Displaying publications 1 - 20 of 29 in total

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  1. Cross-linked enzyme aggregates of polyethylene terephthalate hydrolyse (PETase) from Ideonella sakaiensis for the improvement of plastic degradation
    Lee YL, Jaafar NR, Ling JG, Huyop F, Abu Bakar FD, Rahman RA, et al.
    Int J Biol Macromol, 2024 Apr;263(Pt 1):130284.
    PMID: 38382786 DOI: 10.1016/j.ijbiomac.2024.130284
    Polyethylene terephthalate (PET) is one of the most produced plastics globally and its accumulation in the environment causes harm to the ecosystem. Polyethylene terephthalate hydrolyse (PETase) is an enzyme that can degrade PET into its monomers. However, free PETase lacks operational stabilities and is not reusable. In this study, development of cross-linked enzyme aggregate (CLEA) of PETase using amylopectin (Amy) as cross-linker was introduced to solve the limitations of free PETase. PETase-Amy-CLEA exhibited activity recovery of 81.9 % at its best immobilization condition. Furthermore, PETase-Amy-CLEA exhibited 1.37-, 2.75-, 2.28- and 1.36-fold higher half-lives than free PETase at 50 °C, 45 °C, 40 °C and 35 °C respectively. Moreover, PETase-Amy-CLEA showed broader pH stability from pH 5 to 10 and could be reused up to 5 cycles. PETase-Amy-CLEA retained >70 % of initial activity after 40 days of storage at 4 °C. In addition, lower Km of PETase-Amy-CLEA indicated better substrate affinity than free enzyme. PETase-Amy-CLEA corroded PET better and products yielded was 66.7 % higher than free PETase after 32 h of treatment. Hence, the enhanced operational stabilities, storage stability, reusability and plastic degradation ability are believed to make PETase-Amy-CLEA a promising biocatalyst in plastic degradation.
  2. Biochemical and in silico structural characterization of a cold-active arginase from the psychrophilic yeast, Glaciozyma antarctica PI12
    Yusof NY, Quay DHX, Kamaruddin S, Jonet MA, Md Illias R, Mahadi NM, et al.
    Extremophiles, 2024 Feb 01;28(1):15.
    PMID: 38300354 DOI: 10.1007/s00792-024-01333-7
    Glaciozyma antarctica PI12 is a psychrophilic yeast isolated from Antarctica. In this work, we describe the heterologous production, biochemical properties and in silico structure analysis of an arginase from this yeast (GaArg). GaArg is a metalloenzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea. The cDNA of GaArg was reversed transcribed, cloned, expressed and purified as a recombinant protein in Escherichia coli. The purified protein was active against L-arginine as its substrate in a reaction at 20 °C, pH 9. At 10-35 °C and pH 7-9, the catalytic activity of the protein was still present around 50%. Mn2+, Ni2+, Co2+ and K+ were able to enhance the enzyme activity more than two-fold, while GaArg is most sensitive to SDS, EDTA and DTT. The predicted structure model of GaArg showed a very similar overall fold with other known arginases. GaArg possesses predominantly smaller and uncharged amino acids, fewer salt bridges, hydrogen bonds and hydrophobic interactions compared to the other counterparts. GaArg is the first reported arginase that is cold-active, facilitated by unique structural characteristics for its adaptation of catalytic functions at low-temperature environments. The structure and function of cold-active GaArg provide insights into the potentiality of new applications in various biotechnology and pharmaceutical industries.
  3. Fulltext Structure of the Reductase Domain of a Fungal Carboxylic Acid Reductase and Its Substrate Scope in Thioester and Aldehyde Reduction
    Daniel B, Hashem C, Leithold M, Sagmeister T, Tripp A, Stolterfoht-Stock H, et al.
    ACS Catal, 2022 Dec 16;12(24):15668-15674.
    PMID: 37180375 DOI: 10.1021/acscatal.2c04426
    The synthesis of aldehydes from carboxylic acids has long been a challenge in chemistry. In contrast to the harsh chemically driven reduction, enzymes such as carboxylic acid reductases (CARs) are considered appealing biocatalysts for aldehyde production. Although structures of single- and didomains of microbial CARs have been reported, to date no full-length protein structure has been elucidated. In this study, we aimed to obtain structural and functional information regarding the reductase (R) domain of a CAR from the fungus Neurospora crassa (Nc). The NcCAR R-domain revealed activity for N-acetylcysteamine thioester (S-(2-acetamidoethyl) benzothioate), which mimics the phosphopantetheinylacyl-intermediate and can be anticipated as the minimal substrate for thioester reduction by CARs. The determined crystal structure of the NcCAR R-domain reveals a tunnel that putatively harbors the phosphopantetheinylacyl-intermediate, which is in good agreement with docking experiments performed with the minimal substrate. In vitro studies were performed with this highly purified R-domain and NADPH, demonstrating carbonyl reduction activity. The R-domain was able to accept not only a simple aromatic ketone but also benzaldehyde and octanal, which are typically considered to be the final product of carboxylic acid reduction by CAR. Also, the full-length NcCAR reduced aldehydes to primary alcohols. In conclusion, aldehyde overreduction can no longer be attributed exclusively to the host background.
  4. Fulltext Metagenomic datasets of air samples collected during episodes of severe smoke-haze in Malaysia
    James GL, Latif MT, Isa MNM, Bakar MFA, Yusuf NYM, Broughton W, et al.
    Data Brief, 2021 Jun;36:107124.
    PMID: 34095374 DOI: 10.1016/j.dib.2021.107124
    Transboundary emissions of smoke-haze from land and forest fires have recurred annually during the dry period (June to October, over the past few decades) in South East Asia. Hazardous air quality has been recorded in Malaysia during these episodes. Agricultural practices such as slash-and-burn of biomass and peat fires particularly in Sumatera and Kalimantan, Indonesia, have been implicated as the major causes of the haze. Past findings have shown that a diversity of microbes can thrive in air including in smoke-haze polluted air. In this study, metagenomic data were generated to reveal the diversity of microorganisms in air during days with and without haze. Air samples were collected during non-haze (2013A01) and two haze (2013A04 and 2013A05) periods in the month of June 2013. DNA was extracted from the samples, subjected to Multiple Displacement Amplification and whole genome sequencing (Next Generation Sequencing) using the HiSeq 2000 Platform. Extensive bio-informatic analyses of the raw sequence data then followed. Raw reads from these six air samples were deposited in the NCBI SRA databases under Bioproject PRJNA662021 with accession numbers SRX9087478, SRX9087479 and SRX9087480.
  5. Structural and functional characterisation of a cold-active yet heat-tolerant dehydroquinase from Glaciozyma antarctica PI12
    Jaafar NR, Mahadi NM, Mackeen MM, Illias RM, Murad AMA, Abu Bakar FD
    J Biotechnol, 2021 Mar 10;329:118-127.
    PMID: 33539893 DOI: 10.1016/j.jbiotec.2021.01.019
    Dehydroquinase or 3-dehydroquinate dehydratase (DHQD) reversibly cleaves 3-dehydroquinate to form 3-dehydroshikimate. Here, we describe the functional and structural features of a cold active type II 3-dehydroquinate dehydratase from the psychrophilic yeast, Glaciozyma antarctica PI12 (GaDHQD). Functional studies showed that the enzyme was active at low temperatures (10-30 °C), but displayed maximal activity at 40 °C. Yet the enzyme was stable over a wide range of temperatures (10-70 °C) and between pH 6.0-10.0 with an optimum pH of 8.0. Interestingly, the enzyme was highly thermo-tolerant, denaturing only at approximately 84 °C. Three-dimensional structure analyses showed that the G. antarctica dehydroquinase (GaDHQD) possesses psychrophilic features in comparison with its mesophilic and thermophilic counterparts such as higher numbers of non-polar residues on the surface, lower numbers of arginine and higher numbers of glycine-residues with lower numbers of hydrophobic interactions. On the other hand, GaDHQD shares some traits (i.e. total number of hydrogen bonds, number of proline residues and overall folding) with its mesophilic and thermophilic counterparts. Combined, these features contribute synergistically towards the enzyme's ability to function at both low and high temperatures.
  6. Functional characterisation and product specificity of Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1
    Jaafar NR, Khoiri NM, Ismail NF, Mahmood NAN, Abdul Murad AM, Abu Bakar FD, et al.
    Enzyme Microb Technol, 2020 Oct;140:109625.
    PMID: 32912685 DOI: 10.1016/j.enzmictec.2020.109625
    Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1 (Blg32) composed of 284 amino acids with a predicted molecular mass of 31.6 kDa is expressed in Escherichia coli and purified to homogeneity. Herein, Blg32 characteristics, substrates and product specificity as well as structural traits that might be involved in the production of sugar molecules are analysed. This enzyme functions optimally at the temperature of 70 °C, pH value of 8.0 with its catalytic activity strongly enhanced by Mn2+. Remarkably, the purified enzyme is highly stable in high temperature and alkaline conditions. It exhibits the highest activity on laminarin (376.73 U/mg) followed by curdlan and yeast β-glucan. Blg32 activity increased by 62% towards soluble substrate (laminarin) compared to insoluble substrate (curdlan). Hydrolytic products of laminarin were oligosaccharides with degree of polymerisation (DP) of 1 to 5 with the main product being laminaritriose (DP3). This suggests that the active site of Blg32 could recognise up to five glucose units. High concentration of Blg32 mainly produces glucose whilst low concentration of Blg32 yields oligosaccharides with different DP (predominantly DP3). A theoretical structural model of Blg32 was constructed and structural analysis revealed that Trp156 is involved in multiple hydrophobic stacking interactions. The amino acid was predicted to participate in substrate recognition and binding. It was also exhibited that catalytic groove of Blg32 has a narrow angle, thus limiting the substrate binding reaction. All these properties and knowledge of the subsites are suggested to be related to the possible mode of action of how Blg32 produces glucooligosaccharides.
  7. Novel cross-linked enzyme aggregates of levanase from Bacillus lehensis G1 for short-chain fructooligosaccharides synthesis: Developmental, physicochemical, kinetic and thermodynamic properties
    Abd Rahman NH, Jaafar NR, Abdul Murad AM, Abu Bakar FD, Shamsul Annuar NA, Md Illias R
    Int J Biol Macromol, 2020 Sep 15;159:577-589.
    PMID: 32380107 DOI: 10.1016/j.ijbiomac.2020.04.262
    Short-chain fructooligosaccharides (scFOSs) can be produced from the levan hydrolysis using levanase. Levanase from Bacillus lehensis G1 (rlevblg1) is an enzyme that specifically converts levan to scFOSs. However, the use of free levanase presents a lack of stability and reusability, thus hindering the synthesis of scFOSs for continuous reactions. Here, CLEAs for rlevblg1 were prepared and characterized. Cross-linked levanase aggregates using glutaraldehyde (CLLAs-ga) and bovine albumin serum (CLLAs-ga-bsa) showed the best activity recovery of 92.8% and 121.2%, respectively. The optimum temperature of CLLAs-ga and CLLAs-ga-bsa was increased to 35 °C and 40 °C, respectively, from its free rlevblg1 (30 °C). At high temperature (50 °C), the half-life of CLLAs-ga-bsa was higher than that of free rlevblg1 and CLLAs-ga. Both CLLAs exhibited higher stability at pH 9 and pH 10. Hyperactivation of CLLAs-ga-bsa was achieved with an effectiveness factor of more than 1 and with improved catalytic efficiency. After 3 h reaction, CLLAs-ga-bsa produced the highest total scFOSs yield of 35.4% and total sugar of 60.4% per gram levan. Finally, the reusability of CLLAs for 8 cycles with more than 50% activity retained makes them as a potential synthetic catalyst to be explored for scFOSs synthesis.
  8. In-Silico Characterization of Glycosyl Hydrolase Family 1 β-Glucosidase from Trichoderma asperellum UPM1
    Mohamad Sobri MF, Abd-Aziz S, Abu Bakar FD, Ramli N
    Int J Mol Sci, 2020 Jun 04;21(11).
    PMID: 32512945 DOI: 10.3390/ijms21114035
    β-glucosidases (Bgl) are widely utilized for releasing non-reducing terminal glucosyl residues. Nevertheless, feedback inhibition by glucose end product has limited its application. A noticeable exception has been found for β-glucosidases of the glycoside hydrolase (GH) family 1, which exhibit tolerance and even stimulation by glucose. In this study, using local isolate Trichoderma asperellum UPM1, the gene encoding β-glucosidase from GH family 1, hereafter designated as TaBgl2, was isolated and characterized via in-silico analyses. A comparison of enzyme activity was subsequently made by heterologous expression in Escherichia coli BL21(DE3). The presence of N-terminal signature, cis-peptide bonds, conserved active site motifs, non-proline cis peptide bonds, substrate binding, and a lone conserved stabilizing tryptophan (W) residue confirms the identity of Trichoderma sp. GH family 1 β-glucosidase isolated. Glucose tolerance was suggested by the presence of 14 of 22 known consensus residues, along with corresponding residues L167 and P172, crucial in the retention of the active site's narrow cavity. Retention of 40% of relative hydrolytic activity on ρ-nitrophenyl-β-D-glucopyranoside (ρNPG) in a concentration of 0.2 M glucose was comparable to that of GH family 1 β-glucosidase (Cel1A) from Trichoderma reesei. This research thus underlines the potential in the prediction of enzymatic function, and of industrial importance, glucose tolerance of family 1 β-glucosidases following relevant in-silico analyses.
  9. 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.
  10. A functionally-distinct carboxylic acid reductase PcCAR4 unearthed from a repertoire of type IV CARs in the white-rot fungus Pycnoporus cinnabarinus
    Ling JG, Mansor MH, Abdul Murad AM, Mohd Khalid R, Quay DHX, Winkler M, et al.
    J Biotechnol, 2020 Jan 10;307:55-62.
    PMID: 31545972 DOI: 10.1016/j.jbiotec.2019.09.008
    Carboxylic acid reductases (CARs) are attracting burgeoning attention as biocatalysts for organic synthesis of aldehydes and their follow-up products from economic carboxylic acid precursors. The CAR enzyme class as a whole, however, is still poorly understood. To date, relatively few CAR sequences have been reported, especially from fungal sources. Here, we sought to increase the diversity of the CAR enzyme class. Six new CAR sequences from the white-rot fungus Pycnoporus cinnabarinus were identified from genome-wide mining. Genome and gene clustering analysis suggests that these PcCAR enzymes play different natural roles in Basidiomycete systems, compared to their type II Ascomycete counterparts. The cDNA sequences of all six Pccar genes were deduced and analysis of their corresponding amino acid sequence showed that they encode for proteins of similar properties that possess a conserved modular functional tri-domain arrangement. Phylogenetic analyses showed that all PcCAR enzymes cluster together with the other type IV CARs. One candidate, PcCAR4, was cloned and over-expressed recombinantly in Escherichia coli. Subsequent biotransformation-based screening with a panel of structurally-diverse carboxylic acid substrates suggest that PcCAR4 possessed a more pronounced substrate specificity compared to previously reported CARs, preferring to reduce sterically-rigid carboxylic acids such as benzoic acid. These findings thus present a new functionally-distinct member of the CAR enzyme class.
  11. Fulltext Structural and functional insights into TRiC chaperonin from a psychrophilic yeast, Glaciozyma antarctica
    Yusof NA, Kamaruddin S, Abu Bakar FD, Mahadi NM, Abdul Murad AM
    Cell Stress Chaperones, 2019 Mar;24(2):351-368.
    PMID: 30649671 DOI: 10.1007/s12192-019-00969-1
    Studies on TCP1-1 ring complex (TRiC) chaperonin have shown its indispensable role in folding cytosolic proteins in eukaryotes. In a psychrophilic organism, extreme cold temperature creates a low-energy environment that potentially causes protein denaturation with loss of activity. We hypothesized that TRiC may undergo evolution in terms of its structural molecular adaptation in order to facilitate protein folding in low-energy environment. To test this hypothesis, we isolated G. antarctica TRiC (GaTRiC) and found that the expression of GaTRiC mRNA in G. antarctica was consistently expressed at all temperatures indicating their importance in cell regulation. Moreover, we showed GaTRiC has the ability of a chaperonin whereby denatured luciferase can be folded to the functional stage in its presence. Structurally, three categories of residue substitutions were found in α, β, and δ subunits: (i) bulky/polar side chains to alanine or valine, (ii) charged residues to alanine, and (iii) isoleucine to valine that would be expected to increase intramolecular flexibility within the GaTRiC. The residue substitutions observed in the built structures possibly affect the hydrophobic, hydrogen bonds, and ionic and aromatic interactions which lead to an increase in structural flexibility. Our structural and functional analysis explains some possible structural features which may contribute to cold adaptation of the psychrophilic TRiC folding chamber.
  12. Functional characterisation of cellobiohydrolase I (Cbh1) from Trichoderma virens UKM1 expressed in Aspergillus niger
    Wahab AFFA, Abdul Karim NA, Ling JG, Hasan NS, Yong HY, Bharudin I, et al.
    Protein Expr Purif, 2019 02;154:52-61.
    PMID: 30261309 DOI: 10.1016/j.pep.2018.09.014
    Cellobiohydrolases catalyze the processive hydrolysis of cellulose into cellobiose. Here, a Trichoderma virens cDNA predicted to encode for cellobiohydrolase (cbhI) was cloned and expressed heterologously in Aspergillus niger. The cbhI gene has an open reading frame of 1518 bp, encoding for a putative protein of 505 amino acid residues with a calculated molecular mass of approximately 54 kDa. The predicted CbhI amino acid sequence has a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region and showed high sequence homology with glycoside hydrolase family 7 proteins. The partially purified enzyme has an optimum pH of 4.0 with stability ranging from pH 3.0 to 6.0 and an optimum temperature of 60 °C. The partially purified CbhI has a specific activity of 4.195 Umg-1 and a low Km value of 1.88 mM when p-nitrophenyl-β-D-cellobioside (pNPC) is used as the substrate. The catalytic efficiency (kcat/Km) was 5.68 × 10-4 mM-1s-1, which is comparable to the CbhI enzymes from Trichoderma viridae and Phanaerochaete chrysosporium. CbhI also showed activity towards complex substrates such as Avicel (0.011 Umg-1), which could be useful in complex biomass degradation. Interestingly, CbhI also exhibited a relatively high inhibition constant (Ki) for cellobiose with a value of 8.65 mM, making this enzyme more resistant to end-product inhibition compared to other fungal cellobiohydrolases.
  13. Fulltext De novo transcriptome resources of the lichens, Dirinaria sp. UKM-J1 and UKM-K1 collected from Jerantut and Klang, Malaysia
    Bharudin I, Abdul Rahim SN, Abu Bakar MF, Ibrahim SN, Kamaruddin S, Latif MT, et al.
    Data Brief, 2018 Aug;19:2416-2419.
    PMID: 30229114 DOI: 10.1016/j.dib.2018.07.020
    Lichen is a symbiotic organism that exists as a single composite body consisting of a mycobiont (fungus) and a photobiont (algae or a cyanobacterium). Many lichen species are considered as extremophiles due to their tolerance to radiation, desiccation, temperature and pollution. However, not all lichen species are tolerant to harsh environmental conditions as several species are sensitive for example to nitrogen, sulphur, acidity, heavy metals, halogens (e.g. fluoride) and ozone. Thus, to better understand why some lichens can withstand exposure to pollutants as opposed to those that are susceptible, we focused on the lichen species of Dirinaria known for their wide distribution in the tropics, subtropics and pantropical, and moderate tolerance to air pollution. Their moderate tolerance to air pollution affords them to thrive in good air quality environments as well as polluted air environments. Lichen samples of Dirinaria sp., UKM-J1 and UKM-K1, were respectively collected from two areas with different levels of air quality based on Air Pollutant Index or API (with index pollutant criteria of PM10, carbon monoxide, ozone, nitrogen dioxide and sulfur dioxide) in the outskirt of Jerantut (UKM-J1), a rural area in the middle of Peninsular Malaysia and the township of Klang (UKM-K1), in a busy area of the Klang Valley, Malaysia. API was monitored throughout 2012-2013 whereby the sample collection site in Klang showed markedly higher concentrations of pollutants in all the index pollutant criteria as compared to that of Jerantut. We performed transcriptome sequencing using Illumina RNA-seq technology and de novo assembly of the transcripts from the lichen samples. Raw reads from both libraries were deposited in the NCBI database with the accession number SRP138994.
  14. Biochemical and structural characterization of a novel cold-active esterase-like protein from the psychrophilic yeast Glaciozyma antarctica
    Hashim NHF, Mahadi NM, Illias RM, Feroz SR, Abu Bakar FD, Murad AMA
    Extremophiles, 2018 Jul;22(4):607-616.
    PMID: 29556723 DOI: 10.1007/s00792-018-1021-z
    Dienelactone hydrolase, an α/β hydrolase enzyme, catalyzes the hydrolysis of dienelactone to maleylacetate, an intermediate for the Krebs cycle. Genome sequencing of the psychrophilic yeast, Glaciozyma antarctica predicted a putative open reading frame (ORF) for dienelactone hydrolase (GaDlh) with 52% sequence similarity to that from Coniophora puteana. Phylogenetic tree analysis showed that GaDlh is closely related to other reported dienelactone hydrolases, and distantly related to other α/β hydrolases. Structural prediction using MODELLER 9.14 showed that GaDlh has the same α/β hydrolase fold as other dienelactone hydrolases and esterase/lipase enzymes, with a catalytic triad consisting of Cys-His-Asp and a G-x-C-x-G-G motif. Based on the predicted structure, GaDlh exhibits several characteristics of cold-adapted proteins such as glycine clustering in the binding pocket, reduced protein core hydrophobicity, and the absence of proline residues in loops. The putative ORF was amplified, cloned, and overexpressed in an Escherichia coli expression system. The recombinant protein was overexpressed as soluble proteins and was purified via Ni-NTA affinity chromatography. Biochemical characterization of GaDlh revealed that it has an optimal temperature at 10 °C and that it retained almost 90% of its residual activity when incubated for 90 min at 10 °C. The optimal pH was at pH 8.0 and it was stable between pH 5-9 when incubated for 60 min (more than 50% residual activity). Its Km value was 256 μM and its catalytic efficiency was 81.7 s-1. To our knowledge, this is the first report describing a novel cold-active dienelactone hydrolase-like protein.
  15. Unravelling the adaptation strategies employed by Glaciozyma antarctica PI12 on Antarctic sea ice
    Bharudin I, Abu Bakar MF, Hashim NHF, Mat Isa MN, Alias H, Firdaus-Raih M, et al.
    Mar Environ Res, 2018 Jun;137:169-176.
    PMID: 29598997 DOI: 10.1016/j.marenvres.2018.03.007
    Glaciozyma antarctica PI12, is a psychrophilic yeast isolated from Antarctic sea. In this work, Expressed Sequence Tags (EST) from cells exposed to three different temperatures; 15 °C, 0 °C and -12 °C were generated to identify genes associated with cold adaptation. A total of 5376 clones from each library were randomly picked and sequenced. Comparative analyses from the resulting ESTs in each condition identified several groups of genes required for cold adaptation. Additionally, 319 unique transcripts that encoded uncharacterised functions were identified in the -12 °C library and are currently unique to G. antarctica. Gene expression analysis using RT-qPCR revealed two of the unknown genes to be up-regulated at -12 °C compared to 0 °C and 15 °C. These findings further contribute to the collective knowledge into G. antarctica cold adaptation and as a resource for understanding the ecological and physiological tolerance of psychrophilic microbes in general.
  16. Structure Prediction of a Novel Exo-β-1,3-Glucanase: Insights into the Cold Adaptation of Psychrophilic Yeast Glaciozyma antarctica PI12
    Mohammadi S, Parvizpour S, Razmara J, Abu Bakar FD, Illias RM, Mahadi NM, et al.
    Interdiscip Sci, 2018 Mar;10(1):157-168.
    PMID: 27475956 DOI: 10.1007/s12539-016-0180-9
    We report a detailed structural analysis of the psychrophilic exo-β-1,3-glucanase (GaExg55) from Glaciozyma antarctica PI12. This study elucidates the structural basis of exo-1,3-β-1,3-glucanase from this psychrophilic yeast. The structural prediction of GaExg55 remains a challenge because of its low sequence identity (37 %). A 3D model was constructed for GaExg55. Threading approach was employed to determine a suitable template and generate optimal target-template alignment for establishing the model using MODELLER9v15. The primary sequence analysis of GaExg55 with other mesophilic exo-1,3-β-glucanases indicated that an increased flexibility conferred to the enzyme by a set of amino acids substitutions in the surface and loop regions of GaExg55, thereby facilitating its structure to cold adaptation. A comparison of GaExg55 with other mesophilic exo-β-1,3-glucanases proposed that the catalytic activity and structural flexibility at cold environment were attained through a reduced amount of hydrogen bonds and salt bridges, as well as an increased exposure of the hydrophobic side chains to the solvent. A molecular dynamics simulation was also performed using GROMACS software to evaluate the stability of the GaExg55 structure at varying low temperatures. The simulation result confirmed the above findings for cold adaptation of the psychrophilic GaExg55. Furthermore, the structural analysis of GaExg55 with large catalytic cleft and wide active site pocket confirmed the high activity of GaExg55 to hydrolyze polysaccharide substrates.
  17. Fulltext The Glaciozyma antarctica genome reveals an array of systems that provide sustained responses towards temperature variations in a persistently cold habitat
    Firdaus-Raih M, Hashim NHF, Bharudin I, Abu Bakar MF, Huang KK, Alias H, et al.
    PLoS One, 2018;13(1):e0189947.
    PMID: 29385175 DOI: 10.1371/journal.pone.0189947
    Extremely low temperatures present various challenges to life that include ice formation and effects on metabolic capacity. Psyhcrophilic microorganisms typically have an array of mechanisms to enable survival in cold temperatures. In this study, we sequenced and analysed the genome of a psychrophilic yeast isolated in the Antarctic region, Glaciozyma antarctica. The genome annotation identified 7857 protein coding sequences. From the genome sequence analysis we were able to identify genes that encoded for proteins known to be associated with cold survival, in addition to annotating genes that are unique to G. antarctica. For genes that are known to be involved in cold adaptation such as anti-freeze proteins (AFPs), our gene expression analysis revealed that they were differentially transcribed over time and in response to different temperatures. This indicated the presence of an array of adaptation systems that can respond to a changing but persistent cold environment. We were also able to validate the activity of all the AFPs annotated where the recombinant AFPs demonstrated anti-freeze capacity. This work is an important foundation for further collective exploration into psychrophilic microbiology where among other potential, the genes unique to this species may represent a pool of novel mechanisms for cold survival.
  18. Cloning, Production and Characterization of a Glycoside Hydrolase Family 7 Enzyme from the Gut Microbiota of the Termite Coptotermes curvignathus
    Woon JS, King PJH, Mackeen MM, Mahadi NM, Wan Seman WMK, Broughton WJ, et al.
    Mol Biotechnol, 2017 Jul;59(7):271-283.
    PMID: 28573450 DOI: 10.1007/s12033-017-0015-x
    Coptotermes curvignathus is a termite that, owing to its ability to digest living trees, serves as a gold mine for robust industrial enzymes. This unique characteristic reflects the presence of very efficient hydrolytic enzyme systems including cellulases. Transcriptomic analyses of the gut of C. curvignathus revealed that carbohydrate-active enzymes (CAZy) were encoded by 3254 transcripts and that included 69 transcripts encoding glycoside hydrolase family 7 (GHF7) enzymes. Since GHF7 enzymes are useful to the biomass conversion industry, a gene encoding for a GHF7 enzyme (Gh1254) was synthesized, sub-cloned and expressed in the methylotrophic yeast Pichia pastoris. Expressed GH1254 had an apparent molecular mass of 42 kDa, but purification was hampered by its low expression levels in shaken flasks. To obtain more of the enzyme, GH1254 was produced in a bioreactor that resulted in a fourfold increase in crude enzyme levels. The purified enzyme was active towards soluble synthetic substrates such as 4-methylumbelliferyl-β-D-cellobioside, 4-nitrophenyl-β-D-cellobioside and 4-nitrophenyl-β-D-lactoside but was non-hydrolytic towards Avicel or carboxymethyl cellulose. GH1254 catalyzed optimally at 35 °C and maintained 70% of its activity at 25 °C. This enzyme is thus potentially useful in food industries employing low-temperature conditions.
  19. 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.

  20. 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.
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