Displaying publications 1 - 20 of 38 in total

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  1. Zamani AI, Barig S, Ibrahim S, Mohd Yusof H, Ibrahim J, Low JYS, et al.
    Microb Cell Fact, 2020 Sep 09;19(1):179.
    PMID: 32907579 DOI: 10.1186/s12934-020-01434-w
    BACKGROUND: Sugars and triglycerides are common carbon sources for microorganisms. Nonetheless, a systematic comparative interpretation of metabolic changes upon vegetable oil or glucose as sole carbon source is still lacking. Selected fungi that can grow in acidic mineral salt media (MSM) with vegetable oil had been identified recently. Hence, this study aimed to investigate the overall metabolite changes of an omnipotent fungus and to reveal changes at central carbon metabolism corresponding to both carbon sources.

    RESULTS: Targeted and non-targeted metabolomics for both polar and semi-polar metabolites of Phialemonium curvatum AWO2 (DSM 23903) cultivated in MSM with palm oil (MSM-P) or glucose (MSM-G) as carbon sources were obtained. Targeted metabolomics on central carbon metabolism of tricarboxylic acid (TCA) cycle and glyoxylate cycle were analysed using LC-MS/MS-TripleQ and GC-MS, while untargeted metabolite profiling was performed using LC-MS/MS-QTOF followed by multivariate analysis. Targeted metabolomics analysis showed that glyoxylate pathway and TCA cycle were recruited at central carbon metabolism for triglyceride and glucose catabolism, respectively. Significant differences in organic acids concentration of about 4- to 8-fold were observed for citric acid, succinic acid, malic acid, and oxaloacetic acid. Correlation of organic acids concentration and key enzymes involved in the central carbon metabolism was further determined by enzymatic assays. On the other hand, the untargeted profiling revealed seven metabolites undergoing significant changes between MSM-P and MSM-G cultures.

    CONCLUSIONS: Overall, this study has provided insights on the understanding on the effect of triglycerides and sugar as carbon source in fungi global metabolic pathway, which might become important for future optimization of carbon flux engineering in fungi to improve organic acids production when vegetable oil is applied as the sole carbon source.

  2. Veeramohan R, Azizan KA, Aizat WM, Goh HH, Mansor SM, Yusof NSM, et al.
    Data Brief, 2018 Jun;18:1212-1216.
    PMID: 29900296 DOI: 10.1016/j.dib.2018.04.001
    Mitragyna speciosa is a psychoactive plant known as "ketum" in Malaysia and "kratom" in Thailand. This plant is distinctly known to produce two important alkaloids, namely mitragynine (MG) and 7-hydroxymitragynine (7-OH-MG) that can bind to opioid receptors [1]. MG was reported to exhibit antidepressant properties in animal studies [2]. These compounds were also proposed to have the potential to replace opioid analgesics with much lower risks of side effects [3]. To date, there are only over 40 metabolites identified in M. speciosa [4,5]. To obtain a more complete profile of secondary metabolites in ketum, we performed metabolomics study using mature leaves of the green M. speciosa variety. The leaf samples were extracted using methanol prior to liquid chromatography-electrospray ionization-time of flight-mass spectrometry (LC-ESI-TOF-MS) analysis. This data can be useful to for the identification of unknown metabolites that are associated with alkaloid biosynthesis pathway in M. speciosa.
  3. Veeramohan R, Zamani AI, Azizan KA, Goh HH, Aizat WM, Razak MFA, et al.
    PLoS One, 2023;18(3):e0283147.
    PMID: 36943850 DOI: 10.1371/journal.pone.0283147
    The fresh leaves of Mitragyna speciosa (Korth.) Havil. have been traditionally consumed for centuries in Southeast Asia for its healing properties. Although the alkaloids of M. speciosa have been studied since the 1920s, comparative and systematic studies of metabolite composition based on different leaf maturity levels are still lacking. This study assessed the secondary metabolite composition in two different leaf stages (young and mature) of M. speciosa, using an untargeted liquid chromatography-electrospray ionisation-time-of-flight-mass spectrometry (LC-ESI-TOF-MS) metabolite profiling. The results revealed 86 putatively annotated metabolite features (RT:m/z value) comprising 63 alkaloids, 10 flavonoids, 6 terpenoids, 3 phenylpropanoids, and 1 of each carboxylic acid, glucoside, phenol, and phenolic aldehyde. The alkaloid features were further categorised into 14 subclasses, i.e., the most abundant class of secondary metabolites identified. As per previous reports, indole alkaloids are the most abundant alkaloid subclass in M. speciosa. The result of multivariate analysis (MVA) using principal component analysis (PCA) showed a clear separation of 92.8% between the young and mature leaf samples, indicating a high variance in metabolite levels between them. Akuammidine, alstonine, tryptamine, and yohimbine were tentatively identified among the many new alkaloids reported in this study, depicting the diverse biological activities of M. speciosa. Besides delving into the knowledge of metabolite distribution in different leaf stages, these findings have extended the current alkaloid repository of M. speciosa for a better understanding of its pharmaceutical potential.
  4. Tan CS, Hassan M, Mohamed Hussein ZA, Ismail I, Ho KL, Ng CL, et al.
    Plant Physiol Biochem, 2018 Feb;123:359-368.
    PMID: 29304481 DOI: 10.1016/j.plaphy.2017.12.033
    Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp280, Leu294 and Ala303). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily.
  5. Shaibullah S, Shuhaimi N, Ker DS, Mohd-Sharif N, Ho KL, Teh AH, et al.
    Commun Biol, 2023 Sep 08;6(1):920.
    PMID: 37684342 DOI: 10.1038/s42003-023-05265-4
    Burkholderia pseudomallei is a highly versatile pathogen with ~25% of its genome annotated to encode hypothetical proteins. One such hypothetical protein, BPSL1038, is conserved across seven bacterial genera and 654 Burkholderia spp. Here, we present a 1.55 Å resolution crystal structure of BPSL1038. The overall structure folded into a modified βαββαβα ferredoxin fold similar to known Cas2 nucleases. The Cas2 equivalent catalytic aspartate (D11) pairs are conserved in BPSL1038 although B. pseudomallei has no known CRISPR associated system. Functional analysis revealed that BPSL1038 is a nuclease with endonuclease activity towards double-stranded DNA. The DNase activity is divalent ion independent and optimum at pH 6. The concentration of monovalent ions (Na+ and K+) is crucial for nuclease activity. An active site with a unique D11(X20)SST motif was identified and proposed for BPSL1038 and its orthologs. Structure modelling indicates the catalytic role of the D11(X20)SST motif and that the arginine residues R10 and R30 may interact with the nucleic acid backbone. The structural similarity of BPSL1038 to Cas2 proteins suggests that BPSL1038 may represent a sub-family of nucleases that share a common ancestor with Cas2.
  6. Shaibullah S, Mohd-Sharif N, Ho KL, Firdaus-Raih M, Nathan S, Mohamed R, et al.
    Acta Crystallogr F Struct Biol Commun, 2014 Dec 01;70(Pt 12):1697-700.
    PMID: 25484229 DOI: 10.1107/S2053230X14025278
    Melioidosis is an infectious disease caused by the pathogenic bacterium Burkholderia pseudomallei. Whole-genome sequencing revealed that the B. pseudomallei genome includes 5855 coding DNA sequences (CDSs), of which ∼25% encode hypothetical proteins. A pathogen-associated hypothetical protein, BPSL1038, was overexpressed in Escherichia coli, purified and crystallized using vapour-diffusion methods. A BPSL1038 protein crystal that grew using sodium formate as precipitant diffracted to 1.55 Å resolution. It belonged to space group C2221, with unit-cell parameters a = 85.36, b = 115.63, c = 46.73 Å. The calculated Matthews coefficient (VM) suggests that there are two molecules per asymmetric unit, with a solvent content of 48.8%.
  7. Seman-Kamarulzaman AF, Mohamed-Hussein ZA, Ng CL, Hassan M
    PLoS One, 2016;11(8):e0161707.
    PMID: 27560927 DOI: 10.1371/journal.pone.0161707
    Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that's highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity towards farnesal. Thus, it was suggested that this novel enzyme may be functioning specifically to oxidize farnesal in the later steps of JH III pathway. This report provides a basic understanding for recombinant production of this particular enzyme. Other strategies such as adding His-tag to the protein makes easy the purification of the protein which is completely different to the native protein. Complete sequence, structure and functional analysis of the enzyme will be important for developing insect-resistant crop plants by deployment of transgenic plant.
  8. Selvaraj BA, Mariatulqabtiah AR, Ho KL, Ng CL, Yong CY, Tan WS
    Int J Mol Sci, 2021 Aug 13;22(16).
    PMID: 34445426 DOI: 10.3390/ijms22168725
    The causative agent of white tail disease (WTD) in the giant freshwater prawn is Macrobrachium rosenbergii nodavirus (MrNV). The recombinant capsid protein (CP) of MrNV was previously expressed in Escherichia coli, and it self-assembled into icosahedral virus-like particles (VLPs) with a diameter of approximately 30 nm. Extensive studies on the MrNV CP VLPs have attracted widespread attention in their potential applications as biological nano-containers for targeted drug delivery and antigen display scaffolds for vaccine developments. Despite their advantageous features, the recombinant MrNV CP VLPs produced in E. coli are seriously affected by protease degradations, which significantly affect the yield and stability of the VLPs. Therefore, the aim of this study is to enhance the stability of MrNV CP by modulating the protease degradation activity. Edman degradation amino acid sequencing revealed that the proteolytic cleavage occurred at arginine 26 of the MrNV CP. The potential proteases responsible for the degradation were predicted in silico using the Peptidecutter, Expasy. To circumvent proteolysis, specific protease inhibitors (PMSF, AEBSF and E-64) were tested to reduce the degradation rates. Modulation of proteolytic activity demonstrated that a cysteine protease was responsible for the MrNV CP degradation. The addition of E-64, a cysteine protease inhibitor, remarkably improved the yield of MrNV CP by 2.3-fold compared to the control. This innovative approach generates an economical method to improve the scalability of MrNV CP VLPs using individual protease inhibitors, enabling the protein to retain their structural integrity and stability for prominent downstream applications including drug delivery and vaccine development.
  9. Satyaveanthan MV, Suhaimi SA, Ng CL, Muhd-Noor ND, Awang A, Lam KW, et al.
    Plant Physiol Biochem, 2021 Apr;161:143-155.
    PMID: 33588320 DOI: 10.1016/j.plaphy.2021.01.050
    The juvenile hormones (JH) in plants are suggested to act as a form of plant defensive strategy especially against insect herbivory. The oxidation of farnesol to farnesoic acid is a key step in the juvenile hormone biosynthesis pathway. We herein present the purification and characterisation of the recombinant Theobroma cacao farnesol dehydrogenase enzyme that catalyses oxidation of farnesol to farnesal. The recombinant enzyme was purified to apparent homogeneity by affinity chromatography. The purified enzyme was characterised in terms of its deduced amino acid sequences, phylogeny, substrate specificity, kinetic parameters, structural modeling, and docking simulation. The phylogenetic analysis indicated that the T. cacao farnesol dehydrogenase (TcFolDH) showed a close relationship with A. thaliana farnesol dehydrogenase gene. The TcFolDH monomer had a large N-terminal domain which adopted a typical Rossmann-fold, harboring the GxxGxG motif (NADP(H)-binding domain) and a small C-terminal domain. The enzyme was a homotrimer comprised of subunits with molecular masses of 36 kDa. The TcFolDH was highly specific to NADP+ as coenzyme. The substrate specificity studies showed trans, trans-farnesol was the most preferred substrate for the TcFolDH, suggesting that the purified enzyme was a NADP+-dependent farnesol dehydrogenase. The docking of trans, trans-farnesol and NADP+ into the active site of the enzyme showed the important residues, and their interactions involved in the substrate and coenzyme binding of TcFolDH. Considering the extensive involvement of JH in both insects and plants, an in-depth knowledge on the recombinant production of intermediate enzymes of the JH biosynthesis pathway could help provide a potential method for insect control.
  10. Satyaveanthan MV, Ng CL, Awang A, Lam KW, Hassan M
    Insect Mol Biol, 2023 Apr;32(2):143-159.
    PMID: 36454188 DOI: 10.1111/imb.12820
    In Southeast Asia, Conopomorpha cramerella (Snellen) which is commonly known as the cocoa pod borer (CPB) moth has been identified as the most detrimental pest of Theobroma cacao L. Apart from the various side effects on human health and non-target organisms, heavily relying on synthetic pyrethroid insecticides to control CPB infestations also increases the environmental contamination risks. Thus, developing biorational insecticides that minimally affect the non-target organism and environment by targeting the insect growth regulation process is needed to manage the pest population. In insects, juvenile hormones (JH) regulate critical biological events, especially metamorphosis, development and reproduction. Since the physiological roles of JH III vary among different organisms, the biochemical properties, especially substrate specificity and analogue inhibition, may also be different. Therefore, studies on the JH III biosynthetic pathway enzymes in both plants and insects are beneficial to discover more effective analogues. Bioinformatic analysis and biochemical characterization of a NADP+ -dependent farnesol dehydrogenase, an intermediate enzyme of the JH III pathway, from C. cramerella (CcFolDH), were described in this study. In addition, the farnesol analogues that may act as a potent analogue inhibitor for CcFolDH ware determined using in vitro enzymatic study. The phylogenetic analysis indicated that CcFolDH shared a close phylogenetic relationship to the honeybee's short-chain dehydrogenase/reductase. The 27 kDa CcFolDH has an NADP(H) binding domain with a typical Rossmann fold and is likely a homotetrameric protein in the solution. The enzyme had a greater preference for substrate trans, trans-farnesol and coenzyme NADP+ . In terms of analogue inhibitor inhibition, hexahydroxyfarnesyl acetone showed the highest inhibition (the lowest Ki ) compared to other farnesol analogues. Thus, hexahydroxyfarnesyl acetone would serve as the most potent active ingredient for future biorational pesticide management for C. cramerella infestation. Based on the bioinformatic analyses and biochemical characterizations conducted in this research, we proposed that rCcFolDH differs slightly from other reported farnesol dehydrogenases in terms of molecular weight, substrate preference, coenzymes utilization and analogue inhibitors selection.
  11. Remali J, Loke KK, Ng CL, Aizat WM, Tiong J, Zin NM
    Genom Data, 2017 Sep;13:7-10.
    PMID: 28580299 DOI: 10.1016/j.gdata.2017.05.015
    Streptomyces sp. produces bioactive compounds with a broad spectrum of activities. Streptomyces kebangsaanesis SUK12 has been identified as a novel endophytic bacteria isolated from ethnomedicinal plant Portulaca olerace, and was found to produce the phenazine class of biologically active antimicrobial metabolites. The potential use of the phenazines has led to our research interest in determining the genome sequence of Streptomyces kebangsaanensis SUK12. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number PRJNA269542. The raw sequence data are available [https://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP105770].
  12. Remali J, Aizat WM, Ng CL, Lim YC, Mohamed-Hussein ZA, Fazry S
    PeerJ, 2020;8:e9197.
    PMID: 32509463 DOI: 10.7717/peerj.9197
    Background: DNA double strand break repair is important to preserve the fidelity of our genetic makeup after DNA damage. Rad50 is one of the components in MRN complex important for DNA repair mechanism. Rad50 mutations can lead to microcephaly, mental retardation and growth retardation in human. However, Rad50 mutations in human and other organisms have never been gathered and heuristically compared for their deleterious effects. It is important to assess the conserved region in Rad50 and its homolog to identify vital mutations that can affect functions of the protein.

    Method: In this study, Rad50 mutations were retrieved from SNPeffect 4.0 database and literature. Each of the mutations was analyzed using various bioinformatic analyses such as PredictSNP, MutPred, SNPeffect 4.0, I-Mutant and MuPro to identify its impact on molecular mechanism, biological function and protein stability, respectively.

    Results: We identified 103 mostly occurred mutations in the Rad50 protein domains and motifs, which only 42 mutations were classified as most deleterious. These mutations are mainly situated at the specific motifs such as Walker A, Q-loop, Walker B, D-loop and signature motif of the Rad50 protein. Some of these mutations were predicted to negatively affect several important functional sites that play important roles in DNA repair mechanism and cell cycle signaling pathway, highlighting Rad50 crucial role in this process. Interestingly, mutations located at non-conserved regions were predicted to have neutral/non-damaging effects, in contrast with previous experimental studies that showed deleterious effects. This suggests that software used in this study may have limitations in predicting mutations in non-conserved regions, implying further improvement in their algorithm is needed. In conclusion, this study reveals the priority of acid substitution associated with the genetic disorders. This finding highlights the vital roles of certain residues such as K42E, C681A/S, CC684R/S, S1202R, E1232Q and D1238N/A located in Rad50 conserved regions, which can be considered for a more targeted future studies.

  13. Remali J, Sarmin N'M, Ng CL, Tiong JJL, Aizat WM, Keong LK, et al.
    PeerJ, 2017;5:e3738.
    PMID: 29201559 DOI: 10.7717/peerj.3738
    Background: Streptomyces are well known for their capability to produce many bioactive secondary metabolites with medical and industrial importance. Here we report a novel bioactive phenazine compound, 6-((2-hydroxy-4-methoxyphenoxy) carbonyl) phenazine-1-carboxylic acid (HCPCA) extracted from Streptomyces kebangsaanensis, an endophyte isolated from the ethnomedicinal Portulaca oleracea.

    Methods: The HCPCA chemical structure was determined using nuclear magnetic resonance spectroscopy. We conducted whole genome sequencing for the identification of the gene cluster(s) believed to be responsible for phenazine biosynthesis in order to map its corresponding pathway, in addition to bioinformatics analysis to assess the potential of S. kebangsaanensis in producing other useful secondary metabolites.

    Results: The S. kebangsaanensis genome comprises an 8,328,719 bp linear chromosome with high GC content (71.35%) consisting of 12 rRNA operons, 81 tRNA, and 7,558 protein coding genes. We identified 24 gene clusters involved in polyketide, nonribosomal peptide, terpene, bacteriocin, and siderophore biosynthesis, as well as a gene cluster predicted to be responsible for phenazine biosynthesis.

    Discussion: The HCPCA phenazine structure was hypothesized to derive from the combination of two biosynthetic pathways, phenazine-1,6-dicarboxylic acid and 4-methoxybenzene-1,2-diol, originated from the shikimic acid pathway. The identification of a biosynthesis pathway gene cluster for phenazine antibiotics might facilitate future genetic engineering design of new synthetic phenazine antibiotics. Additionally, these findings confirm the potential of S. kebangsaanensis for producing various antibiotics and secondary metabolites.

  14. Rahaman SN, Mat Yusop J, Mohamed-Hussein ZA, Ho KL, Teh AH, Waterman J, et al.
    Acta Crystallogr F Struct Biol Commun, 2016 Mar;72(Pt 3):207-13.
    PMID: 26919524 DOI: 10.1107/S2053230X16002016
    C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.
  15. Pang SL, Ho KL, Waterman J, Teh AH, Chew FT, Ng CL
    Acta Crystallogr F Struct Biol Commun, 2015 Nov;71(Pt 11):1396-400.
    PMID: 26527267 DOI: 10.1107/S2053230X1501818X
    Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Der f 21 (rDer f 21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDer f 21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19 M Tris-HCl pH 8.0, 32% PEG 400 at 293 K. X-ray diffraction data were collected to 1.49 Å resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25 Å, β = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blo t 5 (45%) and Blo t 21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDer f 21 is likely to be a dimeric protein.
  16. Pang SL, Matta SA, Sio YY, Ng YT, Say YH, Ng CL, et al.
    Sci Rep, 2021 01 13;11(1):921.
    PMID: 33441720 DOI: 10.1038/s41598-020-79820-y
    House dust mites (HDMs) are one of the major causes of allergies in the world. The group 23 allergen, Der p 23, from Dermatophagoides pteronyssinus, is a major allergen amongst HDM-sensitized individuals. This study aims to determine the specific immunoglobulin E (sIgE) binding frequency and IgE-binding residues of recombinant Der p 23 (rDer p 23) allergen amongst a cohort of consecutive atopic individuals in a tropical region. We performed site-directed mutagenesis and carried out immuno-dot blot assays using 65 atopic sera. The immuno-dot blot assays results indicated that the two residues K44 and E46 which are located at the N-terminal region are the major IgE-binding residues. The rDerp-23 sIgE titers are strongly correlated to the number of IgE-binding residues for rDer p 23 (P 
  17. Pang SL, Ho KL, Waterman J, Rambo RP, Teh AH, Mathavan I, et al.
    Sci Rep, 2019 Mar 20;9(1):4933.
    PMID: 30894561 DOI: 10.1038/s41598-019-40879-x
    Group 21 and 5 allergens are homologous house dust mite proteins known as mid-tier allergens. To reveal the biological function of group 21 allergens and to understand better the allergenicity of the rDer f 21 allergen, we determined the 1.5 Å crystal structure of rDer f 21 allergen from Dermatophagoides farinae. The rDer f 21 protein consists of a three helical bundle, similar to available structures of group 21 and homologous group 5 allergens. The rDer f 21 dimer forms a hydrophobic binding pocket similar to the one in the Der p 5 allergen, which indicates that both of the homologous groups could share a similar function. By performing structure-guided mutagenesis, we mutated all 38 surface-exposed polar residues of the rDer f 21 allergen and carried out immuno-dot blot assays using 24 atopic sera. Six residues, K10, K26, K42, E43, K46, and K48, which are located in the region between the N-terminus and the loop 1 of rDer f 21 were identified as the major IgE epitopes of rDer f 21. Epitope mapping of all potential IgE epitopes on the surface of the rDer f 21 crystal structure revealed heterogeneity in the sIgE recognition of the allergen epitopes in atopic individuals. The higher the allergen-sIgE level of an individual, the higher the number of epitope residues that are found in the allergen. The results illustrate the clear correlation between the number of specific major epitope residues in an allergen and the sIgE level of the atopic population.
  18. Nyanasegran PK, Nathan S, Firdaus-Raih M, Muhammad NAN, Ng CL
    J Microbiol Biotechnol, 2023 Jan 28;33(1):15-27.
    PMID: 36451302 DOI: 10.4014/jmb.2207.07032
    The incidence of melioidosis cases caused by the gram-negative pathogen Burkholderia pseudomallei (BP) is seeing an increasing trend that has spread beyond its previously known endemic regions. Biofilms produced by BP have been associated with antimicrobial therapy limitation and relapse melioidosis, thus making it urgently necessary to understand the mechanisms of biofilm formation and their role in BP biology. Microbial cells aggregate and enclose within a self-produced matrix of extracellular polymeric substances (EPSs) to form biofilm. The transition mechanism of bacterial cells from planktonic state to initiate biofilm formation, which involves the formation of surface attachment microcolonies and the maturation of the biofilm matrix, is a dynamic and complex process. Despite the emerging findings on the biofilm formation process, systemic knowledge on the molecular mechanisms of biofilm formation in BP remains fractured. This review provides insights into the signaling systems, matrix composition, and the biosynthesis regulation of EPSs (exopolysaccharide, eDNA and proteins) that facilitate the formation of biofilms in order to present an overview of our current knowledge and the questions that remain regarding BP biofilms.
  19. Noor-Hassim MB, Ng CL, Teo HM, Azmi WA, Muhamad-Zalan NB, Karim NB, et al.
    BioTechnologia (Pozn), 2023;104(2):209-216.
    PMID: 37427028 DOI: 10.5114/bta.2023.127209
    As the global human population continues to grow, the demand for food rises accordingly. Unfortunately, anthropogenic activities, climate change, and the release of gases from the utilization of synthetic fertilizers and pesticides are causing detrimental effects on sustainable food production and agroecosystems. Despite these challenges, there remain underutilized opportunities for sustainable food production. This review discusses the advantages and benefits of utilizing microbes in food production. Microbes can be used as alternative food sources to directly supply nutrients for both humans and livestock. Additionally, microbes offer higher flexibility and diversity in facilitating crop productivity and agri-food production. Microbes function as natural nitrogen fixators, mineral solubilizers, nano-mineral synthesizers, and plant growth regulator inducers, all of which promote plant growth. They are also active organisms in degrading organic materials and remediating heavy metals and pollution in soils, as well as soil-water binders. In addition, microbes that occupy the plant rhizosphere release biochemicals that have nontoxic effects on the host and the environment. These biochemicals could act as biocides in controlling agricultural pests, pathogens, and diseases. Therefore, it is important to consider the use of microbes for sustainable food production.
  20. Nieland S, Barig S, Salzmann J, Gehrau F, Zamani AI, Richter A, et al.
    Microb Biotechnol, 2021 07;14(4):1422-1432.
    PMID: 33421319 DOI: 10.1111/1751-7915.13739
    To set a benchmark in fungal growth rate, a differential analysis of prototrophic Aspergillus fumigatus AR04 with three ascomycetes applied in > 103 t year-1 scale was performed, i.e. Ashbya gosspyii (riboflavin), Aspergillus niger (citric acid) and Aspergillus oryzae (food-processing). While radial colony growth decreased 0.5-fold when A. gossypii was cultivated at 40°C instead of 28°C, A. fumigatus AR04 responded with 1.7-fold faster hyphal growth. A. niger and A. oryzae formed colonies at 40°C, but not at 43°C. Moreover, all A. fumigatus strains tested grew even at 49°C. In chemostat experiments, A. fumigatus AR04 reached steady state at a dilution rate of 0.7 h-1 at 40°C, 120% more than reported for A. gossypii at 28°C. To study mycelial growth rates under unlimited conditions, carbon dioxide increase rates were calculated from concentrations detected online in the exhaust of batch fermentations for 3 h only. All rates calculated suggest that A. fumigatus AR04 approximates Arrhenius' rule when comparing short cultivations at 30°C with those at 40°C. Linearization of the exponential phase and comparison of the slopes revealed an increase to 192% by the 10°C up-shift.
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