purification-identification-antimicrobial-protein-from-green-alga

Purification and Identification of Antimicrobial Protein from the Green Alga <em>Tetraspora</em> Sp. CU2551

Antimicrobial resistance (AMR) is an escalating global health threat, necessitating the discovery of novel antimicrobial agents. Algae-derived proteins ...

King Mongkut's Institute of Technology Ladkrabang

Genetic studies are important to understand the complex biological system of various organisms. Some eukaryotic green organisms have tough cell wall which precludes the efficient extraction of the genetic materials. Here, we developed the method for simple and rapid isolation of high quality DNA from a green alga Tetraspora sp. CU2551. The cell homogenization procedures were combined with physical force plus heat treatment to disrupt the cell envelope of Tetraspora sp. CU2551. Without protease treatment, vortexing with glass bead for 30-105 s at 70 °C led to the isolation of a high purity DNA which was suitable for downstream process. The improved method was successfully developed and could be applied for the rapid isolation of DNA from other unicellular and filamentous green microalgal strains.

Rapid method for DNA isolation from a tough cell wall green alga Tetraspora sp. CU2551.

Dog blood parasites are important tick-borne diseases causing morbidity and mortality in dogs worldwide. Four dog blood parasites species are commonly found in Thailand: , and . They are transmitted easily by tick species. However, there is little prevalence data available in Thailand. Diseases presentation of blood parasites infection is similar, but the treatment of each species is different. Current diagnosis mainly relies on microscopic examination of a stained blood smear, which has low sensitivity. Therefore, accurate diagnosis is important. This study aims to evaluate the efficacy of the conventional polymerase chain reaction (PCR) method and routine blood smears in the detection of four blood parasites species in dogs from Buriram Province, Thailand.

Comparison of conventional polymerase chain reaction and routine blood smear for the detection of , , and  in Buriram Province, Thailand.

Canine monocytic ehrlichiosis caused by Ehrlichia canis infection is a life-threatening vector-borne disease in dogs worldwide. Routine blood smear has very low sensitivity and cannot accurately provide a quantitative result. Conventional PCR (cPCR) and real-time PCR (qPCR) are widely used as molecular methods for E. canis detection. qPCR is quantitative but relies on standard curves of known samples. To overcome this difficulty, this study developed a new E. canis quantitative detection method, using droplet digital polymerase chain reaction (ddPCR). ddPCR was evaluated against cPCR and blood smears. PCR amplicons and genomic DNA (gDNA) from 12 microscopic positive samples were used to identify the limits of detection (LODs) in ddPCR and cPCR. Our ddPCR was assessed in 92 field samples, it was compared with cPCR and blood smears. ddPCR showed LOD=1.6 copies/reaction, or 78 times more sensitive than cPCR (LOD=126 copies/reaction), using PCR amplicons as a template, whereas both ddPCR and cPCR had equal LODs at 0.02 ng gDNA/reaction. In addition, ddPCR had 100% sensitivity and 75% specificity for E. canis detection compared to cPCR and no cross-reaction with other blood pathogens was observed. ddPCR identified more positive samples than cPCR and blood smear. ddPCR improved the overall performance of E. canis detection, with a better LOD and comparable sensitivity and specificity to cPCR. The technique might be helpful for diagnosis of E. canis in light infection, evaluating the number of E. canis and follow-up after treatment.

A newly developed droplet digital PCR for Ehrlichia canis detection: comparisons to conventional PCR and blood smear techniques.

The green algae Tetraspora sp. CU2551 was previously identified as a strain with high potential for biohydrogen production; however, its algal biomass characteristics changed from green to reddish orange within 43 days of biohydrogen production. The crude pigments were extracted, partially purified, and characterized by chemical determination. The present study focused on elucidating the carotenoid composition of the selected green alga Tetraspora sp. CU2551. The pigment extract was partially purified and fractionated using thin layer chromatography, and yielded two major and two minor carotenoid bands. The fractions were confirmed by high-performance liquid chromatography with a diode array detector (HPLC-DAD) before being identified and confirmed using Liquid Chromatograph-Quadrupole Time of Flight-Mass Spectrometry (LC-QTOF-MS). The spectral data of these fractions revealed four sub-fractions of interest that were lutein, canthaxanthin, neochrome, and β-carotene, which had percentages in the crude extracts of 30.57%, 25.47%, 7.89%, and 0.71%, respectively. Lutein and canthaxanthin were found to be the major carotenoid pigments present. Our findings in this present study are the first reporting of Tetraspora sp. CU2551 as a potential alternate source for carotenoid pigment production.

Identification of major carotenoids from green alga Tetraspora sp. CU2551: partial purification and characterization of lutein, canthaxanthin, neochrome, and β-carotene.

Shrimp farming wastewater includes high amounts of phosphate and microbiological contaminants, necessitating further treatment before release into receiving water bodies. After 24 h of shrimp wastewater treatment, alginate beads containing the blue-green algal Synechocystis strain lacking the phosphate regulator gene (mutant strain ΔSphU) at 150 mg L reduced phosphate content from 17.5 mg L to 5.0 mg L, representing 71.5% removal efficiency, with phosphate removal rate reaching 6.9 mg gDW h during photobioreactor operation. For short-term treatment, removal rates of nitrate, ammonium and nitrite were 42.7, 48.5 and 92.9%, respectively. Microalgal encapsulated beads also impacted the bacterial community composition dynamics in shrimp wastewater. Next-generation sequencing targeting the V3-V4 region of the 16S rDNA gene showed significant differences in bacterial community composition after 24 h of treatment. Proteobacteria are the most abundant phylum in shrimp wastewater. After 24 h of bioremediation, reductions of harmful bacteria in the Cellvibrionaceae and Pseudomonadaceae families were recorded at 5.85 and 3.18%, respectively. Engineered microalgal immobilization under optimal conditions can be applied as an alternative short-term bioremediation strategy to remove phosphate and other harmful microbial contamination from shrimp farming wastewater.

Highly effective reduction of phosphate and harmful bacterial community in shrimp wastewater using short-term biological treatment with immobilized engineering microalgae.

 and  are tick-borne, Gram-negative bacteria that cause canine monocytic ehrlichiosis and canine cyclic thrombocytopenia, respectively. These diseases are of great importance and are distributed globally. This study aimed to create new primers for the identification of  and  in naturally infected dogs using polymerase chain reaction (PCR), DNA sequencing, and phylogenetic analysis using the  rDNA and  genes.

Molecular detection and phylogeny of  and  in naturally infected dogs in Central and Northeast Thailand.

Hydrogen gas (H) is one of the potential future sustainable and clean energy carriers that may substitute the use of fossil resources including fuels since it has a high energy content (heating value of 141.65 MJ/kg) when compared to traditional hydrocarbon fuels [1]. Water is a primary product of combustion being a most significant advantage of H being environmentally friendly with the capacity to reduce global greenhouse gas emissions. H is used in various applications. It generates electricity in fuel cells, including applications in transportation, and can be applied as fuel in rocket engines [2]. Moreover, H is an important gas and raw material in many industrial applications. However, the high cost of the H production processes requiring the use of other energy sources is a significant disadvantage. At present, H can be prepared in many conventional ways, such as steam reforming, electrolysis, and biohydrogen production processes. Steam reforming uses high-temperature steam to produce hydrogen gas from fossil resources including natural gas. Electrolysis is an electrolytic process to decompose water molecules into O and H. However, both these two methods are energy-intensive and producing hydrogen from natural gas, which is mostly methane (CH) and in steam reforming generates CO and pollutants as by-products. On the other hand, biological hydrogen production is more environmentally sustainable and less energy intensive than thermochemical and electrochemical processes [3], but most concepts are not yet developed to production scale.

Photobiohydrogen Production and Strategies for H Yield Improvements in Cyanobacteria.

Correction to: Photobiohydrogen Production and Strategies for H Yield Improvements in Cyanobacteria.

Canine babesiosis, caused by the protozoan parasite , is characterized by clinical manifestations, including hemolytic anemia, thrombocytopenia, multiple organ failure, and may result in death. This disease is detected using conventional blood smears, which are time-consuming and have low sensitivity. This study aimed to investigate a more rapid and sensitive method for detecting  infection in dogs by examining the expressed serum protein profiles using proteomics.

Proteomics identification of overexpressed serum proteins in dogs with  infection.

Glycogen and poly-3-hydroxybutyrate (PHB) are excellent biopolymer products from cyanobacteria. In this study, we demonstrate that nitrogen metabolism is positively influenced by the exogenous application of trehalose (Tre) in  under nitrogen-deprived (-N) conditions. Cells were cultivated photoautotrophically for 5 days under -N conditions, with or without the addition of exogenous Tre. The results revealed that biomass and chlorophyll- content of  experienced enhancement with the addition of 0.003 M and 0.03 M Tre in the -N medium after one day, indicating relief from growth inhibition caused by nitrogen deprivation. The highest glycogen content (54.09 ± 1.6% (/) DW) was observed in cells grown for 2 days under the -N + 0.003 M Tre condition ( < 0.05), while the highest PHB content (15.2 ± 0.2% (/) DW) was observed in cells grown for 3 days under the -N + 0.03 M Tre condition ( < 0.05). The RT-PCR analysis showed a significant increase in  and  transcript levels, representing approximately 1.2- and 1.3-fold increases, respectively, in  grown under -N + 0.003 M Tre and -N + 0.03 M Tre conditions. This was accompanied by the induction of enzyme activities, including glycogen synthase and PHA synthase with maximal values of 89.15 and 0.68 µmol min mg protein, respectively. The chemical structure identification of glycogen and PHB from  was confirmed by FTIR and NMR analysis. This research represents the first study examining the performance of trehalose in promoting glycogen and PHB production in cyanobacteria under nitrogen-deprived conditions.

Exogenous Trehalose Improves Growth, Glycogen and Poly-3-Hydroxybutyrate (PHB) Contents in Photoautotrophically Grown  under Nitrogen Deprivation.

Limnophila aromatica, a traditional medicinal plant, has been previously reported to possess notable antimicrobial properties. However, the specific bioactive constituents responsible for this activity remain largely unidentified. This study aimed to isolate, identify, and evaluate the antibacterial potential of compounds from a 100% ethanolic extract of L. aromatica. The crude ethanolic extract exhibited the highest antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA) and was subsequently subjected to Diaion HP-20 column chromatography, followed by preparative HPLC. Seven major peaks were identified using UV-Vis spectra, LC-QTOF-MS, and NMR analyses. The antibacterial efficacy of these isolated compounds was assessed using disc diffusion and broth microdilution assays against Bacillus subtilis, S. aureus, MRSA, Escherichia coli, and Pseudomonas aeruginosa. The isolated compounds were identified as norethindrone acetate, isothymusin, nevadensin, gardenin B, 5,3'-dihydroxy-7,8,2'-trimethoxyisoflavone, jasmolin II, and oleanolic acid. Nevadensin, the predominant compound (79.55%), demonstrated potent bactericidal activity against B. subtilis, S. aureus, and MRSA. Jasmolin II and gardenin B also exhibited promising antibacterial effects. Although the disc diffusion assay was limited by compound diffusion, the broth microdilution method confirmed significant MIC values, ranging from 0.59 to 2.86 mg/ml while the MBC values ranged from 0.59 to 12.72 mg/ml. This study highlights nevadensin and other flavonoids from L. aromatica as potential candidates for the development of alternative antibacterial therapies, particularly against drug-resistant Gram-positive bacteria.

Discovery and characterization of bioactive compounds from Limnophila aromatica: nevadensin and related flavonoids as potent antimicrobial agents.

Chitin, a long-chain polysaccharide, is a major component in the exoskeletons of arthropods and the cell walls of fungi. Its derivative, chitosan, is widely used in various fields due to its solubility and versatility. This study focuses on the purification and biochemical characterization of exo-1,4-β-D-glucosaminidase from Amycolatopsis sp. KLSc63, an enzyme crucial for the degradation of chitin and chitosan. The enzyme was purified using anion exchange chromatography and characterized for its activity on colloidal chitin and chitosan solutions. Optimal activity was observed at pH 5.0 and temperatures of 40-45 °C for colloidal chitin and pH 4.0-6.0 at 55 °C for chitosan solution. The enzyme's molecular weight was approximately 94 kDa. Various metal ions and surfactants significantly influenced enzyme activity, with Mn²⁺ at 1 mM concentration notably enhancing both activities. Structural modeling and docking studies confirmed the enzyme's substrate specificity and binding interactions. These findings highlight the potential applications of exo-1,4-β-D-glucosaminidase in industrial processes, waste management, and the production of bioactive compounds.

Purification, characterization, and structural insights of Exo-1,4-β-D-glucosaminidase from Amycolatopsis sp. KLSc63: predictive modeling with glucosamine and N-acetyl glucosamine dimers.

Article	Year
Rapid method for DNA isolation from a tough cell wall green alga Tetraspora sp. CU2551. World journal of microbiology & biotechnology\| PubMed ID: 27116965	2016
Comparison of conventional polymerase chain reaction and routine blood smear for the detection of , , and in Buriram Province, Thailand. Veterinary world\| PubMed ID: 31327907	2019
A newly developed droplet digital PCR for Ehrlichia canis detection: comparisons to conventional PCR and blood smear techniques. The Journal of veterinary medical science\| PubMed ID: 35473801	2022
Identification of major carotenoids from green alga Tetraspora sp. CU2551: partial purification and characterization of lutein, canthaxanthin, neochrome, and β-carotene. World journal of microbiology & biotechnology\| PubMed ID: 35689122	2022
Highly effective reduction of phosphate and harmful bacterial community in shrimp wastewater using short-term biological treatment with immobilized engineering microalgae. Journal of environmental management\| PubMed ID: 36257228	2023
Molecular detection and phylogeny of and in naturally infected dogs in Central and Northeast Thailand. Veterinary world\| PubMed ID: 36718324	2022
Photobiohydrogen Production and Strategies for H Yield Improvements in Cyanobacteria. Advances in biochemical engineering/biotechnology\| PubMed ID: 37009974	2023
Correction to: Photobiohydrogen Production and Strategies for H Yield Improvements in Cyanobacteria. Advances in biochemical engineering/biotechnology\| PubMed ID: 37650917	2023
Proteomics identification of overexpressed serum proteins in dogs with infection. Veterinary world\| PubMed ID: 38023278	2023
Exogenous Trehalose Improves Growth, Glycogen and Poly-3-Hydroxybutyrate (PHB) Contents in Photoautotrophically Grown under Nitrogen Deprivation. Biology\| PubMed ID: 38392345	2024
Discovery and characterization of bioactive compounds from Limnophila aromatica: nevadensin and related flavonoids as potent antimicrobial agents. World journal of microbiology & biotechnology\| PubMed ID: 40770451	2025
Purification, characterization, and structural insights of Exo-1,4-β-D-glucosaminidase from Amycolatopsis sp. KLSc63: predictive modeling with glucosamine and N-acetyl glucosamine dimers. Molecular biology reports\| PubMed ID: 41222866	2025

Cherdsak Maneeruttanarungroj

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