Displaying publications 1 - 20 of 36 in total

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  1. Fulltext Cold-Active Lipases and Esterases: A Review on Recombinant Overexpression and Other Essential Issues
    Matinja AI, Kamarudin NHA, Leow ATC, Oslan SN, Ali MSM
    Int J Mol Sci, 2022 Dec 06;23(23).
    PMID: 36499718 DOI: 10.3390/ijms232315394
    Cold environments characterised by diverse temperatures close to or below the water freezing point dominate about 80% of the Earth's biosphere. One of the survival strategies adopted by microorganisms living in cold environments is their expression of cold-active enzymes that enable them to perform an efficient metabolic flux at low temperatures necessary to thrive and reproduce under those constraints. Cold-active enzymes are ideal biocatalysts that can reduce the need for heating procedures and improve industrial processes' quality, sustainability, and cost-effectiveness. Despite their wide applications, their industrial usage is still limited, and the major contributing factor is the lack of complete understanding of their structure and cold adaptation mechanisms. The current review looked at the recombinant overexpression, purification, and recent mechanism of cold adaptation, various approaches for purification, and three-dimensional (3D) crystal structure elucidation of cold-active lipases and esterase.
  2. Fulltext Recovery of a Bacteriocin-Like Inhibitory Substance from Lactobacillus bulgaricus FTDC 1211 Using Polyethylene-Glycol Impregnated Amberlite XAD-4 Resins System
    Abdul Aziz NFH, Abbasiliasi S, Ng ZJ, Abu Zarin M, Oslan SN, Tan JS, et al.
    Molecules, 2020 Nov 16;25(22).
    PMID: 33207534 DOI: 10.3390/molecules25225332
    Lactobacillus bulgaricus is a LAB strain which is capable of producing bacteriocin substances to inhibit Staphylococcus aureus. The aim of this study was to purify a bacteriocin-like inhibitory substance (BLIS) produced by L. bulgaricus FTDC 1211 using an aqueous impregnated resins system consisting of polyethylene-glycol (PEG) impregnated on Amberlite XAD4. Important parameters influencing on purification of BLIS, such as the molecular weight and concentration of PEG, the concentration and pH of sodium citrate and the concentration of sodium chloride, were optimized using a response surface methodology. Under optimum conditions of 11% (w/w) of PEG 4000 impregnated Amberlite XAD4 resins and 2% (w/w) of sodium citrate at pH 6, the maximum purification factor (3.26) and recovery yield (82.69% ± 0.06) were obtained. These results demonstrate that AIRS could be used as an alternate purification system in the primary recovery step.
  3. Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris
    Latiffi AA, Salleh AB, Rahman RN, Oslan SN, Basri M
    Genes Genet Syst, 2013;88(2):85-91.
    PMID: 23832300
    The thermostable alkaline protease from Bacillus stearothermophilus F1 has high potential for industrial applications, and attempt to produce the enzyme in yeast for higher yield was undertaken. Secretory expression of F1 protease through yeast system could improve enzyme's capability, thus simplifying the purification steps. Mature and full genes of F1 protease were cloned into Pichia pastoris expression vectors (pGAPZαB and pPICZαB) and transformed into P. pastoris strains (GS115 and SMD1168H) via electroporation method. Recombinant F1 protease under regulation constitutive GAP promoter revealed that the highest expression was achieved after 72 h cultivation. While inducible AOX promoter showed that 0.5% (v/v) methanol was the best to induce expression. It was proven that constitutive expression strategy was better than inducible system. The α-secretion signal from the plasmid demonstrated higher secretory expression level of F1 protease as compared to native Open Reading Frame (ORF) in GS115 strain (GE6GS). Production medium YPTD was found to be the best for F1 protease expression with the highest yield of 4.13 U/mL. The protein was expressed as His-tagged fusion protein with a size about 34 kDa.
  4. Enhancing food preservation with postbiotic metabolites γ-aminobutyric acid (GABA) and bacteriocin-like inhibitory substances (BLIS) produced by Lactobacillus brevis C23 co-cultures in plant-based medium
    Chuah WW, Tan JS, Hazwani Oslan SN, Bothi Raja P
    Prep Biochem Biotechnol, 2024 Apr;54(4):514-525.
    PMID: 37694843 DOI: 10.1080/10826068.2023.2252047
    Lactic acid bacteria (LAB) can produce γ-aminobutyric acid (GABA) with antioxidant properties and sedative effects when it binds to the GABA receptor in the human brain. LAB can also produce bacteriocin-like inhibitory substances (BLIS) with antimicrobial capabilities during carbohydrate fermentation. GABA and BLIS are natural compounds with potential health benefits and food preservation properties. Lactobacillus brevis C23 was co-cultured with three different LABs as inducers, which produced the highest GABA content and BLIS activity. They were cultured in various plant-based media to obtain an edible and better-tasting final product over commercially available media like MRS broth. A coconut-based medium with additives was optimized using response surface methodology (RSM) to increase GABA and BLIS production. The optimized medium for maximum GABA production (3.22 ± 0.01 mg/mL) and BLIS activity (84.40 ± 0.44%) was a 5.5% coconut medium containing 0.23% glucose, 1.44% Tween 20, 0.48% L-glutamic acid, and 0.02% pyridoxine. Due to the presence of GABA, the cell-free supernatant (CFS) as a postbiotic showed higher antioxidant activity than other food preservatives like nisin and potassium sorbate. Finally, microbiological tests on food samples showed that the postbiotic was more effective than other preservatives at combating the growth of LAB, molds and coliform bacteria, making it a possible food preservative.
  5. Locally isolated yeasts from Malaysia: identification, phylogenetic study and characterization
    Oslan SN, Salleh AB, Rahman RN, Basri M, Chor AL
    Acta Biochim. Pol., 2012;59(2):225-9.
    PMID: 22577620
    Yeasts are a convenient platform for many applications. They have been widely used as the expression hosts. There is a need to have a new yeast expression system to contribute the molecular cloning demands. Eight yeast isolates were screened from various environment sources and identified through ribosomal DNA (rDNA) Internal Transcribed Spacer (ITS). Full sequence of the rDNA ITS region for each isolate was BLASTed and phylogenetic study was constructed by using MEGA4. Among the isolates, isolate WB from 'ragi' (used to ferment carbohydrates) could be identified as a new species in order Saccharomycetales according to rDNA ITS region, morphology and biochemical tests. Isolate SO (from spoiled orange), RT (rotten tomato) and RG (different type of 'ragi') were identified as Pichia sp. Isolates R1 and R2, S4 and S5 (from the surrounding of a guava tree) were identified as Issatchenkia sp. and Hanseniaspora sp., respectively. Geneticin, 50 µg/mL, was determined to be the antibiotic marker for all isolates excepted for isolates RT and SO which used 500 µg/mL and 100 µg/mL Zeocin, respectively. Intra-extracellular proteins were screened for lipolytic activity at 30°C and 70°C. Thermostable lipase activity was detected in isolates RT and R1 with 0.6 U/mg and 0.1 U/mg, respectively. In conclusion, a new yeast-vector system for isolate WB can be developed by using phleomycin or geneticin as the drugs resistance marker. Moreover, strains RT and R1 can be investigated as a novel source of a thermostable lipase.
  6. Fulltext Utilization of acid pre-treated coconut dregs as a substrate for production of detergent compatible lipase by Bacillus stratosphericus
    Mohd Zin NB, Mohamad Yusof B, Oslan SN, Wasoh H, Tan JS, Ariff AB, et al.
    AMB Express, 2017 Dec;7(1):131.
    PMID: 28651380 DOI: 10.1186/s13568-017-0433-y
    In recent years, many efforts have been directed to explore the methods to reduce the production costs of industrial lipase by improving the yield and the use of low-cost agricultural wastes. Coconut dregs, which is a lignocellulosic by-product from coconut oil and milk processing plants, is rich in cellulose (36%) and crude fat (9%). A newly isolated Bacillus stratosphericus has been demonstrated to perform cellulose hydrolysis on coconut dregs producing fermentable sugars. The highest extracellular lipase activity of 140 U/mL has been achieved in submerged fermentation with acid pre-treated coconut dregs. The lipase was found to be active over a wide range of temperatures and pHs. The activity of lipase can be generally increased by the presence of detergent ingredients such as Tween-80, cetyltrimethylammonium bromide, hydrogen peroxide and phosphate per sulphate. The great compatibility of lipase in commercial detergents has also underlined its potential as an additive ingredient in biodetergent formulations.
  7. Fulltext Haematococcus pluvialis as a Potential Source of Astaxanthin with Diverse Applications in Industrial Sectors: Current Research and Future Directions
    Oslan SNH, Tan JS, Oslan SN, Matanjun P, Mokhtar RAM, Shapawi R, et al.
    Molecules, 2021 Oct 27;26(21).
    PMID: 34770879 DOI: 10.3390/molecules26216470
    Haematococcus pluvialis, a green microalga, appears to be a rich source of valuable bioactive compounds, such as astaxanthin, carotenoids, proteins, lutein, and fatty acids (FAs). Astaxanthin has a variety of health benefits and is used in the nutraceutical and pharmaceutical industries. Astaxanthin, for example, preserves the redox state and functional integrity of mitochondria and shows advantages despite a low dietary intake. Because of its antioxidant capacity, astaxanthin has recently piqued the interest of researchers due to its potential pharmacological effects, which include anti-diabetic, anti-inflammatory, and antioxidant activities, as well as neuro-, cardiovascular-, ocular, and skin-protective properties. Astaxanthin is a popular nutritional ingredient and a significant component in animal and aquaculture feed. Extensive studies over the last two decades have established the mechanism by which persistent oxidative stress leads to chronic inflammation, which then mediates the majority of serious diseases. This mini-review provides an overview of contemporary research that makes use of the astaxanthin pigment. This mini-review provides insight into the potential of H. pluvialis as a potent antioxidant in the industry, as well as the broad range of applications for astaxanthin molecules as a potent antioxidant in the industrial sector.
  8. Fulltext Reductive evolution in outer membrane protein biogenesis has not compromised cell surface complexity in Helicobacter pylori
    Webb CT, Chandrapala D, Oslan SN, Bamert RS, Grinter RD, Dunstan RA, et al.
    Microbiologyopen, 2017 12;6(6).
    PMID: 29055967 DOI: 10.1002/mbo3.513
    Helicobacter pylori is a gram-negative bacterial pathogen that chronically inhabits the human stomach. To survive and maintain advantage, it has evolved unique host-pathogen interactions mediated by Helicobacter-specific proteins in the bacterial outer membrane. These outer membrane proteins (OMPs) are anchored to the cell surface via a C-terminal β-barrel domain, which requires their assembly by the β-barrel assembly machinery (BAM). Here we have assessed the complexity of the OMP C-terminal β-barrel domains employed by H. pylori, and characterized the H. pyloriBAM complex. Around 50 Helicobacter-specific OMPs were assessed with predictive structural algorithms. The data suggest that H. pylori utilizes a unique β-barrel architecture that might constitute H. pylori-specific Type V secretions system. The structural and functional diversity in these proteins is encompassed by their extramembrane domains. Bioinformatic and biochemical characterization suggests that the low β-barrel-complexity requires only minimalist assembly machinery. The H. pylori proteins BamA and BamD associate to form a BAM complex, with features of BamA enabling an oligomerization that might represent a mechanism by which a minimalist BAM complex forms a larger, sophisticated machinery capable of servicing the outer membrane proteome of H. pylori.
  9. Recent advances in simultaneous thermostability-activity improvement of industrial enzymes through structure modification
    Nezhad NG, Rahman RNZRA, Normi YM, Oslan SN, Shariff FM, Leow TC
    Int J Biol Macromol, 2023 Mar 31;232:123440.
    PMID: 36708895 DOI: 10.1016/j.ijbiomac.2023.123440
    Engineered thermostable microbial enzymes are widely employed to catalyze chemical reactions in numerous industrial sectors. Although high thermostability is a prerequisite of industrial applications, enzyme activity is usually sacrificed during thermostability improvement. Therefore, it is vital to select the common and compatible strategies between thermostability and activity improvement to reduce mutants̕ libraries and screening time. Three functional protein engineering approaches, including directed evolution, rational design, and semi-rational design, are employed to manipulate protein structure on a genetic basis. From a structural standpoint, integrative strategies such as increasing substrate affinity; introducing electrostatic interaction; removing steric hindrance; increasing flexibility of the active site; N- and C-terminal engineering; and increasing intramolecular and intermolecular hydrophobic interactions are well-known to improve simultaneous activity and thermostability. The current review aims to analyze relevant strategies to improve thermostability and activity simultaneously to circumvent the thermostability and activity trade-off of industrial enzymes.
  10. Functional expression, purification, biochemical and biophysical characterizations, and molecular dynamics simulation of a histidine acid phosphatase from Saccharomyces cerevisiae
    Nezhad NG, Jamaludin SZB, Rahman RNZRA, Yahaya NM, Oslan SN, Shariff FM, et al.
    World J Microbiol Biotechnol, 2024 Apr 17;40(6):171.
    PMID: 38630327 DOI: 10.1007/s11274-024-03970-8
    A histidine acid phosphatase (HAP) (PhySc) with 99.50% protein sequence similarity with PHO5 from Saccharomyces cerevisiae was expressed functionally with the molecular mass of ∼110 kDa through co-expression along with the set of molecular chaperones dnaK, dnaJ, GroESL. The purified HAP illustrated the optimum activity of 28.75 ± 0.39 U/mg at pH 5.5 and 40 ˚C. The Km and Kcat values towards calcium phytate were 0.608 ± 0.09 mM and 650.89 ± 3.6 s- 1. The half-lives (T1/2) at 55 and 60 ˚C were 2.75 min and 55 s, respectively. The circular dichroism (CD) demonstrated that PhySc includes 30.5, 28.1, 21.3, and 20.1% of random coils, α-Helix, β-Turns, and β-Sheet, respectively. The Tm recorded by CD for PhySc was 56.5 ± 0.34˚C. The molecular docking illustrated that His59 and Asp322 act as catalytic residues in the PhySc. MD simulation showed that PhySc at 40 ˚C has higher structural stability over those of the temperatures 60 and 80 ˚C that support the thermodynamic in vitro investigations. Secondary structure content results obtained from MD simulation indicated that PhySc consists of 34.03, 33.09, 17.5, 12.31, and 3.05% of coil, helix, turn, sheet, and helix310, respectively, which is almost consistent with the experimental results.
  11. Fulltext A Review on Haematococcus pluvialis Bioprocess Optimization of Green and Red Stage Culture Conditions for the Production of Natural Astaxanthin
    Oslan SNH, Shoparwe NF, Yusoff AH, Rahim AA, Chang CS, Tan JS, et al.
    Biomolecules, 2021 02 10;11(2).
    PMID: 33578851 DOI: 10.3390/biom11020256
    As the most recognizable natural secondary carotenoid astaxanthin producer, the green microalga Haematococcus pluvialis cultivation is performed via a two-stage process. The first is dedicated to biomass accumulation under growth-favoring conditions (green stage), and the second stage is for astaxanthin evolution under various stress conditions (red stage). This mini-review discusses the further improvement made on astaxanthin production by providing an overview of recent works on H. pluvialis, including the valuable ideas for bioprocess optimization on cell growth, and the current stress-exerting strategies for astaxanthin pigment production. The effects of nutrient constituents, especially nitrogen and carbon sources, and illumination intensity are emphasized during the green stage. On the other hand, the significance of the nitrogen depletion strategy and other exogenous factors comprising salinity, illumination, and temperature are considered for the astaxanthin inducement during the red stage. In short, any factor that interferes with the cellular processes that limit the growth or photosynthesis in the green stage could trigger the encystment process and astaxanthin formation during the red stage. This review provides an insight regarding the parameters involved in bioprocess optimization for high-value astaxanthin biosynthesis from H. pluvialis.
  12. In silico identification and characterization of potential druggable targets among hypothetical proteins of Leptospira interrogans serovar Copenhageni: a comprehensive bioinformatics approach
    Siddiqui Q, Ali MSM, Leow ATC, Oslan SN, Mohd Shariff F
    J Biomol Struct Dyn, 2023 Dec;41(20):10347-10367.
    PMID: 36510668 DOI: 10.1080/07391102.2022.2154845
    Leptospirosis is one of the neglected zoonosis, affecting human and animal populations worldwide. Reliable effective therapeutics and concerns to look for more research into the molecular analysis of its genome is therefore needed. In the genomic pool of the Leptospira interrogans many hypothetical proteins are still uncharacterized. In the current research, we performed extensive in silico analysis to prioritize the potential hypothetical proteins of L. interrogans serovar Copenhageni via stepwise reducing the available hypothetical proteins (Total 3606) of the assembly to only 15, based on non-homologous to homosapien, essential, functional, virulent, cellular localization. Out of them, only two proteins WP_000898918.1 (Hypothetical Protein 1) & WP_001014594.1 (Hypothetical Protein 2) were found druggable and involved in protein-protein interaction network. The 3 D structures of these two target proteins were predicted via ab initio homology modeling followed by structures refinement and validation, as no structures were available till date. The analysis also revealed that the functional domains, families and protein-protein interacting partners identified in both proteins are crucial for the survival of the bacteria. The binding cavities were predicted for both the proteins through blind and specific protein-ligand docking with their respective ligands and inhibitors and were found to be in accordance with the druggable sites predicted by DoGSiteScorer. The docking interactions were found within the active functional domains for both the proteins while for Hypothetical Protein 2, the same residues were involved in interactions with Cytidine-5'-triphosphate in blind and specific docking. Furthermore, the simulations of molecular dynamics and free binding energy revealed the stable substrate binding and efficient binding energies, and were in accordance to our docking results. The work predicted two unique hypothetical proteins of L. interrogans as a potential druggable targets for designing of inhibitors for them.Communicated by Ramaswamy H. Sarma.
  13. Versatility of subtilisin: A review on structure, characteristics, and applications
    Azrin NAM, Ali MSM, Rahman RNZRA, Oslan SN, Noor NDM
    Biotechnol Appl Biochem, 2022 Dec;69(6):2599-2616.
    PMID: 35019178 DOI: 10.1002/bab.2309
    Due to its thermostability and high pH compatibility, subtilisin is most known for its role as an additive for detergents in which it is categorized as a serine protease according to MEROPS database. Subtilisin is typically isolated from various bacterial species of the Bacillus genus such as Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, and various other organisms. It is composed of 268-275 amino acid residues and is initially secreted in the precursor form, preprosubtilisin, which is composed of 29-residues signal peptide, 77-residues propeptide, and 275-residues active subtilisin. Subtilisin is known for the presence of high and low affinity calcium binding sites in its structure. Native subtilisin has general properties of thermostability, tolerance to neutral to high pH, broad specificity, and calcium-dependent stability, which contribute to the versatility of subtilisin applicability. Through protein engineering and immobilization technologies, many variants of subtilisin have been generated, which increase the applicability of subtilisin in various industries including detergent, food processing and packaging, synthesis of inhibitory peptides, therapeutic, and waste management applications.
  14. Fulltext Expression and Characterization of Geobacillus stearothermophilus SR74 Recombinant α-Amylase in Pichia pastoris
    Gandhi S, Salleh AB, Rahman RN, Chor Leow T, Oslan SN
    Biomed Res Int, 2015;2015:529059.
    PMID: 26090417 DOI: 10.1155/2015/529059
    Geobacillus stearothermophilus SR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactive α-amylase. Increased production and commercialization of thermostable α-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostable α-amylase in G. stearothermophilus SR74 was amplified, sequenced, and subcloned into P. pastoris GS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinant α-amylase SR74 achieved in shake flask was 28.6 U mL(-1) at 120 h after induction. The recombinant 59 kDa α-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg(-1). The optimum pH of α-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0-8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t₁/₂) of 88 min at 60°C. In conclusion, thermostable α-amylase SR74 from G. stearothermophilus SR74 would be beneficial for industrial applications, especially in liquefying saccrification.
  15. Fulltext A Host-Vector System for the Expression of a Thermostable Bacterial Lipase in a Locally Isolated Meyerozyma guilliermondii SMB
    Salleh AB, Baharuddin SM, Rahman RNZRA, Leow TC, Basri M, Oslan SN
    Microorganisms, 2020 Nov 06;8(11).
    PMID: 33171893 DOI: 10.3390/microorganisms8111738
    Screening for a new yeast as an alternative host is expected to solve the limitations in the present yeast expression system. A yeast sample which was isolated from the traditional food starter 'ragi' from Malaysia was identified to contain Meyerozyma guilliermondii strain SMB. This yeast-like fungus strain SMB was characterized to assess its suitability as an expression host. Lipase activity was absent in this host (when assayed at 30 °C and 70 °C) and Hygromycin B (50 μg/mL) was found to be its best selection marker. Then, the hyg gene (Hygromycin B) was used to replace the sh ble gene (Zeocin) expression cassette in a Komagataella phaffii expression vector (designated as pFLDhα). A gene encoding the mature thermostable lipase from Bacillus sp. L2 was cloned into pFLDhα, followed by transformation into strain SMB. The optimal expression of L2 lipase was achieved using YPTM (Yeast Extract-Peptone-Tryptic-Methanol) medium after 48 h with 0.5% (v/v) methanol induction, which was 3 times faster than another K. phaffii expression system. In conclusion, a new host-vector system was established as a platform to express L2 lipase under the regulation of PFLD1. It could also be promising to express other recombinant proteins without inducers.
  16. Fulltext Antifreeze Proteins and Their Practical Utilization in Industry, Medicine, and Agriculture
    Eskandari A, Leow TC, Rahman MBA, Oslan SN
    Biomolecules, 2020 12 09;10(12).
    PMID: 33317024 DOI: 10.3390/biom10121649
    Antifreeze proteins (AFPs) are specific proteins, glycopeptides, and peptides made by different organisms to allow cells to survive in sub-zero conditions. AFPs function by reducing the water's freezing point and avoiding ice crystals' growth in the frozen stage. Their capability in modifying ice growth leads to the stabilization of ice crystals within a given temperature range and the inhibition of ice recrystallization that decreases the drip loss during thawing. This review presents the potential applications of AFPs from different sources and types. AFPs can be found in diverse sources such as fish, yeast, plants, bacteria, and insects. Various sources reveal different α-helices and β-sheets structures. Recently, analysis of AFPs has been conducted through bioinformatics tools to analyze their functions within proper time. AFPs can be used widely in various aspects of application and have significant industrial functions, encompassing the enhancement of foods' freezing and liquefying properties, protection of frost plants, enhancement of ice cream's texture, cryosurgery, and cryopreservation of cells and tissues. In conclusion, these applications and physical properties of AFPs can be further explored to meet other industrial players. Designing the peptide-based AFP can also be done to subsequently improve its function.
  17. Fulltext Native to designed: microbial -amylases for industrial applications
    Lim SJ, Oslan SN
    PeerJ, 2021;9:e11315.
    PMID: 34046253 DOI: 10.7717/peerj.11315
    Background: -amylases catalyze the endo-hydrolysis of -1,4-D-glycosidic bonds in starch into smaller moieties. While industrial processes are usually performed at harsh conditions, -amylases from mainly the bacteria, fungi and yeasts are preferred for their stabilities (thermal, pH and oxidative) and specificities (substrate and product). Microbial -amylases can be purified and characterized for industrial applications. While exploring novel enzymes with these properties in the nature is time-costly, the advancements in protein engineering techniques including rational design, directed evolution and others have privileged their modifications to exhibit industrially ideal traits. However, the commentary on the strategies and preferably mutated residues are lacking, hindering the design of new mutants especially for enhanced substrate specificity and oxidative stability. Thus, our review ensures wider accessibility of the previously reported experimental findings to facilitate the future engineering work.

    Survey methodology and objectives: A traditional review approach was taken to focus on the engineering of microbial -amylases to enhance industrially favoured characteristics. The action mechanisms of - and -amylases were compared to avoid any bias in the research background. This review aimed to discuss the advances in modifying microbial -amylases via protein engineering to achieve longer half-life in high temperature, improved resistance (acidic, alkaline and oxidative) and enhanced specificities (substrate and product). Captivating results were discussed in depth, including the extended half-life at 100C, pH 3.5 and 10, 1.8 M hydrogen peroxide as well as enhanced substrate (65.3%) and product (42.4%) specificities. These shed light to the future microbial -amylase engineering in achieving paramount biochemical traits ameliorations to apt in the industries.

    Conclusions: Microbial -amylases can be tailored for specific industrial applications through protein engineering (rational design and directed evolution). While the critical mutation points are dependent on respective enzymes, formation of disulfide bridge between cysteine residues after mutations is crucial for elevated thermostability. Amino acids conversion to basic residues was reported for enhanced acidic resistance while hydrophobic interaction resulted from mutated hydrophobic residues in carbohydrate-binding module or surface-binding sites is pivotal for improved substrate specificity. Substitution of oxidation-prone methionine residues with non-polar residues increases the enzyme oxidative stability. Hence, this review provides conceptual advances for the future microbial -amylases designs to exhibit industrially significant characteristics. However, more attention is needed to enhance substrate specificity and oxidative stability since they are least reported.

  18. Opportunistic yeast pathogen Candida spp.: Secreted and membrane-bound virulence factors
    Lim SJ, Mohamad Ali MS, Sabri S, Muhd Noor ND, Salleh AB, Oslan SN
    Med Mycol, 2021 Dec 03;59(12):1127-1144.
    PMID: 34506621 DOI: 10.1093/mmy/myab053
    Candidiasis is a fungal infection caused by Candida spp. especially Candida albicans, C. glabrata, C. parapsilosis and C. tropicalis. Although the medicinal therapeutic strategies have rapidly improved, the mortality rate as candidiasis has continuously increased. The secreted and membrane-bound virulence factors (VFs) are responsible for fungal invasion, damage and translocation through the host enterocytes besides the evasion from host immune system. VFs such as agglutinin-like sequences (Als), heat shock protein 70, phospholipases, secreted aspartyl proteinases (Sap), lipases, enolases and phytases are mostly hydrolases which degrade or interact with the enterocyte membrane components. Candidalysin, however, acts as a peptide toxin to induce necrotic cell lysis. To date, structural studies of the VFs remain underexplored, hindering their functional analyses. Among the VFs, only Sap and Als have their structures deposited in Protein Data Bank (PDB). Therefore, this review scrutinizes the mechanisms of these VFs by discussing the VF-deficient studies of several Candida spp. and their abilities to produce these VFs. Nonetheless, their latest reported sequential and structural analyses are discussed to impart a wider perception of the host-pathogen interactions and potential vaccine or antifungal drug targets. This review signifies that more VFs structural investigations and mining in the emerging Candida spp. are required to decipher their pathogenicity and virulence mechanisms compared to the prominent C. albicans.

    LAY SUMMARY: Candida virulence factors (VFs) including mainly enzymes and proteins play vital roles in breaching the human intestinal barrier and causing deadly invasive candidiasis. Limited VFs' structural studies hinder deeper comprehension of their mechanisms and thus the design of vaccines and antifungal drugs against fungal infections.

  19. Enolase in Meyerozyma guilliermondii strain SO: Sequential and structural insights of MgEno4581 as a putative virulence factor and host-fungal interactions through comprehensive in silico approaches
    Amran AI, Lim SJ, Muhd Noor ND, Salleh AB, Oslan SN
    Microb Pathog, 2023 Mar;176:106025.
    PMID: 36754101 DOI: 10.1016/j.micpath.2023.106025
    Meyerozyma guilliermondii is a rare opportunistic fungal pathogen that causes deadly invasive candidiasis in human. M. guilliermondii strain SO is a local yeast isolate that possesses huge industrial interests but also pathogenic towards zebrafish embryos. Enolases that bind to human extracellular matrix (ECM) proteins are among the fungal virulence factors. To understand its pathogenicity mechanism down to molecular level, especially in the rare M. guilliermondii, this study aimed to identify and characterize the potentially virulence-associated enolase in M. guilliermondii strain SO using bioinformatics approaches. Profile Hidden-Markov model was implemented to identify enolase-related sequences in the fungal proteome. Sequence analysis deciphered only one (MgEno4581) out of nine sequences exhibited potent virulence traits observed similarly in the pathogenic Candida albicans. MgEno4581 structure that was predicted via SWISS-MODEL using C. albicans enolase (CaEno1; PDB ID: 7vrd) as the homology modeling template portrayed a highly identical motif with CaEno1 that facilitates ECM proteins binding. Amino acid substitutions (D234K, K235A, Y238H, K239D, G243K, V248C and Y254F) in ECM-binding motif of Saccharomyces cerevisiae enolase (ScEno) compared to MgEno4581 and CaEno1 caused changes in motif's surface charges. Protein-protein docking indicated F253 in ScEno only interacted hydrophobically with human plasminogen (HPG). Hydrogen linkages were observed for both MgEno4581 and CaEno1, suggesting a stronger interaction with HPG in the hydrophilic host microenvironments. Thus, our in silico characterizations on MgEno4581 provided new perspectives on its potential roles in candidiasis (fungal-host interactions) caused by M. guilliermondii, especially M. guilliermondii strain SO on zebrafish embryos that mimic the immunocompromised individuals as previously evident.
  20. In silico structural exploration of serine protease from a CTG-clade yeast Meyerozyma guilliermondii strain SO
    Lorrine OE, Rahman RNZRA, Joo Shun T, Salleh AB, Oslan SN
    Anal Biochem, 2023 May 01;668:115092.
    PMID: 36889624 DOI: 10.1016/j.ab.2023.115092
    In eukaryotes, serine proteases are cellular localized hydrolases reported to regulate essential biological reactions. Improved industrial applications of proteins are aided by prediction and analysis of their 3-dimensional structures (3D). A serine protease was identified from CTG-clade yeast Meyerozyma guilliermondii strain SO and its 3D structure as well as its catalytic attributes have not been fully understood yet, thus we seek to report on the catalytic mechanism of M. guilliermondii strain SO MgPRB1 using substrate PMSF via in silico docking as well as its stability by way of disulfide bonds formation. Herein, bioinformatics tools and techniques were used to predict, validate and analyze the possible changes of CUG ambiguity (if any) in strain SO using template PDB ID: 3F7O. Structural assessments confirmed the classic catalytic triad Asp305, His337, and Ser499. Superimposition of MgPRB1 and template 3F7O structures revealed the unlinked cysteine residues between Cys341, Cys440, Cys471 and Cys506 of MgPRB1 compared to template 3F7O with two disulfide bonds formation, which confers structural stability. In conclusion, serine protease structure from strain SO was successfully predicted and studies towards understanding at the molecular level may be undertaken for its potential applications in the degradation of peptide bonds.
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