Ice-ice disease caused by pathogenic Vibrio spp. is a major constraint in the aquaculture systems of Kappaphycus alvarezii. The use of beneficial bacteria as biological control agents represents a sustainable alternative to chemical treatments in seaweed aquaculture. In this study, an endophytic bacterium isolated from healthy K. alvarezii thalli, designated VUMS1, was identified as Bacillus altitudinis based on 16 S rRNA gene analysis. VUMS1 exhibited strong antagonistic activity against Vibrio harveyi, V. owensii, and V. alginolyticus, producing inhibition zones ranging from 9.0 to 16.5 mm on Day 1 and increasing to 17.5–19.0 mm by Day 5. The strain also inhibited Staphylococcus haemolyticus, Shigella dysenteriae, and Enterococcus faecalis, with inhibition halos reaching up to 27 mm after 72 h. Co-culture assays demonstrated substantial suppression of Vibrio growth over time. Relative to the control, V. harveyi, V. owensii, and V. alginolyticus exhibited marked reductions in CFU/mL, reaching 90.2

Glaciozyma antarctica is an Antarctic psychrophilic yeast providing an excellent eukaryotic model for dissecting molecular mechanisms of cold adaptation. Yet, its extracellular protein repertoire, the interface between the cell and extreme surroundings, remains unexplored. Here, we present the first integrated bioinformatics–proteomics secretome map of G. antarctica, linking computational prediction with experimental validation under varying temperature stress, yielding 9 high-confidence experimentally supported secreted proteins. Three complementary pipelines (Phobius, SignalP/TMHMM, and WoLF PSORT) predicted 278 high-confidence secreted proteins, predominantly glycosyl hydrolases, lipases, oxidoreductases, and stress-response factors. To validate these predictions, we performed LC–MS/MS analysis of extracellular fractions from cultures exposed to − 12 °C, 0 °C, and 12 °C. A total of 104 secreted proteins was identified, including 55 shared across all temperatures and multiple unique cold-induced candidates such as antifreeze proteins and glycosidases. Comparative analysis uncovered a temperature-dependent remodeling of the secretome and revealed several proteins lacking canonical signal peptides, highlighting potential unconventional secretion mechanisms or moonlighting roles in extracellular environments. These findings demonstrate that G. antarctica adapts to freezing environments by dynamically reshaping its extracellular proteome to maintain structural plasticity and metabolic efficiency. This integrative approach identifies promising cold-active enzymes for potential application in the low-temperature biocatalysis industry.

Arginase is a metal-dependent metalloenzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea and is widely distributed across animals, bacteria, fungi and protozoa. Here, we report the first three-dimensional crystal structure of a cold-active arginase from the psychrophilic yeast Glaciozyma antarctica (GaArg). The apo structure was solved at 2.35 Å resolution in space group H3 by molecular replacement using an AlphaFold-generated model. GaArg adopts a conserved αβα sandwich fold similar to previously characterized arginases. The crystal structure reveals four regions lacking interpretable electron density, three of which are located near the entrance to the active site. Despite conservation of the identities and the coordination geometry of metal-binding residues, the apo GaArg structure exhibits broadly conserved ligand-binding residue orientations relative to homologous arginases, with minor conformational differences and a disordered loop corresponding to a substrate-interacting region. Analytical size-exclusion chromatography and multimer prediction support the hexameric assembly of GaArg in solution. Quantitative analysis of intramolecular interactions indicates that GaArg contains fewer hydrogen bonds than mesophilic and thermophilic homologues, while its salt-bridge content is comparable to that of the mesophilic enzyme but lower than that of the thermophilic homologue. These features are consistent with a modest reduction in structural rigidity associated with cold adaptation. Ligand- and metal-bound structures will be required to establish their contributions to cold-adaptation.

Carboxylic acid reductases (CARs) have been garnering attention in applications for the sustainable synthesis of aldehydes. Despite numerous discoveries, not all characteristics of CAR enzymes have been extensively studied or understood. Herein, we report the discovery and expression of a new CAR enzyme (TvirCAR2) from the ascomycetous fungus, Trichoderma virens. Tvircar2 is one of the five putative CARs identified from analyses of the T. virens genome. In silico, analyses showed that TvirCAR2 has a high hydrophobicity index and that its corresponding gene is part of a biosynthetic gene cluster predicted to synthesize hybrid polyketide synthases-nonribosomal peptide synthetase secondary metabolites. TvirCAR2 was highly expressed as soluble and insoluble forms in an Escherichia coli expression host. The solubility of the purified TvirCAR2 necessitated the addition of glycerol in the purification and assay buffers. Substrate screening via molecular docking showed that benzoic acid was a suitable substrate candidate. The TvirCAR2 enzyme catalyzed the reduction of benzoic acid with a specific activity of around 1.4 µmol/h/mg. Homologs, which are predicted to exhibit similar hydrophobicity, are the CARs from Stachybotrys bisbyi (StbB), which is involved in the production of the meroterpenoid, ilicicolin B, and Trichoderma reesei (TrCAR), which is part of a similar but still uncharacterized biosynthetic gene cluster.

Drought stress is the primary constraint on plant-based food production, particularly paddy production. Several studies have examined plant resistance to heat and osmotic pressure. This study aimed to isolate bacteria with plant growth-promoting properties that could tolerate high temperatures and improve paddy growth during drought. Five isolates with a high optical density value (OD600) at 30% PEG 6000 (equivalent to -1.03MPa) and able to grow at high temperatures were identified based on the 16S rRNA gene sequences as Achromobacter spanius UKM UR10, Bacillus pumillus UKM UR11, Bacillus cereus UKM R66, and Bacillus altitudinis UKM RB11, which were isolated from the root, where as Bacillus sp. UKM S8 was isolated from rhizosphere soil. These isolates exhibited 1-amino cyclopropane-1-carboxylate (ACC) deaminase activities ranging between 1.01 and 1.12 mmol α-ketobutyrate mg-1 protein h-1, which degraded ACC to α-ketobutyrate and ammonia. Other plant growth promoters assessed include indole acetic acid (IAA; concentration between 9.69 and 13.15µg/ml) and phosphate solubilization (concentrations between 31.74 and 51.30 mg/l) production. Subsequently, the selected plant growth-promoting rhizobacteria (PGPR) were incorporated as a consortium and inoculated on paddy seeds, thus increasing total chlorophyll, proline, and soluble sugar content in paddy subjected to drought-stress conditions. Paddy yield components and performances, such as panicle number, spikelet number, dry grain weight, number of leaves, stalk length, and root length increased significantly. This isolated PGPR exhibits heat resistance, promotes plant growth, and can serve as an inoculant for paddy plants under drought conditions.

Proteases are one of the most significant classes of enzymes, holding immense physiological relevance and extensive industrial applications. The genome of Glaciozyma antarctica was fully sequenced, showing 7,857 open reading frames that offer an intriguing opportunity to investigate its proteolytic repertoire. This study aims to unveil the protease landscape of G. antarctica, a psychrophilic yeast that produces cold-active enzymes that offer remarkable benefits, particularly in the food and pharmaceutical industries. In this work, we performed a comprehensive analysis to identify the diverse families of proteases encoded within the G. antarctica genome and compare them with proteases from other mesophilic and thermophilic fungi in the MEROPS database. The sequence similarity searches resulted in the identification of 195 open reading frames predicted to encode for proteases in G. antarctica with a high number of intracellular proteases. These findings suggest an evolved system for protein quality control and turnover, essential for cell viability and adaptation to environmental stressors. The MEROPS classification analysis showed an abundance of metalloproteases, constituting 38% of the total protease genes, a proportion surpassing that found in other yeast and fungal genomes studied. This reflects the vital role of metalloproteases in the cold adaptation of microbes in the Antarctic region. This unique profile not only sheds light on the adaptive mechanisms of psychrophilic organisms but also presents a rich reservoir of potential cold-active proteases for various applications. The findings of this study provide a foundation for targeted enzyme discovery and engineering, unlocking new frontiers in industrial biotechnology and extremophile biology.

Phytopathogens are causing global food security concerns, resulting in approximately 12.5% crop loss. These fungi significantly impact plant physiology, growth, and development. Traditional fungicides used for control are known to be harmful to both humans and the environment. Therefore, this study advocates an eco-friendly approach using biological control agents to curb phytopathogenic fungi growth. This research focuses on identifying potential antagonistic microorganisms capable of inhibiting two common phytopathogenic fungi: Magnaporthe oryzae, responsible for rice blast disease, and Fusarium solani, causing Fusarium wilt disease. The inhibitory strength of the microorganisms isolated from six different locations in Peninsula Malaysia was tested in vitro via dual culture assays. Our findings revealed three actinomycete species isolated from Bangi Forest Reserve, UKM, namely Streptomyces morookaense UKM1, Streptomyces rubrisoli UKM1, and Streptomyces gelaticus UKM1 exhibit a remarkable ability to inhibit the growth of both M. oryzae and F. solani, with a percentage inhibition radial growth (PIRG) exceeding 70%. Additionally, distinct differences in pathogens mycelia were observed after being grown together with the antagonistic microorganisms. In summary, our research identifies promising microorganisms with potent inhibition capabilities against multiple plant pathogens, offering potential solutions for sustainable agriculture and improved food security.

Antarctica is characterized by extreme cold, isolated, and unique ecosystems. Nevertheless, Antarctica harbors diverse species of microorganisms, particularly in its ice-covered lakes and subglacial environments. These microorganisms have special adaptations to extreme cold and low-nutrient conditions. Some extremophiles, like psychrophiles can thrive in these harsh environments. Phenoliferia glacialis USM-PSY62, previously identified as Rhodotorula sp. USM-PSY62 is a psychrophilic yeast isolated from the ice brine of Antarctica. However, there is very little information on this psychrophilic yeast. This study aims to characterize the P. glacialis USM-PSY62 through the identification of the optimum growth parameters in different media (Yeast Peptone Dextrose, YPD & Yeast Malt, YM), temperature (4 degrees C, 15 degrees C, 20 degrees C) and pH (6, 7, 8, 9) as well as their ability in carbon assimilation and extracellular enzyme production. It has an optimal growth in YPD compared to YM broth media. P. glacialis USM-PSY62 grows optimally at 15 degrees C and pH 7.0. This Antarctic yeast enters the stationary phase on day six of incubation under optimum conditions. It appeared mainly as elongated-shape and oval-shaped with budding formation and was found to produce extracellular enzymes such as protease and amylase in the presence of 2% glucose concentration in YM media. P. glacialis USM-PSY62 also can assimilate various types of carbon sources including raffinose, arabinose, and maltose. Interestingly, the psychrophilic yeast presented growth in media supplemented with Persistent Organic Pollutants (POPs) such as dichlorophenyldichloroethylene (DDE) and polychlorinated biphenyl (PCB). These preliminary findings suggest that P. glacialis USM-PSY62 has tremendous potential for bioremediation application in polluted cold regions, as well as deepening our knowledge of its optimal growth conditions.

Glaciozyma antarctica PI12 is a psychrophilic yeast isolated from Antarctica. In this work, we describe the heterologous production, biochemical properties and in silico structure analysis of an arginase from this yeast (GaArg). GaArg is a metalloenzyme that catalyses the hydrolysis of l-arginine to l-ornithine and urea. The cDNA of GaArg was reversed transcribed, cloned, expressed and purified as a recombinant protein in Escherichia coli. The purified protein was active against l-arginine as its substrate in a reaction at 20 °C, pH 9. At 10–35 °C and pH 7–9, the catalytic activity of the protein was still present around 50

G protein-coupled receptors (GPCRs) are integral components of eukaryotic heterotrimeric G proteins, playing crucial roles in detecting extracellular signals and initiating the activity of signaling proteins within cells to activate cellular responses to these signals. The objectives of this study are to identify and characterize the function of Git3, a Class III GPCR protein, in the oil palm pathogen Ganoderma boninense. To identify the potential genes encoded for GPCR in this fungus, intensive data mining on the genome and transcriptome data has been carried out. A total of six classes of GPCRs have been identified. These include Class II pheromone detectors, Class III carbon detectors, Class IV nitrogen detectors, Class VII proteins similar to glycosyltransferase, Class VIII proteins similar to hemolysin, and Class X protein receptors. Among these, the Class III protein Git3, postulated to be involved in glucose sensing and fungal pathogenicity, was selected for gene knockdown using RNA interference (RNAi). A plasmid, designated pUChph-GIT3, was constructed, to target git3 silencing by incorporating a hygromycin resistance gene cassette and antisense sequences of git3. Transformation of G. boninense PER71 with pUChph-GIT3 produced five potential Delta git3 gene-silenced mutants. PCR analysis confirmed the integration of the RNAi expression cassette into the fungal genome. Quantitative PCR (qPCR) analysis revealed significant reductions in git3 expression in three G. boninense mutants, M42, M66, and M5 by 47%, 23%, and 13%, respectively. The Disease Severity Index (DSI) indicated slower disease progression in oil palm plantlets infected with Delta git3 mutants compared to those infected with wild-type G. boninense PER71. In conclusion, this study successfully isolated and characterized the git3 GPCR from G. boninense and demonstrated that it might play a role during the early stages of infection, as the mutants were able to slow the progression of infection in oil palm plantlets.

Plants are known to release various volatile compounds under stress conditions.When inoculated on crops, plant-growth promoting Rhizobacteria (PGPR) produces volatile organic compounds (VOCs), thus enhancing crop seedling production, crop weight, crop yield, and stress resistance.In this study, non-inoculated and inoculated rice plants with a PGPR consortium were set up in glasshouses under drought and non-stress conditions.Drought stress was applied for six days, after which water was added to maintain plant growth.Under both conditions, 68 VOCs were found in rice leaves.The volatile organic compounds (VOCs) were quantified using the solid-phase microextraction technique paired with gas chromatography mass spectrometry (SPME-GCMS).Ethylene oxide (EO) was detected in drought-stressed plants compared to that of ethylene.The percentage of EO in non-inoculated rice was higher relative to inoculated rice plants under drought conditions.The identified VOCs in the inoculated and non-inoculated rice (drought stress and non-stress) belonged to the chemical classes of aldehyde, alcohol, terpene, ketone, ester, ether, amine esters, amides, and others.The enzymatic antioxidant activity of rice leaves was also determined to scavenge reactive oxygen species (ROS).Superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPX) levels significantly increased in their activities in inoculated rice compared to non-inoculated under drought stress.Our research focused on how drought affects plant metabolism above and below ground to adapt to a stressful environment.

Microbial proteases constitute one of the most important groups of industrially relevant enzymes. Proline iminopeptidases (PIPs) that specifically release amino-terminal proline from peptides are of major interest for applications in food biotechnology. Proline iminopeptidase has been extensively characterised in bacteria and filamentous fungi. However, no similar reports exist for yeasts. In this study, a protease gene from Glaciozyma antarctica designated as GaPIP was cloned and overexpressed in Escherichia coli. Sequence analyses of the gene revealed a 960 bp open reading frame encoding a 319 amino acid protein (35,406 Da). The purified recombinant GaPIP showed a specific activity of 3561 Umg−1 towards L-proline-p-nitroanilide, confirming its identity as a proline iminopeptidase. GaPIP is a cold-active enzyme with an optimum activity of 30 °C at pH 7.0. The enzyme is stable between pH 7.0 and 8.0 and able to retain its activity at 10–30 °C. Although GaPIP is a serine protease, only 25% inhibition by the serine protease inhibitor, phenylmethanesulfonylfluoride (PMSF) was recorded. This enzyme is strongly inhibited by the presence of EDTA, suggesting that it is a metalloenzyme. The dimeric structure of GaPIP was determined at a resolution of 2.4 Å. To date, GaPIP is the first characterised PIP from yeasts and the structure of GaPIP is the first structure for PIP from eukaryotes.

The use of cellulase enzymes in the degradation of lignocellulose agriculture biomass has long been studied and various efforts have been made to improve the efficiency of the hydrolysis process. The efficiency of enzymatic degradation of agricultural biomass to simple sugars requires a mixture of enzymes containing various types of cellulolytic activity. In this study, a recombinant multi-enzyme mixture consisting of three basic components of cellulase namely endoglucanase (EglB) and beta-glucosidase (BglA) from Aspergillus niger as well as cellobiohydrolase (CbhII) of Trichoderma virens was created specifically for hydrolysis of oil palm empty fruit bunch (OPEFB). The production of recombinant cellulases has been performed using Pichia pastoris expression host. The enzyme ratio optimisation was determined using Response Surface Methodology (RSM). The results showed that the hydrolysis of OPEFB at 50 degrees C and pH 5.0 using enzymes at 641.4 units CMCase: 10.14 Avicelase units: 93.8 beta-glucosidase units, produced the highest reducing sugar and glucose at 63 mg and 40 mg per gram of OPEFB substrate, respectively. The hydrolysis of OPEFB by a multi-enzyme mixture that has been formed in this study showed that these three combinations of recombinant enzymes have the potential to be used for the degradation of OPEFB.

Penggunaan enzim selulase untuk penguraian biojisim pertanian lignoselulosa telah lama dikaji dan pelbagai usaha telah dilakukan untuk meningkatkan kecekapan proses hidrolisis. Keberkesanan penguraian biojisim pertanian kepada gula ringkas memerlukan satu campuran enzim yang mengandungi pelbagai jenis aktiviti selulolitik. Dalam kajian ini, satu campuran multi-enzim rekombinan yang terdiri daripada tiga komponen asas selulase iaitu endoglukanase (EglB) dan β-glukosidase (BglA) daripada Aspergillus niger serta selobiohidrolase (CbhII) daripada Trichoderma virens telah dibentuk khusus untuk hidrolisis tandan kosong kelapa sawit (TKKS). Penghasilan enzim selulase rekombinan telah dilakukan menggunakan hos pengekspresan Pichia pastoris. Pengoptimuman nisbah enzim untuk tindak balas ditentukan menggunakan Kaedah Gerak Balas Permukaan (RSM). Hasil menunjukkan hidrolisis TKKS pada suhu 50 °C dan pH 5.0 menggunakan enzim pada nisbah 641.4 unit CMCase: 10.14 unit Avicelase: 93.8 unit β-glukosidase, menghasilkan gula terturun dan glukosa tertinggi, masing-masing sebanyak 63 mg dan 40 mg per gram substrat TKKS. Hasil hidrolisis TKKS oleh campuran multi enzim yang telah dibentuk dalam kajian ini menunjukkan ketiga-tiga gabungan enzim rekombinan ini berpotensi untuk digunakan bagi penguraian TKKS.

Basal stem rot (BSR) disease is a devastating threat to Malaysia oil palm plantation, which is caused by Ganoderma boninense fungi. Dikaryon is the dominant stage of G. boninense and the pathogenic form of the fungus as compared to the monokaryon. Mating signalling response lead by mitogen-activated pathway (MAPK) responsible in the transformation of sterile monokaryon to pathogenic dikaryon. Expression of the STE3 gene involved in the autocrine activation of mating pathway. Mutations of STE3 gene in some pathogenic yeast resulted in inhibition of pheromone responses and blocked the signalling in the cell fusion. Identification of pheromone receptor, STE3 gene involved in mating signalling pathway is important to understand the mating response towards pathogenesis of G. boninense. Genome walking and polymerase chain reaction (PCR) were carried out to amplify isolate the full length of STE3 gene. Several in silico sequence analysis were conducted to characterise the STE3 gene. The full-length gene sequence including the upstream and downstream regions obtained could be used in future studies to understand pathogenesis of G. boninense.

Proline iminopeptidase is an exopeptidase which catalyses the cleavage of prolines from the N-termini of peptides. In industries, this enzyme can be used as a biocatalyst especially in food processing such as in debittering of proteolysates and flavour development of cheeses. Thus for the purpose of applications, an enzyme engineering approach was undertaken to enhance the characteristics of Glaciozyma antarctica proline iminopeptidase through development of enzyme variants that possess optimum enzyme activities in lower temperatures. Six amino acid residues of G. antarctica proline iminopeptidase Glu189, Thr143, Ala195, Lue131, Lue144 and Ala277 were selected based on its low B-factor value determined via the B-FITTER programme and alignment guided approach. Sets of mutagenising primers were designed to introduce mutations at the chosen sites by randomisation using NNK degenerate primers. CASTER tool analyses determined that a minimum of 94 colonies should be screened in these focused libraries for a 95% coverage of the possible variants. This study provides valuable information in developing enhanced thermolability of proline iminopeptidase using directed evolution.

Studies on TCP1-1 ring complex (TRiC) chaperonin have shown its indispensable role in folding cytosolic proteins in eukaryotes. In a psychrophilic organism, extreme cold temperature creates a low-energy environment that potentially causes protein denaturation with loss of activity. We hypothesized that TRiC may undergo evolution in terms of its structural molecular adaptation in order to facilitate protein folding in low-energy environment. To test this hypothesis, we isolated G. antarctica TRiC (GaTRiC) and found that the expression of GaTRiC mRNA in G. antarctica was consistently expressed at all temperatures indicating their importance in cell regulation. Moreover, we showed GaTRiC has the ability of a chaperonin whereby denatured luciferase can be folded to the functional stage in its presence. Structurally, three categories of residue substitutions were found in α, β, and δ subunits: (i) bulky/polar side chains to alanine or valine, (ii) charged residues to alanine, and (iii) isoleucine to valine that would be expected to increase intramolecular flexibility within the GaTRiC. The residue substitutions observed in the built structures possibly affect the hydrophobic, hydrogen bonds, and ionic and aromatic interactions which lead to an increase in structural flexibility. Our structural and functional analysis explains some possible structural features which may contribute to cold adaptation of the psychrophilic TRiC folding chamber.

Aims: Subtilisin, a serine protease, is a key player in many industrial applications especially in the detergent industry. Most reported subtilisins originate from mesophilic and thermophilic microorganisms. Only scarce information about cold-active subtilisins from psychrophilic microbes is available. Here we describe the isolation, cloning and in silico characterisation of a gene encoding subtilisin in the obligate psychrophilic yeast, Glaciozyma antarctica PI12. Methodology and results: A full-length cDNA from Glaciozyma antarctica encoding subtilisin (GaSUB) was isolated through Reverse-Transcription-Polymerase Chain Reaction (RT-PCR) techniques. The open reading frame of GaSUB comprised 1,125 nucleotides encoding 375 amino acids. The GaSUB amino acid sequence had 49% sequence identity with a subtilisin from the yeast, Puccinia striiformis. Bioinformatic analyses revealed that the GaSUB protein contains a domain that represents the S8 domain of the largest protease family. The predicted model of GaSUB protein using MODELLER and Pymol software revealed that this enzyme has longer loops and less intramolecular interactions between amino acid residues as compared to its mesophilic and thermophilic counterparts. These characteristics are known to help in protein flexibility and stability in cold-active enzymes. Conclusion, significance and impact of study: Bioinformatics characterisations suggested that this enzyme is uniquely adapted to cold environments. Further work using amplified cDNA will be conducted to confirm the catalytic function of this enzyme.

Cellobiohydrolases catalyze the processive hydrolysis of cellulose into cellobiose. Here, a Trichoderma virens cDNA predicted to encode for cellobiohydrolase (cbhI) was cloned and expressed heterologously in Aspergillus niger. The cbh1 gene has an open reading frame of 1518 bp, encoding for a putative protein of 505 amino acid residues with a calculated molecular mass of approximately 54 kDa. The predicted CbhI amino acid sequence has a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region and showed high sequence homology with glycoside hydrolase family 7 proteins. The partially purified enzyme has an optimum pH of 4.0 with stability ranging from pH 3.0 to 6.0 and an optimum temperature of 60 degrees C. The partially purified CbhI has a specific activity of 4.195 Umg(-1) and a low K-m value of 1.88 mM when p-nitrophenyl-beta-D-cellobioside (pNPC) is used as the substrate. The catalytic efficiency (k(cat)/K-m) was 5.68 x 10(-4) mM(-1) s(-1), which is comparable to the CbhI enzymes from Trichoderma viridae and Phanaerochaete chrysosporiwn. CbhI also showed activity towards complex substrates such as Avicel (0.011 Umg(-1)), which could be useful in complex biomass degradation. Interestingly, CbhI also exhibited a relatively high inhibition constant (K-i) for cellobiose with a value of 8.65 mM, making this enzyme more resistant to end-product inhibition compared to other fungal cellobiohydrolases.