Bioinformatics Analyses of Endo-1,4-beta-xylanase from Bacillus sp. SS3.4

Various types of microorganisms particularly bacteria and fungi are capable of producing xylanolytic enzymes that degrade xylan into simple sugars to be used as their carbon sources. Xylanolytic enzymes, particularly endo-1,4-β-xylanase, has been extensively used in various industrial sectors, including biofuel, livestock, paper, and food. Previously, Bacillus sp. SS3.4 was observed to have xylanolytic activities on wheat bran, suggesting its capability of producing xylanolytic enzymes, endo-1,4-β-xylanase included. With great numbers of data available in nucleotide and protein sequence databases, bioinformatics analyses of protein structure for function prediction have become attractive to supplement wet laboratory experiments to reduce time and money consumption. Therefore, in this study, bioinformatics analyses were performed on Bacillus sp. endo-1,4-β-xylanase sequence in order to further evaluate the enzyme’s function by exploring its protein structure in comparison to other known bacterial endo-1,4-β-xylanase. In this study, Bacillus sp. endo-1,4-β-xylanase sequence was identified from its whole genome sequence. The sequence was then analyzed by using BLAST to perform a homology search for identifying the protein, MUSCLE to perform multiple sequence alignment for comparing with other enzyme sequences, PHYLOGENY-FR to display its relatedness among Bacillus, Aspergillus niger and Trichoderma reesei, and SWISS-MODEL to generate its three-dimensional structure. Results from BLAST confirmed that the identified sequence was endo-1,4-β-xylanase, with greater relatedness to Bacillus velezensis and Bacillus amyloliquefaciens endo- 1,4-β-xylanase. While results from MUSCLE and SWISS-MODEL suggested that the endo-1,4-β-xylanase belongs to the Glycosyl Hydrolase 11 (GH11) family bearing the distinct shape of a jelly roll with well-conserved binding and catalytic residues. These results strongly suggest that Bacillus sp. endo-1,4-β-xylanase is potentially capable of degrading xylan with highly similar mechanism as with other endo-1,4-β-xylanase. Keywords: Bacillus, bioinformatics analysis, endo-1,4

Metabolite Identification from Biodegradation of Congo Red by Pichia sp.

Azo dyes are commonly used in textile and paper industries. However, its improper disposal often results in polluting water bodies. Azo dyes can cause adverse health effects because of its carcinogenic properties. Various methods to remove azo dyes from water have been proposed, including biological methods such as biosorption and biodegradation. Biosorption and biodegradation were done by using bacteria, yeast or mold. In general, yeasts have some advantages for azo dyes degradation due to its faster growth compared to mold and better resistance against unfavorable environment compared to bacteria. Previously, we observed that yeast Pichia sp. have the ability to degrade Congo red, an azo dye. However, information regarding biodegradation of azo dyes by Pichia sp. are still limited. Therefore, in this study, we showed degradation of Congo red by Pichia sp. crude enzyme extract obtained from separating Pichia cells from medium by centrifugation, followed by identification of its biodegradation products. Biodegradation product was separated from enzyme by ethyl acetate and then Gas Chromatography-Mass Spectroscopy (GC-MS) method was employed to identify biodegradation product. Chromatogram results of GC-MS showed that Congo red were degraded into various products such as biphenyl, naphthalene and smaller molecules with 94 m/z and 51 m/z. These results suggest involvement of azo reductase and laccase-like enzymes which cleaves azo bonds and oxidize the dye molecules to smaller molecules. This study implies the use of Pichia sp. as a bioremediation agent for the removal of azo dyes. Keywords: Biodegradation, Congo red, Pichia sp., metabolite identification, GC-M

SEQUENCE ANALYSIS OF 18s DNA OF Melosira sp., Dunaliella sp., Isochrysis sp. AND Porphyridium sp.

Phytoplankton has high level of biodiversity. In previous years phytoplankton was identified by their morphological characters. However, their morphology might change in different environments. These difficulties can be overcome by comparing their 18S rDNA sequences. This research is aimed to verify the identity of Melosira sp., Dunaliella sp., Isochrysis sp. and Porphyridium sp. Here, PCR method was used to amplify 18s DNA sequences. Three primer pairs were used, i.e. 18S-F and 18S-R; 501F and 1700R; 18S-2F and 18S-2R. PCR products were sequenced. MEGA5 was used to make phylogenetic tree. Genus verification for Isochrysis sp., Dunaliella sp. and Melosira sp. were conducted successfully using Blast and phylogenetic tree. 18s DNA sequence of Porphyridium sp. shows an interesting result and needs further verification.Keywords: Phytoplankton, Melosira sp., Dunaliella sp., Isochrysis sp., Porphyridium sp

Safety Assessment of Bacillus subtilis G8 Isolated from Natto for Food Application

Various bacteria are widely used as food-fermenting agents, including Lactobacillus, Bifidobacterium, and Bacillus. Despite they are generally recognized as safe to be consumed by humans, those bacteria could potentially cause antibiotic resistance as they could acquire and transfer antibiotic resistance genes from or to other microbes within the human gastrointestinal tract. Profiling antibiotic resistance pattern in those bacteria is therefore important to control the spread of antibiotic resistance. In this study, antibiotic resistance profile of Bacillus subtilis G8 was assessed. B. subtilis G8 had been isolated from commercialised Japanese natto in Indonesia and had been previously reported for its fibrinolytic characteristics. The antibiotic resistance phenotype and genotype of B. subtilis G8 were assessed through the Kirby-Bauer disk diffusion method and whole-genome analysis, respectively. B. subtilis G8 exhibited resistance towards Oxacillin, Lincomycin and Tiamulin-Lefamulin. The bioinformatics analysis indicated several responsible genes mediating those resistance, i.e., ybxI (for Oxacillin), lmrB (for Lincomycin) and vmlR (for Lincomycin and Tiamulin-Lefamulin). All identified genes were found in the chromosomal DNA. Further analysis found no mobile genetic elements within the genome, therefore reducing a risk of resistance gene transfer via plasmid and subsequently supporting safety profile of B. subtilis G8 in food fermentation usage

Clot Lysis Activity of Bacillus subtilis G8 Isolated from Japanese Fermented Natto Soybeans

 Background and objective: Bacillus spp. with potent fibrin digesting enzymes, especially Bacillus subtilis (natto), has gained favorable insights into the world of cardiovascular health and functional foods. In this study, Bacillus subtilis G8 was isolated from Japanese fermented Natto soybean commercialized in Indonesia. The overall activity of Bacillus subtilis G8 in resolving blood clot was evaluated under variable conditions quantitatively using clot lysis assay.Material and methods: Cell-free crude extracts of Bacillus subtilis G8 culture were partially purified with acetone precipitation method. Fibrinolytic activity of the enzymes was verified using fibrin plate assay and zymography. The measurement of clot liquefying activity under physiological temperature and pH was performed using chicken blood. The measurement of clot liquefying activity under physiological temperature and pH was performed using chicken blood. The measurement of clot liquefying activity under physiological temperature and pH was performed using chicken blood.Results and conclusion: Through zymography, Bacillus subtilis G8 expressed four bands of fibrinolytic proteins sized 19 to 30 kDa. Under controlled conditions, a significantly higher clot lysis was seen at 37 and 40°C, compared to lower temperatures (P≤0.05). In contrast, no significant decrease was seen in blood clot at all tested pH (P>0.05). These findings support the use of functional foods for the prevention of blood clot or as a part of blood health restoration.Conflict of interest: The authors declare no conflict of interest

ANTIANGIOGENESIS AND ANTIBACTERIAL ACTIVITIES FROM AN INDONESIAN MARINE-DERIVED FUNGUS Dactylaria sp

Marine-derived fungi have been proven to be rich sources of chemically diverse natural products with a broad range of biological activities. The aim of this study was to determine the antibacterial and antiangiogenesis activities of marine derived fungi Dactylaria sp. Cultivation of the fungus Dactylaria sp (strain TID 24041021-1) was isolated from the marine invertebrate sponge. Culture of marine fungus was macerated with acetone and partitioned with ethyl acetate. The ethyl acetate extracts with 50, 100, and 200µg/mL concentrations, were assayed for their antiangiogenic activity by using chorioallantoic membrane in vivo. At the same time, ethyl acetate extracts at levels of 5, 10, 15, and 20mg/mL were assayed to pathogenic bacteria Bacillus subtilis, Escherichia coli, and Staphylococcus aureus using well diffusion method. The result of this study showed that ethyl acetate extract at concentration 50µg/mL could inhibit angiogenesis. The best antiangiogenic activity was showed at concentration of 200µg/mL ethyl acetate extract. Antibacterial activity from ethyl acetate extract inhibited the growth of B. subtilis (2.25-5mm), E. coli (0.63-3.50mm) and S. aureus (01.75mm) bacteria. Key words: Marine sponge-derived fungi, antiangiogenesis activity, antibacterial activit

Bioinformatics Analyses of Endo-1,4-beta-xylanase From Bacillus SP. SS3.4

Various types of microorganisms particularly bacteria and fungi are capable of producing xylanolytic enzymes that degrade xylan into simple sugars to be used as their carbon sources. Xylanolytic enzymes, particularly endo-1,4-β-xylanase, has been extensively used in various industrial sectors, including biofuel, livestock, paper, and food. Previously, Bacillus sp. SS3.4 was observed to have xylanolytic activities on wheat bran, suggesting its capability of producing xylanolytic enzymes, endo-1,4-β-xylanase included. With great numbers of data available in nucleotide and protein sequence databases, bioinformatics analyses of protein structure for function prediction have become attractive to supplement wet laboratory experiments to reduce time and money consumption. Therefore, in this study, bioinformatics analyses were performed on Bacillus sp. endo-1,4-β-xylanase sequence in order to further evaluate the enzyme's function by exploring its protein structure in comparison to other known bacterial endo-1,4-β-xylanase. In this study, Bacillus sp. endo-1,4-β-xylanase sequence was identified from its whole genome sequence. The sequence was then analyzed by using BLAST to perform a homology search for identifying the protein, MUSCLE to perform multiple sequence alignment for comparing with other enzyme sequences, PHYLOGENY-FR to display its relatedness among Bacillus, Aspergillus Niger and Trichoderma reesei, and SWISS-MODEL to generate its three-dimensional structure. Results from BLAST confirmed that the identified sequence was endo-1,4-β-xylanase, with greater relatedness to Bacillus velezensis and Bacillus amyloliquefaciens endo- 1,4-β-xylanase. While results from MUSCLE and SWISS-MODEL suggested that the endo-1,4-β-xylanase belongs to the Glycosyl Hydrolase 11 (GH11) family bearing the distinct shape of a jelly roll with well-conserved binding and catalytic residues. These results strongly suggest that Bacillus sp. endo-1,4-β-xylanase is potentially capable of degrading xylan with highly similar mechanism as with other endo-1,4-β-xylanase. Keywords: Bacillus, bioinformatics analysis, endo-1,4

The potential of copper-resistant bacteria Acinetobacter sp. strain CN5 in decolorizing dyes

Irawati W, Pinontoan R, Mouretta B, Yuwono T. 2021. The potential of copper-resistant bacteria Acinetobacter sp. strain CN5 in decolorizing dyes. Biodiversitas 23: 680-686. Bacteria with multi-resistance to copper and dyes may be employed in bioremediation of copper and dye waste more effectively than physical or chemical remediation. This study aims at determining the effect of the addition of various textile dyes and copper on the growth of Acinetobacter sp. CN5 and its ability to decolorize dyes. The dye resistance test was carried out by inoculating bacterial isolates into Luria Bertani media containing 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 ppm dyes. The twelve dyes used in the test were methylene blue, malachite green, congo red, mordant orange, reactive black, direct yellow, basic fuchsine, reactive orange, dispersion orange, remazol red, wantex yellow, and wantex red. The decolorization activity was analyzed by spectrophotometry at a 300-900 nm wavelength. The study results demonstrated that bacteria thrived in media containing 50 ppm of all dyes, except malachite green dye. Acinetobacter sp. CN5 was found to be resistant to up to 500 ppm methylene blue, basic fuchsine, and wantex red and resistant to congo red at 450 ppm. Acinetobacter sp. CN5 was also able to decolorize methylene blue, congo red, basic fuchsine, and red wantex by 57.64, 53.17, 91.37, and 67.50, respectively. The addition of 5 mM CuSO4 to the medium increased the ability of Acinetobacter sp. CN5 to decolorize the congo red from 57.64 to 82.58. © 2022, Society for Indonesian Biodiversity. All rights reserved

Skrining Aktivitas Antioksidan dari Mikroalga

Skrining aktivitas antioksidan dari mikroalga dilakukan untuk mengetahui potensi substansi bioaktif dari mikroalga sebagai alternatif pengganti antioksidan sintetik. Penelitian ini menggunakan lima jenis mikroalga, yaitu Porphyridium sp., Platymonas sp., Dunaliella sp., Chlorella stigmatophora dan Isochrysis sp. (T. iso). Kelima jenis mikroalga dikulturkan dengan menggunakan 10 liter air laut steril dengan salinitas 30 ppm yang ditambahkan medium ekstrak tauge dan vitamin. Mikroalga dimaserasi dengan aseton, selanjutnya dipartisi dengan etil asetat (EtOAc). EtOAc extrak selanjutnya difraksinasi menggunakan metode kromatografi kolom. Studi aktivitas antioksidan dilakukan dengan metode kualitatif dan kuantitatif DPPH. Hasil penelitian secara kualitatif menunjukkan bahwa ekstrak kasar EtOAc dari mikroalga Dunaliella sp. dan C.stigmatophora memiliki aktivitas antioksidan. Uji aktivitas antioksidan secara kuantitatif dari hasil fraksinasi ekstrak EtOAc Dunaliella sp. dan C.stigmatophora menunjukkan bahwa fraksi nomor 7.3 dari Dunaliella sp. memiliki aktivitas antioksidan dengan nilai IC50 445,86 ?g/ml dan fraksi nomor 10 dari C. stigmatophora dengan IC501192,13 ?g/ml