Displaying all 11 publications

Abstract:
Sort:
  1. Fulltext Rational mutagenesis of cyclodextrin glucanotransferase at the calcium binding regions for enhancement of thermostability
    Goh PH, Illias RM, Goh KM
    Int J Mol Sci, 2012;13(5):5307-23.
    PMID: 22754298 DOI: 10.3390/ijms13055307
    Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as "parent CGTase" herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.
    Matched MeSH terms: Glucosyltransferases/genetics*
  2. Fruit characteristics, soluble sugar compositions and transcriptome analysis during the development of Citrus maxima "seedless", and identification of SUS and INV genes involved in sucrose degradation
    Deng S, Mai Y, Niu J
    Gene, 2019 Mar 20;689:131-140.
    PMID: 30576805 DOI: 10.1016/j.gene.2018.12.016
    Citrus maxima "seedless" is originally from Malaysia, and now is widely cultivated in Hainan province, China. The essential features of this cultivar are thin skin, green epicarp and seedless at the ripening stage. Here, using C. maxima "seedless" as experimental material, we investigated the physical and inclusion indicators, and found the accumulation of storage compounds during 120-210 DAF leading to inconsistent increase between volume and weight. Component analysis of soluble sugar indicated that arabinose and xylose have a high content in early development of pummelo juice sacs (PJS), whereas fructose, glucose and sucrose show a significant increase during PJS maturation. To clarify a global overview of the gene expressing profiles, the PJSs from four periods (60, 120, 180 and 240 DAF) were selected for comparative transcriptome analysis. The resulting 8275 unigenes showed differential expression during PJS development. Also, the stability of 11 housekeeping genes were evaluated by geNorm method, resulting in a set of five genes (UBC, ACT, OR23, DWA2 and CYP21D) used as control for normalization of gene expression. Based on transcriptome data, 5 sucrose synthases (SUSs) and 10 invertases (INVs) were identified to be involved in sucrose degradation. Importantly, SUS4 may be responsible for arabinose and xylose biosynthesis to form the cell wall in early development, while SUS3 and VIN2 may be important in the accumulation of soluble hexose leading to cell expansion through an osmotic-independent pathway in late development. The information provides valuable metabolite and genetic resources in C. maxima "seedless", and is important for achieving high fruit yield and quality.
    Matched MeSH terms: Glucosyltransferases/genetics*
  3. An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production
    Low KO, Mahadi NM, Rahim RA, Rabu A, Abu Bakar FD, Murad AM, et al.
    J Ind Microbiol Biotechnol, 2011 Sep;38(9):1587-97.
    PMID: 21336875 DOI: 10.1007/s10295-011-0949-0
    Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.
    Matched MeSH terms: Glucosyltransferases/genetics
  4. Fulltext Optimization of a heterologous signal peptide by site-directed mutagenesis for improved secretion of recombinant proteins in Escherichia coli
    Jonet MA, Mahadi NM, Murad AM, Rabu A, Bakar FD, Rahim RA, et al.
    J. Mol. Microbiol. Biotechnol., 2012;22(1):48-58.
    PMID: 22456489 DOI: 10.1159/000336524
    A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinant cyclodextrin glucanotransferase (CGTase) to the periplasmic and, eventually, extracellular space of Escherichia coli. Eight mutant SPs were constructed using site-directed mutagenesis to improve the secretion of recombinant CGTase. M5 is a mutated SP in which replacement of an isoleucine residue in the h-region to glycine created a helix-breaking or G-turn motif with decreased hydrophobicity. The mutant SP resulted in 110 and 94% increases in periplasmic and extracellular recombinant CGTase, respectively, compared to the wild-type SP at a similar level of cell lysis. The formation of intracellular inclusion bodies was also reduced, as determined by sodium dodecyl sulfate-polyacrylamyde gel electrophoresis, when this mutated SP was used. The addition of as low as 0.08% glycine at the beginning of cell growth improved cell viability of the E. coli host. Secretory production of other proteins, such as mannosidase, also showed similar improvement, as demonstrated by CGTase production, suggesting that the combination of an optimized SP and a suitable chemical additive leads to significant improvements of extracellular recombinant protein production and cell viability. These findings will be valuable for the extracellular production of recombinant proteins in E. coli.
    Matched MeSH terms: Glucosyltransferases/genetics
  5. A mutant L-asparaginase II signal peptide improves the secretion of recombinant cyclodextrin glucanotransferase and the viability of Escherichia coli
    Ismail NF, Hamdan S, Mahadi NM, Murad AM, Rabu A, Bakar FD, et al.
    Biotechnol Lett, 2011 May;33(5):999-1005.
    PMID: 21234789 DOI: 10.1007/s10529-011-0517-8
    L-Asparaginase II signal peptide was used for the secretion of recombinant cyclodextrin glucanotransferase (CGTase) into the periplasmic space of E. coli. Despite its predominant localisation in the periplasm, CGTase activity was also detected in the extracellular medium, followed by cell lysis. Five mutant signal peptides were constructed to improve the periplasmic levels of CGTase. N1R3 is a mutated signal peptide with the number of positively charged amino acid residues in the n-region increased to a net charge of +5. This mutant peptide produced a 1.7-fold enhancement of CGTase activity in the periplasm and significantly decreased cell lysis to 7.8% of the wild-type level. The formation of intracellular inclusion bodies was also reduced when this mutated signal peptide was used as judged by SDS-PAGE. Therefore, these results provide evidence of a cost-effective means of expression of recombinant proteins in E. coli.
    Matched MeSH terms: Glucosyltransferases/genetics
  6. Effect of signal peptides on the secretion of β-cyclodextrin glucanotransferase in Lactococcus lactis NZ9000
    Subramaniam M, Baradaran A, Rosli MI, Rosfarizan M, Khatijah Y, Raha AR
    J. Mol. Microbiol. Biotechnol., 2012;22(6):361-72.
    PMID: 23295307 DOI: 10.1159/000343921
    Cyclodextrin glucanotransferase (CGTase) is an extracellular enzyme which catalyzes the formation of cyclodextrin from starch. The production of CGTase using lactic acid bacterium is an attractive alternative and safer strategy to produce CGTase. In this study, we report the construction of genetically modified Lactococcus lactis strains harboring plasmids that secrete the Bacillus sp. G1 β-CGTase, with the aid of the signal peptides (SPs) SPK1, USP45 and native SP (NSP). Three constructed vectors, pNZ:NSP:CGT, pNZ:USP:CGT and pNZ:SPK1:CGT, were developed in this study. Each vector harbored a different SP fused to the CGTase. The formation of halo zones on starch plates indicated the production and secretion of β-CGTase by the recombinants. The expression of this enzyme is shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis. A band size of ∼75 kDa corresponding to β-CGTase is identified in the intracellular and the extracellular environments of the host after medium modification. The replacement of glucose by starch in the medium was shown to induce β-CGTase production in L. lactis. Although β-CGTase production is comparatively low in NZ:SPK1:CGT, the SP SPK1 was shown to have higher secretion efficiency compared to the other SPs used in this study.
    Matched MeSH terms: Glucosyltransferases/genetics*
  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: Glucosyltransferases/genetics*
  8. Use of Megaprimer and Overlapping Extension PCR (OE-PCR) to Mutagenize and Enhance Cyclodextrin Glucosyltransferase (CGTase) Function
    Goh KM, Liew KJ, Chai KP, Illias RM
    Methods Mol Biol, 2017;1498:385-396.
    PMID: 27709591
    Protein engineering is a very useful tool for probing structure-function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.
    Matched MeSH terms: Glucosyltransferases/genetics*
  9. Improvement of cyclodextrin glycosyltransferase gene expression in Escherichia coli by insertion of regulatory sequences involved in the promotion of RNA transcription
    Ramli N, Abd-Aziz S, Alitheen NB, Hassan MA, Maeda T
    Mol Biotechnol, 2013 Jul;54(3):961-8.
    PMID: 23338983 DOI: 10.1007/s12033-013-9647-7
    Regulation of RNA transcription in controlling the expression of genes at promoter and terminator regions is crucial as the interaction of RNA polymerase occurred at both sites. Gene encoding cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. NR5 UPM isolated in the previous study was used for further construction of pTZCGT-SS, pTZCGT-BS and pTZCGT-BT expression systems for enhancement of CGTase production. The putative promoter regions, -35 and -10 sequences were found in the upstream of the mature gene start codon. Whereas, long inverted repeats sequences which can form a stable stem and loop structure was found downstream of the open reading frame (ORF) of Bacillus sp. NR5 UPM CGTase. The construction of E. coli strain harbouring pTZCGT-BS showed increment of 3.2-fold in CGTase activity compared to the wild type producer. However, insertion of terminator downstream of CGTase gene in E. coli strain harbouring pTZCGT-BT only resulted in 4.42 % increment of CGTase production compared to E. coli strain containing pTZCGT-BS, perhaps due to low intrinsic termination efficiency. Thus, it is suggested that the insertion of the putative promoter regions upstream of the coding sequence for the construction of CGTase expression system will further enhance in the recombinant enzyme production.
    Matched MeSH terms: Glucosyltransferases/genetics*
  10. Cloning, extracellular expression and characterization of a predominant beta-CGTase from Bacillus sp. G1 in E. coli
    Ong RM, Goh KM, Mahadi NM, Hassan O, Rahman RN, Illias RM
    J Ind Microbiol Biotechnol, 2008 Dec;35(12):1705-14.
    PMID: 18726621 DOI: 10.1007/s10295-008-0462-2
    The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60 degrees C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% beta-cyclodextrin (CD) and 10% gamma-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of beta-CD.
    Matched MeSH terms: Glucosyltransferases/genetics
  11. Fulltext Shigella flexneri serotype 1c derived from serotype 1a by acquisition of gtrIC gene cluster via a bacteriophage
    Tang SS, Carlin NI, Talukder KA, Cam PD, Verma NK
    BMC Microbiol, 2016 Jun 27;16(1):127.
    PMID: 27349637 DOI: 10.1186/s12866-016-0746-z
    BACKGROUND: Shigella spp. are the primary causative agents of bacillary dysentery. Since its emergence in the late 1980s, the S. flexneri serotype 1c remains poorly understood, particularly with regard to its origin and genetic evolution. This article provides a molecular insight into this novel serotype and the gtrIC gene cluster that determines its unique immune recognition.

    RESULTS: A PCR of the gtrIC cluster showed that serotype 1c isolates from different geographical origins were genetically conserved. An analysis of sequences flanking the gtrIC cluster revealed remnants of a prophage genome, in particular integrase and tRNA(Pro) genes. Meanwhile, Southern blot analyses on serotype 1c, 1a and 1b strains indicated that all the tested serotype 1c strains may have had a common origin that has since remained distinct from the closely related 1a and 1b serotypes. The identification of prophage genes upstream of the gtrIC cluster is consistent with the notion of bacteriophage-mediated integration of the gtrIC cluster into a pre-existing serotype.

    CONCLUSIONS: This is the first study to show that serotype 1c isolates from different geographical origins share an identical pattern of genetic arrangement, suggesting that serotype 1c strains may have originated from a single parental strain. Analysis of the sequence around the gtrIC cluster revealed a new site for the integration of the serotype converting phages of S. flexneri. Understanding the origin of new pathogenic serotypes and the molecular basis of serotype conversion in S. flexneri would provide information for developing cross-reactive Shigella vaccines.

    Matched MeSH terms: Glucosyltransferases/genetics
Related Terms
Filters
Contact Us

Please provide feedback to Administrator (afdal@afpm.org.my)

External Links