A new method for gene synthesis and its high-level expression.

Abstract:

:An optimized Citrobacter braakii phytase gene, appA-c, was chemically synthesized by oligonucleotides synthesis and over-lap PCR method. The appA-c gene encoding 423 amino acids was cloned into expression vector pPIC9 and transformed into methylotropic yeast Pichia pastoris. From about 2000 transformants, 400 transformants exhibiting phytase activity were obtained. One transformant showing the strongest phytase activity was selected for detailed analyses in 5 L bioreactor. Under control of the highly-inducible alcohol oxidase gene (AOX1) promoter, the transformant was able to secrete 3.85 mg/ml protein to the culture supernatant in about 110 h methanol induction, which comprises of 12,116 U ml(-1) phytase activity. Further characterization of the recombinant phytase was conducted. The optimal pH and temperature for this recombinant phytase was about 4.0 and 50 degrees C, respectively. Fe3+, Zn2+ and Cu2+ could significantly inhibit the recombinant phytase enzyme activity. The specific activity of this recombinant enzyme was 3147 U mg(-1). The K(m) and V(max) values for sodium phytate were determined to be 0.5 mM and 3085 U/mg, respectively. To our knowledge, this is the first report of a chemically synthesized C. braakii appA gene heterologous expression with the highest expression level and highest phytase activity achieved. The novel gene optimization and synthesis method can be applied to other related researches.

journal_name

J Microbiol Methods

authors

Cao SS,Hu ZQ

doi

10.1016/j.mimet.2009.08.018

subject

Has Abstract

pub_date

2009-10-01 00:00:00

pages

106-10

issue

1

eissn

0167-7012

issn

1872-8359

pii

S0167-7012(09)00267-X

journal_volume

79

pub_type

杂志文章
  • 3D-printed microfluidic magnetic preconcentrator for the detection of bacterial pathogen using an ATP luminometer and antibody-conjugated magnetic nanoparticles.

    abstract::Various types of microfluidic systems have been developed to detect bacterial pathogens. However, most of these require enrichment steps that take at least several hours when detecting bacteria that are present with a low number of cells and, in addition, fabrication requires complicated assembly steps. In this study,...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2016.12.001

    authors: Park C,Lee J,Kim Y,Kim J,Lee J,Park S

    更新日期:2017-01-01 00:00:00

  • The use of a rapid assay to detect the neuraminidase production in oral Porphyromonas spp. isolated from dogs and humans.

    abstract::Neuraminidase was produced by 32.1% and 28.5% of Porphyromonas from dogs with and without periodontitis, respectively; and by 31.8% of bacteria from humans. The presence of neuraminidase in Porphyromonas spp. suggests that this enzyme can be involved with the pathogenesis of the periodontal disease, and the use of thi...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2013.06.017

    authors: de Assis PR,Nakano V,Senhorinho GN,Avila-Campos MJ

    更新日期:2013-09-01 00:00:00

  • Evaluation of 5'-nuclease and hybridization probe assays for the detection of shiga toxin-producing Escherichia coli in human stools.

    abstract::5'-Nuclease and a hybridization probe assays for the detection of shiga toxin-producing Escherichia coli were validated with regard to selectivity, analytical sensitivity, reproducibility and clinical performance. Both assays were capable of detecting the classical stx(1) and stx(2) genes when challenged with referenc...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2007.05.016

    authors: Schuurman T,Roovers A,van der Zwaluw WK,van Zwet AA,Sabbe LJ,Kooistra-Smid AM,van Duynhoven YT

    更新日期:2007-09-01 00:00:00

  • Gene transfection of Toxoplasma gondii using PEI/DNA polyplexes.

    abstract::The purpose of this study was to explore the potential of using cationic polyethylenimine (PEI) to deliver green fluorescent protein (GFP) to protozoan parasite Toxoplasma gondii. PEI/DNA polyplexes were formed using branched PEI and pEGFP-N1 plasmid with various N/P ratios that ranged from 5 to 50. With the increment...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2012.08.001

    authors: Salehi N,Peng CA

    更新日期:2012-10-01 00:00:00

  • In situ identification of symbiotic dinoflagellates, the genus Symbiodinium with fluorescence-labeled rRNA-targeted oligonucleotide probes.

    abstract::Fluorescence in situ hybridization has been used for the identification and analysis of populations of the dinoflagellate Symbiodinium that lives symbiotically in marine invertebrates. Conditions for in situ hybridization of Symbiodinium were optimized and used to identify the clade to which the isolate belongs using ...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/s0167-7012(02)00250-6

    authors: Yokouchi H,Takeyama H,Miyashita H,Maruyama T,Matsunaga T

    更新日期:2003-06-01 00:00:00

  • Method for evaluating mold growth on ceiling tile.

    abstract::A method to extract mold spores from porous ceiling tiles was developed using a masticator blender. Ceiling tiles were inoculated and analyzed using four species of mold. Statistical analysis comparing results obtained by masticator extraction and the swab method was performed. The masticator method was demonstrated a...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2004.12.013

    authors: Betancourt DA,Dean TR,Menetrez MY

    更新日期:2005-06-01 00:00:00

  • Use of competitive DNA hybridization to identify differences in the genomes of bacteria.

    abstract::Although recent technological advances in DNA sequencing and computational biology now allow scientists to compare entire microbial genomes, comparisons of closely related bacterial species and individual isolates by whole-genome sequencing approaches remains prohibitively expensive for most laboratories. Here we repo...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2005.12.006

    authors: Shanks OC,Santo Domingo JW,Graham JE

    更新日期:2006-08-01 00:00:00

  • High-throughput analysis of the impact of antibiotics on the human intestinal microbiota composition.

    abstract::Antibiotic treatments can lead to a disruption of the human microbiota. In this in-vitro study, the impact of antibiotics on adult intestinal microbiota was monitored in a new high-throughput approach: a fermentation screening-platform was coupled with a phylogenetic microarray analysis (Intestinal-chip). Fecal inocul...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2012.12.011

    authors: Ladirat SE,Schols HA,Nauta A,Schoterman MH,Keijser BJ,Montijn RC,Gruppen H,Schuren FH

    更新日期:2013-03-01 00:00:00

  • Utility of the cytochrome c oxidase subunit I gene for the diagnosis of toxoplasmosis using PCR.

    abstract::Toxoplasmosis is caused by the protozoan parasite Toxoplasma gondii, which belongs to the phylum Apicomplexa. Since this parasite causes severe clinical symptoms in immunocompromised patients, early diagnosis of toxoplasmosis is essential. PCR is currently used for early diagnosis, but there is no consensus regarding ...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2017.08.001

    authors: Feng X,Norose K,Li K,Hikosaka K

    更新日期:2017-10-01 00:00:00

  • Attenuated colicin-based screening to discover and create novel resistance genes.

    abstract::I formulated a systematic approach to screen for colicin resistance-associated genes by using an attenuated colicin mutant and demonstrated its utility in a screen of genes related to colicin E5 resistance. Screening of an Escherichia coli genome library revealed rstA as a partial resistance gene to colicin E5. Transc...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2014.03.003

    authors: Futai K

    更新日期:2014-05-01 00:00:00

  • The identification of carbapenemase types in Enterobacteriaceae by using molecular assay and phenotyping confirmation tests.

    abstract::In this study, we aimed to identify the molecular carbapenemase types of the Enterobacteriaceae isolates and to evaluate the performance of manually prepared and commercially available combination disc methods and the modified Hodge test. One hundred and forty carbapenemase producing isolates and 45 isolates as contro...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2016.03.010

    authors: Genc O,Aksu E,Gulcan A

    更新日期:2016-06-01 00:00:00

  • Permeabilization and hybridization protocols for rapid detection of Bacillus spores using fluorescence in situ hybridization.

    abstract:BACKGROUND:Fluorescence in situ hybridization (FISH) is not adapted for the detection of bacterial spores because of their resistance to conventional permeabilization treatments. Since spore-forming bacteria have important ecological, economical, and medical roles, their in situ detection needs to be improved. The aim ...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2008.12.009

    authors: Filion G,Laflamme C,Turgeon N,Ho J,Duchaine C

    更新日期:2009-04-01 00:00:00

  • Comparative diagnostic efficacy of recombinant LLO and PI-PLC-based ELISAs for detection of listeriosis in animals.

    abstract::The present study for the first time evaluates the serodiagnostic efficacy of two recombinant antigens namely, listeriolysin O (rLLO) and phosphatidyl-inositol phospholipase C (rPI-PLC). Indirect ELISA with the above recombinant antigens was used on samples collected from bovines (n=106), goats (n=138) and pigs (n=92)...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2017.04.005

    authors: Suryawanshi RD,Malik SVS,Jayarao B,Chaudhari SP,Savage E,Vergis J,Kurkure NV,Barbuddhe SB,Rawool DB

    更新日期:2017-06-01 00:00:00

  • Quantifying fungal infection of plant leaves by digital image analysis using Scion Image software.

    abstract::A digital image analysis method previously used to evaluate leaf color changes due to nutritional changes was modified to measure the severity of several foliar fungal diseases. Images captured with a flatbed scanner or digital camera were analyzed with a freely available software package, Scion Image, to measure chan...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2008.03.008

    authors: Wijekoon CP,Goodwin PH,Hsiang T

    更新日期:2008-08-01 00:00:00

  • Plasmid integration method: a new tool for analysis of the essentiality and function of genes in S. aureus.

    abstract::Staphylococcus aureus is a Gram-positive coccus and one of the major causes of community-acquired and hospital-acquired infections. We established the convenient and reliable experimental system for analyzing the essentiality and function of genes, the plasmid integration (PI) method. This method is based on plasmid i...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2012.05.017

    authors: Yamachika S,Onodera Y,Hiramatsu K,Takase H

    更新日期:2012-09-01 00:00:00

  • A multi-amplicon 16S rRNA sequencing and analysis method for improved taxonomic profiling of bacterial communities.

    abstract::Metagenomic sequencing of bacterial samples has become the gold standard for profiling microbial populations, but 16S rRNA profiling remains widely used due to advantages in sample throughput, cost, and sensitivity even though the approach is hampered by primer bias and lack of specificity. We hypothesized that a hybr...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2018.09.019

    authors: Schriefer AE,Cliften PF,Hibberd MC,Sawyer C,Brown-Kennerly V,Burcea L,Klotz E,Crosby SD,Gordon JI,Head RD

    更新日期:2018-11-01 00:00:00

  • Analysis of the genetic distribution among members of Clostridium botulinum group I using a novel multilocus sequence typing (MLST) assay.

    abstract::Clostridium botulinum is the etiological agent of botulism. Due to food-borne poisoning and the potential use of the extremely toxic botulinum neurotoxin (BoNT) from C. botulinum in bioterror or biocrime related actions, reliable high resolution typing methods for discriminating C. botulinum strains are needed. Partia...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2013.11.003

    authors: Olsen JS,Scholz H,Fillo S,Ramisse V,Lista F,Trømborg AK,Aarskaug T,Thrane I,Blatny JM

    更新日期:2014-01-01 00:00:00

  • Microanalysis of antimicrobial properties of human fluids.

    abstract::Host defense responses of animals and plants to pathogenic microbes are mediated in part by the release of antimicrobial substances into tissue fluids. Exploration of the antimicrobial properties of tissue fluids is often limited by their small quantity. We have developed assays of antimicrobial activity that require ...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/s0167-7012(00)00140-8

    authors: Cole AM,Wu M,Kim YH,Ganz T

    更新日期:2000-07-01 00:00:00

  • Fluorescence in situ hybridisation coupled to ultra small immunogold detection to identify prokaryotic cells using transmission and scanning electron microscopy.

    abstract::We describe a method based on fluorescence in situ hybridisation (FISH) that allows the identification of individual cells by electron microscopy. We hybridised universal and specific fluorescein-labelled oligonucleotide probes to the ribosomal RNA of prokaryotic microorganisms in heterogeneous cell mixtures. We then ...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2005.02.018

    authors: Gérard E,Guyot F,Philippot P,López-García P

    更新日期:2005-10-01 00:00:00

  • Monitoring of multiple bacteriocins through a developed dual extraction protocol and comparison of HPLC-DAD with turbidometry as their quantification system.

    abstract::The present study describes the development of a simple and efficient screening system that allows identification and quantification of nine bacteriocins produced by Lactococcus lactis. Cell-free L. lactis extracts presented a broad spectrum of antibacterial activity, including Gram-negative bacteria, Gram-positive ba...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2016.06.003

    authors: Katharopoulos E,Touloupi K,Touraki M

    更新日期:2016-08-01 00:00:00

  • Construction of a nontypeable Haemophilus influenzae-specific ectopic delivery vector.

    abstract::Complementation of chromosomal mutations in trans can introduce artifacts due to the number of episomal copies of the gene in question. One solution is to study the gene expressed at a single ectopic site in cis. We have designed and constructed a vector that allows homologous recombination into a gene encoding a fram...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2004.02.014

    authors: Daines DA,Smith AL

    更新日期:2004-06-01 00:00:00

  • Performance of PCR-based and Bioluminescent assays for mycoplasma detection.

    abstract::Contaminated eukaryotic cell cultures are frequently responsible for unreliable results. Regulatory entities request that cell cultures must be mycoplasma-free. Mycoplasma contamination remains a significant problem for cell cultures and may have an impact on biological analysis since they affect many cell parameters....

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2015.08.010

    authors: Falagan-Lotsch P,Lopes TS,Ferreira N,Balthazar N,Monteiro AM,Borojevic R,Granjeiro JM

    更新日期:2015-11-01 00:00:00

  • Codon-improved fluorescent proteins in investigation of Staphylococcus aureus host pathogen interactions.

    abstract::Here we present the use of three fluorescent proteins in Staphylococcus aureus, Cerulean, PA-GFP, and mRFPmars. All molecules have an improved codon adaptation for expression in the A + T rich organisms and extend the fluorescent protein portfolio in staphylococcal research. ...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2010.07.022

    authors: Paprotka K,Giese B,Fraunholz MJ

    更新日期:2010-10-01 00:00:00

  • A preliminary evaluation of a new enzyme immunoassay to detect Chlamydia pneumoniae-specific antibodies.

    abstract::New enzyme immunoassays (EIAs) for determination of specific IgG, IgA, and IgM antibody titers to Chlamydia pneumoniae were evaluated independently in three research laboratories. Specificity of the EIAs was enhanced by removing LPS from the chlamydial antigen. The performance of these EIAs was evaluated in comparison...

    journal_title:Journal of microbiological methods

    pub_type: 临床试验,杂志文章,多中心研究

    doi:10.1016/s0167-7012(00)00220-7

    authors: Ossewaarde JM,Tuuminen T,Boersma WG,Sandström M,Palomäki P,Boman J

    更新日期:2000-12-15 00:00:00

  • Direct identification of mycobacteria from liquid media using a triplex real-time PCR coupled with pyrosequencing method.

    abstract::Culture in enriched broth, as well as on a solid medium, is recommended for primary isolation of mycobacteria. With the introduction of liquid mycobacterial culture methods, a substantial workload regarding the identification of culture-recovered mycobacterial species, particularly Mycobacterium tuberculosis complex (...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2015.10.011

    authors: Kim JU,Cha CH,Park SH

    更新日期:2015-12-01 00:00:00

  • Direct common gram-negative bacterial identification from positive blood culture bottles by SELDI-TOF MS.

    abstract::A protein database was constructed and validated with identification rate over 90% for the 4 most common Gram-negative bacteria on agar plates. By protein masses comparison, 120 bacteria of the 4 species from blood culture bottles were identified. The concordance was high (Kappa=0.906) between our method and conventio...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2014.07.021

    authors: Xiao D,Yang Y,Jiang W,Zhang H,Liu H,Yu H,Xie C,Zhong M,Chen L,Huang W

    更新日期:2014-10-01 00:00:00

  • Development and standardization of an in-house indirect ELISA for detection of duck antibody to fowl cholera.

    abstract::Serological tests, such as agglutination and indirect hemagglutination assay (IHA), have been used to identify antibodies against Pasteurella multocida in poultry sera, but none are highly sensitive. An enzyme-linked immunosorbent assays (ELISA) has been used with varying degrees of success in attempts to monitor sero...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2017.08.018

    authors: Poolperm P,Varinrak T,Kataoka Y,Tragoolpua K,Sawada T,Sthitmatee N

    更新日期:2017-11-01 00:00:00

  • Construction of a conditional lethal Salmonella mutant via genetic recombination using the ara system and asd gene.

    abstract::In order to construct a conditional lethal Salmonella mutant, an arabinose-regulated recombinant genetic system was used. The Salmonella aspartate semialdehyde dehydrogenase (asd) gene was localized under the control of araC P(araBAD) in a plasmid to create the araC P(araBAD)::asd cassette. The cassette was cloned int...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2011.08.004

    authors: Kim SW,Kang HY,Hur J,Gal SW,Bang WY,Cho KK,Kim CW,Bahk JD,Lee JH

    更新日期:2011-11-01 00:00:00

  • Fast identification of Escherichia coli in urinary tract infections using a virulence gene based PCR approach in a novel thermal cycler.

    abstract::Uropathogenic Escherichia coli (UPEC) is the most common causal agent of urinary tract infections (UTIs) in humans. Currently, clinical detection methods take hours (dipsticks) to days (culturing methods), limiting rapid intervention. As an alternative, the use of molecular methods could improve speed and accuracy, bu...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2019.105799

    authors: Brons JK,Vink SN,de Vos MGJ,Reuter S,Dobrindt U,van Elsas JD

    更新日期:2020-02-01 00:00:00

  • MALDI-TOF MS based identification of food-borne yeast isolates.

    abstract::In this study, food-borne yeast isolates (n=96), comprising at least 33 species, were identified using MALDI-TOF MS and conventional methods (API ID 32 C and Phoenix Yeast ID). Discrepancies of both methods were resolved by sequencing the ITS1-5.8S-rRNA-ITS2 region. For ten isolates, mainly classified to Rhodotorula a...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章

    doi:10.1016/j.mimet.2014.08.021

    authors: Pavlovic M,Mewes A,Maggipinto M,Schmidt W,Messelhäußer U,Balsliemke J,Hörmansdorfer S,Busch U,Huber I

    更新日期:2014-11-01 00:00:00