As a type of prebiotics and dietary fiber, inulin performs plenty of significant physiological functions and is applied in food and pharmaceutical fields. Inulosucrase from microorganisms can use sucrose as the substrate to synthesize inulin possessing higher molecular weight than that from plants. In this work, a hypothetical gene coding inulosucrase was selected from the GenBank database. The catalytic domain was remained by N- and C- truncation strategies, constructing the recombinant plasmid. The recombinant plasmid was expressed in E. coli expression system, and after purifying the crude enzyme by Ni²⁺ affinity chromatography, a recombinant enzyme with a molecular weight of approximately 65 kDa was obtained. The optimal pH and temperature of the recombinant enzyme were 5.5 and 45 °C, respectively, when sucrose was used as the sole substrate. The activity of this enzyme was inhibited by various metal ions at different degrees. After purifying the produced polysaccharide, nuclear magnetic resonance analysis was used to determine that the polysaccharide was inulin connected by β-(2,1) linkages. Finally, the conditions for the production of inulin were optimized. The results showed that the inulin production reached the maximum, approximately 287 g/L after 7 h, when sucrose concentration and enzyme dosage were 700 g/L and 4 U/mL, respectively. The conversion rate from sucrose to inulin was approximately 41%.
Escherichia coli/genetics* ; Hexosyltransferases/genetics* ; Inulin ; Oligosaccharides ; Sucrose

In order to study the expression and the activity of inositol phosphorylceramide synthase (BcAUR1 gene) in Botrytis cinerea, we amplified BcAUR1 by RT-PCR from Botrytis cinerea, using the special primers with FLAG and BamH I/Xho I restriction sites. Recombinant pYES2-BcAUR1 was constructed to transform into Saccharomyces cerevisae deltayorl by LiAC. The expression of inositol phosphorylceramide (IPC) synthase and its activity were detected by Western blotting and HPLC, respectively. The results show that pYES2-BcAUR1 could express in uracil mutant deltayorl of Saccharomyces cerevisae. IPC synthase enzyme activity of pYES2-BcAUR1 transformants significantly increased and was approximately double than no-load BcAUR1 transformants. The low concentration of Aureobasidin A could inhibit growth of no-load BcAUR1 transformants, but pYES2-BcAUR1 transformants could resist fungal growth inhibition which was induced by Aureobasidin A.
Botrytis ; enzymology ; genetics ; Depsipeptides ; pharmacology ; Fungal Proteins ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Hexosyltransferases ; genetics ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism

To investigate the function of STT3a gene in salt adaptation and flagellar regeneration of Dunaliella salina (D. salina), a pair of degenerate primers was designed according to conserved homologous amino acid sequences of VCVFTA and DVDYVL of STT3a from Chlamydomonas, Arabidopsis thaliana and other organisms. A cDNA sequence of 1 650 bp encoding a whole functional domain of STT3a was amplified from D. salina by RT-PCR and 3' Rapid Amplification of cDNA Ends (RACE), which shared homology with Chlamydomonas (48%), Arabidopsis thaliana (50%), Homo sapiens (46%), etc. Real-time fluorescence quantitative PCR (real-time Q-PCR) demonstrated that the STT3a mRNAs from D. salina were induced by increased concentration of NaCl, and increased to 11-fold higher by 3.5 mol/L NaCl than that by 1.5 mol/L NaCl (P < 0.01). Also, STT3a mRNA of D. salina maintained at a higher level in the process of flagellar regeneration with than without experiencing deflagellar treatment. In conclusion, the findings of this study demonstrate that the high expression of the STT3a gene enhances the capability of salt adaptation and flagellar regeneration in D. salina.
Adaptation, Physiological ; physiology ; Arabidopsis ; enzymology ; Chlamydomonas ; enzymology ; Chlorophyta ; enzymology ; genetics ; Cloning, Molecular ; Flagella ; metabolism ; Hexosyltransferases ; chemistry ; genetics ; metabolism ; Membrane Proteins ; chemistry ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism ; Sodium Chloride ; pharmacology

The timely detection of blood-borne pathogens is one of the most important functions of the microbiology laboratory. Recently, methicillin-resistant staphylococci have become the most important pathogens seen by the laboratory. The purpose of this study was to evaluate Staphy agar, a novel screening medium, for the detection methicillin-resistant Staphylococcus aureus, S. epidermidis, or other coagulase-negative staphylococci (CNS) from positive blood cultures showing Gram-positive cocci in clusters. Eighty-six blood cultures that yielded Gram-positive cocci in clusters were included in this study. The organisms were finally identified by the Vitek system, and oxacillin resistance was confirmed by polymerase chain reaction (PCR)-based mecA gene detection. The identification and oxacillin resistance of all S. aureus strains showed complete agreement with the Vitek and PCR results. The presumptive detection of S. epidermidis and other CNS were consistent with the Vitek system in 94.7%, and the screening of oxacillin resistance was consistent with the result of PCR in 92.1% of 38 strains. The Staphy agar method is reliable and rapid for differentiating Gram-positive cocci in clusters in blood and for determining their methicillin resistance.
*Bacterial Proteins ; Carrier Proteins/genetics ; Drug Resistance, Microbial ; *Hexosyltransferases ; Muramoylpentapeptide Carboxypeptidase/genetics ; Oxacillin/*pharmacology ; Penicillin-Binding Proteins ; *Peptidyl Transferases ; Staphylococcus aureus/*drug effects/genetics ; Staphylococcus epidermidis/*drug effects/genetics

The Bacillus subtilis Sac B gene with the vacuolar targeting signal sequence driven by 35S promotor was transferred into Lespedeza thunbergii by Agrobacterium mediated method. Total 62 Kan-resistant plants were obtained, of which 5 plants were proved to be transgenic plants. The transgenic plants were characterized by PCR amplification, PCR-Southern hybridization and RT-PCR. The physiological assay results showed that the transgenic plants were more tolerant to stress than the controls under the condition of 200mmol/L NaCl and 5% PEG, respectively, and that the content of soluble sugar in trnsgenic plants was significantly higher than that of controls in the period of tests (5-15 days) under salt and PEG stress.
Bacillus subtilis ; enzymology ; genetics ; Carbohydrate Metabolism ; Carbohydrates ; chemistry ; Hexosyltransferases ; genetics ; Lespedeza ; drug effects ; genetics ; growth & development ; metabolism ; Plants, Genetically Modified ; Reverse Transcriptase Polymerase Chain Reaction ; Sodium Chloride ; pharmacology ; Solubility ; Stress, Physiological ; drug effects ; Transformation, Genetic ; Transgenes ; genetics

The main commercial production of fructooligosaccharides (FOS) comes from enzymatic transformation using sucrose as substrate by microbial enzyme fructosyltransferase. A fructosyltransferase genomic DNA was isolated from Aspergillus niger QU10 by PCR. The nucleotide sequence showed a 1 941 bp size, and has been submitted to GenBank (KF699529). The cDNA of the fructosyltransferase, containing an open reading frame of 1 887 bp, was further cloned by RT-PCR. The fructosyltransferase gene from Aspergillus niger was functionally expressed both in Escherichia coli and Pichia pastoris GS 115. The highest activity value for the construction with the α-factor signal peptide reached 431 U/mL after 3 days of incubation. The recombinant enzyme is extensively glycosylated, and the active form is probably represented by a homodimer with an apparent molecular mass of 200 kDa as judged from mobility in seminative PAGE gels. The extracellular recombinant enzyme converted sucrose mostly to FOS, mainly 1-kestose and nystose, liberating glucose. FOS reached a maximal value and represented about 58% of total sugars present in the reaction mixture after 4 h reaction. The results suggest that the availability of recombinant Pichia pastoris as a new source of a FOS-producing enzyme might result of biotechnology interest for industrial application.
Aspergillus niger ; enzymology ; genetics ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; Fungal Proteins ; genetics ; metabolism ; Glycosylation ; Hexosyltransferases ; genetics ; metabolism ; Molecular Sequence Data ; Molecular Weight ; Pichia ; Sucrose ; metabolism ; Trisaccharides ; metabolism

Rhamnolipid biosurfactant is mainly produced by Pseudomonas aeruginosa that is the opportunistic pathogenic strain and not suitable for future industrial development. In order to develop a relatively safe microbial strain for the production of rhamnolipid biosurfactant, we constructed engineered Escherichia coli strains for rhamnolipid production by expressing different copy numbers of rhamnosyltransferase (rhlAB) gene with the constitutive synthetic promoters of different strengths in E. coli ATCC 8739. We further studied the combinatorial regulation of rhlAB gene and rhaBDAC gene cluster for dTDP-1-rhamnose biosynthesis with different synthetic promoters, and obtained the best engineered strain-E. coli TIB-RAB226. Through the optimization of culture temperature, the titer of rhamnolipd reached 124.3 mg/L, 1.17 fold higher than that under the original condition. Fed-batch fermentation further improved the production of rhamnolipid and the titer reached the highest 209.2 mg/L within 12 h. High performance liquid chromatography-mass spectrometry (LC-MS) analysis showed that there are total 5 mono-rhamnolipid congeners with different nuclear mass ratio and relative abundance. This study laid foundation for heterologous biosynthesis of rhanomilipd.
Bacterial Proteins ; genetics ; Batch Cell Culture Techniques ; Decanoates ; Escherichia coli ; metabolism ; Fermentation ; Glycolipids ; biosynthesis ; Hexosyltransferases ; genetics ; Industrial Microbiology ; methods ; Multigene Family ; Promoter Regions, Genetic ; Pseudomonas aeruginosa ; Rhamnose ; analogs & derivatives ; biosynthesis ; Surface-Active Agents ; metabolism

UNLABELLEDStreptococcus pneumoniae is a common cause of potentially life-threatening infections such as meningitis, bacteraemia, pneumonia worldwide, for which children of preschool age are at particularly high risk. Since the late 1970s and 1980s, antibiotic resistance among pneumococci has become an emerging problem. Several multidrug-resistant clones have rapidly spread throughout the world.

OBJECTIVE(1) To investigate the prevalence of penicillin and other antibiotics nonsusceptibility among pneumococci. (2) To analyze the correlation of pbp2b amplicon profiles with penicillin resistance. (3) To serotype 31 isolates of penicillin-resistant pneumococci by latex agglutination. (4) To analyze the chromosomal relatedness of serotype 23F and 6 isolates of penicillin-resistant pneumococci by using pulsed-field gel electrophoresis (PFGE) and characterize these isolates in molecular epidemiology.

METHODS(1) Susceptibility was determined by using broth microdilution, E-test, and K-B disk. (2) The correlation of pbp2b amplicon profiles with penicillin resistance was assessed by restriction fragment length polymorphism (RFLP). (3) Serotyping of penicillin-resistant pneumococcal isolates was performed by using latex agglutination. (4) The properties of serotype 23F and 6 isolates of penicillin-resistant pneumococci were assessed by PFGE.

RESULTSS. pneumoniae with increased nonsusceptibility (including intermediate strains and resistant strains) to penicillin G was 9.9% in 1997, 12.6% in 1998, 14.6% in 2000; to cefuroxime 4.2%, 1.5%, 8.2%; to cefotaxime 0.0%, 1.7%, 1.0% respectively. There were no statistically significant differences (P > 0.05). While resistance to erythromycin, trimethoprim-sulfamethoxazole and chloramphenicol increased significantly from 76.8% in 1997 to 87.4% in 2000, from 74.7% to 88.3%, and from 22.6% to 40.8%, respectively (P < 0.05). RFLP analysis of pneumococcal pbp2b-specific amplicons was effective for screening penicillin resistance. Of the 31 strains of penicillin-resistant pneumococci (MICs 0.12 - 2.0 micro g/ml) studied, 6 (19.4%) strains (MICs 0.12 - 0.19 micro g/ml) were serotype 23F and 3 (9.7%) strains (MICs 0.5 - 1.5 micro g/ml) were serotype 6. There were nearly identical susceptibility to antibiotics and identical PFGE patterns in the former, and there were different susceptibility to antibiotics and different PFGE patterns in the latter. Three serotype 6 strains had different susceptibility to antibiotics and different PFGE patterns, which suggested that those strains may be scattered.

CONCLUSIONGenerally beta-lactams retained their activity against S. pneumoniae in Beijing. Resistance to erythromycin, trimethoprim-sulfamethoxazole, and chloramphenicol increased drastically. RFLP analysis of pneumococcal pbp2b-specific amplicons was effective for screening penicillin resistance. In 6 strains of serotype 23 F there were nearly identical susceptibility to antibiotics and identical PFGE patterns, which suggested the probability that there was a spread of serotype 23F isolates with low-level penicillin resistance in local area.

Aminoacyltransferases ; Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; blood ; genetics ; Carrier Proteins ; blood ; genetics ; Drug Resistance, Bacterial ; genetics ; Electrophoresis, Gel, Pulsed-Field ; Hexosyltransferases ; blood ; genetics ; Muramoylpentapeptide Carboxypeptidase ; blood ; genetics ; Penicillin-Binding Proteins ; Peptidyl Transferases ; blood ; genetics ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Streptococcus pneumoniae ; drug effects ; genetics

The tissue-specific and MeJA-induced transcriptional levels of BcUGT3 and BcUGT6 in Bupleurum chinense were analyzed in the present study. The transcriptional levels of BcUGT3 in root, leaf, flower and fruit were similar and they all were higher than those in stem. The transcriptional level of BcUGT6 was the highest in leaf and the lowest in flower among in all tested tissues. With non-treated adventitious roots as control, BcUGT6's transcriptional levels were elevated to nearly 2 folds for 2 h, 8 h, 24 h, 2 d and 4 d in MeJA-treated adventitious roots of B. chinense. It showed that the transcriptional level of BcUGT6 was slightly affected by MeJA. While, BcUGT3's transcriptional levels were gradually elevated, and till 4 d after MeJA treatment, the expression level was about 7 folds than that of non-treated control. Using pET-28a (+), the expressions of two genes was investigated. Induced by IPTG, the target proteins were expressed in E. coli and then purified. All the results obtained in the present study will be helpful for follow-up bio-function analysis of BcUGT3 and BcUGT6.
Acetates ; pharmacology ; Bupleurum ; cytology ; enzymology ; genetics ; Cell Membrane ; metabolism ; Cyclopentanes ; pharmacology ; Escherichia coli ; genetics ; Gene Expression ; Gene Expression Regulation, Plant ; drug effects ; Hexosyltransferases ; chemistry ; genetics ; isolation & purification ; metabolism ; Intracellular Space ; metabolism ; Oxylipins ; pharmacology ; Protein Sorting Signals ; Protein Structure, Secondary ; Protein Transport ; Sequence Analysis ; Transcription, Genetic ; drug effects