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

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

Protein engineering of selected residues from conserved sequence regions of a novel Anoxybacillus α-amylase

The α-amylases from Anoxybacillus species (ASKA and ADTA), Bacillus aquimaris (BaqA) and Geobacillus thermoleovorans (GTA, Pizzo and GtamyII) were proposed as a novel group of the α-amylase family GH13. An ASKA yielding a high percentage of maltose upon its reaction on starch was chosen as a model to study the residues responsible for the biochemical properties. Four residues from conserved sequence regions (CSRs) were thus selected, and the mutants F113V (CSR-I), Y187F and L189I (CSR-II) and A161D (CSR-V) were characterised. Few changes in the optimum reaction temperature and pH were observed for all mutants. Whereas the Y187F (t1/2 43 h) and L189I (t1/2 36 h) mutants had a lower thermostability at 65°C than the native ASKA (t1/2 48 h), the mutants F113V and A161D exhibited an improved t1/2 of 51 h and 53 h, respectively. Among the mutants, only the A161D had a specific activity, kcat and kcat/Km higher (1.23-, 1.17- and 2.88-times, respectively) than the values determined for the ASKA. The replacement of the Ala-161 in the CSR-V with an aspartic acid also caused a significant reduction in the ratio of maltose formed. This finding suggests the Ala-161 may contribute to the high maltose production of the ASKA

Editorial: Genetics, genomics and -omics of thermophiles

Presentación de los contenidos de la revista.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Fermentaciones Industriale

Strep-tag ii mutant maltose-binding protein for reagentless fluorescence sensing

Maltose-binding protein (MBP) is a periplasmic binding protein found in Gram negative bacteria. MBP is involved in maltose transport and bacterial chemotaxis; it binds to maltose and maltodextrins comprising α(1-4)-glucosidically linked linear glucose polymers and α(1-4)-glucosidically linked cyclodextrins. Upon ligand binding, MBP changes its conformation from an open to a closed form. This molecular recognition-transducing a ligand-binding event into a physical one-renders MBP an ideal candidate for biosensor development. Here, we describe the construction of a Strep-tag II mutant MBP for reagentless fluorescence sensing. malE, which encodes MBP, was amplified. A cysteine residue was introduced by site-directed mutagenesis to ensure a single label attachment at a specific site with a thiol-specific fluorescent probe. An environmentally sensitive fluorophore (IANBD amide) was covalently attached to the introduced thiol group and analysed by fluorescence sensing. The tagged mutant MBP (D95C) was purified (molecular size, ∼42 kDa). The fluorescence measurements of the IANBD-labelled Strep-tag II-D95C in the solution phase showed an appreciable change in fluorescence intensity (dissociation constant, 7.6±1.75 μM). Our mutant MBP retains maltose-binding activity and is suitable for reagentless fluorescence sensin

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

Editorial: Genetics, genomics and -omics of thermophiles

Presentación de los contenidos de la revista.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Fermentaciones Industriale

Rational Mutagenesis of Cyclodextrin Glucanotransferase at the Calcium Binding Regions for Enhancement of Thermostability

Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as “parent CGTase” herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected

Impact of sterilization and chemical fertilizer on the microbiota of oil palm seedlings

Soil nutrients and microbiota are known as essential components for healthy plant growth and crop productivity. However, limited studies have been conducted on the importance of soil microbiota in the early growth of oil palm seedlings (Elaeis guineensis Jacq.) under the influence of nitrogen, phosphorus and potassium (NPK) compound fertilizer (nitrogen, phosphorus, and potassium). In this study, we analyzed the root microbial community associated with seedlings grown under normal and sterilized soil conditions to ascertain the microbial strains potentially associated with soil, plant health and chemical fertilizer efficiency. Oil palm seedlings were grown under four treatments: (i) fertilized normal soil (+FN), (ii) unfertilized normal soil (−FN), (iii) fertilized sterilized soil (+FS) and (iv) unfertilized sterilized soil (−FS). Our findings revealed that chemical fertilizer promoted the growth of the copiotrophs Pseudomonadota and Bacteroidota in the control +FN, which are known to degrade complex polysaccharides. After autoclaving, the soil macronutrient content did not change, but soil sterilization reduced microbial diversity in the +FS and −FS treatments and altered the soil microbiota composition. Sterilized soil with a depleted microbial population adversely affected crop growth, which was exacerbated by fertilizer use. In the rhizosphere and rhizoplane compartments, a total of 412 and 868 amplicon sequence variances (ASVs) were found depleted in the +FS and −FS treatments, respectively. Several genera were identified in the ASVs with diminished abundance, including Humibacter, Microbacterium, Mycobacterium, 1921-2, HSB OF53-F07, Mucilaginibacter, Bacillus, Paenibacillus, and several unclassified genera, suggesting their possible roles in promoting the plant growth of oil palm seedlings. Soil sterilization might remove these beneficial microbes from the bulk soil pool, affecting the colonization ability in the rhizocompartments as well as their role in nutrient transformation. Therefore, this study provides useful insights concerning the benefits of a soil microbiome survey before making fertilizer recommendations

Draft genome sequence of Cellulomonas sp. PS-H5, isolated from Sekinchan beach in Selangor, Malaysia

Cellulomonas sp. PS-H5 was isolated from Sekinchan Beach in Selangor, Malaysia, using an ex situ cultivation method. The present work reports a high-quality draft annotated genome sequence of this strain and suggests its potential glycoside hydrolase enzymes for cellulose, hemicellulose, and starch degradations