Displaying publications 21 - 40 of 133 in total

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  1. Fulltext Preparation of kenaf stem hemicellulosic hydrolysate and its fermentability in microbial production of xylitol by Escherichia coli BL21
    Shah SSM, Luthfi AAI, Low KO, Harun S, Manaf SFA, Illias RM, et al.
    Sci Rep, 2019 03 11;9(1):4080.
    PMID: 30858467 DOI: 10.1038/s41598-019-40807-z
    Kenaf (Hibiscus cannabinus L.), a potential fibre crop with a desirably high growth rate, could serve as a sustainable feedstock in the production of xylitol. In this work, the extraction of soluble products of kenaf through dilute nitric-acid hydrolysis was elucidated with respect to three parameters, namely temperature, residence time, and acid concentration. The study will assist in evaluating the performance in terms of xylose recovery. The result point out that the maximum xylose yield of 30.7 g per 100 g of dry kenaf was attained from 2% (v/v) HNO3 at 130 °C for 60 min. The detoxified hydrolysate was incorporated as the primary carbon source for subsequent fermentation by recombinant Escherichia coli and the performance of strain on five different semi-synthetic media on xylitol production were evaluated herein. Among these media, batch cultivation in a basal salt medium (BSM) afforded the highest xylitol yield of 0.35 g/g based on xylose consumption, which corresponded to 92.8% substrate utilization after 38 h. Subsequently, fermentation by E. coli in the xylose-based kenaf hydrolysate supplemented with BSM resulting in 6.8 g/L xylitol which corresponding to xylitol yield of 0.38 g/g. These findings suggested that the use of kenaf as the fermentation feedstock could be advantageous for the development of sustainable xylitol production.
    Matched MeSH terms: Escherichia coli/metabolism
  2. Strategies for humanizing glycosylation pathways and producing recombinant glycoproteins in microbial expression systems
    Khan AH, Noordin R
    Biotechnol Prog, 2019 03;35(2):e2752.
    PMID: 30457225 DOI: 10.1002/btpr.2752
    Homogeneously glycosylated proteins are essential for analyzing the structure of N-glycans, studying their consequences inside cells, and developing therapeutic glycoproteins. However, the isolation of glycoproteins with homogeneous glycans from human is difficult since glycoforms slightly differ from each other with respect to molecular weight and charge. Microbial expression systems have numerous benefits in expression technology and have gained great attention, because they are more adaptable to the biotechnology industry. While selecting an expression host, the glycosylation pattern must be taken into account, because glycosylation strongly depends on cellular production system and selected production clone. This review discussed the technological developments in glycoengineering of microbial expression systems for humanizing the glycosylation profile and highlighted the expression potential of Leishmania expression system. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2752, 2019.
    Matched MeSH terms: Escherichia coli/metabolism*
  3. Fulltext Characterization and safety evaluation of partially purified bacteriocin produced by Escherichia coli E isolated from fermented pineapple Ananas comosus (L.) Merr
    Le VT, Leelakriangsak M, Lee SW, Panphon S, Utispan K, Koontongkaew S
    Braz J Microbiol, 2019 Jan;50(1):33-42.
    PMID: 30637641 DOI: 10.1007/s42770-018-0014-5
    Antibacterial activity of cell-free supernatant from Escherichia coli E against selected pathogenic bacteria in food and aquaculture was the highest against Edwardsiella tarda 3, a significant aquaculture pathogen. Biochemical properties of the bacteriocins were studied and bacteriocin was found to be sensitive to proteinase K, demonstrating its proteinaceous nature. In addition, pH and temperature affected bacteriocin activity and stability. The bacteriocins were partially purified by ammonium sulfate precipitation. The antibacterial activity was only detected in 20% ammonium sulfate fraction and direct detection of its activity was performed by overlaying on the indicator strains. The inhibition zone associated with the antibacterial activity was detected in the sample overlaid by E. tarda 3 and Staphylococcus aureus DMST8840 with the relative molecular mass of about 27 kDa and 10 kDa, respectively. Bacteriocin showed no cytotoxic effect on NIH-3T3 cell line; however, two virulence genes, aer and sfa, were detected in the genome of E. coli E by PCR. The characteristics of bacteriocins produced by E. coli E exhibited the antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria and the safe use determined by cytotoxicity test which may have interesting biotechnological applications.
    Matched MeSH terms: Escherichia coli/metabolism*
  4. Fulltext Isolation and characterization of malaria PfHRP2 specific VNAR antibody fragments from immunized shark phage display library
    Leow CH, Fischer K, Leow CY, Braet K, Cheng Q, McCarthy J
    Malar J, 2018 Oct 24;17(1):383.
    PMID: 30355309 DOI: 10.1186/s12936-018-2531-y
    BACKGROUND: Malaria rapid diagnostic tests (RDTs) represent an important antibody based immunoassay platform. Unfortunately, conventional monoclonal antibodies are subject to degradation shortening shelf lives of RDTs. The variable region of the receptor (VNAR) from shark has a potential as alternative to monoclonal antibodies in RDTs due to high thermal stability.

    METHODS: In this study, new binders derived from shark VNAR domains library were investigated. Following immunization of a wobbegong shark (Orectolobus ornatus) with three recombinant malaria biomarker proteins (PfHRP2, PfpLDH and Pvaldolase), a single domain antibody (sdAb) library was constructed from splenocytes. Target-specific VNAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken.

    RESULTS: The primary VNAR domain library possessed a titre of 1.16 × 106 pfu/mL. DNA sequence analysis showed 82.5% of isolated fragments appearing to contain an in-frame sequence. After multiple rounds of biopanning, a highly dominant clone specific to PfHRP2 was identified and selected for protein production in an E. coli expression system. Biological characterization showed the recombinant protein expressed in periplasmic has better detection sensitivity than that of cytoplasmic proteins. Assays of binding activity indicated that its reactivity was inferior to the positive control mAb C1-13.

    CONCLUSIONS: Target-specific bacteriophage VNARs were successfully isolated after a series of immunization, demonstrating that phage display technology is a useful tool for selection of antigen binders. Generation of new binding reagents such as VNAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.

    Matched MeSH terms: Escherichia coli/metabolism
  5. Process intensification of core streptavidin production through high-cell-density cultivation of recombinant E. coli and a temperature-based refolding method
    Chua LH, Tan SC, Liew MWO
    J Biotechnol, 2018 Jun 20;276-277:34-41.
    PMID: 29679607 DOI: 10.1016/j.jbiotec.2018.04.012
    An intensified process was developed that enables high level production of recombinant core streptavidin (cSAV), a non-glycosylated tetrameric protein utilised in a wide range of applications. A pH-stat fed-batch feeding strategy was employed to achieve high-cell-density and improve volumetric yield of cSAV which was expressed as inclusion bodies (IBs). The effect of induction at different cell densities (OD 20, 60 and 100) on volumetric and specific yield were then studied. Highest volumetric yield of cSAV (1550 mg L-1) was obtained from induction at OD 100 without significant reductions in specific yield. To recover active cSAV from IBs, the possibility of refolding using a temperature-based refolding method was investigated. Refolded cSAV obtained from temperature-based refolding were then compared against cSAV refolded with conventional dialysis and dilution methods using quantitative and qualitative metrics. The temperature-based refolding method was found to improve the yield of cSAV by 6-18% in comparison to conventional methods without compromising quality. Intensification was achieved by reductions in process volumes and a more concentrated product stream. Using the newly developed process, the volumetric yield of cSAV IBs was improved by thirty-six fold in comparison to low-cell-density shake flask cultivation, and 33% of cSAV can be recovered from IBs at 90% purity.
    Matched MeSH terms: Escherichia coli/metabolism*
  6. Fulltext Microtiter miniature shaken bioreactor system as a scale-down model for process development of production of therapeutic alpha-interferon2b by recombinant Escherichia coli
    Tan JS, Abbasiliasi S, Kadkhodaei S, Tam YJ, Tang TK, Lee YY, et al.
    BMC Microbiol, 2018 01 04;18(1):3.
    PMID: 29439680 DOI: 10.1186/s12866-017-1145-9
    BACKGROUND: Demand for high-throughput bioprocessing has dramatically increased especially in the biopharmaceutical industry because the technologies are of vital importance to process optimization and media development. This can be efficiently boosted by using microtiter plate (MTP) cultivation setup embedded into an automated liquid-handling system. The objective of this study was to establish an automated microscale method for upstream and downstream bioprocessing of α-IFN2b production by recombinant Escherichia coli. The extraction performance of α-IFN2b by osmotic shock using two different systems, automated microscale platform and manual extraction in MTP was compared.

    RESULTS: The amount of α-IFN2b extracted using automated microscale platform (49.2 μg/L) was comparable to manual osmotic shock method (48.8 μg/L), but the standard deviation was 2 times lower as compared to manual osmotic shock method. Fermentation parameters in MTP involving inoculum size, agitation speed, working volume and induction profiling revealed that the fermentation conditions for the highest production of α-IFN2b (85.5 μg/L) was attained at inoculum size of 8%, working volume of 40% and agitation speed of 1000 rpm with induction at 4 h after the inoculation.

    CONCLUSION: Although the findings at MTP scale did not show perfect scalable results as compared to shake flask culture, but microscale technique development would serve as a convenient and low-cost solution in process optimization for recombinant protein.

    Matched MeSH terms: Escherichia coli/metabolism*
  7. Site-saturation mutagenesis of mutant L-asparaginase II signal peptide hydrophobic region for improved excretion of cyclodextrin glucanotransferase
    Ismail A, Illias RM
    J Ind Microbiol Biotechnol, 2017 Dec;44(12):1627-1641.
    PMID: 28921081 DOI: 10.1007/s10295-017-1980-6
    The excretion of cyclodextrin glucanotransferase (CGTase) into the culture medium offers significant advantages over cytoplasmic expression. However, the limitation of Escherichia coli is its inability to excrete high amount of CGTase outside the cells. In this study, modification of the hydrophobic region of the N1R3 signal peptide using site-saturation mutagenesis improved the excretion of CGTase. Signal peptide mutants designated M9F, V10L and A15Y enhanced the excretion of CGTase three-fold and demonstrated two-fold higher secretion rate than the wild type. However, high secretion rate of these mutants was non-productive for recombinant protein production because it caused up to a seven-fold increase in cell death compared to the wild type. Our results indicated that the excretion of CGTase is highly dependent on hydrophobicity, secondary conformation and the type and position of amino acids at the region boundary and core segment of the h-region.
    Matched MeSH terms: Escherichia coli/metabolism
  8. Fulltext Genome-Wide Discovery of Genes Required for Capsule Production by Uropathogenic Escherichia coli
    Goh KGK, Phan MD, Forde BM, Chong TM, Yin WF, Chan KG, et al.
    mBio, 2017 10 24;8(5).
    PMID: 29066548 DOI: 10.1128/mBio.01558-17
    Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections and possesses an array of virulence factors for colonization, survival, and persistence. One such factor is the polysaccharide K capsule. Among the different K capsule types, the K1 serotype is strongly associated with UPEC infection. In this study, we completely sequenced the K1 UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1 capsule-specific phage, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing (TraDIS) to identify the complement of genes required for capsule production. Our analysis identified known genes involved in capsule biosynthesis, as well as two additional regulatory genes (mprA and lrhA) that we characterized at the molecular level. Mutation of mprA resulted in protection against K1 phage-mediated killing, a phenotype restored by complementation. We also identified a significantly increased unidirectional Tn5 insertion frequency upstream of the lrhA gene and showed that strong expression of LrhA induced by a constitutive Pcl promoter led to loss of capsule production. Further analysis revealed loss of MprA or overexpression of LrhA affected the transcription of capsule biosynthesis genes in PA45B and increased sensitivity to killing in whole blood. Similar phenotypes were also observed in UPEC strains UTI89 (K1) and CFT073 (K2), demonstrating that the effects were neither strain nor capsule type specific. Overall, this study defined the genome of a UPEC urosepsis isolate and identified and characterized two new regulatory factors that affect UPEC capsule production.IMPORTANCE Urinary tract infections (UTIs) are among the most common bacterial infections in humans and are primarily caused by uropathogenic Escherichia coli (UPEC). Many UPEC strains express a polysaccharide K capsule that provides protection against host innate immune factors and contributes to survival and persistence during infection. The K1 serotype is one example of a polysaccharide capsule type and is strongly associated with UPEC strains that cause UTIs, bloodstream infections, and meningitis. The number of UTIs caused by antibiotic-resistant UPEC is steadily increasing, highlighting the need to better understand factors (e.g., the capsule) that contribute to UPEC pathogenesis. This study describes the original and novel application of lytic capsule-specific phage killing, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing to define the entire complement of genes required for capsule production in UPEC. Our comprehensive approach uncovered new genes involved in the regulation of this key virulence determinant.
    Matched MeSH terms: Uropathogenic Escherichia coli/metabolism
  9. Genome-scale metabolic models as platforms for strain design and biological discovery
    Mienda BS
    J Biomol Struct Dyn, 2017 Jul;35(9):1863-1873.
    PMID: 27251747 DOI: 10.1080/07391102.2016.1197153
    Genome-scale metabolic models (GEMs) have been developed and used in guiding systems' metabolic engineering strategies for strain design and development. This strategy has been used in fermentative production of bio-based industrial chemicals and fuels from alternative carbon sources. However, computer-aided hypotheses building using established algorithms and software platforms for biological discovery can be integrated into the pipeline for strain design strategy to create superior strains of microorganisms for targeted biosynthetic goals. Here, I described an integrated workflow strategy using GEMs for strain design and biological discovery. Specific case studies of strain design and biological discovery using Escherichia coli genome-scale model are presented and discussed. The integrated workflow presented herein, when applied carefully would help guide future design strategies for high-performance microbial strains that have existing and forthcoming genome-scale metabolic models.
    Matched MeSH terms: Escherichia coli/metabolism
  10. Production of refolded Toxoplasma gondii recombinant SAG1-related sequence 3 (SRS3) and its use for serodiagnosis of human toxoplasmosis
    Mirzadeh A, Saadatnia G, Golkar M, Babaie J, Noordin R
    Protein Expr Purif, 2017 05;133:66-74.
    PMID: 28263855 DOI: 10.1016/j.pep.2017.03.001
    SAG1-related sequence 3 (SRS3) is one of the major Toxoplasma gondii tachyzoite surface antigens and has been shown to be potentially useful for the detection of toxoplasmosis. This protein is highly conformational due to the presence of six disulfide bonds. To achieve solubility and antigenicity, SRS3 depends on proper disulfide bond formation. The aim of this study was to over-express the SRS3 protein with correct folding for use in serodiagnosis of the disease. To achieve this, a truncated SRS3 fusion protein (rtSRS3) was produced, containing six histidyl residues at both terminals and purified by immobilized metal affinity chromatography. The refolding process was performed through three methods, namely dialysis in the presence of chemical additives along with reduced/oxidized glutathione and drop-wise dilution methods with reduced/oxidized glutathione or reduced DTT/oxidized glutathione. Ellman's assay and ELISA showed that the protein folding obtained by the dialysis method was the most favorable, probably due to the correct folding. Subsequently, serum samples from individuals with chronic infection (n = 76), probable acute infection (n = 14), and healthy controls (n = 81) were used to determine the usefulness of the refolded rtSRS3 for Toxoplasma serodiagnosis. The results of the developed IgG-ELISA showed a diagnostic specificity of 91% and a sensitivity of 82.89% and 100% for chronic and acute serum samples, respectively. In conclusion, correctly folded rtSRS3 has the potential to be used as a soluble antigen for the detection of human toxoplasmosis.
    Matched MeSH terms: Escherichia coli/metabolism
  11. Virus-like particle of Macrobrachium rosenbergii nodavirus produced in Spodoptera frugiperda (Sf9) cells is distinctive from that produced in Escherichia coli
    Kueh CL, Yong CY, Masoomi Dezfooli S, Bhassu S, Tan SG, Tan WS
    Biotechnol Prog, 2017 Mar;33(2):549-557.
    PMID: 27860432 DOI: 10.1002/btpr.2409
    Macrobrachium rosenbergii nodavirus (MrNV) is a virus native to giant freshwater prawn. Recombinant MrNV capsid protein has been produced in Escherichia coli, which self-assembled into virus-like particles (VLPs). However, this recombinant protein is unstable, degrading and forming heterogenous VLPs. In this study, MrNV capsid protein was produced in insect Spodoptera frugiperda (Sf9) cells through a baculovirus system. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the recombinant protein produced by the insect cells self-assembled into highly stable, homogenous VLPs each of approximately 40 nm in diameter. Enzyme-linked immunosorbent assay (ELISA) showed that the VLPs produced in Sf9 cells were highly antigenic and comparable to those produced in E. coli. In addition, the Sf9 produced VLPs were highly stable across a wide pH range (2-12). Interestingly, the Sf9 produced VLPs contained DNA of approximately 48 kilo base pairs and RNA molecules. This study is the first report on the production and characterization of MrNV VLPs produced in a eukaryotic system. The MrNV VLPs produced in Sf9 cells were about 10 nm bigger and had a uniform morphology compared with the VLPs produced in E. coli. The insect cell production system provides a good source of MrNV VLPs for structural and immunological studies as well as for host-pathogen interaction studies. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:549-557, 2017.
    Matched MeSH terms: Escherichia coli/metabolism*
  12. Fulltext AidP, a novel N-Acyl homoserine lactonase gene from Antarctic Planococcus sp
    See-Too WS, Ee R, Lim YL, Convey P, Pearce DA, Yin WF, et al.
    Sci Rep, 2017 02 22;7:42968.
    PMID: 28225085 DOI: 10.1038/srep42968
    Planococcus is a Gram-positive halotolerant bacterial genus in the phylum Firmicutes, commonly found in various habitats in Antarctica. Quorum quenching (QQ) is the disruption of bacterial cell-to-cell communication (known as quorum sensing), which has previously been described in mesophilic bacteria. This study demonstrated the QQ activity of a psychrotolerant strain, Planococcus versutus strain L10.15T, isolated from a soil sample obtained near an elephant seal wallow in Antarctica. Whole genome analysis of this bacterial strain revealed the presence of an N-acyl homoserine lactonase, an enzyme that hydrolyzes the ester bond of the homoserine lactone of N-acyl homoserine lactone (AHLs). Heterologous gene expression in E. coli confirmed its functions for hydrolysis of AHLs, and the gene was designated as aidP (autoinducer degrading gene from Planococcus sp.). The low temperature activity of this enzyme suggested that it is a novel and uncharacterized class of AHL lactonase. This study is the first report on QQ activity of bacteria isolated from the polar regions.
    Matched MeSH terms: Escherichia coli/metabolism
  13. Crystallization and X-ray crystallographic analysis of recombinant TylP, a putative γ-butyrolactone receptor protein from Streptomyces fradiae
    Mohd-Sharif N, Shaibullah S, Givajothi V, Tan CS, Ho KL, Teh AH, et al.
    Acta Crystallogr F Struct Biol Commun, 2017 02 01;73(Pt 2):109-115.
    PMID: 28177322 DOI: 10.1107/S2053230X17001212
    TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced in Escherichia coli and purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%(v/v) ethylene glycol and 5%(v/v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space group P3121, with unit-cell parameters a = b = 126.62, c = 95.63 Å.
    Matched MeSH terms: Escherichia coli/metabolism
  14. Fulltext Serum from Nipah Virus Patients Recognises Recombinant Viral Proteins Produced in Escherichia coli
    Tiong V, Lam CW, Phoon WH, AbuBakar S, Chang LY
    Jpn J Infect Dis, 2017 Jan 24;70(1):26-31.
    PMID: 27169942 DOI: 10.7883/yoken.JJID.2015.501
    The genes for Nipah virus (NiV) proteins were amplified from viral RNA, cloned into the plasmid pTriEx-3 Hygro, expressed, and purified using immobilized metal affinity chromatography. The recombinant N, F, and G NiV proteins (rNiV-N, rNiV-F, and rNiV-G), were successfully expressed in Escherichia coli and purified with a yield of 4, 16, and 4 mg/L, respectively. All 3 recombinant viral proteins reacted with all 19 samples of NiV-positive human sera. The rNiV-N and rNiV-G proteins were the most immunogenic. The recombinant viral proteins did not react with any of the 12 NiV-negative sera. However, serum from a patient with a late-onset relapsing NiV infection complication was found to be primarily reactive to rNiV-G only. Additionally, there is a distinctive variation in the profile of antigen-reactive bands between the sample from a case of relapsing NiV encephalitis and that of acute NiV infection. The overall findings of this study suggest that the recombinant viral proteins have the potential to be developed further for use in the detection of NiV infection, and continuous biosurveillance of NiV infection in resource-limited settings.
    Matched MeSH terms: Escherichia coli/metabolism
  15. Model-assisted formate dehydrogenase-O (fdoH) gene knockout for enhanced succinate production in Escherichia coli from glucose and glycerol carbon sources
    Mienda BS, Shamsir MS, Md Illias R
    J Biomol Struct Dyn, 2016 Nov;34(11):2305-16.
    PMID: 26510527 DOI: 10.1080/07391102.2015.1113387
    Succinic acid is an important platform chemical that has broad applications and is been listed as one of the top twelve bio-based chemicals produced from biomass by the US Department of Energy. The metabolic role of Escherichia coli formate dehydrogenase-O (fdoH) under anaerobic conditions in relation to succinic acid production remained largely unspecified. Herein we report, what are to our knowledge, the first metabolic fdoH gene knockout that have enhanced succinate production using glucose and glycerol substrates in E. coli. Using the most recent E. coli reconstruction iJO1366, we engineered its host metabolism to enhance the anaerobic succinate production by deleting the fdoH gene, which blocked H(+) conduction across the mutant cell membrane for the enhanced succinate production. The engineered mutant strain BMS4 showed succinate production of 2.05 g l(-1) (41.2-fold in 7 days) from glycerol and .39 g l(-1) (6.2-fold in 1 day) from glucose. This work revealed that a single deletion of the fdoH gene is sufficient to increase succinate production in E. coli from both glucose and glycerol substrates.
    Matched MeSH terms: Escherichia coli/metabolism*
  16. Cold-adapted organic solvent tolerant alkalophilic family I.3 lipase from an Antarctic Pseudomonas
    Ganasen M, Yaacob N, Rahman RN, Leow AT, Basri M, Salleh AB, et al.
    Int J Biol Macromol, 2016 Nov;92:1266-1276.
    PMID: 27506122 DOI: 10.1016/j.ijbiomac.2016.06.095
    Lipolytic enzymes with cold adaptation are gaining increasing interest due to their biotechnological prospective. Previously, a cold adapted family I.3 lipase (AMS8 lipase) was isolated from an Antarctic Pseudomonas. AMS8 lipase was largely expressed in insoluble form. The refolded His-tagged recombinant AMS8 lipase was purified with 23.0% total recovery and purification factor of 9.7. The purified AMS8 lipase migrated as a single band with a molecular weight approximately 65kDa via electrophoresis. AMS8 lipase was highly active at 30°C at pH 10. The half-life of AMS8 lipase was reported at 4 and 2h under the incubation of 30 and 40°C, respectively. The lipase was stable over a broad range of pH. It showed enhancement effect in its relative activity under the presence of Li(+), Na(+), K(+), Rb(+) and Cs(+) after 30min treatment. Heavy metal ions such as Cu(2+), Fe(3+) and Zn(2+) inhibited AMS8 activity. This cold adapted alkalophilic AMS lipase was also active in various organic solvent of different polarity. These unique properties of this biological macromolecule will provide considerable potential for many biotechnological applications and organic synthesis at low temperature.
    Matched MeSH terms: Escherichia coli/metabolism
  17. Crystal structure of fuculose aldolase from the Antarctic psychrophilic yeast Glaciozyma antarctica PI12
    Jaafar NR, Littler D, Beddoe T, Rossjohn J, Illias RM, Mahadi NM, et al.
    Acta Crystallogr F Struct Biol Commun, 2016 11 01;72(Pt 11):831-839.
    PMID: 27827354
    Fuculose-1-phosphate aldolase (FucA) catalyses the reversible cleavage of L-fuculose 1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde. This enzyme from mesophiles and thermophiles has been extensively studied; however, there is no report on this enzyme from a psychrophile. In this study, the gene encoding FucA from Glaciozyma antarctica PI12 (GaFucA) was cloned and the enzyme was overexpressed in Escherichia coli, purified and crystallized. The tetrameric structure of GaFucA was determined to 1.34 Å resolution. The overall architecture of GaFucA and its catalytically essential histidine triad are highly conserved among other fuculose aldolases. Comparisons of structural features between GaFucA and its mesophilic and thermophilic homologues revealed that the enzyme has typical psychrophilic attributes, indicated by the presence of a high number of nonpolar residues at the surface and a lower number of arginine residues.
    Matched MeSH terms: Escherichia coli/metabolism
  18. Fulltext Risk factors and spatial distribution of extended spectrum β-lactamase-producing- Escherichia coli at retail poultry meat markets in Malaysia: a cross-sectional study
    Aliyu AB, Saleha AA, Jalila A, Zunita Z
    BMC Public Health, 2016 08 02;16:699.
    PMID: 27484086 DOI: 10.1186/s12889-016-3377-2
    BACKGROUND: The significant role of retail poultry meat as an important exposure pathway for the acquisition and transmission of extended spectrum β-lactamase-producing Escherichia coli (ESBL-EC) into the human population warrants understanding concerning those operational practices associated with dissemination of ESBL-EC in poultry meat retailing. Hence, the objective of this study was to determine the prevalence, spatial distribution and potential risk factors associated with the dissemination of ESBL-EC in poultry meat retail at wet-markets in Selangor, Malaysia.

    METHODS: Poultry meat (breast, wing, thigh, and keel) as well as the contact surfaces of weighing scales and cutting boards were sampled to detect ESBL-EC by using culture and disk combination methods and polymerase chain reaction assays. Besides, questionnaire was used to obtain data and information pertaining to those operational practices that may possibly explain the occurrence of ESBL-EC. The data were analysed using logistic regression analysis at 95 % CI.

    RESULTS: The overall prevalence of ESBL-EC was 48.8 % (95 % CI, 42 - 55 %). Among the risk factors that were explored, type of countertop, sanitation of the stall environment, source of cleaning water, and type of cutting board were found to be significantly associated with the presence of ESBL-EC.

    CONCLUSIONS: Thus, in order to prevent or reduce the presence of ESBL-EC and other contaminants at the retail-outlet, there is a need to design a process control system based on the current prevailing practices in order to reduce cross contamination, as well as to improve food safety and consumer health.

    Matched MeSH terms: Escherichia coli/metabolism
  19. Model-aided atpE gene knockout strategy in Escherichia coli for enhanced succinic acid production from glycerol
    Mienda BS, Shamsir MS, Md Illias R
    J Biomol Struct Dyn, 2016 Aug;34(8):1705-16.
    PMID: 26513379 DOI: 10.1080/07391102.2015.1090341
    Succinic acid is an important platform chemical with a variety of applications. Model-guided metabolic engineering strategies in Escherichia coli for strain improvement to increase succinic acid production using glucose and glycerol remain largely unexplored. Herein, we report what are, to our knowledge, the first metabolic knockout of the atpE gene to have increased succinic acid production using both glucose and alternative glycerol carbon sources in E. coli. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic production of succinic acid by deleting the atpE gene, thereby generating additional reducing equivalents by blocking H(+) conduction across the mutant cell membrane. This strategy produced 1.58 and .49 g l(-1) of succinic acid from glycerol and glucose substrate, respectively. This work further elucidates a model-guided and/or system-based metabolic engineering, involving only a single-gene deletion strategy for enhanced succinic acid production in E. coli.
    Matched MeSH terms: Escherichia coli/metabolism*
  20. Production of the virus-like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents
    Joseph NM, Ho KL, Tey BT, Tan CS, Shafee N, Tan WS
    Biotechnol Prog, 2016 Jul 08;32(4):1038-45.
    PMID: 27088434 DOI: 10.1002/btpr.2279
    The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus-like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti-myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high-performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038-1045, 2016.
    Matched MeSH terms: Escherichia coli/metabolism
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