Dairong Qiao

Sichuan University, Hua-yang, Sichuan, China

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Publications (44)54.76 Total impact

  • Huoxiang Zhou · Xi Li · Mingyue Guo · Qingrui Xu · Yu Cao · Dairong Qiao · Yi Cao · Hui Xu ·
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    ABSTRACT: The endo-polygalacturonase gene (endo-pgaA) was cloned from DNA of Aspergillus niger SC323 using the cDNA synthesized by overlapping PCR, and successfully expressed in Saccharomyces cerevisiae EBY100 through fusing α-factor signal peptide of yeast. The full-length cDNA consists of 1113 bp and encodes a protein of 370 amino acids with a calculated molecular mass of 38.8 kDa. After induction by galactose for 48 h, the activity of recombinant endo-PgaA in culture supernatant can reach up to 1448.48 U/mg. The recombinant protein was purified to homogeneity by ammonium sulfate precipitation and gel filtration column chromatography and subsequently characterized. The optimal pH and temperature of the purified recombinant enzyme were 5.0 and 50 °C, respectively. The Michaelis-Menten constant (Km) and maximal velocity (Vmax) of the enzyme for pectin were 88.54 μmol/ml and 175.44 μmol/mg/min, respectively. The enzyme activity was enhanced by Ca(2+), Cu(2+), Na(+) and strongly inhibited by Pb(2+), Mn(2+). The pectin hydrolysates were mainly galacturonic acid and other oligo-galacturonates. Therefore, these characteristics suggest that the recombinant endo-PgaA may be of potential use in food and feed industry.
    Journal of Microbiology and Biotechnology 03/2015; 25(7). DOI:10.4014/jmb.1501.01024 · 1.53 Impact Factor
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    Tao Song · Weijia Zhang · Congchong Wei · Tengfei Jiang · Hui Xu · Yi Cao · Yu Cao · Dairong Qiao ·
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    ABSTRACT: Agar is a polysaccharide extracted from the cell walls of some macro-algaes. Among the reported agarases, most of them come from marine environment. In order to better understand different sources of agarases, it is important to search new non-marine native ones. In this study, seven agar-degrading bacteria were first isolated from the tissues of plants, belonging to three genera, i.e., Paenibacillus sp., Pseudomonas sp., and Klebsiella sp. Among them, the genus Klebsiella was first reported to have agarolytic ability and the genus Pseudomonas was first isolated from non-marine environment with agarase activity. Besides, seven strains were characterized by investigating the growth and agarase production in the presence of various polysaccharides. The results showed that they could grow on several polysaccharides such as araban, carrageenan, chitin, starch, and xylan. Besides, they could also produce agarase in the presence of different polysaccharides other than agar. Extracellular agarases from seven strains were further analyzed by SDS-PAGE combined with activity staining and estimated to be 75 kDa which has great difference from most reported agarases.
    Current Microbiology 10/2014; 70(2). DOI:10.1007/s00284-014-0713-6 · 1.42 Impact Factor
  • Yu Cao · Hui Xu · Li Xie · Yi Yi · Yingpeng Yu · Shunli Feng · Dairong Qiao · Yi Cao ·
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    ABSTRACT: A general model of the catalytic mechanism for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) has already been proposed. But whether shikimate-3-phosphate (S3P) alone can cause EPSPs' conformation changes, and whether the binding site of phosphoenolpyruvate (PEP) and glyphosate is the same are still in debate. In this paper, DsaroA gene amplified and cloned from Dunaliella salina (our laboratory's early study) was used for DsEPSPs expression and purification. Then the DsEPSP conformation changes as it bind with different substrates were detected by fluorimetry. The results show that we obtained the DsEPSPs by prokaryotic expression and purification, and the S3P binding with DsEPSPs alone cannot cause DsEPSPs to form "close" conformation directly. However, when S3P exits, DsEPSPs did have a trend to change to the "close" conformation. Then the "close" conformation can be formed completely with the addition of phosphoenolpyruvate (PEP) or glyphosate. The inorganic phosphorus can help S3P to induce two domains of DsEPSPs to form "close" conformation. Besides, when DsEPSPs binds with S3P, in 295 nm, only the intensity of emission peak decreases, however, in 280 nm, not only the peak intensity reduces but also the blue-shift phenomenon takes place. The reason for blue-shift phenomenon was the distribution of aromatic amino acids in EPSPs. EPSPs is a good target for novel antibiotics and herbicides, because of shikimic acid pathway is only present in plants and microorganisms, completely absent in mammals, fish, birds, reptiles, and insects. The results demonstrate that the binding of substrates to EPSPs causes a conformational change from an open form to a closed form, that might be important for designing of novel antimicrobial and herbicidal agents that block closure of the enzyme.
    Journal of Basic Microbiology 09/2014; 54(9). DOI:10.1002/jobm.201300324 · 1.82 Impact Factor
  • Tao Song · Yu Cao · Hui Xu · Weijia Zhang · Baojin Fei · Dairong Qiao · Yi Cao ·
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    ABSTRACT: Agar is a polysaccharide polymer material, generally extracted from seaweed. Most agar degradation strains were isolated from seawater. In order to find new species resources and novel agarase from soil, an agar-degrading bacterium Paenibacillus sp. SSG-1 was isolated from soil. Agarase SSG-1a was purified to homogeneity by 30.2 fold with a yield of 4.8% through ammonium sulfate precipitation, DEAE FF chromatography and native-PAGE separation. The tandem mass spectrometry (MS/MS) results indicated that purified SSG-1a should be a novel β-agarase. The molecular mass of SSG-1a was estimated to be 77 kDa. The optimal temperature and pH for SSG-1a were 50°C and pH 6.0, respectively. Moreover, SSG-1a was stable in pH range of 4.0–10.0 and at temperature up to 40°C. It could hydrolyze the β-1,4 linkage of agarose to produce neoagarohexaose (95 mol%) and neoagarooctaose (5 mol%). Metal ion Mn2+ and reducing reagents (β-Me and DTT) could increase its activity by 150% and 60%, respectively.
    Journal of Bioscience and Bioengineering 08/2014; 118(2). DOI:10.1016/j.jbiosc.2014.02.008 · 1.88 Impact Factor
  • Shu Zhang · Xin Ran Li · Hui Xu · Yu Cao · Shu Han Ma · Yi Cao · Dairong Qiao ·
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    ABSTRACT: Dunaliella salina, a unicellular green alga, has the potential to grow in hypersaline environments via one of its gene products, superoxide dismutase (SOD). The superoxide radicals (O2 (-) ) produced by environmental stresses can cause damage to cells, and SOD catalyzes the turnover of such free radicals to protect cells. In this study, the gene coding for SOD in D. salina was cloned and the product was further identified and characterized. The open reading frame of this gene was 651 bp long, encoding for 217 amino acids. According to the sequence alignment using BLAST, native polyacrylamide electrophoresis for SOD activity analysis, and atomic absorption spectroscopy analysis, this protein belongs to the manganese-containing superoxide dismutase (MnSOD) family. Complementation analysis, performed by introducing plasmids carrying an inducible version of the D. salina gene encoding for MnSOD into an SOD-deficient mutant of E.coli, revealed that this gene could not only complement the defects in SOD activity, but was also capable of providing a stronger tolerance to restrictive growth conditions, such as high salt and prolonged UV exposure, compared to the tolerance of wild-type strains.
    Journal of Basic Microbiology 05/2014; 54(5). DOI:10.1002/jobm.201200483 · 1.82 Impact Factor
  • Jiafu Lin · Bohan Zhao · Yu Cao · Hui Xu · Shuhan Ma · Mingyue Guo · Dairong Qiao · Yi Cao ·
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    ABSTRACT: A three-dimensional structure of lipase protein was constructed by using homology modeling. Six different Fe-MCM-41 carriers were synthesized with different pore size based on the properties of the lipase examined. The relative activity of lipase from Yarrowia lipolytica (YYL) immobilized on Fe-MCM-41 with a pore size of 4.27 nm (FM-4-YYL) reached 197% when compared with free lipase. This result was notably higher than that of YYL encapsulated in other forms of Fe-MCM-41. Moreover, FM-4-YYL has excellent thermal stability in that it can preserve nearly 80% of the initial activity after incubation at 60 C for 1 h. In addition, immobilized lipases were used as catalysts for the transesterification of olive oil with methanol. The highest conversion yield (98%) was observed when FM-4-YYL was used as a biocatalyst for biodiesel (10 mL olive oil, 1.66 mL methanol, and 1.5 mL water at 30 C for 4h). FM-4-YYL can be reused for nine cycles without significant loss in activity. The work demonstrates that the selection and modification of adsorbents based on enzyme protein properties is a very promising strategy for increasing stability and enhancing active the performance of biocatalysts for industrial production.
    Applied Catalysis A General 05/2014; 478:175–185. DOI:10.1016/j.apcata.2014.03.034 · 3.94 Impact Factor
  • Shu Zhang · Yu Cao · Li Xie · Dairong Qiao · Yi Cao ·
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    ABSTRACT: Superoxide dismutases (SOD) are able to remove the superoxide anion free radicals produced by environmental stress and thereby protect cells from being injured by reactive oxygen species. However, SOD is unable to transduce automatically across cell membranes. Protein transduction domains (PTDs) are peptides able to mediate protein delivery into cells and were first observed in the HIV‑1 Tat protein. In the present study, PTD (RKKRRQRRR) was fused to Dunaliella salina (Ds)MnSOD to form PTD‑DsMnSOD. This was inserted into pET32a to construct the recombinant plasmid pET32a‑PTD‑DsMnSOD and transduced into E. coli BL21(DE3) to obtain purified PTD‑DsMnSOD proteins. Liposome‑encapsulated proteins are also able to cross cell membranes. In this study, DsMnSOD proteins were purified and encapsulated by liposomes. The obtained MnSOD, PTD‑MnSOD and liposome MnSOD were used to protect human umbilical vein endothelial cells (HUVECs) from injury under oxygen pressure. A cell counting kit 8 was used to test the survival rate of HUVECs and results indicated that the protective effect of MnSOD was limited compared with that of PTD‑MnSOD and liposome MnSOD. Thus, PTD and liposomes exhibited improved effects when MnSOD was present in cells.
    Molecular Medicine Reports 04/2014; 9(4):1427-33. DOI:10.3892/mmr.2014.1970 · 1.55 Impact Factor
  • Xinran Li · Hui Xu · Jie Xie · Qiaofu Yi · Wei Li · Dairong Qiao · Yi Cao · Yu Cao ·
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    ABSTRACT: In order to improve the expression of heat-resistant xylanase XYNB from Aspergillus niger SCTCC 400264, XynB has been cloned into Pichia pastoris secretary vector pPIC9K. The XynB production of recombinant P. pastoris was four times as E. coli, the Vmax and specific activity of XynB reached 2547.7μmol/mg and 4757 U/mg, respectively. And the XynB still had 74% residual enzyme activity after 30 minute-heat treatment at 80 °C. The van der Waals force analysis in XYNB (ACN89393 and AAS67299), there is one more oxygen radicals in AAS67299 in their catalytic site, indicating that the local cavity is much more free, and it is more optimal for substrate binding, affinity reaction, and proton transfer etc, and eventually increasing enzyme activity. The H-bonds analysis of XYNB indicated that there are two more H-bonds in 33rd Ser of XYNB ( AAS67299) than 33rd Ala(ACN89393 ), two H-bonds between Ser70 and Asp67.
    Journal of Microbiology and Biotechnology 01/2014; 24(4). DOI:10.4014/jmb.1307.07086 · 1.53 Impact Factor
  • Xian Sun · Yu Cao · Hui Xu · Yan Liu · Jianrui Sun · Dairong Qiao · Yi Cao ·
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    ABSTRACT: Triacylglyceride (TAG) and carbohydrate are potential feedstock for biofuels production. In this study, a two-stage process was applied for enhancing TAG/carbohydrate production in the selected microalgae - Neochloris oleoabundans HK-129. In stage I, effects of nitrogen, light intensity and iron on cell growth were investigated, and the highest biomass productivity of 292.83±5.83mg/L/d was achieved. In stage II, different nitrogen-starvation periods, light intensities and iron concentrations were employed to trigger accumulation of TAG and carbohydrate. The culture under 2-day N-starvation, 200μmol/m(2)/s light intensity and 0.037mM Fe(3+) concentration produced the maximum TAG and carbohydrate productivity of 51.58mg/L/d and 90.70mg/L/d, respectively. Nitrogen starvation period and light intensity had marked effects on TAG/carbohydrate accumulation and fatty acids profile, compared to iron concentration. The microalgal lipid was mainly composed of C16/C18 fatty acids (90.02%), saturated fatty acids (29.82%), and monounsaturated fatty acids (32.67%), which is suitable for biodiesel synthesis.
    Bioresource Technology 01/2014; 155C:204-212. DOI:10.1016/j.biortech.2013.12.109 · 4.49 Impact Factor
  • Yang Liu · Yu Yang · Feng Zhao · Xuejun Fan · Wei Zhong · Dairong Qiao · Yi Cao ·
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    ABSTRACT: We collected flies from Chengdu Shuangliu International Airport to examine for the presence of bacteria and to determine the sensitivity patterns of those bacteria. A total of 1,228 flies were collected from 6 sites around Chengdu Shuangliu International Airport from April to September 2011. The predominant species was Chrysomya megacephala (n=276, 22.5%). Antimicrobial-resistant gram-negative enteric bacteria (n=48) were isolated from flies using MacConkey agar supplemented with cephalothin (20 microg/ml). These were identified as Escherichia coli (n=37), Klebsiella pneumoniae (n=6), Pseudomonas aeruginosa (n=3) and Aeromonas hydrophila (n=2). All isolated bacteria were tested for resistance to 21 commonly used antimicrobials: amoxicillin (100%), ticarcillin (100%), cephalothin (100%), cefuroxime (100%), ceftazidime 1 (93.8%), piperacillin (93.8%), cefotaxime (89.6%), ticarcillin-clavulanate (81.3%), trimethoprim-sulfamethoxazole (62.5%), ciprofloxacin (54.2%), gentamicin (45.8%), cefepime (39.6%), tobramycin (39.6%), ceftazidime (22.9%), cefoxitin (16.7%), amikacin (16.7%), netilmicin (14.6%), amoxicillin-clavulanate (6.3%) and piperacillin-tazobactam (2.1%). No resistance to meropenem or imipenem was observed. Antibiotic resistance genes among the isolated bacteria were analyzed for by polymerase chain reaction. Thirty of the 48 bacteria with resistance (62.5%) possessed the blaTEM gene.
    The Southeast Asian journal of tropical medicine and public health 11/2013; 44(6):988-96. · 0.72 Impact Factor
  • Yang Liu · Wei Zhong · Zhongan Fei · Feng Zhao · Dairong Qiao · Yi Cao ·
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    ABSTRACT: Housefly (Musca domestica) is an important mechanical and biological vector harboring and transmitting human and animal pathogens. To study the composition of bacterial community in housefly midgut and assess the role of housefly in the epidemiology of diseases, 16S rRNA gene clone library of bacteria in housefly midgut was constructed. The library compromised of 88 sequences, which were grouped into 22 OTUs, including Proteobacteria (12 OTUs), Firmicutes (9 OTUs), and Bacteroidetes (1 OTU). Twelve OTUs of Proteobacteria belonged to Betaproteobacteria (1 OTU), Epsinoproteobacteria (1 OTU) and Gammaproteobacteria (10 OTUs) respectively. No Alphaproteobacteria or Deltaproteobacteria was found. The 9 Firmicutes included Lactobacillales (6 OTUs), Clostridia (1 OTU) and Bacillales (2 OTUs). In the library, Gammaproteobacteria was the dominant phylotype, which compromised 71 sequences (80.7%). Some species in Enterobacter, Klebsiella, Providencia, Proteus, Aeromonas, and Staphylococcus in the library were pathogens or opportunistic pathogens to human and animal. These results help to understand the significant roles of housefly in disease transmitting.
    Chinese Journal of Applied and Environmental Biology 10/2013; 19(5):800-804. DOI:10.3724/SP.J.1145.2013.00800
  • Baojin Fei · Hui Xu · Yu Cao · Shuhan Ma · Hongxiu Guo · Tao Song · Dairong Qiao · Yi Cao ·
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    ABSTRACT: Despite recent advances in our understanding of the importance of protein surface properties for protein thermostability,there are seldom studies on multi-factors rational design strategy, so a more scientific, simple and effective rational strategy is urgent for protein engineering. Here, we first attempted to use a three-factors rational design strategy combining three common structural features, protein flexibility, protein surface, and salt bridges. Escherichia coli AppA phytase was used as a model enzyme to improve its thermostability. Moreover, the structure and enzyme features of the thermostable mutants designed by our strategy were analyzed roundly. For the single mutants, two (Q206E and Y311K), in five exhibited thermostable property with a higher success rate of prediction (40 %). For the multiple mutants, the themostable sites were combined with another site, I427L, we obtained by directed evolution, Q206E/I427L, Y311K/I427L, and Q206E/Y311K/I427L, all exhibited thermostable property. The Y311K/I427L doubled thermostability (61.7 %, and was compared to 30.97 % after being heated at 80 °C for 10 min) and catalytic efficiency (4.46 was compared to 2.37) improved more than the wild-type AppA phytase almost without hampering catalytic activity. These multi-factors of rational design strategy can be applied practically as a thermostabilization strategy instead of the conventional single-factor approach.
    Journal of Industrial Microbiology 03/2013; 40(5). DOI:10.1007/s10295-013-1260-z · 2.44 Impact Factor
  • Yuzhi Miao · Hui Xu · Baojin Fei · Dairong Qiao · Yi Cao ·
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    ABSTRACT: The major objective of this study was to engineer lactic acid bacteria to produce the enzyme phytase from a gene native to Bacillus subtilis GYPB04. The phytase gene (phyC) of B. subtilis GYPB04 was cloned into the plasmid pMG36e for expression in Lactococcus lactis. The enzyme activity in L. lactis cultured in GM17 broth was 20.25 U/mL at 36°C. The expressed phytase was characterized as active in a pH range of 2.0-9.0 at a temperature range of 20-80°C, with an optimum pH of 5.5-6.5 and temperature of 60°C. When cultured in food-grade milk broth, the transformed L. lactis grew to an OD600 nm value of 1.05 and had a phytase yield of 13.58 U/mL. In same broth under optimized conditions for cell growth and phytase production, the transformant reached an OD600 nm value of 1.68 and a phytase yield of 42.12 U/mL, representing approximately 1.6-fold and 3.1-fold increases, respectively, compared to growth in natural milk broth. Fermentation was scaled to 5 L under optimized conditions, and product analysis revealed a final OD600 nm value of 1.89 and an extracellular enzyme activity of 24.23 U/mL. The results of this study may be used in the dairy fermentation industry for the development of functional, healthy yogurts and other fermented dairy foods that provide both active phytase and viable probiotics to the consumer.
    Journal of Bioscience and Bioengineering 02/2013; 116(1). DOI:10.1016/j.jbiosc.2013.01.021 · 1.88 Impact Factor
  • Baojin Fei · Hui Xu · Feiwei Zhang · Xinran Li · Shuhan Ma · Yu Cao · Jie Xie · Dairong Qiao · Yi Cao ·
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    ABSTRACT: In order to study on the relationship between Escherichia coli AppA phytase's thermostability and salt bridges, and indicate an effective technical route of which factor to think about and where to modify at AppA for enhancing its thermostability, a salt bridge subtraction mutant E31Q and a salt bridge addition mutant Q307D were constructed by site-directed mutagenesis. The residual activities of the wild-type AppA phytase, E31Q and Q307D were 31.42%, 17.46%, and 40.57%, respectively, after being heated at 80°C for 10 min. The salt bridge subtraction mutant E31Q showed 13.96% thermostability decreasement, and the salt bridge addition mutant Q307D showed 9.15% thermostability enhancement than the wild-type both without the pH and temperature optimum changed. It proved salt bridges play a key role in E. coli AppA phytase's thermostability and the α/β-domain of AppA may be sensitive to heat. Salt bridges and the α/β-domain of AppA should have high priority to think about to enhance AppA's thermostability for commercial application. Besides, molecular dynamics simulation was used for salt bridges analysis.
    Journal of Bioscience and Bioengineering 01/2013; 115(6). DOI:10.1016/j.jbiosc.2012.12.010 · 1.88 Impact Factor
  • Hui Xu · Feiwei Zhang · Huiyi Shang · Xinran Li · Jing Wang · Dairong Qiao · Yi Cao ·
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    ABSTRACT: Based on the strategy of changing pH-stability profiles by altering pKa values of catalytic residues, rational protein engineering was applied to improve alkalophilic adaptation of Aspergillus niger endoxylanase XynB. Computational predictions and molecular modeling were carried out using PROPKA server and SWISS-MODEL server, respectively. Three endoxylanase mutant of S108V, N151E, and Q178R, in which the pKa values of either catalytic glutamate residues shifted, were generated. In agreement with expectation, the variant of Q178R improved alkalophilic performances. The mutant Q178R raised the optimum pH of XynB from 5.5 to 6.0 and retained 37% of the maximum activity at pH 8.0. Interestingly, the pKa values of Glu84 and Glu175 in Q178R are 7.91 and 6.32, respectively. The pKa of Glu175 is lower than that of Glu84, as opposed to the fact that the pKa of Glu84 is lower than that of Glu175 in other GH11 xylanases. It indicated that Glu175 may convert into a nucleophile residue and Glu84 into an acid/base residue.
    Journal of Bioscience and Bioengineering 01/2013; 115(6). DOI:10.1016/j.jbiosc.2012.12.006 · 1.88 Impact Factor
  • Huoxiang Zhou · Xiaohu Feng · Tao Song · Yan Liu · Xian Sun · Hui Xu · Dairong Qiao · Yi Cao ·
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    ABSTRACT: A filamentous fungal strain 7-4, which was screened out from 45 strains, was capable of producing high-yield lipids up to 46.8%, and identified by rDNA-ITS2 sequence and morphological observation. The sequence data was compared with the published sequences of relative filamentous fungal strains in Genbank. The results indicated that the homology of the rDNA-ITS2 sequences of strain 7-4 with those of several Mucor strains was more than 98.0%. As shown in the phylogenetic tree, strain7-4 and M. fragilis constituted a branch and they constituted a branch together with M. circinelloides. Furthermore, the strain 7-4 was similar to M. circinelloides in morphological character. Thus, the strain 7-4 was preliminarily determined to be belonged to the species of M. fragilis. The results showed that its composition of lipids was similar to vegetable oil and its unsaturated fatty acid content was 91.77%. This research therefore suggested that these microbes could be provided as raw material resource for microbial oil production.
    Chinese Journal of Applied and Environmental Biology 12/2012; 18(6):988. DOI:10.3724/SP.J.1145.2012.00988
  • Baojin Fei · Yu Cao · Hui Xu · Xinran Li · Tao Song · Zhongan Fei · Dairong Qiao · Yi Cao ·
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    ABSTRACT: Due to our previous research, mainly the thermostable mutants Q307D, Y311K, and I427L, we conjectured that Escherichia coli AppA phytase's C-terminal plays an important role in its thermostability, and AppA begins to collapse from the C-terminal when at a higher temperature. So here we constructed C-lose mutant to prove it. The residual activities of the wild-type AppA phytase and C-lose were 31.42 and 70.49 %, respectively, after being heated at 80 °C for 10 min. The C-terminal deletion mutant C-lose showed 39.07 % thermostability enhancement than the wild-type both without the pH and temperature optimum changed. It proved the C-lose plays a key role in E. coli AppA phytase's thermostability.
    Current Microbiology 12/2012; 66(4). DOI:10.1007/s00284-012-0283-4 · 1.42 Impact Factor
  • Hui Xu · Jiafu Lin · Zhong’an Fei · Baojin Fei · Yingpeng Yu · Dairong Qiao · Yi Cao ·
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    ABSTRACT: In order to explore lipase-producing microbes with different characteristics, 54 lipase-producing strains were screened out using pure culture method from 108 samples from the Qinghai alpine meadow - Ya'an Mountainous Regions Chengdu Plain, and only one strain is alkaline lipase-producing strain. All the ERIC-PCR fingerprints were obtained, and the tree diagram was divided into five operation units according to the fingerprints. The results showed that there existed more rich genetic diversity in all the isolates from Chengdu Plain than from Tibetan Plateau and Ya'an Mountainous Regions. The isolated strains distributed in Bacillus, Klebsiella, Pseudomonas, Staphylococcus and Enterobacter. Moreover, the fingerprints displayed different specific bands in different strains, appearing two conservative bands (300 bp and 1 300 bp) which indicated that these two bands might be the SCAR marker of the lipase-producing strains.
    Chinese Journal of Applied and Environmental Biology 06/2012; 18(3):455. DOI:10.3724/SP.J.1145.2012.00455
  • Zengting Li · Li Xie · Shunli Feng · Fangfang You · Guihua He · Qiang Lin · Yi Cao · Dairong Qiao ·
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    ABSTRACT: To construct recombinant yeast strains which can produce hybrid antimicrobial peptides with natural activity and determine the optimum conditions for expression, the Sac I -linearized recombinant expression vectors pPICZα-A-CecropinB (I∼10)-Magainin2 (1-12) (pPICZa-A-CBMA) and pPICZα-A-Lfcin-Pro-CecropinB (pPICZa-A-LPCB) were transformed into Pichia pastoris X-33 by electroporation, respectively. The positive clone strains were screened with Zeocin and identified by PCR. The protein expression was induced by methanol and the antibacterial activity was tested through antibacterial experiments. Furthermore, the expression conditions of hybrid peptide LPCB were optimized and the stability of plasmid was tested by culture. The results showed that the genetic engineering strains P. pastoris X-33/pPICZα-A-CBMA and P. pastoris X-33/pPICZα-A-LPCB were successfully constructed. The expression product of the former exhibited little antimicrobial activity, while the latter exhibited relatively stronger activity. And the optimum expression conditions of LPCB were as follows: Temperature 25 °C, pH neutral, adding 0.5% (V/V, ψ) methanol to culture every 24 h, induced expression time 72 h. P. pastoris X-33/pPICZα-A-LPCB was cultured for 100 generations and no plasmid was lost.
    Chinese Journal of Applied and Environmental Biology 02/2012; 18(1). DOI:10.3724/SP.J.1145.2012.00065
  • Linfeng Lu · Jianyu Yue · Zhong’an Fei · Rui Zhao · Dairong Qiao · Yi Cao ·
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    ABSTRACT: Sclerotinia sclerotiorum is one of the most terrible diseases for rapeseeds in production. Its pathogenicity may probably get from horizontal gene transfer (HGT). For understanding its pathogenic mechanism and finding targets for new fungal inhibitors, firstly in BLASTp comparison results of gene XM-001585458.1-coded proteins XP-001585508.1 of different species, low E value 3.23e-109 and high SCORE value 436 appeared in the results of bacteria, showing HGT happened probably. Then via phylogenetic method, gene XM-001585458.1 was found out the closest to bacterium. At the same time, by nucleotide composition analysis, XM-001585458.1 also appeared quite different from the genome of S. sclerotiorum, since GC-content increased 14.95%. To sum up, HGT of gene XM-001585458.1 was found out. And structure and COG analysis indicated that there were two distinct transmembrane domains in 14-171 and 465∼485 of protein XP-001585508.1, and this protein also consisted of 6 β-sheets, 1 α-helix, some turns, and random coils. So the candidate protein encoded by the transferring gene should be further predicted to be a membrane protein participating in polysaccharide metabolic. And it can be assumed that the gene transferring is related to the pathogenicity of S. sclerotiorum and its activity of hydrolyzing the cell wall when it is infecting the plant.
    Chinese Journal of Applied and Environmental Biology 02/2012; 18(1). DOI:10.3724/SP.J.1145.2012.00042

Publication Stats

178 Citations
54.76 Total Impact Points


  • 2004-2015
    • Sichuan University
      • • College of Life Sciences
      • • Key Laboratory of Food Engineering of Sichuan Province
      • • Key Laboratory of Bio-resource and Eco-environment (MOE)
      Hua-yang, Sichuan, China