Hairul Azman Roslan

Sago palm (Metroxylon sagu Rottb.) is one of the most important cash crops grown abundantly in Mukah, Sarawak, Malaysia. The palm’s versatility and high starch content led the Sarawak government to introduce large scale commercial sago estate plantations in efforts to boost the local economy. However, several challenges were encountered, including stunted growth (non-trunking) despite the plants reaching maturity. In spite of many researches to address this, information on the molecular mechanisms of sago palms is relatively low. This study compares the transcriptome datasets expressed in trunking sago palms of different localities. The RNA extracted was subjected to RNA sequencing. Raw data was processed with fastp, generating 85.61 Gb clean data, averaging 7 Gb data from each sample. The clean reads were subjected to assembly and mapping using Salmon. Expression count tables were used for DEGs identification using idep.96 webserver. A total of 718 DEGs were commonly expressed in all datasets. Enrichment analysis revealed gene products responsible for maintaining cell stability were enriched in GO, whereas the MAPK and ribosome pathways were significantly enriched in KEGG. The data obtained from this study can be considered for further analysis and understanding,especially toward the genes responsible for normal development in sago palms.

Fermentation food has been widely consumed since ancient times due to its many beneficial properties as well as improved texture and taste. Raw sago starch “lemantak” (RSS “lemantak”) is one of the staple foods in the state of Sarawak. Derived from the bark of Metroxylon sagu, this starch has yet to be investigated in terms of its phytochemical and nutraceutical properties after being subjected to fermentation. This study investigated the nutraceutical of fermented RSS “lemantak” in its phenolic content and antioxidant activity in different fermentation conditions by selected endophytic fungi, Aspergillus niger, Penicillium chermesinum and Fusarium equiseti. This study compared two different fermentation modes and also investigated phenolic production by manipulating the fungal consortia, incubation period as well as the solvent used. It was determined that the best endophyte culture was consortia of Aspergillus niger and Penicillium chermesinum under solid state fermentation (SSF) while monoculture Aspergillus niger was the best fungal culture under SMF. It was found that the dual cultures of Aspergillus niger and Penicillium chermesinum (AP) showed high total phenolic content of 174.95 mg GAE/g and high antioxidant activity at different concentrations ranging from 90.21-76.17% under solid-state fermentation at day 7. In contrast, under submerged fermentation, it was found that consortia of Aspergillus niger and Penicillium chermesinum produced low phenolic content of 11.54 mg GAE/g and had lower antioxidant capacity with the range values of 39.26-34.03%. On the other hand, monoculture Aspergillus niger strives best under SMF at 203.65 mg GAE/g and has good antioxidant activity of 90.72-14.15% of scavenging activity. Nevertheless, it was determined that the dual culture of AP showed high potential based on the phenolic content antioxidant activity under SSF while monoculture A. niger showed potential as a phenolic content producer and antioxidant activity. This study ascertained that the best conditions for fermented RSS “lemantak” with higher phenolic content and antioxidant properties are the methanol extract of fermented RSS inoculated with dual cultures Aspergillus niger and Penicillium chermesinum for a period of 7 days of incubation under SSF.

This research evaluates the bioactivity of twelve endophytic fungi successfully isolated and characterised from Gynura procumbens. The fungal extracts displayed inhibitory activity against Staphylococcus aureus, Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli and Salmonella typhi with the MIC and MBC of 5000 µg/mL. High antioxidant activity using DPPH free radical scavenging assay with inhibition of 86.6% and IC₅₀ value of 104.25 ± 18.51 µg/mL were exhibited by ethyl acetate extract of Macrophomina phaseolina SN6. In contrast, the highest scavenging activity percentage of methanolic extract was exhibited by Mycoleptodiscus indicus SN4 (50.0%). Besides that, the highest ferric reducing antioxidant power (FRAP) value of ethyl acetate and methanolic extract was recorded from M. phaseolina SN6 (239.9 mg Fe (II)/g) and M. indicus SN4 (44.7 mg Fe (II)/g), respectively. Total phenolic content (TPC) and total flavonoid content (TFC) of ethyl acetate and methanolic fungal extracts were determined using Folin-Ciocalteu and aluminium chloride, respectively. The highest TPC for ethyl acetate and methanolic extracts were exhibited by Colletotrichum gloeosporioides SN11 (87.0 mg GAE/g) and M. indicus SN4 (35.0 mg GAE/g), whereas the highest TFC of ethyl acetate and methanolic extracts were showed by M. phaseolina SN6 (122.8 mg QCE/g) and M. indicus SN4 (60.4 mg QCE/g), respectively. Bioactive metabolites of isoelemicin (50.8%), terpinen-4-ol (21.5%), eucalyptol (24.3%), oleic acid (19.8%) and β-pinene (10.9%) were detected. Owing to the higher content of phytochemicals represented in the ethyl acetate extract of M. phaseolina, SN6 is therefore identified to be a superior candidate in exhibiting strong antioxidant and antimicrobial properties be fit for further pharmaceutical studies.

In this study, an alpha-amylase enzyme from a locally isolated Aspergillus flavus NSH9 was purified and characterized. The extracellular α-amylase was purified by ammonium sulfate precipitation and anion-exchange chromatography at a final yield of 2.55-fold and recovery of 11.73%. The molecular mass of the purified α-amylase was estimated to be 54 kDa using SDS-PAGE and the enzyme exhibited optimal catalytic activity at pH 5.0 and temperature of 50 °C. The enzyme was also thermally stable at 50 °C, with 87% residual activity after 60 min. As a metalloenzymes containing calcium, the purified α-amylase showed significantly increased enzyme activity in the presence of Ca²⁺ ions. Further gene isolation and characterization shows that the α-amylase gene of A. flavus NSH9 contained eight introns and an open reading frame that encodes for 499 amino acids with the first 21 amino acids presumed to be a signal peptide. Analysis of the deduced peptide sequence showed the presence of three conserved catalytic residues of α-amylase, two Ca²⁺-binding sites, seven conserved peptide sequences, and several other properties that indicates the protein belongs to glycosyl hydrolase family 13 capable of acting on α-1,4-bonds only. Based on sequence similarity, the deduced peptide sequence of A. flavus NSH9 α-amylase was also found to carry two potential surface/secondary-binding site (SBS) residues (Trp 237 and Tyr 409) that might be playing crucial roles in both the enzyme activity and also the binding of starch granules.

Fructose-1,6-bisphosphate aldolase (FBAld) is an enzyme that catalyzes the cleavage of D-fructose-1,6-phosphate (FBP) to D-glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), and plays vital role in glycolysis and gluconeogenesis. However, molecular characterization and functional roles of FBAld remain unknown in sago palm. Here we report a modified CTAB-RNA extraction method was developed for the isolation of good quality RNA (RIN>8) from sago leaves and the isolation of FBAld cDNA from sago palm. The isolated sago FBAld (msFBAld) cDNA has total length of 1288 bp with an open reading frame of 1020 bp and a predicted to encode for a protein of 340 amino acid resides. The predicted protein shared a high degree of homology with Class-I FBAld from other plants. Meanwhile, the msFBAld gene spanned 2322 bp and consisted of five exons. Conserved domain search identified fifteen catalytically important amino acids at the active site and phylogenetic tree revealed localization of msFBAld in the chloroplast. A molecular 3D-structure of msFBAld was generated by homology modeling and a Ramachandran plot with 86.7% of the residues in the core region, 13.4% in the allowed region with no residues in the disallowed region. The modeled structure is a homotetramer containing an (/(-TIM-barrel at the center. Superimposition of the model with Class-I aldolases identified a catalytic dyad, Lys209-Glu167, which could be involved in the Schiff's base formation and aldol condensation. Apart from that, overproduction of the recombinant msFBAld in Escherichia coli resulted in increased tolerance towards salinity.

Enrichment culture technique leads to the discovery of six presumptive TPH-degrading bacteria. Identification and characterization tests using morphological, biochemical and molecular techniques have successfully isolated Pseudomonas aeruginosa (UMAS1PF), Serratia marcescens (UMAS2SF) and Klebsiella spp. (UMAS3KF). All strains were able to use crude oil as sole carbon and energy source for their growth since they were able to survive in Minimal Salt medium supplemented with 1% (v/v) crude oil. Growth study showed that they produced the highest cell counts on the third or fourth day by 108 – 1011 CFU/ml. Six artificial consortium inoculums have been produced from the growth study. Gas chromatography analysis showed that all isolates had the ability to degrade aliphatic hydrocarbon with 100% degradation of nC19 – C24. Among the isolates, UMAS2SF was the best and fastest n-alkane degrader with degradation percentage between 55 – 90% of n-C14 – C18 in 14 days. This was followed by UMAS1PF and UMAS3KF with 11 – 82% and 1.3% degradation, respectively. Enhancement study showed that plot with inoculum and NPK addition successfully enhanced n-alkane degradation. Plot A2:B3+NPK degraded n-alkane the fastest followed by plot treated by C+NPK, A1:B2, B+NPK and A2:B3. Result showed that UMAS1PF was the best PAHs degrader as most of the high molecular weight PAHs was degraded. In the enhancement study, the plot amended with A2:B3 showed the highest PAHs degradation, followed by plots A1:B2, A3:B1:C2 and A1:C3 that was assigned as the third, fourth and fifth best in mineralizing PAHs, respectively.