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  1. 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.

  2. 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.
  3. Fulltext Optimization of physical conditions for the production of thermostable T1 lipase in Pichia guilliermondii strain SO using response surface methodology
    Abu ML, Nooh HM, Oslan SN, Salleh AB
    BMC Biotechnol, 2017 Nov 10;17(1):78.
    PMID: 29126403 DOI: 10.1186/s12896-017-0397-7
    BACKGROUND: Pichia guilliermondii was found capable of expressing the recombinant thermostable lipase without methanol under the control of methanol dependent alcohol oxidase 1 promoter (AOXp 1). In this study, statistical approaches were employed for the screening and optimisation of physical conditions for T1 lipase production in P. guilliermondii.

    RESULT: The screening of six physical conditions by Plackett-Burman Design has identified pH, inoculum size and incubation time as exerting significant effects on lipase production. These three conditions were further optimised using, Box-Behnken Design of Response Surface Methodology, which predicted an optimum medium comprising pH 6, 24 h incubation time and 2% inoculum size. T1 lipase activity of 2.0 U/mL was produced with a biomass of OD600 23.0.

    CONCLUSION: The process of using RSM for optimisation yielded a 3-fold increase of T1 lipase over medium before optimisation. Therefore, this result has proven that T1 lipase can be produced at a higher yield in P. guilliermondii.

  4. The use of response surface methodology for enhanced production of a thermostable bacterial lipase in a novel yeast system
    Abu ML, Mohammad R, Oslan SN, Salleh AB
    Prep Biochem Biotechnol, 2021;51(4):350-360.
    PMID: 32940138 DOI: 10.1080/10826068.2020.1818256
    A thermostable bacterial lipase from Geobacillus zalihae was expressed in a novel yeast Pichia sp. strain SO. The preliminary expression was too low and discourages industrial production. This study sought to investigate the optimum conditions for T1 lipase production in Pichia sp. strain SO. Seven medium conditions were investigated and optimized using Response Surface Methodology (RSM). Five responding conditions namely; temperature, inoculum size, incubation time, culture volume and agitation speed observed through Plackett-Burman Design (PBD) method had a significant effect on T1 lipase production. The medium conditions were optimized using Box-Behnken Design (BBD). Investigations reveal that the optimum conditions for T1 lipase production and Biomass concentration (OD600) were; Temperature 31.76 °C, incubation time 39.33 h, culture volume 132.19 mL, inoculum size 3.64%, and agitation speed of 288.2 rpm with a 95% PI low as; 12.41 U/mL and 95% PI high of 13.65 U/mL with an OD600 of; 95% PI low as; 19.62 and 95% PI high as; 22.62 as generated by the software was also validated. These predicted parameters were investigated experimentally and the experimental result for lipase activity observed was 13.72 U/mL with an OD600 of 24.5. At these optimum conditions, there was a 3-fold increase on T1 lipase activity. This study is the first to develop a statistical model for T1 lipase production and biomass concentration in Pichia sp. Strain SO. The optimized production of T1 lipase presents a choice for its industrial application.
  5. A Novel Mini Protein Design of Haloalkane Dehalogenase
    Daud NH, Leow TC, Oslan SN, Salleh AB
    Mol Biotechnol, 2019 Mar 27.
    PMID: 30919327 DOI: 10.1007/s12033-019-00169-3
    The application of native enzymes may not be economical owing to the stability factor. A smaller protein molecule may be less susceptible to external stresses. Haloalkane dehalogenases (HLDs) that act on toxic haloalkanes may be incorporated as bioreceptors to detect haloalkane contaminants. Therefore, this study aims to develop mini proteins of HLD as an alternative bioreceptor which was able to withstand extreme conditions. Initially, the mini proteins were designed through computer modeling. Based on the results, five designed mini proteins were deemed to be viable stable mini proteins. They were then validated through experimental study. The smallest mini protein (model 5) was chosen for subsequent analysis as it was expressed in soluble form. No dehalogenase activity was detected, thus the specific binding interaction of between 1,3-dibromopropane with mini protein was investigated using isothermal titration calorimetry. Higher binding affinity between 1,3-dibromopropane and mini protein was obtained than the native. Thermal stability study with circular dichroism had proven that the mini protein possessed two times higher Tm value at 83.73 °C than the native at 43.97 °C. In conclusion, a stable mini protein was successfully designed and may be used as bioreceptors in the haloalkane sensor that is suitable for industrial application.
  6. Recent insight into the advances and prospects of microbial lipases and their potential applications in industry
    Eskandari A, Leow TC, Rahman MBA, Oslan SN
    Int Microbiol, 2024 Mar 15.
    PMID: 38489100 DOI: 10.1007/s10123-024-00498-7
    Enzymes play a crucial role in various industrial sectors. These biocatalysts not only ensure sustainability and safety but also enhance process efficiency through their unique specificity. Lipases possess versatility as biocatalysts and find utilization in diverse bioconversion reactions. Presently, microbial lipases are gaining significant focus owing to the rapid progress in enzyme technology and their widespread implementation in multiple industrial procedures. This updated review presents new knowledge about various origins of microbial lipases, such as fungi, bacteria, and yeast. It highlights both the traditional and modern purification methods, including precipitation and chromatographic separation, the immunopurification technique, the reversed micellar system, the aqueous two-phase system (ATPS), and aqueous two-phase flotation (ATPF), moreover, delves into the diverse applications of microbial lipases across several industries, such as food, vitamin esters, textile, detergent, biodiesel, and bioremediation. Furthermore, the present research unveils the obstacles encountered in employing lipase, the patterns observed in lipase engineering, and the application of CRISPR/Cas genome editing technology for altering the genes responsible for lipase production. Additionally, the immobilization of microorganisms' lipases onto various carriers also contributes to enhancing the effectiveness and efficiencies of lipases in terms of their catalytic activities. This is achieved by boosting their resilience to heat and ionic conditions (such as inorganic solvents, high-level pH, and temperature). The process also facilitates the ease of recycling them and enables a more concentrated deposition of the enzyme onto the supporting material. Consequently, these characteristics have demonstrated their suitability for application as biocatalysts in diverse industries.
  7. 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.
  8. Advances in Therapeutic Cancer Vaccines, Their Obstacles, and Prospects Toward Tumor Immunotherapy
    Eskandari A, Leow TC, Rahman MBA, Oslan SN
    Mol Biotechnol, 2024 Apr 16.
    PMID: 38625508 DOI: 10.1007/s12033-024-01144-3
    Over the past few decades, cancer immunotherapy has experienced a significant revolution due to the advancements in immune checkpoint inhibitors (ICIs) and adoptive cell therapies (ACTs), along with their regulatory approvals. In recent times, there has been hope in the effectiveness of cancer vaccines for therapy as they have been able to stimulate de novo T-cell reactions against tumor antigens. These tumor antigens include both tumor-associated antigen (TAA) and tumor-specific antigen (TSA). Nevertheless, the constant quest to fully achieve these abilities persists. Therefore, this review offers a broad perspective on the existing status of cancer immunizations. Cancer vaccine design has been revolutionized due to the advancements made in antigen selection, the development of antigen delivery systems, and a deeper understanding of the strategic intricacies involved in effective antigen presentation. In addition, this review addresses the present condition of clinical tests and deliberates on their approaches, with a particular emphasis on the immunogenicity specific to tumors and the evaluation of effectiveness against tumors. Nevertheless, the ongoing clinical endeavors to create cancer vaccines have failed to produce remarkable clinical results as a result of substantial obstacles, such as the suppression of the tumor immune microenvironment, the identification of suitable candidates, the assessment of immune responses, and the acceleration of vaccine production. Hence, there are possibilities for the industry to overcome challenges and enhance patient results in the coming years. This can be achieved by recognizing the intricate nature of clinical issues and continuously working toward surpassing existing limitations.
  9. 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.
  10. 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.
  11. 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.
  12. 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.
  13. Heterologous Expression of PA8FAD9 and Functional Characterization of a Δ9-Fatty Acid Desaturase from a Cold-Tolerant Pseudomonas sp. A8
    Garba L, Ali MS, Oslan SN, Rahman RN
    Mol Biotechnol, 2016 Nov;58(11):718-728.
    PMID: 27629791
    Fatty acid desaturase enzymes are capable of inserting double bonds between carbon atoms of saturated fatty acyl-chains to produce unsaturated fatty acids. A gene coding for a putative Δ9-fatty acid desaturase-like protein was isolated from a cold-tolerant Pseudomonas sp. A8, cloned and heterologously expressed in Escherichia coli. The gene named as PA8FAD9 has an open reading frame of 1185 bp and codes for 394 amino acids with a predicted molecular weight of 45 kDa. The enzyme showed high Δ9-fatty acid desaturase-like protein activity and increased overall levels of cellular unsaturated fatty acids in the recombinant E. coli cells upon expression at different temperatures. The results showed that the ratio of palmitoleic to palmitic acid in the recombinant E. coli cells increased by more than twice the amount observed in the control cells at 20 °C using 0.4 mM IPTG. GCMS analysis confirmed the ability of this enzyme to convert exogenous stearic acid to oleic acid incorporated into the recombinant E. coli membrane phospholipids. It may be concluded that the PA8FAD9 gene from Pseudomonas sp. A8 codes for a putative Δ9-fatty acid desaturase protein actively expressed in E. coli under the influence of temperature and an inducer.
  14. Genomic and phenomic analysis of a marine bacterium, Photobacterium marinum J15
    Roslan NN, Ngalimat MS, Leow ATC, Oslan SN, Baharum SN, Sabri S
    Microbiol Res, 2020 Mar;233:126410.
    PMID: 31945517 DOI: 10.1016/j.micres.2020.126410
    Photobacterium species are widely distributed in the marine environment. The overall metabolism of this genus remains largely unknown. In order to improve our knowledge on this bacterium, the relationship between the genome and phenome of the Photobacterium isolate was analyzed. The cream colored, Gram-negative, rod-shaped and motile bacterial strain, J15, was isolated from marine water of Tanjung Pelepas, Johor, Malaysia. The 5,684,538 bp genome of strain J15 comprised 3 contigs (2 chromosomes and 1 plasmid) with G + C content of 46.39 % and contained 4924 protein-coding genes including 180 tRNAs and 40 rRNAs. The phenotypic microarray (PM) as analyzed using BIOLOG showed the utilization of; i) 93 of the 190 carbon sources tested, where 61 compounds were used efficiently; ii) 41 of the 95 nitrogen sources tested, where 22 compounds were used efficiently; and iii) 3 of the 94 phosphorous and sulphur sources tested. Furthermore, high tolerance to osmotic stress, basic pH and toxic compounds as well as resistance to antibiotics of strain J15 were determined by BIOLOG PM. The ANI and kSNP analyses revealed that strain J15 to be the same species with Photobacterium marinum AK15 with ANI value of 96.93 % and bootstrapping value of 100 in kSNP. Based on the ANI and kSNP analyses, strain J15 was identified as P. marinum J15.
  15. 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.
  16. 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.

  17. 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.
  18. 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.
  19. 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.
  20. 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.
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