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A number of protocols have been reported for efficient fungal DNA and RNA isolation. However, many of these methods are often designed for certain groups or morphological forms of fungi and, in some cases, are species dependent. In this report, we compared four published protocols for DNA isolation from a locally isolated oleaginous fungus, Cunninghamella bainieri strain 2a1. These protocols either involved the use of polyvinyl pyrrolidone (PVP), hexacetyltrimethylammonium bromide (CTAB) or without using PVB or CTAB. For RNA isolation, we tested two published protocols, one of which is based on TRI REAGENT (Molecular Research Center, USA) and another is simple method employing phenol for RNA extraction and LiCl for precipitation. We found that the protocol involving the use of CTAB produced the highest genomic DNA yield with the best quality compared to other protocols. In the presence of CTAB, unwanted polysaccharides were removed and this method yielded an average amount of 816 ± 12.2 µg DNA/g mycelia with UV absorbance ratios A260/280 and A260/230 of 1.67 ± 0.64 and 1.97 ± 0.23, respectively. The genomic DNA isolated via this protocol is also suitable for PCR amplification and restriction enzyme digestion. As for RNA isolation, the method involving phenol extraction and LiCl precipitation produced the highest yield of RNA with an average amount of 372 ± 6.0 µg RNA/g mycelia. The RNA appears to be relatively pure since it has UV absorbance ratios A260/280 and A260/230 of 1.89 ± 2.00 and 1.99 ± 0.03, respectively. Finally, we have demonstrated that this method could produce RNA of sufficient quality for RT-PCR that amplified a 600 bp fragment of ∆12-fatty acid desaturase gene in C. bainieri.

In this study, types of porous metal-organic framework materials namely MOF-5 (robust framework), MOF-199 (open framework) in the non ion-exchanged condition was compared in terms of hydrophobicity-hydrophilicity of the surface. The investigations were done using microcalorimetric measurements of heat of immersions in water. MOF- 5 and MOF-199 were synthesized using plant-based polymer of palm oil derived fatty alcohols (PODFA) as porous template using “green” sol-gel approach. The synthesis approach applied was environmental-friendly by eliminating the use of organic solvents and surfactants substituting with water and PODFA. X-ray diffraction patterns of MOF-199 exhibited prominent reflection peaks assigned to octahedral structure with small peak shift while MOF-5 exhibited truncated and full cubic morphology. PODFA with longer aliphatic carbon chain exhibited higher porosity and pore volume. The addition of PODFA increased the homogeneity of the cubic and octahedral single crystals typically observed in MOF-5 and MOF-199 morphology. This was due to the increased in immiscibility of metal oxide precursors and organic linkers hence allowing reactions to occur from all directions. PODFA molecules were suggested to form self-assemblies at the air-water interface governed by surface tension auto-oscillations and formed clusters on which metal oxide and organic linker precursors bind with. The water adsorption isotherms of MOF-5 and MOF-199 prepared in this study exhibited “gate-opening” effect at lower relative pressure not observed in other nanoporous framework structure made up of two-components such as organosilica materials. [ABSTRACT FROM AUTHOR]

Ice recrystallization during thawing post-cryopreservation results in extensive cellular damage and ultimately leads to cell death and reduced cell viability. Antifreeze proteins (AFPs) are a group of proteins that allow organisms to survive in subzero environments. These proteins have thermal hysteresis and ice recrystallization inhibitory activities. In this present study, we demonstrated the efficiency of a recombinant antifreeze protein from the Antarctic yeast, Glaciozyma antarctica, as a recrystallization inhibitor (RI) of ice growth and assessed its application as a cryopreservative of the fungal cutinase enzyme against freeze-thaw cycles. Recombinant Afp1 from G. antarctica, a psychrophilic yeast, has been produced in a methylotrophic yeast, Pichia pastoris, system that results in the expression of a hyper-glycoprotein (~55 kDa). Recombinant Afp1 exhibits antifreeze functions: thermal hysteresis (TH) and recrystallization inhibition where the highest TH values recorded for ~0.5°C at 10 mg/mL. The cryoprotective effects of Afp1 on purified recombinant cutinase showed that Afp1 can retain enzymatic activity up to ~20% when subjected to several cycles of freeze thawing. These findings indicate that Afp1 might act as a cryoprotective agent and thus, has great potential in biotechnology applications. [ABSTRACT FROM AUTHOR]

Chitinases are a group of glycosyl hydrolases that are essential to recycle chitin presence in nature. The aim of this work is to characterise the sequence of a chitinase isolated from a psychrophilic yeast, Glaciozyma antarctica PI12 and to predict and analyse the 3-dimensional protein structure. The cDNA for the G. antarctica chitinase gene, GaCHT43, with a length of 1,176 bp was reverse transcribed from mRNA, cloned and sequenced. The gene encodes a mature protein of 391 amino acids with an expected molecular weight of 43 kDa. Sequence analysis showed that GaCh43 has high similarity to the endochitinase family 18 proteins of other fungi. A three-dimensional (3D) model of GaCht43 was built by homology modelling with Aspergillus fumigatus chitinase (1W9P) as the template. Validation analysis via PROCHECK, VERIFY3D and ERRAT showed that the GaCht43 model surpassed the quality requirements and was accepted for further analysis. GaCht43 contained chitinase conserved regions, SxGG and DxxDxDxE that are required in the catalytic mechanism. Analysis of the GaCht43 structure showed the presence of extra loop regions compared to mesophilic chitinases, which might contribute to the flexibility of the protein. [ABSTRACT FROM AUTHOR]

Glycoside hydrolases are enzymes that hydrolyse glycosidic bonds in carbohydrate chains to produce simple molecules. In fungi, glycoside hydrolases are important enzymes that hydrolyse complex carbohydrates into simple sugars that can subsequently be consumed for energy metabolism. Glaciozyma antarctica is a psychrophilic yeast isolated from sea ice in Antarctica. The G. antarctica genome has been completely sequenced, and a total of 7,857 genes have been predicted. The objective of the present study was to determine different classes of glycoside hydrolases from the G. antarctica genome and predict the localisation of these enzymes. Using genome mining, a total of 97 G. antarctica genes were predicted to encode glycoside hydrolases. The majority of the enzymes, including endoglucanases, xylanases, and chitinases, were identified from GH family 5 (12 genes), followed by GH family 45 (11 genes), GH family 10 (11 genes) and GH family 18 (9 genes). The secreted glycoside hydrolase enzymes were primarily endoglucanases from GH family 45, and these enzymes degrade celluloses in the cell walls of plants and algae. Extracellular glycoside hydrolases have been implicated as important in nutrient scavenging and organic decomposition in Antarctic sea ice. [ABSTRACT FROM AUTHOR]

Glaciozyma antarctica is a psychrophilic yeast isolated from the Antarctic sea ice. In this study, we performed a de novo characterisation of molecular chaperones from G. antarctica genome datasets. A total of 7857 genes that code for various types of proteins have been predicted from the G. antarctica genome sequence. From these genes, we identified 89 possible molecular chaperones that matched known molecular chaperones from other organisms available in various databases such as Uniprot, Gene Ontology, cpnDB and NCBI. For an in-depth analysis of molecular functions, we used homologous clustering to transfer knowledge from unknown to known functions using Cluster Analysis of Sequences (CLANS) bioinformatics software. The results reveal 12 major groups of chaperones that contribute to the cold-adaptation mechanism through their molecular function, biological processes and cellular components. The findings lay the foundation for future functional genomics studies on this organism and shed light on how lower eukaryotic cells respond to low temperature. [ABSTRACT FROM AUTHOR]

To survive in extremely cold environments, psychrophilic microorganisms produce exopolysaccharides (EPS), which are carbohydrate polymers that constitute a substantial component of the extracellular polymers surrounding cells of these microorganisms. EPS can interfere with RNA extraction and decrease the purity of the RNA extracted from EPS producing microorganisms. In this work, six commercial RNA extraction kits and two published protocols for RNA extraction were evaluated for total RNA extraction from the psychrophilic yeast, Glaciozyma antarctica. All the protocols were optimised to obtain the highest quality of total RNA. The results show that all of the tested commercial kits and the tested conventional methods yielded RNA from G. antarctica, albeit with varying quality. The protocol that utilises TRIzol® reagent was the most effective method for isolating total RNA from G. antarctica of which this protocol resulted in the highest RNA yield and purity compared to other methods. This method of RNA extraction produced RNA of sufficient quality for reverse transcriptase PCR (RT-PCR) to detect the expression of the G. antarctica delta 9-fatty acid desaturase gene as well as for the construction of a G. antarctica cDNA library. [ABSTRACT FROM AUTHOR]

A Pichia pastoris transformant carrying the cutinase cDNA of Glomerella cingulata was over-expressed in a 5 L bioreactor (2.0 L working volume) under fed-batch conditions. Bioreactor experiments rely on varying selected parameters in repeated rounds of optimisation: here these included duration of induction, pH and temperature. Highest cell densities (320 g L-1 wet cell weight) with a cutinase production of 3800 mg L-1 and an activity of 434 U mL-1 were achieved 24 h after induction with methanol in basal salt medium (at pH 5 and 28 °C). Characterisation of the cutinase showed that it was stable between pH 6 and pH 11, had an optimum pH of 8.0 and retained activity for 30 min at 50 °C (optimum temperature 25 °C).The preferred substrates of G. cingulata cutinase were the medium- to long-chain ρ-nitrophenyl esters of ρ-nitrophenylcaprylate (C8), ρ-nitrophenyllaurate (C12) and ρ-nitrophenylmyristate (C14), with the highest catalytic efficiency, kcat/Km of 7.7 ± 0.7 mM-1 s-1 for ρ-nitrophenylcaprylate. Microscopic analyses showed that the G. cingulata cutinase was also capable of depolymerising the high molecular weight synthetic polyester, polyethylene terephthalate. [ABSTRACT FROM AUTHOR]

Cellulose nanocrystal is a nanomaterial that has a large specific surface area, high surface energy, and high strength. As well, it is biocompatible, environmentally friendly, nontoxic, and can be extracted from biomass resources. Because of these features, cellulose nanocrystals can be used to improve the mechanical properties of polymer matrices with a shape memory effect and as a shape memory switch. In this study, a polytrimethylene ether glycol-based thermoplastic polyurethane (TPU)/cellulose nanocrystal (CNC) composite was prepared via an in-situ polymerization process to create a self-healing polymer matrix. Also, the effect of CNC doses in low concentrations (≤2 wt%) on the different properties of the resulting bio-nanocomposite was investigated. The results showed that the introduction of CNCs affects the hydrogen bonding within the polymer matrix and provides better thermal stability in the high temperature range than pure TPU. Furthermore, the samples with 0 wt%, 0.75 wt%, 1 wt%, and 2 wt% of CNC exhibited an increasing trend in tensile strength with values of 11.71 MPa, 18.95 MPa, 17.88 MPa, and 26.18 MPa, respectively, which indicates a remarkable improvement in mechanical strength. The shape memory behavior was also notably prominent in this polymer composite, where the composite containing 2 wt% of CNC showed the fastest recovery time (240 s) at 75 °C with the highest shape retention. Moreover, their flow behavior and deformation capacity were examined through rheology tests. Besides, docking simulations were conducted in silico to assess the interaction of the TPU/CNC composite with the DNA gyrase enzyme. The interaction between CNC/TPU composite and DNA gyrase was meticulously analyzed across 10 distinct conformations, yielding docking scores ranging from -6.5 Kcal/mol to -5.3 Kcal/mol. Overall, the physico-mechanical properties of the TPU/CNC composites were substantially enhanced with the incorporation of nanofillers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Conventional culture method and biochemical tests remain as the 'gold standard' method for the identification of S. sonnei which are time-consuming. We have discovered previously the potential of three OMPs of S. sonnei (33.3 kDa, 43.8 kDa and 100.3 kDa) as biomarkers in the diagnostic test for shigellosis. Here, we evaluated the performance of the outer membrane proteins of S. sonnei for the development of an antibody-based immunoassay for the detection of S. sonnei infections. All threetarget proteins were specifically recognized when probed with S. sonnei sera. In addition, another two potential proteins of molecular weight 29.0 kDa and 88.2 kDa in size were also exclusively recognized by the IgA when probed with S. sonnei sera. The optimized ELISA demonstrated higher sensitivity and specificity which exceeded 86.0%. In conclusion, the identified target proteins showed great potential as diagnostic biomarkers for the detection of S. sonnei infections in patients.

The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.

Escherichia coli-the powerhouse for recombinant protein production-is rapidly gaining status as a reliable and efficient host for secretory expression. An improved understanding of protein translocation processes and its mechanisms has inspired and accelerated the development of new tools and applications in this field and, in particular, a more efficient secretion signal. Several important characteristics and requirements are summarised for the design of a more efficient signal peptide for the production of recombinant proteins in E. coli. General approaches and strategies to optimise the signal peptide, including the selection and modification of the signal peptide components, are included. Several challenges in the secretory production of recombinant proteins are discussed, and research approaches designed to meet these challenges are proposed.