Aims: The white rot fungus Pycnoporus cinnabarinus MUCL 39533 is able to reduce vanillic acid to vanillin. Reduction of vanillic acid to vanillin catalysed by the key enzyme aryl-aldehyde dehydrogenase has been reported. Here we report the isolation and cloning of aryl-aldehyde dehydrogenase from P. cinnabarinus strain MUCL 39533. Methodology and results: An aryl-aldehyde dehydrogenase gene (PcALDH) was isolated from P. cinnabarinus by producing a partial cDNA sequence fragment of an aryl-aldehyde dehydrogenase gene through PCR. Degenerate PCR primers were designed based on codons corresponding to conserved amino acid regions of aryl-aldehyde dehydrogenases of several fungi and bacteria. The full-length PcALDH cDNA was obtained through ReverseTranscription-Polymerase Chain Reaction (RT-PCR) and Rapid Amplification cDNA Ends (RACE) PCR. PcALDH cDNA comprises an open reading frame of 1,506 bp that encodes a protein of 501 amino acids. The PcALDH predicted protein showed the highest amino acid sequence identity (84%) to ALDH from Trametes versicolor. In silico analysis of PcALDH indicated that it belongs to the ALDH super-family and Class 3 ALDH. Conclusion, significance and impact study: PcALDH cDNA was successfully isolated and characterized. Important motifs identified from the highly conserved PcALDH protein indicated that it belongs to the aldehyde dehydrogenase superfamily. The cDNA clone will be used in expression studies to confirm the catalytic function of the enzyme.
Vanillic Acid ; Flavoring Agents

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.

Aims: The cellulase complexes of Trichoderma reesei are relatively low in endoglucanase and β-glucosidase activity compared with exoglucanase. The aim of this study is to determine the effect of Humicola insolens recombinant endoglucanase on the activities of commercial cellulases, Celluclast® and Acellerase® BG, during the hydrolysis of pretreated oil palm empty fruit bunch (OPEFB) fibres to simple sugars. Methodology and results: An endoglucanase (CMC3) from H. insolens ATTC 16454 was expressed in Pichia pastoris. The recombinant protein was purified and verified by SDS-PAGE and Western blot. The enzymatic hydrolysis of OPEFB fibres was carried out at 55 °C for 72 h and 1:40 and 1:100 mixtures of CMC3 and Celluclast® were used. All reaction mixtures were added with commercial β-glucosidase, Accelerase® BG, at a fixed concentration of 116 mg/mL. The sugars produced were analysed by high-performance liquid chromatography. Two sugar peaks were successfully resolved at different retention times and were identified as xylose and glucose. At Celluclast®-to-CMC3 activity ratio of 1:100, the highest reducing sugar concentration was obtained, whereby the glucose and xylose production increased by ~59% and ~27%, respectively. Conclusion, significance and impact of study Recombinant CMC3 can act synergistically with Celluclast® and Accelerase® BG to increase the production of glucose and xylose from pretreated OPEFB fibre. This study contributes greatly towards the development of efficient cellulase enzyme cocktail for the efficient hydrolysis of OPEFB biomass and the production of simple and fermentable sugars.

Aims: Short-chain fructo-oligosaccharides (scFOSs) are good prebiotics that enhance the growth of probiotic bacteria. The aim of this study is to determine the effect of scFOSs produced by levan hydrolysis using recombinant endo-levanase from B. lehensis G1 on the growth of probiotics isolated from commercially cultured milk drinks. Methodology and results: Two probiotic bacteria, Lactobacillus casei and L. rhamnosus, were isolated from commercially cultured milk drinks. ScFOSs were produced by levan hydrolysis using recombinant endo-levanase from B. lehensis G1. The scFOS and levan (control) were added independently to the growth medium, and the growth rates of the probiotic bacteria were determined. Results showed that the growth rate of L. casei decreased in the presence of levan compared with the control medium but increased by approximately 20% when supplemented with scFOS produced by Levblg1-N28S. Similarly, the growth rate of L. rhamnosus increased by approximately 20% when supplemented with scFOS produced by Levblg1 and Levblg1-N28S. Conclusion, significance and impact of study The scFOSs produced by the enzymatic hydrolysis of levan using a recombinant endo-levanase from B. lehensis G1 have significant potential as prebiotics because they were able to promote the growth of the probiotic bacteria.

Aims: The methylotrophic yeast Pichia pastoris is widely used to express foreign proteins fused to secretion signals. As the effect of the expression host on the final protein product is unclear, we compared the properties of an endoglucanase (eglB of Aspergillus niger) expressed in two different P. pastoris strains. Methodology and results: Full-length cDNA encoding endoglucanase of A. niger strain ATCC10574 was isolated and expressed in P. pastoris X33 (the methanol utilisation plus phenotype, Mut+) and P. pastoris GS115 (slow methanol utilisation, MutS). EglB-GS115 showed the highest activity and stability at 60 °C while EglB-X33 was most active at 50 °C. EglB-X33 was active towards other substrates such as arabinogalactan, guar gum and locust bean gum besides its specific substrate, carboxymethyl cellulose (CMC). However, EglB-GS115 was only active on CMC. The affinity of EglB-X33 towards CMC (Km = 7.5 mg/mL and specific activity 658 U/mg) was higher than that of EglB-GS115 (Km = 11.57 mg/mL, specific activity 144 U/mg). Conclusion, significance and impact of study Although eglB was cloned in the same expression vector (pPICZαC), two different characteristics of enzymes were recovered from the supernatant of the different hosts. Thus, expression of recombinant enzyme in different P. pastoris strains greatly affects the physical structure and biochemical properties of the enzyme.

Aims: The basal stem rot disease in oil palm is caused by the pathogenic Ganoderma boninense, which is infectious after mating and forming dikaryotic hyphae. This study was aimed to generate a mating-type biomarker for the detection of pathogenic Ganoderma species. Methodology and results: Mating-type region of Ganoderma was amplified using polymerase chain reaction (PCR) and primers flanking the mating-type region of other basidiomycetes. Amplified fragments were sequenced and were identified as the Ganoderma pheromone receptor gene of matB locus called the gprb2 gene. Using this biomarker, the pheromone receptor gene was detected in a total of 107 pathogenic Ganoderma spp. while the gene was not detected in the non-pathogenic Ganoderma lucidum. Phylogenetic tree analyses of the gene fragment encoding the partial amino acid sequence of gprb2 showed clades of close evolutionary relationship among the 107 pathogenic Ganoderma spp. Phylogenetic analyses using deduced amino acid sequences of the Ganoderma pheromone receptor b2 gene, gprb2 with homologous pheromone receptors of other basidiomycetous fungi revealed high conservation of this pheromone receptor within their respective taxonomy. Conclusion, significance and impact of study A potential mating-type biomarker was successfully identified that could detect pathogenic Ganoderma spp. The research findings will be helpful in oil palm screening to detect pathogenic Ganoderma spp. and gain further insight into the role of the mating-type loci of Ganoderma towards its pathogenesis in causing the basal stem rot disease of oil palm.
Genes, Mating Type, Fungal ; Ganoderma