EXPRESSION AND ENZYMATIC PROPERTIES OF A UNIQUE RECOMBINANT ANTICOAGULANT AND FIBRINOLYTIC ENZYME FROM ACINETOBACTER BAUMANNII TU04

Objective: The objective of this research is to clone and express a new fibrinolytic enzyme encoding serine protease gene in Escherichia coli thus, characterize such purified recombinant.Methods: The recombinant clone was successfully expressed in Lemo21 system and purified using immobilized nickel cation affinity chromatography on a His•bind resin®, followed by ammonium sulfate precipitation and protein filtration in combination. General properties of the purified enzyme were investigated, including the molecular weight, effects of inhibitors and metal ions, substrate specificity, amylolytic activity, fibrinolytic activity and effect of anticoagulant activity in-vitro.Results: The recombinant clone was expressed in Lemo21 system in the cytoplasm in a soluble and active form. The resulting enzyme, SERpro was purified to homogeneity with a purification of 19.35-fold and recovery yield of 4.85%. The enzyme exhibited maximal activity at 37 °C and at pH7.4, respectively. The molecular weight of the purified enzyme was 82 kDa, determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fibrinogenolysis peptide sequence analysis revealed that SERpro degraded Bβ chain of Fibrin at a much lower rate but cleaved Aα and γ-chain extensively. The enzyme was activated by metal ions such as Mg2+, Fe3+and Zn2+, and was inhibited strongly by PMSF. The clotting time of human blood serum in the presence of 1U SERpro reached a relative partial thromboplastin time of 13.9% with a 1.14-fold increase.Conclusion: The study deduced SERpro as a new protease with anti-thrombotic activity from Acinetobacter baumannii TU04.Â

Identification and characterization of potential useful bacteria from marine environment

Marine environment remained as largely unexplored source for researchers to discover novel properties from marine organisms which can benefits human kind. The study aims to isolate marine bacterium from various source of marine environment. Six bacterial strains were successfully isolated from marine samples from seashore of the Desaru, Malaysia and identified by 16S rRNA sequencing. The characterizations of bacterial strains were also performed based morphological tests, Gram staining, biochemical tests and antibiotic sensitivity against several antibiotics by disc diffusion method. The 16S rRNA sequence of D-2, D-4, D-7, D-15, D-31 and D-33 revealed a high identity of 97 to 99% with 16S rRNA sequence belong to genera Pseudomonas, Marinomonas, Exiquobacterium,Micrococcus, Pseudoalteromonasand Shewanella respectively. Strain D-31 exhibited higher tolerance towards antibiotic with resistance to Kanamycin, Ampicillin and Erythromycin. However, the growth of other strains was retarded by at least two of the antibiotics on their normal growth. The isolation of marine bacterial strain belongs to Marinomonas sp. and Pseudoalteromonassp. create of interest for further biological characterization as the strains from these two genera have been proven for the discovery of new antimicrobial substances, enzymes for industry application and unique secondary metabolites

Antimicrobial activity of copper kaolinite and surfactant modified copper kaolinite against gram positive and gram negative bacteria

The aim of this research was to determine the antimicrobial activity of kaolinite modified with antimicrobial compounds against Gram positive and Gram negative bacteria. Copper kaolinite (Cu-kaolinite) was prepared by loading raw kaolinite with copper nitrate trihydrate (CuNO3) while surfactant modified Cu-kaolinite (SM-Cu-kaolinite) was prepared by adding cationic surfactants hexadecyltrimethyl ammonium (HDTMA) on Cu-Kaolinite. Samples was characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analyzer. The antimicrobial activity of the samples was tested against Gram negative bacteria (Escherichia coli ATCC 11229 and Pseudomonas aeruginosa ATCC 15442), and Gram positive bacteria (Staphylococcus aureus ATCC 6538 and Enterococcus faecalis ATCC 29212) through disc diffusion technique (DDT) and minimum inhibition concentration (MIC). The results showed that the antimicrobial activity of Cu-kaolinite increased after modified with HDTMA due to the synergistic effects of Cu ions and HDTMA molecules on the kaolinite. The antimicrobial activity for surfactant modified Cu-kaolinite was greater for Gram positive bacteria compared to Gram negative bacteria. In conclusion, the attachment of HDTMA on Cu-kaolinite contributed to the enhanced antimicrobial activity against wide spectrum of bacteria (Gram positive and Gram negative bacteria)

Insights into microbial community structure and diversity in oil palm waste compost

Empty fruit bunch (EFB) and palm oil mill effluent (POME) are the major wastes generated by the oil palm industry in Malaysia. The practice of EFB and POME digester sludge co-composting has shown positive results, both in mitigating otherwise environmentally damaging waste streams and in producing a useful product (compost) from these streams. In this study, the bacterial ecosystems of 12 week-old EFB-POME co-compost and POME biogas sludge from Felda Maokil, Johor were analysed using 16S metagenome sequencing. Over 10 phyla were detected with Chloroflexi being the predominant phylum, representing approximately 53% of compost and 23% of the POME microbiome reads. The main bacterial lineage found in compost and POME was Anaerolinaceae (Chloroflexi) with 30% and 18% of the total gene fragments, respectively. The significant differences between compost and POME communities were abundances of Syntrophobacter, Sulfuricurvum, and Coprococcus. No methanogens were identified due to the bias of general 16S primers to eubacteria. The preponderance of anaerobic species in the compost, and high abundance of secondary metabolite fermenting bacteria is due to an extended composting time, with anaerobic collapse of the pile in the tropical heat. Predictive functional profiles of the metagenomes using 16S rRNA marker genes suggest the presence of enzymes involved in polysaccharide degradation such as glucoamylase, endoglucanase, arabinofuranosidase, all of which were strongly active in POME. Eubacterial species associated with cellulytic methanogenesis were present in both samples

Global Transcriptomic Responses of Roseithermus sacchariphilus Strain RA in Media Supplemented with Beechwood Xylan

The majority of the members in order Rhodothermales are underexplored prokaryotic extremophiles. Roseithermus, a new genus within Rhodothermales, was first described in 2019. Roseithermus sacchariphilus is the only species in this genus. The current report aims to evaluate the transcriptomic responses of R. sacchariphilus strain RA when cultivated on beechwood xylan. Strain RA doubled its growth in Marine Broth (MB) containing xylan compared to Marine Broth (MB) alone. Strain RA harbors 54 potential glycosyl hydrolases (GHs) that are affiliated with 30 families, including cellulases (families GH 3, 5, 9, and 44) and hemicellulases (GH 2, 10, 16, 29, 31,43, 51, 53, 67, 78, 92, 106, 113, 130, and 154). The majority of these GHs were upregulated when the cells were grown in MB containing xylan medium and enzymatic activities for xylanase, endoglucanase, β-xylosidase, and β-glucosidase were elevated. Interestingly, with the introduction of xylan, five out of six cellulolytic genes were upregulated. Furthermore, approximately 1122 genes equivalent to one-third of the total genes for strain RA were upregulated. These upregulated genes were mostly involved in transportation, chemotaxis, and membrane components synthesis

Characterization of β-glucosidases from Meridianimaribacter sp. CL38

The production of second-generation biofuel requires a huge amount of freshwater. It is estimated that at least three gal of freshwater is used to produce one gal of biofuel. The replacement of freshwater with seawater serves as a potential alternative in biofuel generation. Therefore, salt-tolerant enzymes play an important role in saccharification and fermentation process. Halophilic β-glucosidase is one of the key enzymes for the process. In this study, the β-glucosidase of halophile Meridianimaribacter sp. CL38 isolated from mangrove soil was characterized. Strain CL38 achieved maximum production of β-glucosidase at 12th hour of growth. The β-glucosidase showed highest activity at 2% (w/v) NaCl while highly stable at salt concentration ranging from 1-2% (w/v) (more than 96% of relative activity). Its β-glucosidase activity remained active in the presence of 5mM Mn2+, Mg2+, Ca2+ ions, and 1% (v/v) Tween-20 and Tween-80. The draft genome sequence of strain CL38 was retrieved from GenBank database and submitted to dbCAN meta server for CAZymes annotation. Strain CL38 harbors 44 GHs and GH3 are annotated as β-glucosidases. The β-glucosidases of Meridianimaribacter flavus (99.61%) and Mesoflavibacter sabulilitoris (97.44%) showed the closest identity with Bgl3a and Bgl3b protein sequences from strain CL38, respectively. Glycoside hydrolase family 3 domain was identified in both enzymes via InterPro scan server. The presence of signal peptides indicated that both enzymes were secreted extracellularly. Five motifs were identified in Bgl3a and Bgl3b, with the active site (nucleophile) found at Asp296 and Asp297, respectively. Collectively, these β-glucosidases could be potentially used in the biofuel production, in particular the lignocellulosic biomass pretreatment process. This is the first attempt to characterize the β-glucosidase in genus Meridianimaribacter as so far none of the lignocellulolytic enzymes from this genus were characterized

Revealing the potential of xylanase from a new halophilic microbulbifer sp. CL37 with paper de-inking ability

Paper de-inking is one of the critical processes in pulp and paper industry as it is ecofriendly and energy saving. This process requires microbial enzymes such as xylanases with ability to withstand harsh bioprocess conditions. Microbulbifer is a halophilic genus with ability to produce hydrolytic enzymes that could be applied in the biotechnological industry. So far, none of the xylanases from this genus have been studied, particularly in paper de-inking process. Therefore, in this study, the xylanase of a new halophilic bacterium, Microbulbifer sp. strain CL37, was characterized. Strain CL37 produced maximum amount of xylanase at 14th hour of incubation at 30 °C. The xylanase demonstrated optimal activity at 70 °C and pH 7. The xylanase was stable at wide range of NaCl (0–14%, w/v), in the presence of Al3+, Ca2+, Co2+, Cu+, Cu2+, Fe2+, Fe3+, Mn2+, Zn2+, acetone, chloroform, ethanol, sodium deoxycholate, Triton X-100, Tween 20, 40, 60, and 80, indicating that it is a halotolerant enzyme with high stability in various additives. The xylanase also demonstrated its ability to de-ink paper with considerably high efficiency (159%) as compared to other strains. The valuable characteristics possessed by xylanase of strain CL37 could potentially benefit to de-inking process in paper industry

Characterizing a Halo-Tolerant GH10 Xylanase from Roseithermus sacchariphilus Strain RA and Its CBM-Truncated Variant

A halo-thermophilic bacterium, Roseithermus sacchariphilus strain RA (previously known as Rhodothermaceae bacterium RA), was isolated from a hot spring in Langkawi, Malaysia. A complete genome analysis showed that the bacterium harbors 57 glycoside hydrolases (GHs), including a multi-domain xylanase (XynRA2). The full-length XynRA2 of 813 amino acids comprises a family 4_9 carbohydrate-binding module (CBM4_9), a family 10 glycoside hydrolase catalytic domain (GH10), and a C-terminal domain (CTD) for type IX secretion system (T9SS). This study aims to describe the biochemical properties of XynRA2 and the effects of CBM truncation on this xylanase. XynRA2 and its CBM-truncated variant (XynRA2∆CBM) was expressed, purified, and characterized. The purified XynRA2 and XynRA2∆CBM had an identical optimum temperature at 70 ◦C, but different optimum pHs of 8.5 and 6.0 respectively. Furthermore, XynRA2 retained 94% and 71% of activity at 4.0 M and 5.0 M NaCl respectively, whereas XynRA2∆CBM showed a lower activity (79% and 54%). XynRA2 exhibited a turnover rate (kcat) of 24.8 s−1, but this was reduced by 40% for XynRA2∆CBM. Both the xylanases hydrolyzed beechwood xylan predominantly into xylobiose, and oat-spelt xylan into a mixture of xylo-oligosaccharides (XOs). Collectively, this work suggested CBM4_9 of XynRA2 has a role in enzyme performance

Physical and antioxidative responses of Orthosiphon stamineus towards various copper and lead concentrations

Plants normally change their physiological and biochemical properties when exposed to heavy metal stress. We investigated the response of Orthosiphon stamineus towards different concentrations of Pb (0, 2, 5, 8 mg/L) and Cu (1, 2, 4, 5 mg/L). Heavy metals left in soil, plant physical characteristics, and the level of antioxidants in O. stamineus were determined. Our results showed that the tested Pb concentrations did not significantly affect stem elongation, but at 2 mg/L, Pb increased the leaf growth. Pb at 5 and 8 mg/L increased the total plant biomass. In contrast, 5 mg/L Cu treatment affected stem elongation and the root length of O. stamineus. The concentrations of Pb and Cu in soil were significantly reduced after the plants were harvested. Biochemically, 5 mg/L Pb had significantly increased the activity of catalase, while Cu at 5 mg/L significantly reduced the activity of superoxide dismutase and ascorbate peroxidase. Total flavonoid content increased in Pb-treated plants, but the total phenolics content decreased. Cu treatment at 2 mg/L, on the other hand increased the total phenolics content. Our results demonstrated that O. stamineus adapt to metal stress via physical changes, and scavenge oxygen radicals through enzymatic and non-enzymatic antioxidant productions