Displaying publications 1 - 20 of 38 in total

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  1. A facile enzymatic synthesis of geranyl propionate by physically adsorbed Candida rugosa lipase onto multi-walled carbon nanotubes
    Mohamad NR, Buang NA, Mahat NA, Lok YY, Huyop F, Aboul-Enein HY, et al.
    Enzyme Microb Technol, 2015 May;72:49-55.
    PMID: 25837507 DOI: 10.1016/j.enzmictec.2015.02.007
    In view of several disadvantages as well as adverse effects associated with the use of chemical processes for producing esters, alternative techniques such as the utilization of enzymes on multi-walled carbon nanotubes (MWCNTs), have been suggested. In this study, the oxidative MWCNTs prepared using a mixture of HNO3 and H2SO4 (1:3 v/v) were used as a supportive material for the immobilization of Candida rugosa lipase (CRL) through physical adsorption process. The resulting CRL-MWCNTs biocatalysts were utilized for synthesizing geranyl propionate, an important ester for flavoring agent as well as in fragrances. Enzymatic esterification of geraniol with propionic acid was carried out using heptane as a solvent and the efficiency of CRL-MWCNTs as a biocatalyst was compared with the free CRL, considering the incubation time, temperature, molar ratio of acid:alcohol, presence of desiccant as well as its reusability. It was found that the CRL-MWCNTs resulted in a 2-fold improvement in the percentage of conversion of geranyl propionate when compared with the free CRL, demonstrating the highest yield of geranyl propionate at 6h at 55°C, molar ratio acid: alcohol of 1:5 and with the presence of 1.0g desiccant. It was evident that the CRL-MWCNTs biocatalyst could be reused for up to 6 times before a 50% reduction in catalytic efficiency was observed. Hence, it appears that the facile physical adsorption of CRL onto F-MWCNTs has improved the activity and stability of CRL as well as served as an alternative method for the synthesis of geranyl propionate.
  2. An Overview of Crosslinked Enzyme Aggregates: Concept of Development and Trends of Applications
    Bouguerra OM, Wahab RA, Huyop F, Al-Fakih AM, Mahmood WMAW, Mahat NA, et al.
    Appl Biochem Biotechnol, 2024 Jan 05.
    PMID: 38180645 DOI: 10.1007/s12010-023-04809-y
    Enzymes are commonly used as biocatalysts for various biological and chemical processes in industrial applications. However, their limited operational stability, catalytic efficiency, poor reusability, and high-cost hamper further industrial usage. Thus, crosslinked enzyme aggregates (CLEAs) are developed as a better enzyme immobilization tool to extend the enzymes' operational stability. This immobilization method is appealing because it is simpler due to the absence of ballast and permits the collective use of crude enzyme cocktails. CLEAs, so far, have been successfully developed using a variety of enzymes, viz., hydrolases, proteases, amidases, lipases, esterases, and oxidoreductase. Recent years have seen the emergence of novel strategies for preparing better CLEAs, which include the combi- and multi-CLEAs, magnetics CLEAs, and porous CLEAs for various industrial applications, viz., laundry detergents, organic synthesis, food industries, pharmaceutical applications, oils, and biodiesel production. To better understand the different strategies for CLEAs' development, this review explores these strategies and highlights the relevant concerns in designing innovative CLEAs. This article also details the challenges faced during CLEAs preparation and solutions for overcoming them. Finally, the trending strategies to improve the preparation of CLEAs alongside their industrial application trends are also discussed.
  3. Fulltext An S188V mutation alters substrate specificity of non-stereospecific α-haloalkanoic acid dehalogenase E (DehE)
    Hamid AA, Hamid TH, Wahab RA, Omar MS, Huyop F
    PLoS One, 2015;10(3):e0121687.
    PMID: 25816329 DOI: 10.1371/journal.pone.0121687
    The non-stereospecific α-haloalkanoic acid dehalogenase E (DehE) degrades many halogenated compounds but is ineffective against β-halogenated compounds such as 3-chloropropionic acid (3CP). Using molecular dynamics (MD) simulations and site-directed mutagenesis we show here that introducing the mutation S188V into DehE improves substrate specificity towards 3CP. MD simulations showed that residues W34, F37, and S188 of DehE were crucial for substrate binding. DehE showed strong binding ability for D-2-chloropropionic acid (D-2CP) and L-2-chloropropionic acid (L-2CP) but less affinity for 3CP. This reduced affinity was attributed to weak hydrogen bonding between 3CP and residue S188, as the carboxylate of 3CP forms rapidly interconverting hydrogen bonds with the backbone amide and side chain hydroxyl group of S188. By replacing S188 with a valine residue, we reduced the inter-molecular distance and stabilised bonding of the carboxylate of 3CP to hydrogens of the substrate-binding residues. Therefore, the S188V can act on 3CP, although its affinity is less strong than for D-2CP and L-2CP as assessed by Km. This successful alteration of DehE substrate specificity may promote the application of protein engineering strategies to other dehalogenases, thereby generating valuable tools for future bioremediation technologies.
  4. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes
    Mohamad NR, Marzuki NH, Buang NA, Huyop F, Wahab RA
    Biotechnology, biotechnological equipment, 2015 Mar 04;29(2):205-220.
    PMID: 26019635
    The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies.
  5. Fulltext Baseline amplicon sequencing data for the ITS2 region in the green honey of Banggi Island, Sabah
    Ullah S, Huda N, Wahab RA, Hamid AAA, Nasir MHM, Mohamad MAN, et al.
    Data Brief, 2024 Feb;52:110044.
    PMID: 38328502 DOI: 10.1016/j.dib.2024.110044
    Green honey, was discovered on Banggi Island, Sabah, showing high in essential amino acids and chlorophyll derivatives. Despite its lucrative market potential owing to its distinctive color, uncertainties persist regarding its nature. This study leverages amplicon sequencing by targeting micro- and macro-organisms present in honey environmental DNA (eDNA) using Internal Transcribed Spacer 2 (ITS2) region, enabling the identification of floral and microorganism sources that represent the honey's composition. The investigation into green honey from Banggi Island concerns the prevalence of honey adulteration and authenticity for economic gain. Adulteration methods, such as the addition of sugar syrups, compromise honey purity. Using a sequencing approach would help in determining the geographic origin and verifying the authenticity of the honey. The study aims to identify plant species or microorganisms in honey's eDNA. To authenticate honey, we utilized ITS2 with Illumina sequencing, exploring the diversity of green honey samples. Raw sequence reads obtained for the green honey sample revealed 1,438,627 raw reads, with a GC average of 49.22 %. A total of 44 amplicon sequence variances (ASVs) were identified, including three genera: Zygosaccharomyces with two species, Fraxinus with three species, and the genus Ficaria with only one species. Their respective relative abundances were 98.55%, 0.94%, and 0.51%. Zygosaccharomyces rouxii and Zygosaccharomyces mellis were identified as the pre-dominant yeast species in honey, while the Fraxinus and Ficaria genus represent common plant species in Sabah, particularly in Banggi Island. The dominance of Zygosaccharomyces species aligns with their known prevalence in honey, affirming the reliability of our findings. The presence of Fraxinus and Ficaria in the honey sample correlates with its abundance in the local environment. This amplicon sequencing approach not only contributes to our understanding of green honey composition but also serves as a valuable resource for authenticating honey origin in Malaysia, particularly for green honey from Banggi Island, Sabah. Our study pioneers the application of ITS2 amplicon sequencing for green honey amplicon sequencing, providing valuable insights into its composition and origin. This methodology, with a focus on eDNA, contributes to the authentication and quality determination of honey in Malaysia, addressing the pressing concerns of adulteration and variability in production practices.
  6. Biodegradation of 3-chloropropionic acid (3-CP) by Bacillus cereus WH2 and its in silico enzyme-substrate docking analysis
    Muslem WH, Edbeib MF, Aksoy HM, Kaya Y, Hamid AAA, Hood MHM, et al.
    J Biomol Struct Dyn, 2020 07;38(11):3432-3441.
    PMID: 31401940 DOI: 10.1080/07391102.2019.1655482
  7. Biological and molecular approaches of the degradation or decolorization potential of the hypersaline Lake Tuz Bacillus megaterium H2 isolate
    Oyewusi HA, Adedamola Akinyede K, Wahab RA, Susanti E, Syed Yaacob SN, Huyop F
    J Biomol Struct Dyn, 2023 Jul 16.
    PMID: 37455463 DOI: 10.1080/07391102.2023.2234040
    The presence of synthetic dyes in water bodies and soil is one of the major issues affecting the global ecology, possibly impacting societal well-being adversely due to the colorants' recalcitrance and toxicity. Herein, the study spectrophotometrically monitored the ability of the Bacillus megaterium H2 azoreductase (AzrBmH2) to degrade four synthetic dyes, reactive blue 4, remazol brilliant red, thymol blue, and methyl red, followed by in-silico assessment using GROMACS. We found that the bacterium degraded as much as 60% of all four synthetic dyes at various tested concentrations. The genome analysis revealed five different azoreductase genes, which were then modeled into the AzrBmH21, AzrBmH22/3, and AzrBmH24/5 templates. The AzrBmH2-substrate complexes showed binding energies with all the dyes of between -10.6 to -6.9 kcal/mol and formed 4-6 hydrogen bonds with the predicted catalytic binding residues (His10, Glu 14, Ser 58, Met 99, Val 107, His 183, Asn184 and Gln 191). In contrast, the lowest binding energies were observed for the AzrBmH21-substrates (-10.6 to -7.9). Molecular dynamic simulations revealed that the AzrBmH21-substrate complexes were more stable (RMSD 0.2-0.25 nm, RMSF 0.05 - 0.3 nm) and implied strong bonding with the dyes. The Molecular Mechanics Poisson-Boltzmann Surface Area results also mirrored this outcome, showing the lowest azoreductase-dye binding energy in the order of AzrBmH21-RB4 (-78.18 ± 8.92 kcal/mol), AzrBmH21-RBR (-67.51 ± 7.74 kcal/mol), AzrBmH21-TB (-46.62 ± 5.23 kcal/mol) and AzrBmH21-MR (-40.78 ± 7.87 kcal/mol). In short, the study demonstrated the ability of the B. megaterium H2 to efficiently decolorize the above-said synthetic dyes, conveying the bacterium's promising use for large-scale dye remediation.Communicated by Ramaswamy H. Sarma.
  8. Biophysical characterization of a recombinant lipase KV1 from Acinetobacter haemolyticus in relation to pH and temperature
    Batumalaie K, Khalili E, Mahat NA, Huyop F, Wahab RA
    Biochimie, 2018 Sep;152:198-210.
    PMID: 30036604 DOI: 10.1016/j.biochi.2018.07.011
    Spectroscopic and calorimetric methods were employed to assess the stability and the folding aspect of a novel recombinant alkaline-stable lipase KV1 from Acinetobacter haemolyticus under varying pH and temperature. Data on far ultraviolet-circular dichroism of recombinant lipase KV1 under two alkaline conditions (pH 8.0 and 12.0) at 40 °C reveal strong negative ellipticities at 208, 217, 222 nm, implying its secondary structure belonging to a α + β class with 47.3 and 39.0% ellipticity, respectively. Results demonstrate that lipase KV1 adopts its most stable conformation at pH 8.0 and 40 °C. Conversely, the protein assumes a random coil structure at pH 4.0 and 80 °C, evident from a strong negative peak at ∼ 200 nm. This blue shift suggests a general decline in enzyme activity in conjunction with the partially or fully unfolded state that invariably exposed more hydrophobic surfaces of the lipase protein. The maximum emission at ∼335 nm for pH 8.0 and 40 °C indicates the adoption of a favorable protein conformation with a high number of buried tryptophan residues, reducing solvent exposure. Appearance of an intense Amide I absorption band at pH 8.0 corroborates an intact secondary structure. A lower enthalpy value for pH 4.0 over pH 8.0 and 12.0 in the differential scanning calorimetric data corroborates the stability of the lipase at alkaline conditions, while a low Km (0.68 ± 0.03 mM) for tributyrin verifies the high affinity of lipase KV1 for the substrate. The data, herein offer useful insights into future structure-based tunable catalytic activity of lipase KV1.
  9. Candida rugosa Lipase Immobilized onto Acid-Functionalized Multi-walled Carbon Nanotubes for Sustainable Production of Methyl Oleate
    Che Marzuki NH, Mahat NA, Huyop F, Buang NA, Wahab RA
    Appl Biochem Biotechnol, 2015 Oct;177(4):967-84.
    PMID: 26267406 DOI: 10.1007/s12010-015-1791-z
    The chemical production of methyl oleate using chemically synthesized fatty acid alcohols and other toxic chemicals may lead to significant environmental hazards to mankind. Being a highly valuable fatty acid replacement raw material in oleochemical industry, the mass production of methyl oleate via environmentally favorable processes is of concern. In this context, an alternative technique utilizing Candida rugosa lipase (CRL) physically adsorbed on multi-walled carbon nanotubes (MWCNTs) has been suggested. In this study, the acid-functionalized MWCNTs prepared using a mixture of HNO3 and H2SO4 (1:3 v/v) was used as support for immobilizing CRL onto MWCNTs (CRL-MWCNTs) as biocatalysts. Enzymatic esterification was performed and the efficiency of CRL-MWCNTs was evaluated against the free CRL under varying conditions, viz. temperature, molar ratio of acid/alcohol, solvent log P, and enzyme loading. The CRL-MWCNTs resulted in 30-110 % improvement in the production of methyl oleate over the free CRL. The CRL-MWCNTs attained its highest yield (84.17 %) at 50 °C, molar ratio of acid/alcohol of 1:3, 3 mg/mL of enzyme loading, and iso-octane (log P 4.5) as solvent. Consequently, physical adsorption of CRL onto acid-functionalized MWCNTs has improved the activity and stability of CRL and hence provides an environmentally friendly means for the production of methyl oleate.
  10. Comparative modeling and enzymatic affinity of novel haloacid dehalogenase from Bacillus megaterium strain BHS1 isolated from alkaline Blue Lake in Turkey
    Wahhab BH, Oyewusi HA, Wahab RA, Mohammad Hood MH, Abdul Hamid AA, Al-Nimer MS, et al.
    J Biomol Struct Dyn, 2024;42(3):1429-1442.
    PMID: 37038649 DOI: 10.1080/07391102.2023.2199870
    This study presents the initial structural model of L-haloacid dehalogenase (DehLBHS1) from Bacillus megaterium BHS1, an alkalotolerant bacterium known for its ability to degrade halogenated environmental pollutants. The model provides insights into the structural features of DehLBHS1 and expands our understanding of the enzymatic mechanisms involved in the degradation of these hazardous pollutants. Key amino acid residues (Arg40, Phe59, Asn118, Asn176, and Trp178) in DehLBHS1 were identified to play critical roles in catalysis and molecular recognition of haloalkanoic acid, essential for efficient binding and transformation of haloalkanoic acid molecules. DehLBHS1 was modeled using I-TASSER, yielding a best TM-score of 0.986 and an RMSD of 0.53 Å. Validation of the model using PROCHECK revealed that 89.2% of the residues were located in the most favored region, providing confidence in its structural accuracy. Molecular docking simulations showed that the non-simulated DehLBHS1 preferred 2,2DCP over other substrates, forming one hydrogen bond with Arg40 and exhibiting a minimum energy of -2.5 kJ/mol. The simulated DehLBHS1 exhibited a minimum energy of -4.3 kJ/mol and formed four hydrogen bonds with Arg40, Asn176, Asp9, and Tyr11, further confirming the preference for 2,2DCP. Molecular dynamics simulations supported this preference, based on various metrics, including RMSD, RMSF, gyration, hydrogen bonding, and molecular distance. MM-PBSA calculations showed that the DehLBHS1-2,2-DCP complex had a markedly lower binding energy (-21.363 ± 1.26 kcal/mol) than the DehLBHS1-3CP complex (-14.327 ± 1.738 kcal/mol). This finding has important implications for the substrate specificity and catalytic function of DehLBHS1, particularly in the bioremediation of 2,2-DCP in contaminated alkaline environments. These results provide a detailed view of the molecular interactions between the enzyme and its substrate and may aid in the development of more efficient biocatalytic strategies for the degradation of halogenated compounds.Communicated by Ramaswamy H. Sarma.
  11. 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.
  12. Fulltext Customised structural, optical and antibacterial characteristics of cinnamon nanoclusters produced inside organic solvent using 532 nm Q-switched Nd:YAG-pulse laser ablation
    Salim AA, Bakhtiar H, Ghoshal SK, Huyop F
    Opt Laser Technol, 2020 Oct;130:106331.
    PMID: 32457554 DOI: 10.1016/j.optlastec.2020.106331
    Biomedical values of organic natural cinnamon that are buried in their bulk counterpart can be exposed and customised via nanosizing. Based on this factor, a new type of spherical cinnamon nanoclusters (Cin-NCs) were synthesised using eco-friendly nanosecond pulse laser ablation in liquid (PLAL) approach. As-grown nontoxic Cin-NCs suspended in the citric acid of pH 4.5 (acted as organic solvent) were characterised thoroughly to evaluate their structural, optical and bactericidal properties. The effects of various laser fluences (LF) at the fixed wavelength (532 nm) on the physiochemical properties of these Cin-NCs were determined. The FTIR spectra of the Cin-NCs displayed the symmetric-asymmetric stretching of the functional groups attached to the heterocyclic/cinnamaldehyde compounds. The HR-TEM image of the optimum sample revealed the nucleation of the crystalline spherical Cin-NCs with a mean diameter of approximately 10 ± 0.3 nm and lattice fringe spacing around 0.14 nm. In addition, the inhibition zone diameter (IZD) and optical density (OD600) of the proposed Cin-NCs were measured to assess their antibacterial potency against the Staphylococcus aureus (IZD ≈ 24 mm) and Escherichia coli (IZD ≈ 25 mm) bacterial strains. The strong UV absorption (in the range of 269 and 310 nm) shown by these NCs was established to be useful for the antibacterial drug development and food treatment.
  13. Deciphering the catalytic amino acid residues of l-2-haloacid dehalogenase (DehL) from Rhizobium sp. RC1: An in silico analysis
    Adamu A, Wahab RA, Shamsir MS, Aliyu F, Huyop F
    Comput Biol Chem, 2017 Oct;70:125-132.
    PMID: 28873365 DOI: 10.1016/j.compbiolchem.2017.08.007
    The l-2-haloacid dehalogenases (EC 3.8.1.2) specifically cleave carbon-halogen bonds in the L-isomers of halogenated organic acids. These enzymes have potential applications for the bioremediation and synthesis of various industrial products. One such enzyme is DehL, the l-2-haloacid dehalogenase from Rhizobium sp. RC1, which converts the L-isomers of 2-halocarboxylic acids into the corresponding D-hydroxycarboxylic acids. However, its catalytic mechanism has not been delineated, and to enhance its efficiency and utility for environmental and industrial applications, knowledge of its catalytic mechanism, which includes identification of its catalytic residues, is required. Using ab initio fragment molecular orbital calculations, molecular mechanics Poisson-Boltzmann surface area calculations, and classical molecular dynamic simulation of a three-dimensional model of DehL-l-2-chloropropionic acid complex, we predicted the catalytic residues of DehL and propose its catalytic mechanism. We found that when Asp13, Thr17, Met48, Arg51, and His184 were individually replaced with an alanine in silico, a significant decrease in the free energy of binding for the DehL-l-2-chloropropionic acid model complex was seen, indicating the involvement of these residues in catalysis and/or structural integrity of the active site. Furthermore, strong inter-fragment interaction energies calculated for Asp13 and L-2-chloropropionic acid, and for a water molecule and His184, and maintenance of the distances between atoms in the aforementioned pairs during the molecular dynamics run suggest that Asp13 acts as the nucleophile and His184 activates the water involved in DehL catalysis. The results of this study should be important for the rational design of a DehL mutant with improved catalytic efficiency.
  14. Dehalogenase-producing halophiles and their potential role in bioremediation
    Oyewusi HA, Wahab RA, Huyop F
    Mar Pollut Bull, 2020 Nov;160:111603.
    PMID: 32919122 DOI: 10.1016/j.marpolbul.2020.111603
    This review aims to briefly describe the potential role of dehalogenase-producing halophilic bacteria in decontamination of organohalide pollutants. Hypersaline habitats pose challenges to life because of low water activity (water content) and is considered as the largest and ultimate sink for pollutants due to naturally and anthropogenic activities in which a substantial amount of ecological contaminants are organohalides. Several such environments appear to host and support substantial diversity of extremely halophilic and halotolerant bacteria as well as halophilic archaea. Biodegradation of several toxic inorganic and organic compounds in both aerobic and anaerobic conditions are carried out by halophilic microbes. Therefore, remediation of polluted marine/hypersaline environments are the main scorching issues in the field of biotechnology. Although many microbial species are reported as effective pollutants degrader, but little has been isolated from marine/hypersaline environments. Therefore, more novel microbial species with dehalogenase-producing ability are still desired.
  15. Fulltext Exploring the genome of Lactobacillaceae spp. Sy-1 isolated from Heterotrigona itama honey
    Syed Yaacob SN, Huyop F, Misson M, Abdul Wahab R, Huda N
    PeerJ, 2022;10:e13053.
    PMID: 35345581 DOI: 10.7717/peerj.13053
    BACKGROUND: Honey produced by Heterotrigona itama is highly preferred among consumers due to its high-value as a functional food and beneficial lactic acid bacteria (LAB) reservoir. Fructophilic lactic acid bacteria (FLAB) are a group of LAB with unique growth characteristics and are regarded as promising producers of bioactive compounds. Hence, it is not surprising that LAB, especially FLAB, may be involved with the excellent bioactivity of H. itama honey. With the trending consumer preference for H. itama honey coupled with increasing awareness for healthy food, the genomic background of FLAB isolated from this honey must, therefore, be clearly understood. In this study, one FLAB strain designated as Sy-1 was isolated from freshly collected H. itama honey. Its FLAB behavior and genomic features were investigated to uncover functional genes that could add value to functional food.

    METHODS: The fructophilic characteristics of strain Sy-1 were determined, and the genome was sequenced using Illumina iSeq100 and Oxford Nanopore. The average nucleotide identity and phylogenetic analyses based on 16S rRNA, 92 core genes, and whole-genome sequence were performed to unravel the phylogenetic position of strain Sy-1. NCBI Prokaryotic Genome Annotation Pipeline annotated the genome, while the EggNOG-mapper, BLASTKoala, and GHOSTKoala were used to add functional genes and pathways information.

    RESULTS: Strain Sy-1 prefers D-fructose over D-glucose and actively metabolizes D-glucose in the presence of electron acceptors. Genomic annotation of strain Sy-1 revealed few genes involved in carbohydrate transport and metabolism, and partial deletion of adhE gene, in line with the characteristic of FLAB. The 16S rRNA gene sequence of strain Sy-1 showed the highest similarity to unknown LAB species isolated from the gut of honeybees. The phylogenetic analyses discovered that strain Sy-1 belonged to the Lactobacillaceae family and formed a separate branch closer to type strain from the genera of Acetilactobacillus and Apilactobacillus. The ANI analysis showed the similarity of the closest relative, Apilactobacillus micheneri Hlig3T. The assembled genome of Sy-1 contains 3 contigs with 2.03 Mbp and a 41% GC content. A total of 1,785 genes were identified, including 1,685 protein-coding genes, 68 tRNA, and 15 rRNA. Interestingly, strain Sy-1 encoded complete genes for the biosynthesis of folate and riboflavin. High-performance liquid chromatography analysis further confirmed the high production of folic acid (1.346 mg/L) by Sy-1.

    DISCUSSION: Based on phylogenetic and biochemical characteristics, strain Sy-1 should be classified as a novel genus in the family of Lactobacillaceae and a new member of FLAB. The genome information coupled with experimental studies supported the ability of strain Sy-1 to produce high folic acid. Our collective findings support the suitable application of FLAB strain Sy-1 in the functional food and pharmaceutical industries.

  16. Fulltext Green honey of Banggi Island: A preliminary anti-diabetic study on zebrafish model
    Ullah S, Huyop F, Huda N, Ab Wahab R, Hamid AAA, Mohamad MAN, et al.
    Heliyon, 2024 Feb 29;10(4):e26469.
    PMID: 38404777 DOI: 10.1016/j.heliyon.2024.e26469
    Zebrafish is a developing vertebrate model with several advantages, including its small size, and high experimental efficiency. Malaysia exhibit one of the highest diabetes rates in the Western Pacific and incurring an annual cost of 600 million US dollars. The objective of the study is to determine the antidiabetic properties of green honey (GH) using a zebrafish model. Adult zebrafish, aged 3-4 months, were subjected to overfeeding and treated with streptozotocin (STZ) through intraperitoneal injection (IP) on days 7 and 9. The study assessed the oral sucrose tolerance test (OSTT) and the anti-diabetic effects of green honey. The evaluation was conducted at three time points: 30, 60, and 120 min after treatment and sucrose administration. The study utilised a model with a sample size of 5. The study was performed in six groups. These groups are (1) Normal control (non-diabetic, no intervention), (2) Normal control + GH (non-diabetic, supplemented with GH 3 μl), (3) DM control (diabetic, no intervention), (4) DM Gp1 (diabetic, 3 μL GH), (5) DM Gp2 (diabetic, 6 μ L GH), (6) DM Acarbose (diabetic, treated with acarbose). Fasting blood glucose levels for non-diabetic (non-DM) and diabetic (DM) groups were evaluated before and after the 10 days of diabetic induction. DM groups (excess of food and two injections of STZ) have caused a significant increment in the fasting blood glucose to 11.55 mmol/l (p 
  17. Haloadaptation: insights from comparative modeling studies between halotolerant and non-halotolerant dehalogenases
    Edbeib MF, Aksoy HM, Kaya Y, Wahab RA, Huyop F
    J Biomol Struct Dyn, 2020 Aug;38(12):3452-3461.
    PMID: 31422756 DOI: 10.1080/07391102.2019.1657498
    Halophiles are extremophilic microorganisms that grow optimally at high salt concentrations by producing a myriad of equally halotolerant enzymes. Structural haloadaptation of these enzymes adept to thriving under high-salt environments, though are not fully understood. Herein, the study attempts an in silico investigation to identify and comprehend the evolutionary structural adaptation of a halotolerant dehalogenase, DehHX (GenBank accession number: KR297065) of the halotolerant Pseudomonas halophila, over its non-halotolerant counterpart, DehMX1 (GenBank accession number KY129692) produced by Pseudomonas aeruginosa. GC content of the halotolerant DehHX DNA sequence was distinctively higher (58.9%) than the non-halotolerant dehalogenases (55% average GC). Its acidic residues, Asp and Glu were 8.27% and 12.06%, respectively, compared to an average 5.5% Asp and 7% Glu, in the latter; but lower contents of basic and hydrophobic residues in the DehHX. The secondary structure of DehHX interestingly revealed a lower incidence of α-helix forming regions (29%) and a higher percentage of coils (57%), compared to 49% and 29% in the non-halotolerant homologues, respectively. Simulation models showed the DehHX is stable under a highly saline environment (25% w/v) by adopting a highly negative-charged surface with a concomitant weakly interacting hydrophobic core. The study thus, established that a halotolerant dehalogenase undergoes notable evolutionary structural changes related to GC content over its non-halotolerant counterpart, in order to adapt and thrive under highly saline environments.Communicated by Ramaswamy H. Sarma.
  18. Halophiles: biology, adaptation, and their role in decontamination of hypersaline environments
    Edbeib MF, Wahab RA, Huyop F
    World J Microbiol Biotechnol, 2016 Aug;32(8):135.
    PMID: 27344438 DOI: 10.1007/s11274-016-2081-9
    The unique cellular enzymatic machinery of halophilic microbes allows them to thrive in extreme saline environments. That these microorganisms can prosper in hypersaline environments has been correlated with the elevated acidic amino acid content in their proteins, which increase the negative protein surface potential. Because these microorganisms effectively use hydrocarbons as their sole carbon and energy sources, they may prove to be valuable bioremediation agents for the treatment of saline effluents and hypersaline waters contaminated with toxic compounds that are resistant to degradation. This review highlights the various strategies adopted by halophiles to compensate for their saline surroundings and includes descriptions of recent studies that have used these microorganisms for bioremediation of environments contaminated by petroleum hydrocarbons. The known halotolerant dehalogenase-producing microbes, their dehalogenation mechanisms, and how their proteins are stabilized is also reviewed. In view of their robustness in saline environments, efforts to document their full potential regarding remediation of contaminated hypersaline ecosystems merits further exploration.
  19. Identification of functional residues essential for dehalogenation by the non-stereospecific α-haloalkanoic acid dehalogenase from Rhizobium sp. RC1
    Hamid AA, Hamid TH, Wahab RA, Huyop F
    J Basic Microbiol, 2015 Mar;55(3):324-30.
    PMID: 25727054 DOI: 10.1002/jobm.201570031
    The non-stereospecific α-haloalkanoic acid dehalogenase DehE from Rhizobium sp. RC1 catalyzes the removal of the halide from α-haloalkanoic acid D,L-stereoisomers and, by doing so, converts them into hydroxyalkanoic acid L,D-stereoisomers, respectively. DehE has been extensively studied to determine its potential to act as a bioremediation agent, but its structure/function relationship has not been characterized. For this study, we explored the functional relevance of several putative active-site amino acids by site-specific mutagenesis. Ten active-site residues were mutated individually, and the dehalogenase activity of each of the 10 resulting mutants in soluble cell lysates against D- and L-2-chloropropionic acid was assessed. Interestingly, the mutants W34→A,F37→A, and S188→A had diminished activity, suggesting that these residues are functionally relevant. Notably, the D189→N mutant had no activity, which strongly implies that it is a catalytically important residue. Given our data, we propose a dehalogenation mechanism for DehE, which is the same as that suggested for other non-stereospecific α-haloalkanoic acid dehalogenases. To the best of our knowledge, this is the first report detailing a functional aspect for DehE, and our results could help pave the way for the bioengineering of haloalkanoic acid dehalogenases with improved catalytic properties.
  20. In silico analysis of a putative dehalogenase from the genome of halophilic bacterium Halomonas smyrnensis AAD6T
    Oyewusi HA, Akinyede KA, Abdul Wahab R, Huyop F
    J Biomol Struct Dyn, 2023 Jan;41(1):319-335.
    PMID: 34854349 DOI: 10.1080/07391102.2021.2006085
    Microbial-assisted removal of natural or synthetic pollutants is the prevailing green, low-cost technology to treat polluted environments. However, the challenge with enzyme-assisted bioremediation is the laborious nature of dehalogenase-producing microorganisms' bioprospecting. This bottleneck could be circumvented by in-silico analysis of certain microorganisms' whole-genome sequences to predict their protein functions and enzyme versatility for improved biotechnological applications. Herein, this study performed structural analysis on a dehalogenase (DehHsAAD6) from the genome of Halomonas smyrnensis AAD6 by molecular docking and molecular dynamic (MD) simulations. Other bioinformatics tools were also employed to identify substrate preference (haloacids and haloacetates) of the DehHsAAD6. The DehHsAAD6 preferentially degraded haloacids and haloacetates (-3.2-4.8 kcal/mol) and which formed three hydrogen bonds with Tyr12, Lys46, and Asp182. MD simulations data revealed the higher stability of DehHsAAD6-haloacid- (RMSD 0.22-0.3 nm) and DehHsAAD6-haloacetates (RMSF 0.05-0.14 nm) complexes, with the DehHsAAD6-L-2CP complex being the most stable. The detail of molecular docking calculations ranked complexes with the lowest binding free energies as: DehHsAAD6-L-2CP complex (-4.8 kcal/mol) = DehHsAAD6-MCA (-4.8 kcal/mol) < DehHsAAD6-TCA (-4.5 kcal/mol) < DehHsAAD6-2,3-DCP (-4.1 kcal/mol) < DehHsAAD6-D-2CP (-3.9 kcal/mol) < DehHsAAD6-2,2-DCP (-3.5 kcal/mol) < DehHsAAD6-3CP (-3.2 kcal/mol). In a nutshell, the study findings offer valuable perceptions into the elucidation of possible reaction mechanisms of dehalogenases for extended substrate specificity and higher catalytic activity.Communicated by Ramaswamy H. Sarma.
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