Sequence identification, structure prediction and validation of tannase from Aspergillusniger N5-5

Tannases produced by filamentous fungi are in a family of important hydrolases of gallotannins and have broad industry applications.But until now,the 3-D structures of fungi tannases have not been reported.The protein sequence deduced from the cDNA sequence obtained using RT-PCR amplification was identified as tannase through sequence alignment and phylogenetic analysis.Structure models based on the tannase sequence were collected using I-TASSER,and the model with the best match to the surface charge density-pH titration profile was selected as the final structure for tannase from Aspergillusniger N5-5.This work provides an effective method for protein structure research.The structure constructed in this work should be very important to understand the enzyme bioactivities and further developments of fungi tannases.

The homology analysis of internal transcribed spacer sequence of ribosomal DNA in common dermatophytes

In order to analyze the sequences of the internal transcribed spacer （ITS） including the 5.8 S ribosomal DNA （rDNA） of common dermatophytes, so as to obtain a rapid and accurate method to identify the species of dermatophytes and to establish the phylogenetic tree of these species to understand their relationship, 16 strains of dermatophytes were collected and preliminarily identified by morphological characteristics. General primers for fungi ITS1 and ITS4 were used to amplify the ITS rDNA of each strains with PCR. The PCR products after purification were sequenced directly and were analyzed through internet. In the results, 11 strains were identified by means of morphological features, among which 5 strains were Trichophyton, 5 strains were Microsporum and 1 was Epidermophytoa, which was consistent with the results by molecular biology. In the 5 unidentifiable strains, 1 strain was proved to be Chrysosporium by molecular biology. These strains studied could be divided into 3 different classes as indicated in the analysis of the phylogenetic tree of the sequences in ITS, which were quite different from those of morphological classification. It is evident from the above observations that the molecular method of analysis on the ITS sequences is a rapid, highly sensitive and accurate approach for the detection of dematophyte species, however, it still exhibits some limitations needing the supplementation with morphological identification.

Isolation and Identification of Staphylococcus chromogenes and 3 D Structural Analysis of the katA Gene

[Objective]Staphylococcus arthritis became an increasingly significant health problem in intensive chicken farming in China.[Method]In this study,a bacteria strain was isolated from the broiler chicken suffering from arthritis and named as the strain Gg1.[Result]It was then identified as Staphylococcus chromogenes by the biochemical tests and phylogenetic tree analysis based on 16S rDNA sequence.Furthermore,the catalase（katA）gene was amplified by PCR using the designed primers,and the expected fragment was 1 232 bp long encoding a protein of 410 amino acids that shares the conserved motifs including catalase,heme-binding ligand and active center motif.Six phosphorylation sites（Ser95,Thr96,Ser241,Ser242,Thr281,Ser338）,four conserved residues（Ser95,His216,Tyr281,Asp341）and two active sites（His56,Asn129）were demonstrated by multiple sequence alignment and homology comparisons.The homology modeling of 3D structure of katA protein was done by SWISSMODEL server based on the template retrieved from the catalase（PDB：2ISA_A）of Vibrio salmonicida.The katA protein represents a four-domain globular protein,the quality and reliability of the resulting protein structure was further verified by Ramachandran plot.[Conclusion]To our knowledge,this is the first report of S.chromogenes linked to arthritis in chicken and the bioinformatic characterization of its katA gene.

Fast infectious diseases diagnostics based on microfuidic biochip system

Molecular diagnostics is one of the most important tools currently in use for clinical pathogen detection due to its high sensitivity,specificity,and low consume of sample and reagent is keyword to low cost molecular diagnostics.In this paper,a sensitive DNA isothermal amplifi-cation method for fast clinical infectious diseases diagnostics at aM concentrations of DNA was developed using a polycarbonate(PC)microfuidic chip.A portable confocal optical fuo-rescence detector was specifically developed for the microfuidic chip that was capable of highly sensitive real-time detection of amplified products for sequence-specific molecular identification near the optical diffraction limit with low background.The molecular diagnostics of Listeria monocytogenes with nucleic acid extracted from stool samples was performed at a minimum DNA template concentration of 3.65 aM,and a detection limit of less than five copies of genomic DNA.Contrast to the general polymerase chain reaction(PCR)at eppendorf(EP)tube,the detection time in our developed method was reduced from 1.5h to 45 min for multi-target parallel detection,the consume of sample and reagent was dropped from 25μL to 1.45μL.This novel microfuidic chip system and method can be used to develop a micro total analysis system as a clinically relevant pathogen molecular diagnostics method via the amplification of targets,with potential applications in biotechnology,medicine,and clinical molecular diagnostics.

Amino acid identification and sequence analysis of peptides by reaction mass spectrometry

Fast atom bombardment mass spectrometry (FAB-MS) is applied to distinguish N- terminal series ions from C-terminal series ions of a peptide by on-probe acetylation, it provides valuable information about the sequence of an unknown peptide. The FAB mass spectra contain a number of characteristic ions at low-mass region in addition to the sequence ions at high-mass region. It was found that the ions below m/z 200 are characteristic of the amino acid composition of the peptide, from which the amino acid composition of the peptide could be estimated. Additionally, mixture analysis is also discussed.