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ABSTRACT: Phytases play a very important role in increasing phytate digestion and reducing phosphorus pollution in the environment, and phytate-degrading bacteria have a ubiquitous distribution in the environment. Due to its extremely harsh environment, the Tibetan Plateau breeds possibly abundant, extreme microorganisms. In this research, 67 phytate-degrading bacteria were isolated from different habitats in the Tibetan Plateau. Among all isolates, 40.3% were screened from farmland, 25.3% from wetland, 4.5% from saline-alkaline soil, 7.5% from hot springs, and 22.4% from lawns, which showed that the distribution of the phytate-degrading bacteria varied with habitats. By the PCR-RFLP method, 16 different species were identified and named, 4 of which are reported for the first time as phytate-degrading bacteria, that is, Uncultured Enterococcus sp. GYPB01, Bacillaceae bacterium strain GYPB05, Endophytic bacterium strain GYPB16, and Shigella dysenteria strain GYPB22. Through the assay of phytase activity of 16 strains, Klebsiella sp. strain GYPB15 displayed the highest capability of phytase production. Through analysis of the optimum pH, the optimum temperature, and the thermal stability of enzyme from 16 strains, some especial phytate-degrading bacteria were obtained. Our findings clearly indicate a good relation between the composition of the soils from the different environments in the Tibetan Plateau and populations of cultivable phytate-degrading bacteria. Moreover, extreme harsh soils are logically the best soils in which to find some strains of phytate-degrading bacteria for exploiting in the fields of biotechnology and industry.
Canadian Journal of Microbiology 04/2013; 59(4):245-51. · 1.36 Impact Factor
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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; · 1.80 Impact Factor
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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; · 1.79 Impact Factor
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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; · 1.79 Impact Factor
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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; · 1.79 Impact Factor
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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; · 1.82 Impact Factor
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ABSTRACT: Mitogen-activated protein kinase (MAPK) is conserved gene family in all eukaryotic cells. MAPKs play an important role in
abiotic stress responses. In green algae, few MAPK genes have been cloned, although MAPKs have been extensively studied in
yeasts and humans. The microalga Dunaliella is a model for the study of a variety of abiotic stress tolerances. Dunaliella salina exhibits excellent adaptation to the hypersaline environment without a rigid cell wall. The cDNA of MAPK in D. salina was cloned using degenerate oligonucleotide primers in a polymerase chain reaction (PCR) amplification approach and rapid
amplification of cDNA ends method. An open reading frame encoding a 53 kDa protein was identified. The deduced amino acid
sequence showed high homology with other reported MAPKs. The conserved regions, including TEY activation loop and the common
docking domain, were characterized in the deduced amino acid sequence. To our knowledge, we presented the initial full-length
sequence of MAPK in D. salina. The quantitative real-time PCR demonstrated DsMPK expression level gradually decreases under the hyperosmotic and low temperature
environments. The results provide valuable information about the response of DsMPK at the transcription level.
Journal of Applied Phycology 04/2012; 20(1):13-17. · 2.41 Impact Factor
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ABSTRACT: His(354) and His(358), two highly conserved histidines in Xenopus laevis (6-4) photolyase [equivalent to His(401) and His(405), in Dunaliella salina (6-4) photolyase], are critical for photoreactivation. They act as a base and an acid, respectively. However, the remaining high repair activity when the pH value is higher than the pKa of histidine suggests the involvement of other basic amino acids in photoreactivation. According to the results of in vivo enzyme assay and three-dimension structural model of Dunaliella salina (6-4) photolyase we hypothesized that Lys(281) might be involved in the photoreactivation over the pH range from 10.0 to 11.0. To test this, we generated two mutant forms of the (6-4) photolyase, K281G and K281R mutant, by overlap extension polymerase chain reaction, and performed the enzyme assay with these mutants. From these results we conclude that the Lys(281), which is highly conserved in (6-4) photolyases, participates in the photoreactivation and acts as an acid to donate a proton to His(401) when the environmental pH is higher than the pKa value of histidine.
Current Microbiology 01/2011; 62(1):146-51. · 1.82 Impact Factor
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ABSTRACT: Xylanase is one of the most important hemicellulases in industry. However, its low thermostability limits its applications. In this study, one thermostable xylanase-producing strain 400264 was obtained from screening 11 Aspergillus niger strains (producing thermotolerant xylanase), and the optimum temperature of crude xylanase extracted from it was 55°C. Original activity of the crude xylanase is 64% at 60°C and 55% at 85°C with an incubation time of 30 min, respectively. After the expression of recombinant xylanase gene (xynA/xynB), the XYNB (xylanase B) showed higher thermostability than XYNA (xylanase A). Recombinant enzyme XYNB retained 94% of its activity for 10 min at 85°C, while XYNA with no activity left. Site-directed mutagenesis was performed to replace Ala33 of XYNB by Ser33 resulting 19% decrease in enzyme activity after incubating at 85°C for 30 min. It suggested that the Ala33 residue may have a certain effect on the thermophilic adaptation of xylanase.
Current Microbiology 01/2011; 62(1):242-8. · 1.82 Impact Factor
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ABSTRACT: In order to evaluate their antibacterial activities and toxicities, the cecropins-melittin hybrid antimicrobial peptide, CA(1-7)-M(4-11) (CAM) and CB(1-7)-M(4-11) (CBM), were designed by APD2 database. The recombinant hybrid antimicrobial peptides were successfully expressed and purified in Pichia pastoris. Antimicrobial activity assay showed that both of the two hybrid antimicrobial peptides had strong antibacterial abilities against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis, Bacillus thuringiensis, and Salmonella derby. The potency of CAM and CBM to E. coli 25922 were 0.862 and 0.849, respectively, slightly lower than Amp's 0.957. The hemolytic assays indicated CAM and CBM had no hemolytic in vivo and in vitro, and so they had a good application prospect.
Current Microbiology 09/2010; 61(3):169-75. · 1.82 Impact Factor
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ABSTRACT: In order to improve the thermostability of Escherichia coli AppA phytase, Error-prone PCR was used to randomize mutagenesis appA gene, and a gene mutation library was constructed. A mutant I408L was selected from the library by the method of high-throughput screening with 4-methyl-umbelliferylphosphate (4-MUP). The appA gene of the mutant was cloned and expressed in E. coli Origami (DE3). The recombinant protein was purified by Ni-affinity chromatography, and the enzymatic features were analyzed. The results indicated that AppA phytase activities of mutant I408L and wild-type (WT) strain remained at 51.3 and 28%, respectively, after treatment at 85°C for 5 min. It means that the thermostability enhancement of AppA phytase I408L was 23.3% more as compared with WT. The K (m) of both phytase were 0.18 and 0.25 mM, respectively, which indicated that the catalyzing efficiency of I408L was improved. AppA phytase of mutant I408L showed a significant enhancement against trypsin, which was nearly three times compared with WT. In addition, AppA phytase of mutant could be activated by Mg(2+) and Mn(2+); in contrast, it could be inhibited by Ca(2+), Co(2+), Cu(2+), and K(+) in varying degrees, and the enzymatic activity was almost lost the presence of Fe(3+) and Zn(2+). It appears that screening thermotolerant phytase of E. coli by high throughput screening with a fluorescence substrate is a fast, simple, and effective method. The mutant I408L obtained in this study could be used for the large-scale commercial production of phytase.
Current Microbiology 03/2010; 61(4):267-73. · 1.82 Impact Factor
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ABSTRACT: Complex I is the first enzyme in the mitochondrial respiratory chain. It extracts energy from NADH, which is produced by the oxidation of sugars and fats, and traps the energy by virtue of a potential difference or voltage across the mitochondrial inner membrane. Herein, the genomic sequence and four splice variants encoding the complex I 19-kD subunit were isolated from Dunaliella salina. There were four transcripts coding for the complex I 19-kD subunit due to alternative splicing in algae, and the four transcripts were translated to two protein isoforms with varying C-terminals. We report the splicing pattern in the 3'-region of the D. salina 19-kD subunit, in which three of the exons (5, 6, and 7) could be alternatively spliced. Moreover, we found that four alternatively spliced variants were subject to coordinated transcription in response to different stresses by real-time quantitative PCR.
Bioscience Biotechnology and Biochemistry 01/2010; 74(5):1073-8. · 1.28 Impact Factor
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ABSTRACT: Xylose reductase (XR) is a key enzyme in xylose metabolism because it catalyzes the reduction of xylose to xylitol. In order to study the characteristics of XR from Candida tropicalis SCTCC 300249, its XR gene (xyll) was cloned and expressed in Escherichia coli BL21 (DE3). The fusion protein was purified effectively by Ni2+-chelating chromatography, and the kinetics of the recombinant XR was investigated. The Km values of the C. tropicalis XR for NADPH and NADH were 45.5 microM and 161.9 microM, respectively, which demonstrated that this XR had dual coenzyme specificity. Moreover, this XR showed the highest catalytic efficiency (kcat =1.44 x 10(4) min(-1)) for xylose among the characterized aldose reductases. Batch fermentation was performed with Saccharomyces serivisiae W303-lA:pYES2XR, and resulted in 7.63 g/L cell mass, 93.67 g/L xylitol, and 2.34 g/L x h xylitol productivity. This XR coupled with its dual coenzyme specificity, high activity, and catalytic efficiency proved its utility in in vitro xylitol production.
The Journal of Microbiology 07/2009; 47(3):351-7. · 1.10 Impact Factor
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ABSTRACT: Staphylococcus aureus is a member of Gram positive bacteria,but is also one of common pathogens that are most difficult to treat. It infects human skin, soft tissue, mucous membrane, bone and joint, especially in the nosocomial environment. Because studies on Staphylococcus aureus before were largely based on a single gene or protein, it is necessary to study this organism from the whole genome.
We used bioinformatics methods including five computational methods (phylogenetic profile, gene neighbor method, operon method, gene fusion method, interolog) to predict the protein interaction network of Staphylococcus aureus.
We constructed the protein interaction network of Staphylococcus aureus and studied its function.
Through the network analysis, we found that the protein interaction network of Staphylococcus aureus was subject to scale-free property and a number of very important proteins, such as SA0939, SA0868, rpID. Through the analysis of the important cell wall synthesis and signal transduction and regulation local networks, we also found some very important proteins. Such information will help us better understand pathogenic mechanism and develop new drug targets of Staphylococcus aureus.
ACTA MICROBIOLOGICA SINICA 02/2009; 49(1):56-63.
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ABSTRACT: Dunaliella salina is a unicellular green alga and possesses two types of photolyase: Class II cyclobutane pyrimidine dimers (CPD) photolyase and (6-4) photolyase. The gene of D. salina (6-4) photolyase is the first one found in unicellular organisms. CPD photolyases have been extensively studied but (6-4) photolyases are less understood. Because of the data observed in this study, D. salina (6-4) photolyase is insensitive to high salinity; whether it can tolerate a higher level of salinity than other (6-4) photolyases needs to be studied further. However, evidence is provided that (6-4) photolyases might be highly conserved among different species, not only in the sequence identity but also in the photorepair mechanism.
FEMS Microbiology Letters 07/2008; 283(1):42-6. · 2.04 Impact Factor
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ABSTRACT: There are four LhcII genes of Dunaliella salina have been submitted to the database of GenBank. However, little is known about Lhca genes of this green alga, although this knowledge might be available to study the composition and phylogenesis of Lhc gene family. Recently, one Lhca gene was been cloned from the green alga D. salina by PCR amplification using degenerate primers. This cDNA, designated as DsLhca1, contains an open reading frame encoded a protein of 222 amino acids with a calculated molecular mass of 27.8 kDa. DsLhca1 is predicted to contain three transmembrane domains and a N-terminal chloroplast transit peptide (cTP) with length of 33 amino acids. The genomic sequence of DsLhca1 is composed of five introns. The deduced polypeptide sequence of this gene showed a lower degree of identity (less than 30%) with LHCII proteins from D. salina. But its homology to Lhca proteins of other algae (Volvox carteri Lhca_AF110786) was higher with pairwise identities of up to 67.1%. Phylogenetic analysis indicated that DsLhcal protein cannot be assigned to any types of Lhca proteins in higher plants or in Chlamydomonas reinhardtii.
DNA Sequence 08/2007; 19(2):137-45. · 0.75 Impact Factor
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ABSTRACT: Dunaliella salina, a unicelluar green alga that can tolerate an extreme variation of salt concentration is being studied as a model system to analyze the tolerance of abiotic stresses at the molecular level. Upon abnormal NaCl levels, new transcripts were abundantly expressed in cells of the alga. EST gene discovery efforts utilizing salt-shock cells had identified one cDNA designated Dscbr (GenBank accession no. DQ867041) with significant similarity to a carotene biosynthesis related gene (cbr) from Dunaliella bardawil and to early light inducible genes (elip) of higher plants. Dscbr was 976 bp in length, encoding a 190 amino acid deduced polypeptide (DsCBR) with a predicted molecular mass of 19.9 kDa and pI of 9.0. The three dimensional structure of DsCBR modeled by computer homology modeling techniques showed that the protein possessed three predicted transmembrane helices and six conserved pigment-binding residues. Real-Time Quantitative PCR clearly demonstrated that Dscbr mRNA can be rapidly induced by high light intensity and salt shocks. The results presented in this work are consistent with the earlier proposal (Jin et al. 2001 Biochim Biophys Acta 1506:244-259, 2003 Plant Physiol 132:352-364) that the DsCBR protein is an adaptive response to stress-induced photodamage within the alga chloroplast, and plays a key role in the protection and/or repair of the photosynthetic apparatus.
Molecular Biology Reports 07/2007; 35(3):321-7. · 2.93 Impact Factor
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ABSTRACT: The mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (FAD-GPDH), recently reported in plants, has been detailed in yeast and animal systems. It oxidizes glycerol-3-phosphate (G-3-P) to dihydroxyacetone phosphate (DHAP) on the outer surface of mitochondrial inner membrane. A cDNA encoding the Dunaliella salina mitochondrial glycerol-3-phosphate dehydrogenase (DsFAD-GPDH) has been cloned and sequenced. The full length cDNA is 2791 bp, with an open reading frame (ORF) encoding 650 predicted amino acids, which show strong homology to reported FAD-GPDHs and have an apparent mitochondrial targeting sequence in the N-terminal. The sequence has been submitted to the GenBank database under Accession No. DQ916107. Results of Real-Time Quantitative PCR and enzymatic assays show that expression of DsFAD-GPDH is enhanced at first by salt treatment, and repressed by oxygen deficiency and cold stress.
Journal of Basic Microbiology 07/2007; 47(3):266-74. · 1.27 Impact Factor
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ABSTRACT: NADH:ubiquinone oxidoreductase (complex I ) of the mitochondrial respiratory chain catalyzes the transfer of electrons from NADH to ubiquinone coupled to proton translocation across the membrane. The cDNA sequence of Dunaliella salina mitochondrial NADH: ubiquinone oxidoreductase 19-kD subunit contains a 682-bp ORF encoding a protein with an apparent molecular mass of 19 kD. The sequence has been submitted to the GenBank database under Accession No. EF566890 (cDNA sequences) and EF566891 (genomic sequence). The deduced amino-acid sequence is 74% identical to Chlamydomonas reinhardtii mitochondrial NADH:ubiquinone oxidoreductase 18-kD subunit. The 19-kD subunit mRNA expression was observed in oxygen deficiency, salt treatment, and rotenone treatment with lower levels. It demonstrate that the 19-kD subunit of Complex I from Dunaliella salina is regulated by these stresses.
Molecular Biology Reports 06/2007; 35(3):397-403. · 2.93 Impact Factor
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ABSTRACT: Ultraviolet light induces photoproducts, cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs), in cellular DNA, which cause cytotoxic and genotoxic effects on the cells. Cells have several DNA repair mechanisms to repair the damage and to maintain genetic information of the cells. Photoreactivation is one of the DNA repair mechanism to remove UV-induced DNA damage from cellular DNA catalyzed by photolyase under visible light. Two types of photolyase, CPD photolyase and (6-4) photolyase, are specific for CPDs and for (6-4)PPs. We have isolated a gene product encoding CPD photolyase, named PHR2, from Dunaliella salina which is a kind of unicellular alga. Sequence analysis showed that PHR2 encodes a protein that has 529 amino acids and is similar to other Class II CPD photolyase. The complementation assay of the photoreactivation deficiency of the Escherichia coli SY2 by PHR2 cDNA showed a significant increase in survival rate when cells were irradiated with UV-C. Real-time PCR analysis indicated that the transcription of PHR2 was induced by UV-C, white light, high salinity, and H(2)O(2).
Journal of Photochemistry and Photobiology B Biology 06/2007; 87(2):137-43. · 2.81 Impact Factor
