Displaying publications 21 - 40 of 52 in total

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  1. Cgl-SLT2 is required for appressorium formation, sporulation and pathogenicity in Colletotrichum gloeosporioides
    Yong HY, Bakar FD, Illias RM, Mahadi NM, Murad AM
    Braz J Microbiol, 2013 Dec;44(4):1241-50.
    PMID: 24688518
    The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.
  2. Fulltext The multiple roles of hypothetical gene BPSS1356 in Burkholderia pseudomallei
    Yam H, Rahim AA, Mohamad S, Mahadi NM, Manaf UA, Shu-Chien AC, et al.
    PLoS One, 2014;9(6):e99218.
    PMID: 24927285 DOI: 10.1371/journal.pone.0099218
    Burkholderia pseudomallei is an opportunistic pathogen and the causative agent of melioidosis. It is able to adapt to harsh environments and can live intracellularly in its infected hosts. In this study, identification of transcriptional factors that associate with the β' subunit (RpoC) of RNA polymerase was performed. The N-terminal region of this subunit is known to trigger promoter melting when associated with a sigma factor. A pull-down assay using histidine-tagged B. pseudomallei RpoC N-terminal region as bait showed that a hypothetical protein BPSS1356 was one of the proteins bound. This hypothetical protein is conserved in all B. pseudomallei strains and present only in the Burkholderia genus. A BPSS1356 deletion mutant was generated to investigate its biological function. The mutant strain exhibited reduced biofilm formation and a lower cell density during the stationary phase of growth in LB medium. Electron microscopic analysis revealed that the ΔBPSS1356 mutant cells had a shrunken cytoplasm indicative of cell plasmolysis and a rougher surface when compared to the wild type. An RNA microarray result showed that a total of 63 genes were transcriptionally affected by the BPSS1356 deletion with fold change values of higher than 4. The expression of a group of genes encoding membrane located transporters was concurrently down-regulated in ΔBPSS1356 mutant. Amongst the affected genes, the putative ion transportation genes were the most severely suppressed. Deprivation of BPSS1356 also down-regulated the transcriptions of genes for the arginine deiminase system, glycerol metabolism, type III secretion system cluster 2, cytochrome bd oxidase and arsenic resistance. It is therefore obvious that BPSS1356 plays a multiple regulatory roles on many genes.
  3. Fulltext Growth phase-dependent proteomes of the Malaysian isolated Lactococcus lactis dairy strain M4 using label-free qualitative shotgun proteomics analysis
    Yap TW, Rabu A, Abu Bakar FD, Rahim RA, Mahadi NM, Illias RM, et al.
    ScientificWorldJournal, 2014;2014:642891.
    PMID: 24982972 DOI: 10.1155/2014/642891
    Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MS(E)) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications.
  4. Fulltext Development of a pyrG mutant of Aspergillus oryzae strain S1 as a host for the production of heterologous proteins
    Ling SO, Storms R, Zheng Y, Rodzi MR, Mahadi NM, Illias RM, et al.
    ScientificWorldJournal, 2013;2013:634317.
    PMID: 24381522 DOI: 10.1155/2013/634317
    The ease with which auxotrophic strains and genes that complement them can be manipulated, as well as the stability of auxotrophic selection systems, are amongst the advantages of using auxotrophic markers to produce heterologous proteins. Most auxotrophic markers in Aspergillus oryzae originate from chemical or physical mutagenesis that may yield undesirable mutations along with the mutation of interest. An auxotrophic A. oryzae strain S1 was generated by deleting the orotidine-5'-monophosphate decarboxylase gene (pyrG) by targeted gene replacement. The uridine requirement of the resulting strain GR6 pyrGΔ0 was complemented by plasmids carrying a pyrG gene from either Aspergillus nidulans or A. oryzae. β -Galactosidase expression by strain GR6 pyrGΔ0 transformed with an A. niger plasmid encoding a heterologous β -galactosidase was at least 150 times more than that obtained with the untransformed strain. Targeted gene replacement is thus an efficient way of developing auxotrophic mutants in A. oryzae and the auxotrophic strain GR6 pyrGΔ0 facilitated the production of a heterologous protein in this fungus.
  5. Sequence and structural investigation of a novel psychrophilic α-amylase from Glaciozyma antarctica PI12 for cold-adaptation analysis
    Ramli AN, Azhar MA, Shamsir MS, Rabu A, Murad AM, Mahadi NM, et al.
    J Mol Model, 2013 Aug;19(8):3369-83.
    PMID: 23686283 DOI: 10.1007/s00894-013-1861-5
    A novel α-amylase was isolated successfully from Glaciozyma antarctica PI12 using DNA walking and reverse transcription-polymerase chain reaction (RT-PCR) methods. The structure of this psychrophilic α-amylase (AmyPI12) from G. antarctica PI12 has yet to be studied in detail. A 3D model of AmyPI12 was built using a homology modelling approach to search for a suitable template and to generate an optimum target-template alignment, followed by model building using MODELLER9.9. Analysis of the AmyPI12 model revealed the presence of binding sites for a conserved calcium ion (CaI), non-conserved calcium ions (CaII and CaIII) and a sodium ion (Na). Compared with its template-the thermostable α-amylase from Bacillus stearothermophilus (BSTA)-the binding of CaII, CaIII and Na ions in AmyPI12 was observed to be looser, which suggests that the low stability of AmyPI12 allows the protein to work at different temperature scales. The AmyPI12 amino acid sequence and model were compared with thermophilic α-amylases from Bacillus species that provided the highest structural similarities with AmyPI12. These comparative studies will enable identification of possible determinants of cold adaptation.
  6. 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.
  7. Structural prediction of a novel chitinase from the psychrophilic Glaciozyma antarctica PI12 and an analysis of its structural properties and function
    Ramli AN, Mahadi NM, Shamsir MS, Rabu A, Joyce-Tan KH, Murad AM, et al.
    J Comput Aided Mol Des, 2012 Aug;26(8):947-61.
    PMID: 22710891 DOI: 10.1007/s10822-012-9585-7
    The structure of psychrophilic chitinase (CHI II) from Glaciozyma antarctica PI12 has yet to be studied in detail. Due to its low sequence identity (<30 %), the structural prediction of CHI II is a challenge. A 3D model of CHI II was built by first using a threading approach to search for a suitable template and to generate an optimum target-template alignment, followed by model building using MODELLER9v7. Analysis of the catalytic insertion domain structure in CHI II revealed an increase in the number of aromatic residues and longer loops compared to mesophilic and thermophilic chitinases. A molecular dynamics simulation was used to examine the stability of the CHI II structure at 273, 288 and 300 K. Structural analysis of the substrate-binding cleft revealed a few exposed aromatic residues. Substitutions of certain amino acids in the surface and loop regions of CHI II conferred an increased flexibility to the enzyme, allowing for an adaptation to cold temperatures. A substrate binding comparison of CHI II with the mesophilic chitinase from Coccidioides immitis, 1D2K, suggested that the psychrophilic adaptation and catalytic activity at low temperatures were achieved through a reduction in the number of salt bridges, fewer hydrogen bonds and an increase in the exposure of the hydrophobic side chains to the solvent.
  8. 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.
  9. Production of an oligosaccharide-specific cellobiohydrolase from the thermophilic fungus Thielavia terrestris
    Woon JS, Mackeen MM, Sudin AH, Mahadi NM, Illias RM, Murad AM, et al.
    Biotechnol Lett, 2016 May;38(5):825-32.
    PMID: 26830095 DOI: 10.1007/s10529-016-2045-z
    To express and determine the hydrolytic activity of a cellobiohydrolase (TTCBH6B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris.
  10. Erratum to: Production of an oligosaccharide-specific cellobiohydrolase from the thermophilic fungus Thielavia terrestris
    Woon JS, Mackeen MM, Sudin AH, Mahadi NM, Illias RM, Murad AM, et al.
    Biotechnol Lett, 2016 May;38(5):833.
    PMID: 26914687
  11. 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.
  12. High level expression of Glomerella cingulata cutinase in dense cultures of Pichia pastoris grown under fed-batch conditions
    Seman WM, Bakar SA, Bukhari NA, Gaspar SM, Othman R, Nathan S, et al.
    J Biotechnol, 2014 Aug 20;184:219-28.
    PMID: 24910973 DOI: 10.1016/j.jbiotec.2014.05.034
    A Pichia pastoris transformant carrying the cutinase cDNA of Glomerella cingulata was over-expressed in a 5L bioreactor (2.0L working volume) under fed-batch conditions. Bioreactor experiments rely on varying selected parameters in repeated rounds of optimisation: here these included duration of induction, pH and temperature. Highest cell densities (320gL(-1) wet cell weight) with a cutinase production of 3800mgL(-1) and an activity of 434UmL(-1) were achieved 24h after induction with methanol in basal salt medium (at pH 5 and 28°C). Characterisation of the cutinase showed that it was stable between pH 6 and pH 11, had an optimum pH of 8.0 and retained activity for 30min at 50°C (optimum temperature 25°C).The preferred substrates of G. cingulata cutinase were the medium- to long-chain ρ-nitrophenyl esters of ρ-nitrophenylcaprylate (C8), ρ-nitrophenyllaurate (C12) and ρ-nitrophenylmyristate (C14), with the highest catalytic efficiency, kcat/Km of 7.7±0.7mM(-1)s(-1) for ρ-nitrophenylcaprylate. Microscopic analyses showed that the G. cingulata cutinase was also capable of depolymerising the high molecular weight synthetic polyester, polyethylene terephthalate.
  13. Enhanced secretory production of hemolysin-mediated cyclodextrin glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system
    Low KO, Mahadi NM, Abdul Rahim R, Rabu A, Abu Bakar FD, Abdul Murad AM, et al.
    J Biotechnol, 2010 Dec;150(4):453-9.
    PMID: 20959127 DOI: 10.1016/j.jbiotec.2010.10.001
    The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the C-terminal 61 amino acids of HlyA (HlyAs(61)). To produce an improved-secretion variant, the hly components (hlyAs, hlyB and hlyD) were engineered by directed evolution using error-prone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 × 10(4) Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs(61) relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli.
  14. Danger lurking in the "unknowns": structure-to-function studies of hypothetical protein Bleg1_2437 from Bacillus lehensis G1 alkaliphile revealed an evolutionary divergent B3 metallo-beta-lactamase
    Tan SH, Normi YM, Leow AT, Salleh AB, Murad AM, Mahadi NM, et al.
    J. Biochem., 2017 02 01;161(2):167-186.
    PMID: 28175318 DOI: 10.1093/jb/mvw058
    The effectiveness of β-lactam antibiotics as chemotherapeutic agents to treat bacterial infections is gradually threatened with the emergence of antibiotic resistance mechanism among pathogenic bacteria through the production metallo-β-lactamase (MBL). In this study, we discovered a novel hypothetical protein (HP) termed Bleg1_2437 from the genome of alkaliphilic Bacillus lehensis G1 which exhibited MBL-like properties of B3 subclass; but evolutionary divergent from other circulating B3 MBLs. Domain and sequence analysis of HP Bleg1_2437 revealed that it contains highly conserved Zn2+-binding residues such as H54, H56, D58, H59, H131 and H191, important for catalysis, similar with the subclass B3 of MBL. Built 3-D Bleg1_2437 structure exhibited an αββα sandwich layer similar to the well-conserved global topology of MBL superfamily. Other features include a ceiling and floor in the model which are important for accommodation and orientation of β-lactam antibiotics docked to the protein model showed interactions at varying degrees with residues in the binding pocket of Bleg1_2437. Hydrolysis activity towards several β-lactam antibiotics was proven through an in vitro assay using purified recombinant Bleg1_2437 protein. These findings highlight the presence of a clinically important and evolutionary divergent antibiotics-degrading enzyme within the pools of uncharacterized HPs.
  15. Fulltext Emergence of the Asian lineage dengue virus type 3 genotype III in Malaysia
    Tan KK, Zulkifle NI, Sulaiman S, Pang SP, NorAmdan N, MatRahim N, et al.
    BMC Evol. Biol., 2018 04 24;18(1):58.
    PMID: 29699483 DOI: 10.1186/s12862-018-1175-4
    BACKGROUND: Dengue virus type 3 genotype III (DENV3/III) is associated with increased number of severe infections when it emerged in the Americas and Asia. We had previously demonstrated that the DENV3/III was introduced into Malaysia in the late 2000s. We investigated the genetic diversity of DENV3/III strains recovered from Malaysia and examined their phylogenetic relationships against other DENV3/III strains isolated globally.

    RESULTS: Phylogenetic analysis revealed at least four distinct DENV3/III lineages. Two of the lineages (DENV3/III-B and DENV3/III-C) are current actively circulating whereas the DENV3/III-A and DENV3/III-D were no longer recovered since the 1980s. Selection pressure analysis revealed strong evidence of positive selection on a number of amino acid sites in PrM, E, NS1, NS2a, NS2b, NS3, NS4a, and NS5. The Malaysian DENV3/III isolates recovered in the 1980s (MY.59538/1987) clustered into DENV3/III-B, which was the lineage with cosmopolitan distribution consisting of strains actively circulating in the Americas, Africa, and Asia. The Malaysian isolates recovered after the 2000s clustered within DENV3/III-C. This DENV3/III-C lineage displayed a more restricted geographical distribution and consisted of isolates recovered from Asia, denoted as the Asian lineage. Amino acid variation sites in NS5 (NS5-553I/M, NS5-629 T, and NS5-820E) differentiated the DENV3/III-C from other DENV3 viruses. The codon 629 of NS5 was identified as a positively selected site. While the NS5-698R was identified as unique to the genome of DENV3/III-C3. Phylogeographic results suggested that the recent Malaysian DENV3/III-C was likely to have been introduced from Singapore in 2008 and became endemic. From Malaysia, the virus subsequently spread into Taiwan and Thailand in the early part of the 2010s and later reintroduced into Singapore in 2013.

    CONCLUSIONS: Distinct clustering of the Malaysian old and new DENV3/III isolates suggests that the currently circulating DENV3/III in Malaysia did not descend directly from the strains recovered during the 1980s. Phylogenetic analyses and common genetic traits in the genome of the strains and those from the neighboring countries suggest that the Malaysian DENV3/III is likely to have been introduced from the neighboring regions. Malaysia, however, serves as one of the sources of the recent regional spread of DENV3/III-C3 within the Asia region.

  16. Functional and structural analyses of an expansin-like protein from the antarctic yeast Glaciozyma antarctica PI12 reveal strategies of nutrient scavenging in the sea ice environment
    Mohamad Nor N, Hashim NHF, Quay DHX, Mahadi NM, Illias RM, Abu Bakar FD, et al.
    Int J Biol Macromol, 2020 Feb 01;144:231-241.
    PMID: 31843615 DOI: 10.1016/j.ijbiomac.2019.12.099
    Genome data mining of the Antarctic yeast, Glaciozyma antarctica PI12 revealed an expansin-like protein encoding sequence (GaEXLX1). The GaEXLX1 protein is 24.8 kDa with a high alkaline pI of 9.81. Homology modeling of GaEXLX1 showed complete D1 and D2 domains of a conventional expansin. The protein exhibited 36% sequence similarity to Clavibacter michiganensis EXLX1 (PDB: 4JCW). Subsequently, a recombinant GaEXLX1 protein was produced using Escherichia coli expression system. Incubation with Avicel, filter paper and cotton fiber showed that the protein can disrupt the surface of crystalline and pure cellulose, suggesting a cell wall modification activity usually exhibited by expansin-like proteins. Binding assays displayed that GaEXLX1 can bind to polymeric substrates, including those postulated to be present in the sea ice ecosystem such as crab chitin and moss lichenan. GaEXLX1 may assist in the recognition and loosening of these substrates in the sea ice prior to hydrolysis by other extracellular enzymes. Similar loosening mechanism to classical expansin-like protein has been postulated for this psychrophilic protein based on several conserved residues of GaEXLX1 involved in binding interaction identified by docking analyses.
  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.
  18. Fulltext Cellobiohydrolase B of Aspergillus niger over-expressed in Pichia pastoris stimulates hydrolysis of oil palm empty fruit bunches
    Woon JS, Mackeen MM, Illias RM, Mahadi NM, Broughton WJ, Murad AMA, et al.
    PeerJ, 2017;5:e3909.
    PMID: 29038760 DOI: 10.7717/peerj.3909
    BACKGROUND: Aspergillus niger, along with many other lignocellulolytic fungi, has been widely used as a commercial workhorse for cellulase production. A fungal cellulase system generally includes three major classes of enzymes i.e., β-glucosidases, endoglucanases and cellobiohydrolases. Cellobiohydrolases (CBH) are vital to the degradation of crystalline cellulose present in lignocellulosic biomass. However, A. niger naturally secretes low levels of CBH. Hence, recombinant production of A. niger CBH is desirable to increase CBH production yield and also to allow biochemical characterisation of the recombinant CBH from A. niger.

    METHODS: In this study, the gene encoding a cellobiohydrolase B (cbhB) from A. niger ATCC 10574 was cloned and expressed in the methylotrophic yeast Pichia pastoris X-33. The recombinant CBHB was purified and characterised to study its biochemical and kinetic characteristics. To evaluate the potential of CBHB in assisting biomass conversion, CBHB was supplemented into a commercial cellulase preparation (Cellic(®) CTec2) and was used to hydrolyse oil palm empty fruit bunch (OPEFB), one of the most abundant lignocellulosic waste from the palm oil industry. To attain maximum saccharification, enzyme loadings were optimised by response surface methodology and the optimum point was validated experimentally. Hydrolysed OPEFB samples were analysed using attenuated total reflectance FTIR spectroscopy (ATR-FTIR) to screen for any compositional changes upon enzymatic treatment.

    RESULTS: Recombinant CBHB was over-expressed as a hyperglycosylated protein attached to N-glycans. CBHB was enzymatically active towards soluble substrates such as 4-methylumbelliferyl-β-D-cellobioside (MUC), p-nitrophenyl-cellobioside (pNPC) and p-nitrophenyl-cellobiotrioside (pNPG3) but was not active towards crystalline substrates like Avicel(®) and Sigmacell cellulose. Characterisation of purified CBHB using MUC as the model substrate revealed that optimum catalysis occurred at 50 °C and pH 4 but the enzyme was stable between pH 3 to 10 and 30 to 80 °C. Although CBHB on its own was unable to digest crystalline substrates, supplementation of CBHB (0.37%) with Cellic(®) CTec2 (30%) increased saccharification of OPEFB by 27%. Compositional analyses of the treated OPEFB samples revealed that CBHB supplementation reduced peak intensities of both crystalline cellulose Iα and Iβ in the treated OPEFB samples.

    DISCUSSION: Since CBHB alone was inactive against crystalline cellulose, these data suggested that it might work synergistically with other components of Cellic(®) CTec2. CBHB supplements were desirable as they further increased hydrolysis of OPEFB when the performance of Cellic(®) CTec2 was theoretically capped at an enzyme loading of 34% in this study. Hence, A. niger CBHB was identified as a potential supplementary enzyme for the enzymatic hydrolysis of OPEFB.

  19. Fulltext Bioinformatics in Malaysia: hope, initiative, effort, reality, and challenges
    Zeti AM, Shamsir MS, Tajul-Arifin K, Merican AF, Mohamed R, Nathan S, et al.
    PLoS Comput Biol, 2009 Aug;5(8):e1000457.
    PMID: 19714208 DOI: 10.1371/journal.pcbi.1000457
  20. Fulltext Thermotolerance and molecular chaperone function of an SGT1-like protein from the psychrophilic yeast, Glaciozyma antarctica
    Yusof NA, Hashim NH, Beddoe T, Mahadi NM, Illias RM, Bakar FD, et al.
    Cell Stress Chaperones, 2016 Jul;21(4):707-15.
    PMID: 27154490 DOI: 10.1007/s12192-016-0696-2
    The ability of eukaryotes to adapt to an extreme range of temperatures is critically important for survival. Although adaptation to extreme high temperatures is well understood, reflecting the action of molecular chaperones, it is unclear whether these molecules play a role in survival at extremely low temperatures. The recent genome sequencing of the yeast Glaciozyma antarctica, isolated from Antarctic sea ice near Casey Station, provides an opportunity to investigate the role of molecular chaperones in adaptation to cold temperatures. We isolated a G. antarctica homologue of small heat shock protein 20 (HSP20), GaSGT1, and observed that the GaSGT1 mRNA expression in G. antarctica was markedly increased following culture exposure at low temperatures. Additionally, we demonstrated that GaSGT1 overexpression in Escherichia coli protected these bacteria from exposure to both high and low temperatures, which are lethal for growth. The recombinant GaSGT1 retained up to 60 % of its native luciferase activity after exposure to luciferase-denaturing temperatures. These results suggest that GaSGT1 promotes cell thermotolerance and employs molecular chaperone-like activity toward temperature assaults.
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