol from renewable feedstocks has been drawn attention to many countries. However, the cost of producing biobutanol remains significantly higher than the cost of petro-chemical derived butanol. In order to reduce costs in butanol production, the studies should be conducted as the following: using low-cost non-grain feedstocks, developing new processes with high butanol productivity and lower energy intensity breeding new strains with high butanol yield. In the near future, it is believed that the butanol production process of economic competitiveness and sustainability can be established.

Abstract:Recently, harmful algae blooms (HABs) always frequently outbreaks on eutrophication water body and it has been recognized as a serious worldwide environmental problem. It is very important and useful method that algae-lysing microorganisms as biocontrol agents to control HABs and to purify to water. This article reviews on microbial to control harmful algal blooms and discuses application prospect in the future. In additionally, the results are expected to have a certain reference value for researching algae lysing and application in other aspects.

Abstract:Flow cytometer, an advanced instrument integrating fluidics, optics, electronics, biology, immunology, etc., measures cells in suspension that flow in single-file through laser beam. Flow cytometry (FCM), as the core of flow cytometer, is a multidisciplinary technique for analysing and sorting cells or other particles. FCM can provide a multitude of information at the single-cell level, including (but not limited to) total counts, size measurements, nucleic acid content, cell viability and activity, and detection of specific bacterial groups or species. The main advantage of FCM is that it is fast, sensitive, accurate and easy to perform. Here, we briefly review the basic principle of flow cytometer, focusing on its recent applications to rapid detection of bacteria in laboratory research, industrial biotechnology, clinical diagnosis and environment assessment.

Abstract:Preventing foods from the contamination of foodborne pathogens is an important strategy to ensure food safety. Reports have demonstrated that microorganisms could enter the viable but nonculturable (VBNC) state when they respond to various environmental stresses. This paper gives an overview of recent findings on the VBNC state of foodborne pathogens with emphasis on the universality, induced conditions and characteristics of VBNC state of bacteria cells as a response to different environmental stresses (including natural stresses and those during food process and preservation). In addition, the limit of current bacteria detection methods based on bacteria’s culturability but not on their viability and its potential challenge to food safety assessment were discussed.

Abstract:[Objective] To study the lipid compositions changes of Isochrysis zhangjiangensis from nitrogen-sufficient to nitrogen-limited cultivation process. [Methods] High performance thin layer chromatography (HPTLC) was used to analysis lipids of microalga Isochrysis zhanjiangensis. [Results] With the consumption of nutrition in the medium, the cells were gradually subjected to stress conditions, under which condition a large quantity of triacylglycerol (TAG) was accumulated. Opposed the storage of TAG, the lipids composed the biomembrane, monogalactosyldiacylglycerol (MGDG), sulfoquinovosyldiacylglycerol (SQDG), phosphadidylglycerol (PG) and phosphatidylcholine (PC) gradually reduced. The content of free fatty acid (FFA), diacylglycerol (DAG), digalactosyldiacylglycerol (DGDG), phosphatilinosotol (PI) and phosphatidylethanolamine (PE) kept relatively stable. [Conclusion] As a robust and rapid tool for separation and analysis of lipids, HPTLC can be used in the study of lipid metabolism and regulatory accumulation of TAG in microalgae.

Abstract:[Objective] Establish a simple and rapid molecular detection of Yellow fever virus. [Methods] The reserve transcription loop-mediated isothermal amplification (RT-LAMP) that amplifies RNA with high specificity and rapidity under an isothermal condition was applied for rapid detection of Yellow fever virus. A set of primers were designed specifically to identify six special areas of Yellow fever virus’s E gene. [Results] The results indicated that the method had high specificity, and no cross amplification occurred with other arbovirus RNAs. The sensitivity of the RT-LAMP assay was 0.066 pg, which was 100 times higher than that of RT-PCR. The detection results of simulation samples were consistent with expected. [Conclusion] The established rapid detection method for Yellow fever virus that can be carried out in a simple experimental environment has been proved to be specific and sensitive.

Abstract:[Objective] The aim of this study was to obtain DNA with high purity, high yield and good integrity from the soil, for the analysis of the microbial diversity of heavy metal contaminated soil. [Methods] Accompanied by different soil pretreatments, AlNH4(SO4)2 was added in the DNA extraction buffer to analyze the yield, purity and integrity of the total DNA extracted from the soil. [Results] The DNA fragments extracted from the four different methods, TENP-AlNH4(SO4)2, ABG-AlNH4(SO4)2, Wash-AlNH4(SO4)2 and Reagent Kit, were all intact, and the sizes of them were all about 23 kb. The Wash-AlNH4(SO4)2 method gave the purest DNA with 2.13 at A260/A230 and 1.62 at A260/A280, while the yield from ABG-AlNH4(SO4)2 method was the highest with 1 010 μg DNA per gram soil. Both methods could provide DNA with sufficient purity and concentration for the following molecular experiments such as PCR. Through the amplification of 16S rRNA gene from the extracted soil DNA, PCR inhibitors in the soil could be efficiently removed by AlNH4(SO4)2 with appropriate concentrations. [Conclusion] By combining soil pretreatment and utilization of AlNH4(SO4)2, we obtained the desired total DNA from the soil contaminated with heavy metals.