A novel Bacillus ligniniphilus catechol 2,3-dioxygenase shows unique substrate preference and metal requirement

Identification of novel enzymes from lignin degrading microorganisms will help to develop biotechnologies for biomass valorization and aromatic hydrocarbons degradation. Bacillus ligniniphilus L1 grows with alkaline lignin as the single carbon source and is a great candidate for ligninolytic enzyme identification. The first dioxygenase from strain L1 was heterologously expressed, purified, and characterized with an optimal temperature and pH of 32.5 °C and 7.4, respectively. It showed the highest activity with 3-ethylcatechol and significant activities with other substrates in the decreasing order of 3-ethylcatechol > 3-methylcatechol > 3-isopropyl catechol > 2, 3-dihydroxybiphenyl > 4-methylcatechol > catechol. It did not show activities against other tested substrates with similar structures. Most reported catechol 2,3-dioxygenases (C23Os) are Fe2+-dependent whereas Bacillus ligniniphilus catechol 2,3-dioxygenase (BLC23O) is more Mn2+- dependent. At 1 mM, Mn2+ led to 230-fold activity increase and Fe2+ led to 22-fold increase. Sequence comparison and phylogenetic analyses suggested that BL23O is different from other Mn-dependent enzymes and uniquely grouped with an uncharacterized vicinal oxygen chelate (VOC) family protein from Paenibacillus apiaries. Gel filtration analysis showed that BLC23O is a monomer under native condition. This is the first report of a C23O from Bacillus ligniniphilus L1 with unique substrate preference, metal-dependency, and monomeric structure.

Poly-β-Hydroxybutyrate Production by Rhodopseudomonas sp. Grown in Semi-Continuous Mode in a 4 L Photobioreactor

The synthesis of polyhydroxybutyrate (PHB) by photosynthetic non-sulfur bacteria is a potential approach for producing biodegradable plastics. In this work, acetate was used as a single carbon source to study the effect on PHB formation in Rhodopseudomonas sp. cultured in a cylindrical four-liter photobioreactor under semi-continuous mode. The cultivation process is divided into a symmetrical growth phase and a PHB accumulation phase separated temporally. The symmetrical growth phase (nutrient sufficient conditions) was followed by a sulfur-limited phase to promote PHB accumulation. The main novelty is the progressive lowering of the sulfur concentration into Rhodopseudomonas culture, which was obtained by two concomitant conditions: (1) sulfur consumption during the bacterial growth and (2) semi-continuous growth strategy. This caused a progressive lowering of the sulfur concentration into Rhodopseudomonas culturedue to the sulfur-free medium used to replace 2 L of culture (50% of the total) that was withdrawn from the photobioreactor at each dilution. The PHB content ranged from 9.26% to 15.24% of cell dry weight. At the steady state phase, the average cumulative PHB was >210 mg/L. Sulfur deficiency proved to be one of the most suitable conditions to obtain high cumulative PHB in Rhodopseudomonas culture.

Preparation of biokit to treatment the Petrol spots in water

Microorganism isolates used in plant protection were obtained from the College ofAgriculture / University of Baghdad. Its purity was assured. The microbial biokit was preparedthrough using several unsatisfactory and environmentally friendly isolate. The bioactivity ofbiokit were studied in laboratory and showed susceptibility in removing contaminants. Theresults showed that the biomass of the isolates prior to the loading process on a mediumcontaining crude oil as a single carbon source of 5 g/ L, which indicates growth ability andefficiency in the decomposition of pollutants when increase the isolates biomass more than 3 g /L, as well as the appearance of turbidity in the media compared to the control sample (mediawithout oil), and obtained bioemulsifier with a concentration of 2.3 g / L. The chemical analysisof the samples under study was carried out using Gas Chromatography (GC) and GC-MScompared with a non-treatment (control). The results showed the appearance of 43 topographic peaks each indicating a chemicalcompound in the control sample, while a number of these peaks disappeared and a space wasreduced in other peaks that refers to the concentration of compounds, in the biomass sample, andreached the number of peaks in 26, indicating the efficiency of the biokit in the oildecomposition and dismantling of hydrocarbons. The increase in the number of livemicroorganisms and the incubation period is more than 28 days by increasing the growthintensity in the third week may be due to decomposing oil compounds and their exploitation innutrition, where crude oil provides a source of carbon and energy for microorganism’s isolates.In other results showed that the mass of crude oil was converted to a mass of gel andemulsification compared to the control sample after seven days period at a temperature of 28±2°C, Which is characterized by low toxicity and easy decomposition and reduces the surfacetension of hydrocarbons and makes them more solubility and preparation for the dismantling oflife and increase the rate of disintegration. The biokit was tested for retention in storageconditions for a period of one year. at alone prior to the production of the biokit, which showed poor storage capacity. We concludethat biomass has the potential to treat hydrocarbons (oil contaminants) in the environment.

Optimization of β-1,4-Endoxylanase Production by an Aspergillus niger Strain Growing on Wheat Straw and Application in Xylooligosaccharides Production

Plant biomass constitutes the main source of renewable carbon on the planet. Its valorization has traditionally been focused on the use of cellulose, although hemicellulose is the second most abundant group of polysaccharides on Earth. The main enzymes involved in plant biomass degradation are glycosyl hydrolases, and filamentous fungi are good producers of these enzymes. In this study, a new strain of Aspergillus niger was used for hemicellulase production under solid-state fermentation using wheat straw as single-carbon source. Physicochemical parameters for the production of an endoxylanase were optimized by using a One-Factor-at-a-Time (OFAT) approach and response surface methodology (RSM). Maximum xylanase yield after RSM optimization was increased 3-fold, and 1.41- fold purification was achieved after ultrafiltration and ion-exchange chromatography, with about 6.2% yield. The highest activity of the purified xylanase was observed at 50 °C and pH 6. The enzyme displayed high thermal and pH stability, with more than 90% residual activity between pH 3.0–9.0 and between 30–40 °C, after 24 h of incubation, with half-lives of 30 min at 50 and 60 °C. The enzyme was mostly active against wheat arabinoxylan, and its kinetic parameters were analyzed (Km = 26.06 mg·mL−1 and Vmax = 5.647 U·mg−1). Wheat straw xylan hydrolysis with the purified β-1,4 endoxylanase showed that it was able to release xylooligosaccharides, making it suitable for different applications in food technology.

Isolation and Characterization of Novel Bacteria Capable of Degrading 1,4-Dioxane in the Presence of Diverse Co-Occurring Compounds

Biodegradation is found to be a promising, cost-effective and eco-friendly option for the treatment of industrial wastewater contaminated by 1,4-dioxane (1,4-D), a highly stable synthetic chemical and probable human carcinogen. This study aimed to isolate, identify, and characterize metabolic 1,4-D-degrading bacteria from a stable 1,4-D-degrading microbial consortium. Three bacterial strains (designated as strains TS28, TS32, and TS43) capable of degrading 1,4-D as a sole carbon and energy source were isolated and identified as Gram-positive Pseudonocardia sp. (TS28) and Gram-negative Dokdonella sp. (TS32) and Afipia sp. (TS43). This study, for the first time, confirmed that the genus Dokdonella is involved in the biodegradation of 1,4-D. The results reveal that all of the isolated strains possess inducible 1,4-D-degrading enzymes and also confirm the presence of a gene encoding tetrahydrofuran/dioxane monooxygenase (thmA/dxmA) belonging to group 5 soluble di-iron monooxygenases (SDIMOs) in both genomic and plasmid DNA of each of the strains, which is possibly responsible for the initial oxidation of 1,4-D. Moreover, the isolated strains showed a broad substrate range and are capable of degrading 1,4-D in the presence of additional substrates, including easy-to-degrade compounds, 1,4-D biodegradation intermediates, structural analogs, and co-contaminants of 1,4-D. This indicates the potential of the isolated strains, especially strain TS32, in removing 1,4-D from contaminated industrial wastewater containing additional organic load. Additionally, the results will help to improve our understanding of how multiple 1,4-D-degraders stably co-exist and interact in the consortium, relying on a single carbon source (1,4-D) in order to develop an efficient biological 1,4-D treatment system.

Optimization of β-1,4-endoxylanase production by a new Aspergillus niger strain growing on wheat straw and application in xylooligosaccharides production

Plant biomass constitutes the main resource of renewable carbon in the planet and its valorization has traditionally been focused on the use of cellulose, although hemicellulose is the second most abundant group of polysaccharides on earth. Enzymes involved in its degradation are usually glycosyl hydrolases and filamentous fungi are good producers of these enzymes. In this study, a new strain of Aspergillus niger was utilized for hemicellulase production under solid state fermentation using wheat straw as a single carbon source. Physicochemical parameters for production of an endoxylanase were optimized by using one factor at a time approach and response surface methodology (RSM). Maximum xylanase yield after RSM optimization was increased 3-fold. The enzyme was purified by ultrafiltration and ion-exchange chromatography1.41-fold, with 6.2 % yield. Highest xylanase activity was observed at 50 °C and pH 6. A high pH and thermal stability were found, greater than 90% residual activity between pH 3.0-9.0 and between 30-40°C, after 24 h of incubation, presenting half-lives of 30 min at 50 and 60°C. Enzyme was mostly active for wheat arabinoxylan, and displayed the following kinetic parameters Km of 26.06 mg•ml-1 and Vmax of 5,647 U•mg-1min-1.Wheat straw xylan hydrolysis with the purified β-1,4 endoxylanase showed that it was able to release xylooligosaccharides, making it suitable for different applications in food technology.

DEGRADASI DINDING SEL Phytophthora capsici OLEH ENZIM KARBOSI METIL SELULASE ASAL Trichoderma harzianum

<p>Phytophthora capsici Leonian adalah patogen penyebab penyakit busuk pangkal batang lada (Piper nigrum L). Trichoderma harzianum Rifai merupakan agen hayati yang cfektif dan menyebabkan lisis miselia P. capsici. Penelitian ini bertujuan unluk mcngc(ahui peran enzim karboksimetilselulase (CMC-ase) yang diproduksi oleh T. harzianum dalam mendegradasi dinding sel P capsici. Penelitian terdiri atas tiga aktivitas yaitu (a) deteksi produksi enzim CMC-ase, (b) hidrolisis dinding sel P. capsici. dan (c) penggunaan siapan kasar dinding sel (SKDS) P. capsici oleh T. harzianum sebagai satu-satunya sumber karbon dalam media tumbuhnya Aktivitas enzim dideleksi secara kualitatif dengan membandingkan zona bening yang terbentuk pada medium karboksi metil selulosa (CMC) yang diperlakukan dengan satu tetcs iltrat kultur 7 harzianum dan diwarnai dengan larulan congo red, sedangkan aktivitas CMC-ase secara kuantitatif diukur sebagai nilai setara glukosa yang terlepas dari substrat setelah diinkubasi dengan ekstrak kasar CMC-ase. Enzim CMC-ase dickstrak dari kultur filtrat T. harzianum F.51 melalui pengendapan protein dengan 85% ammonium sulfat pada suhu 4°C diikuti dengan sentrifusi dan dialisis. Aktivitas spcsifik enzim tersebut dalam mendegradasi CMC (30,29 unif//g protein) lebih rendah dari aktivitas enzim selulase komersial (97.18 unit/^g protein). Enzim selulase komcrsial dan ekstrak enzim dari kultur 7 harzianum juga dapat menghidrolisis SKDS P. capsici N2 dengan aktivitas masing-masing 31.18 unit dan 19.26 unit. Isolat F-51 tumbuh dan menghasilkan aktivitas enzim serupa manakala karboksimclil selulosa pada media tumbuh diganti dengan SKDS sebagai sumber karbon tunggal Hasil ini menunjukkan bahwa CMC-ase berperan penting dalam mckanisme antagonis T. harzianum terhadap /' capsici. Penelitian lebih lanjut dalam mckanisme produksi enzim ini berpeluang untuk meningkatkan potensi agen hayati.</p><p>Kata kunci : Piper nigrum L., Trichoderma harzianum Rifai, Phytophthora capsici. karbosimctilsclulasc, agen hayati</p><p> </p><p><strong>ABSTRACT</strong></p><p><strong>Degradation of cell wall of Phytophthora capsici N2 by carboxy methyl cellulose &lt;;/ Trichoderma harzianum ES1</strong></p><p>Phytophthora capsici l-eonian is a causal agent of foot rot disease on black pepper (Piper nigrum L ). Trichoderma harzianum Rifai is an effective biocontrol agent and causes lyscs on P. capsici mycelium This experiment was aimed to study the role of carboxymethylccllulose (CMC- ase) produced by T. harzianum in degrading P. capsici cell wall. The experiment was composed in three activities (a) detection of the CMC-ase enzyme production, (b) degradation of P. capsici cell wall by crude extract of the enzyme, and (c) utilization of crude cell wall preparation (cwp) of P. capsici by T. harzianum as single carbon source in its growth medium CMC-ase activity was detected qualitatively on carboxymethylccllulose (CMC) medium, and quantitatively it was measured as glucose equivalent released from substrate after treated with crude extract of CMC-ase. CMC-ase was extracted from culture iltrate of T. harzianum F-51 by precipitation of protein with 85% ammonium sulphate at 4°C followed by dialysis with distilled water and liophyllizcd. Activity of the extracted enzyme on degradation of CMC (30.29 unil-'/jg protein) was lower than activity of commercial cellulose (97.18 united g protein) Commercial cclullosc and the extracted enzyme also degraded crude cell wall prepared (CWP) from P. capsici N2 as indicated with the presence of glucose equivalent released from CWP after incubation with the enzyme; with specific activity of 31.18 unit and 19.26 unit respectively. Trichoderma harzianum grown in medium suplemented with the crude cell wall of P. capsici as a single carbon source produced CMC-ase. Those results indicated that CMC-ase has important role on parasitism of T. harzianum on P. capsici Further investigation is required to elucidate mechanism of production of CMC-ase in T. harzianum for improvement of biocontrol activity of the fungus.</p><p>Key words: Piper nigrum L., Trichoderma harzianum Rifai, Phytophthora capsici, carboxymethylcellulosc, biocontrol</p>

Isolasi Jamur Selulolitik Trichoderma pada Beberapa Limbah Organik

Cellulolytic fungi have an important role for the largest material flow in the biosphere. Trichoderma fungi are ubiquitous and have the ability to degrade cellulose. The study was conducted to isolate dan screen Trichoderma cellulolytic fungi from soil heaps of some organic waste. Soil samples were taken from soil heaps of some organic waste to isolate Trichoderma cellulolytic fungi. The sampling method is carried out randomly from top soil layers of organic waste litter (bagasse, sugar cane leaf, rice straw, and garbage). Isolation and screening of Trichoderma fungi are determined based on observations of macroscopic and microscopic characteristics referring to the key of fungal determination. Cellulolytic ability of Trichoderma isolates was carried out by growing on Carboxy Methyl Cellulose (CMC) media as a single carbon source by measuring the diameter of the colony and the diameter of the clear zone formed. There were 7 Trichoderma fungal isolates that showed cellulolytic activity. Based on clear zone formation, Trichoderma harzianum showed highest cellulolytic than Trichoderma viride.