High Enzyme Research Articles

ВЫДЕЛЕНИЕ ДЕГАЛОГЕНАЗ ПЕРФТОРКАРБОНОВЫХ КИСЛОТ ШТАММОВ PSEUDOMONAS PLECOGLOSSICIDA 2,4 Д И PSEUDOMONAS MOSSELII 5(3)

The aim of this study was to isolate and investigate enzymes involved in the biodegradation of perfluorocarboxylic acids (PFCAs) in Pseudomonas plecoglossicida strain 2,4-D and Pseudomonas mosselii strain 5(3). In this research, enzymes from these strains were concentrated from the culture medium by ultrafiltration and ammonium sulfate precipitation, followed by purification through size-exclusion chromatography. The enzyme activity after ultrafiltration was higher Enzymes from strain 2,4-D released 2 ppm of fluoride ions after 24 hours, increasing to 40,7 ppm after 48 hours. For strain 5(3), fluoride release reached 3 ppm at 24 hours and 46 ppm at 48 hours, confirming the activity of the isolated dehalogenases and their ability to degrade PFCAs. Size-exclusion chromatography revealed that the molecular weight of enzymes from strain 5(3) is approximately 80 kDa, while for strain 2,4-D, it is 76 kDa. The study of these enzymes holds both applied significance for environmental remediation technology development and fundamental importance. Understanding the molecular structure and mechanisms of dehalogenase activity not only provides insights into the cleavage of robust C-F bonds but also sheds light on enzyme evolution as a whole.

Depolymerisation of Kraft Lignin by Tailor-Made Alkaliphilic Fungal Laccases.

Lignins released in the black liquors of kraft pulp mills are an underutilised source of aromatics. Due to their phenol oxidase activity, laccases from ligninolytic fungi are suitable biocatalysts to depolymerise kraft lignins, which are characterised by their elevated phenolic content. However, the alkaline conditions necessary to solubilise kraft lignins make it difficult to use fungal laccases whose activity is inherently acidic. We recently developed through enzyme-directed evolution high-redox potential laccases active and stable at pH 10. Here, the ability of these tailor-made alkaliphilic fungal laccases to oxidise, demethylate, and depolymerise eucalyptus kraft lignin at pH 10 is evidenced by the increment in the content of phenolic hydroxyl and carbonyl groups, the methanol released, and the appearance of lower molecular weight moieties after laccase treatment. Nonetheless, in a second assay carried out with higher enzyme and lignin concentrations, these changes were accompanied by a strong increase in the molecular weight and content of β-O-4 and β-5 linkages of the main lignin fraction, indicating that repolymerisation of the oxidised products prevails in one-pot reactions. To prevent it, we finally conducted the enzymatic reaction in a bench-scale reactor coupled to a membrane separation system and were able to prove the depolymerisation of kraft lignin by high-redox alkaliphilic laccase.

Overcoming methanogenesis barrier to acid inhibition and enhancing PAHs removal by granular biochar during anaerobic digestion

Alleviating acid inhibition is the most important for optimizing anaerobic digestion (AD), but the recalcitrant organic pollutants in digestates have long been ignored. In this study, we aimed to simultaneously enhance both organics bio-methanation and recalcitrant polycyclic aromatic hydrocarbons (PAHs) removal in AD system by using granule biochar and short-term ethanol stimulation. The results showed that adding biochar effectively alleviated volatile fatty acids (VFAs) accumulation and achieved 313.6 mL CH4/d•g COD average yield. Meanwhile, the efficiency of phenanthrene (PHE) biodegradation, as the model PAHs pollutant, was significantly promoted by 37.1%. Additionally, more and compact extracellular polymeric substances improved electron transfer and mass transfer among microbes, and higher enzymes (ATPase, NADH and α-Amylase) activities met the physiological demanding of efficient microbial metabolism in biochar-added reactor. Microbiological analysis revealed that biochar-mediated direct interspecies electron transfer (DIET) selectively enriched exoelectrogens (Bacteroides, Proteiniphilum, f_Christensenellaceae, etc.) and acetoclastic Methanosaeta, which contributed to increasing biomethane production. The promoted PAHs biodegradation was mainly dependent on the cooperation between fermenting f_Paludibacteraceae and hydrogenotrophic Methanolinea, and potentially related to biochar-mediated DIET. Metabolic analysis indicated that the metabolic pathways of acetogenesis and methanogenesis were significantly strengthened by biochar, thus alleviating acid inhibition. Also, biochar stimulated functional genes expression in PAHs anaerobic biodegradation. These findings were further verified by a series of short-term metabolism tests.

Topography-driven differences in soil N transformation constrain N availability in karst ecosystems

Fragile karst ecosystems are characterized by complex topographic landscapes associated with high variations in vegetation restoration. Identifying the characteristics and driving factors of nitrogen (N) availability across the topographic gradient is essential to guide vegetation restoration in karst regions. In this study, we collected soil samples and plant leaves along the topographic gradient (ridge, upper slope, middle slope, and foot slope) of convex slopes in the karst fault basin of southwest China, and determined the indicators reflecting soil N availability, N transformation rates, and their controlling factors. Our results showed that foliar N content and δ15N value, soil inorganic N content and δ15N value, and foliar N:P ratio were substantially lower on the steep hillslopes than on the flat top ridge. Steep slope soils also had a lower enzyme C:N ratio but a higher enzyme N:P ratio than the flat ridge soils. Furthermore, the vector angles calculated by soil extracellular enzyme analysis were below 45o in all studied soils and decreased significantly with increasing slope, indicating that microbial growth was generally limited by N. These results jointly suggest the declines in soil N availability across the topographic gradient, which are further explained by the changes in soil inherent N transformation processes. As the slope became steeper, soil mineralization and autotrophic nitrification (ONH4) rates decreased significantly, while ratio of microbial NH4+ immobilization to ONH4 and NH4+ adsorption rate increased significantly, indicating the decrease in soil inorganic N supply capacity. We further found that deteriorated soil structure, decreased soil organic matter and calcium content, altered microbial abundance, and increased ratios of fungi to bacteria and gram-positive bacteria to gram-negative bacteria were the primary drivers of reduced N transformation rates and N availability across the topographic gradient. Overall, this study highlights the critical role of the topography in controlling soil N availability by regulating N transformation processes in karst regions. The topography should be considered an important factor affecting the functions and services of karst ecosystems.

Vegetation restoration of abandoned cropland improves soil ecosystem multifunctionality through alleviating nitrogen-limitation in the China Danxia.

The microbial requirement for nutrient resources can be estimated by soil extracellular enzyme stoichiometry (EES) and their stoichiometries. Implementing the Grain for Green Program has significantly impacted land use and soil nutrient management in the China Danxia. However, drivers of soil microbial nutrient limitation changes in abandoned cropland (AC) remained unclear after vegetation restoration. Here, according to vector analysis, we evaluated microbial nutrient limitation by studying soil EES across vegetation restoration types (naturally restored secondary forests (NF) and artificially planted forests (AF)) with AC as a control. Results showed both NF and AF soils averaged higher C- and P- acquiring enzyme, indicating rapid C and P turnover rates after vegetation restoration. However, vegetation restoration resulted in higher C requirement for microorganisms with higher enzyme C:N and vector length. In addition, microorganisms shifted from N- (< 45°) to P-limited (> 45°) conditions with enzyme N:P less than 1 after vegetation restoration, and NF exacerbated microbial P limitation compared to AF. Decreased N limitation following vegetation restoration could be contributed to improving soil ecosystem multifunctionality. The greater variation of EES was explained by the interaction of pH, soil nutrient, and microbial biomass than by any one of these factors alone, suggesting that both abiotic and biotic factors regulate microbial nutrient limitation and microbial process. Overall, our results revealed vegetation restoration could alleviate N limitation in the China Danxia, and thus enhance soil ecosystem by regulating lower microbial N limitation, which provide insight into nutrient management strategies under ecological restoration of degraded areas.

Bioreactor and process design for 2G ethanol production from xylose using industrial S. cerevisiae and commercial xylose isomerase

Second-generation ethanol has gained attention since the increase in demand for renewable fuels, but some issues still impair its feasibility, such as the conversion of xylose from biomass. Many routes can be explored for this purpose, but Simultaneous Isomerization and Fermentation (SIF) stand out due to the possibility of working with robust, non-genetically modified Saccharomyces cerevisiae strains. This work addresses bioreactor and process designs for SIF, using high enzyme and yeast load in their commercial forms, that can be introduced in the current ethanol-sugar industry. Characterization of two xylose isomerases (IGI-HF and Sweetzyme) in SIF conditions showed that both are suitable for the process. Between two industrial yeast strains evaluated, S. cerevisiae “Itaiquara” (baker’s yeast) was selected due to its superior xylulose assimilation. Problems with mass transfer and cell damage due to the presence of enzyme particles at high loads were faced, and different strategies were evaluated. Promising ethanol productivity (0.87 gEtOH/L/h, for 50 gDCW/L and 53,000 IU/L) was achieved using an enzyme-multiple-packed bed reactor. In addition, it allowed easy enzyme recovery and reuse, enabling its application in repeated batch operations integrated into the 1G ethanol process.

Acid and alkali pretreatment of corn stalk for fractal‐like kinetic analysis of enzymatic saccharification

AbstractBACKGROUNDThe conversion of lignocellulose to sugar is part of the process of lignocellulose biorefining and, therefore, is crucial for effective lignocellulose utilization.RESULTSIn this study, alkali and acidic pretreatments were carried out to deconstruct corn stalk (CS) material for efficient subsequent enzymatic saccharification. Most xylan and lignin can be removed from CS material by acid and alkali pretreatments, respectively, as verified by XRD (X‐ray diffractometer) and FT‐IR (Fourier Transform Infrared Spectroscopy) analyses. Fractal‐like kinetics were assessed to analyze the effect of enzyme loading on the glucose and xylose sugar yields from the two pretreated CS materials, with fractal dimension considered as a dominant factor in rate constant decrease. Fractal dimension analysis demonstrated that the alkali pretreated CS was more easily hydrolyzed that the acid pretreated CS. For glucan hydrolysis, the effect of enzyme loading on the rate constant of the alkali pretreated CS was marginally higher than that of the acid pretreated CS. For xylan hydrolysis, enzyme loading was shown to significantly improve the rate constant of the alkali pretreated CS compared to the acid pretreated CS. Furthermore, the developed fractal‐like model exhibits high prediction accuracy for sugar yield at high enzyme loadings.CONCLUSIONThe application of the fractal‐like theory was quite successful for studying enzymatic kinetics. © 2022 Society of Chemical Industry (SCI).

Cyanide bioremediation potential of Klebsiella oxytoca JCM 1665 rhodanese immobilized on alginate-glutaraldehyde beads

Rhodanese, a cyanide detoxifying enzyme synthesized by Klebsiella oxytoca was immobilized on alginate-glutaraldehyde beads. K. oxytoca was isolated from industrial wastewater and identified using 16S rRNA gene sequencing with gene bank accession number MN590525. Rhodanese was produced from the bacterium through submerged fermentation. The rhodanese produced was immobilized on alginate-glutaraldehyde beads; its physicochemical properties and cyanide bioremediation potential were compared with the free enzyme. In this study, the optimum concentrations of glutaraldehyde and sodium alginate that resulted in the highest enzyme immobilization yield (89.71%) and lower leakage (1.45 ± 0.2%) were 4.5% (v/v) and 2.5 (%) respectively. The optimum temperature for free and immobilized rhodanese was observed at 50 °C and 60 °C respectively while the optimum pH for both preparations was 6.0. The free rhodanese retained 31% and 23% of relative activity at 60 °C and 70 °C respectively after 30 minutes of incubation while immobilized rhodanese retained about 95% and 70% at the same condition. The entrapped rhodanese showed activity until the 10th cycle and maintained about 70% of its activity after the fifth cycle. After 180 minutes of incubation, the free and immobilized rhodanese was able to biodegrade 115 mg/L cyanide to 77 mg/L and 45 mg/L respectively with degradation efficiency of 33 and 64.34%. These results suggest that immobilized K. oxytoca rhodanese may be profitably exploited in bioremediation of cyanide polluted environment due to its thermal stability and its reusability.

Abstract 3946: Development of selective small molecule AR-V7 inhibitors for prostate cancer treatment

Abstract Castration resistance prostate cancer (CRPC) is a lethal disease in which the expression of ligand-independent androgen receptor (AR) splice variants (AR-Vs) is associated with worse clinical outcomes. AR-V7 is the most prevalent variant in CRPC, it lacks the ligand binding domain and is constitutively active in the nucleus. We and others have showed that, AR-V7 expression confers resistance to the AR signaling inhibitors (abiraterone/enzalutamide) and to taxanes in vivo and in patients with metastatic CRPC. Since abiraterone/enzalutamide and taxanes represent the two most effective therapeutic modalities for men with CRPC, the development of selective AR-V7 inhibitors is a high priority, clinically unmet need. AR-V7 shares with full-length AR (AR-fl), high sequence homology, largely overlapping cistromes and gene transactivation profiles. To develop selective AR-V7 inhibitors, we sought to identify unique biological features of AR-V7, that differentiate it from AR-fl and therapeutically exploit them. Mechanistic studies showed that AR-V7 utilizes a unique nuclear import pathway, not shared by AR-fl. Using fluorescently tagged-AR-fl or AR-V7 proteins in conjunction with live cell imaging, FRAP assays and pathway inhibitors, we showed that AR-V7 exhibits fast nuclear import kinetics partially mediated by the dimerization D-box domain, independently of microtubules and importin α/β. Taken together, these data suggest that AR-V7 nuclear import mechanism is distinct providing a window of therapeutic opportunity to selectively target it. To identify AR-V7 selective inhibitors, we designed and performed a high throughput enzyme complementation screening (HTS) assay using nuclear AR-V7 as a surrogate for AR-V7 activity. We screened a chemical library of ~170K compounds and identified hit compounds inhibiting AR-V7 by proteasomal degradation. Among the degraders, we observed 2 main modes of action: I. compounds selectively degrading AR-V7 and II. compounds degrading both AR-fl and AR-V7. Ongoing efforts include medicinal chemistry for lead compound optimization and target validation experiments.In conclusion, we identified first-in-class selective AR-V7 inhibitors, with the potential to be clinically combined with existing, but mechanistically unrelated AR signaling inhibitors. Further mechanistic studies will elucidate their potential for future clinical development. Citation Format: CheukMan Cherie Au, Seaho Kim, Prerna Vatsa, Mohd Azrin Bin Jamalruddin, Michael Miller, Peter T. Meinke, Paraskevi Giannakakou. Development of selective small molecule AR-V7 inhibitors for prostate cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3946.

Cross-sectional metabolic subgroups and 10-year follow-up of cardiometabolic multimorbidity in the UK Biobank

We assigned 329,908 UK Biobank participants into six subgroups based on a self-organizing map of 51 biochemical measures (blinded for clinical outcomes). The subgroup with the most favorable metabolic traits was chosen as the reference. Hazard ratios (HR) for incident disease were modeled by Cox regression. Enrichment ratios (ER) of incident multi-morbidity versus randomly expected co-occurrence were evaluated by permutation tests; ER is like HR but captures co-occurrence rather than event frequency. The subgroup with high urinary excretion without kidney stress (HR = 1.24) and the subgroup with the highest apolipoprotein B and blood pressure (HR = 1.52) were associated with ischemic heart disease (IHD). The subgroup with kidney stress, high adiposity and inflammation was associated with IHD (HR = 2.11), cancer (HR = 1.29), dementia (HR = 1.70) and mortality (HR = 2.12). The subgroup with high liver enzymes and triglycerides was at risk of diabetes (HR = 15.6). Multimorbidity was enriched in metabolically favorable subgroups (3.4 ≤ ER ≤ 4.0) despite lower disease burden overall; the relative risk of co-occurring disease was higher in the absence of obvious metabolic dysfunction. These results provide synergistic insight into metabolic health and its associations with cardiovascular disease in a large population sample.

Effects of endophytic fungi on parasitic process of Taxillus chinensis

Taxillus chinensis (DC.) Danser is an extensively used medicinal shrub in the traditional as well as modern systems of medicines. It is a perennial hemiparasitic plant, which is difficult to propagate artificially because of its low parasitic rate. Successful parasitism of parasitic plants is to fuse their tissues and connect their vasculature to the host vasculature building a physiological bridge, which can efficiently withdraw water, sugars and nutrients from their host plants. It is reported that endophytic fungi play an important role in cell wall degradation and fusion, which is the key forming process of the physiological bridge. Therefore, in this study, the endophytic fungi from T. chinensis of different hosts were isolated, and then the organisms that could degrade the main components of the cell walls were screened out using a medium consisting of guaihuol and cellulose degradation capacity. The results showed that five strains were screened out from 72 endophytic fungi of T. chinensis which with high enzyme activities for lignocellulosic degradation. The laccase and cellulase activities of five strains reached their peaks at day 7, and the highest enzyme activities of these two enzymes were found in strain P6, which was 117.66 and 1.66 U/mL, respectively. Manganese peroxidase of strain 4 and lignin peroxidase of strain N6 also reached their peaks at day 7 and were the highest among the 5 strains, with enzyme activities of 11.61 and 6.64 U/mL, respectively. Strains 4, 15, 31, N6 and P6 were identified as Colletotrichum sp., Nigerrospora sphaerica, Exserohilum sp., Diaporthe phaseolorum and Pestalotiopsis sp., respectively, according to their morphological and molecular biology properties. The endophytic fungi may secrete efficient cell wall degradation enzymes, which promote the dissolution and relaxation of the cell wall between T. chinensis and host, thus contributing to the parasitism of T. chinensis.

Efficient D-allulose synthesis under acidic conditions by auto-inducing expression of the tandem D-allulose 3-epimerase genes in Bacillus subtilis

BackgroundD-allulose, a hexulose monosaccharide with low calorie content and high sweetness, is commonly used as a functional sugar in food and nutrition. However, enzyme preparation of D-allulose from D-frutose was severely hindered by the non-enzymatic browning under alkaline and high-temperature, and the unnecessary by-products further increased the difficulties in separation and extraction for industrial applications. Here, to address the above issue during the production process, a tandem D-allulose 3-epimerase (DPEases) isomerase synergistic expression strategy and an auto-inducible promoter engineering were levered in Bacillus subtilis 168 (Bs168) for efficient synthesis of D-allulose under the acidic conditions without browning.ResultsFirst, based on the dicistron expression system, two DPEases with complementary functional characteristics from Dorea sp. CAG:317 (DSdpe) and Clostridium cellulolyticum H10 (RCdpe) were expressed in tandem under the promoter HpaII in one cell. A better potential strain Bs168/pMA5-DSdpe-RCdpe increases enzyme activity to 18.9 U/mL at acidic conditions (pH 6.5), much higher than 17.2 and 16.7 U/mL of Bs168/pMA5-DSdpe and Bs168/pMA5-RCdpe, respectively. Subsequently, six recombinant strains based on four constitutive promoters were constructed in variable expression cassettes for improving the expression level of protein. Among those engineered strains, Bs168/pMA5-PspoVG-DSdpe-PsrfA-RCdpe exhibited the highest enzyme activity with 480.1 U/mL on fed-batch fermentation process in a 5 L fermenter at pH 6.5, about 2.1-times higher than the 228.5 U/mL of flask fermentation. Finally, the maximum yield of D-allulose reached as high as 163.5 g/L at the fructose concentration (50% w/v) by whole-cell biocatalyst.ConclusionIn this work, the engineered recombinant strain Bs168/pMA5-PspoVG-DSdpe-PsrfA-RCdpe was demonstrated as an effective microbial cell factory for the high-efficient synthesis of D-allulose without browning under acidic conditions. Based on the perspectives from this research, this strategy presented here also made it possible to meet the requirements of the industrial hyper-production of other rare sugars under more acidic conditions in theory.

Directed evolution driving the generation of an efficient keratinase variant to facilitate the feather degradation

Keratinases can specifically degrade keratins, which widely exist in hair, horns, claws and human skin. There is a great interest in developing keratinase to manage keratin waste generated by the poultry industry and reusing keratin products in agriculture, medical treatment and feed industries. Degradation of keratin waste by keratinase is more environmentally friendly and more sustainable compared with chemical and physical methods. However, the wild-type keratinase-producing strains usually cannot meet the requirements of industrial production, and some are pathogenic, limiting their development and utilization. The main purpose of this study is to improve the catalytic performance of keratinase via directed evolution technology for the degradation of feathers. We first constructed a mutant library through error-prone PCR and screened variants with enhanced enzyme activity. The keratinase activity was further improved through fermentation conditions optimization and fed-batch strategies in a 7-L bioreactor. As a result, nine mutants with enhanced activity were identified and the highest enzyme activity was improved from 1150 to 8448 U/mL finally. The mutant achieved efficient biodegradation of feathers, increasing the degradation rate from 49 to 88%. Moreover, a large number of amino acids and soluble peptides were obtained as degradation products, which were excellent protein resources to feed. Therefore, the study provided a keratinase mutant with application potential in the management of feather waste and preparation of protein feed additive.Graphical

Production Pectinases from Some Species of Aspergillus by Solid State Fermentation and Determination of Optimum Parameters for Production

بعد أن تم اختيار عشرين عزلة فطرية تتبع جنس Aspergillus من أربع وستين عينة من ثمار الفاكهة والخضار، وانتخاب الأفضل من حيث إنتاجها للإنزيمات البكتينية على الوسط الصلبplate assay method، تم اختيار الخمس عزلات الأقوى من بينها لإنتاج الإنزيمات البكتينية باستخدام نظام تخمير الوسط الصلب Solid state fermentation، وذلك بهدف معرفة إمكانية العزلات المنتخبة على إنتاج الإنزيمات البكتينية وتحديد العزلة الأكثر إنتاجاً لتلك الإنزيمات، إضافة إلى أمثلة إنتاج الإنزيمات من تلك العزلة من حيث درجة حرارة التحضين، مدة التحضين، تركيز البكتين في وسط التخمير، تركيز المعلق الفطري من الأبواغ، حجم اللقاح الفطري، بالإضافة إلى تحديد المصدر الكربوني والمصدر الآزوتي الأفضل للوصول إلى الإنتاج الأعظمي من الإنزيمات البكتينية، وكانت أربع عزلات من نوع A.niger وواحدة من نوع A.fumigatus. وقد استخدمت قشور البرتقال في عملية التخمير كمصدر للبكتين بعد أن تم حساب البكتين فيها وكان 5%، كما تبين أن فعالية الإنزيمات البكتينية المنتجة من العزلات المأخوذة من ثمار الحمضيات أعلى بالمقارنة مع باقي العزلات وقد وصلت الفعالية إلى51، U/ g 52، كما تبين من الدراسة المتبعة لتحديد الظروف المثلى لإنتاج هذه الإنزيمات أنه يمكن الحصول على أعلى فعالية إنزيمية عند درجة حرارة تحضين 40°C، ومدة تحضين 96ساعة، وعند استخدام كمية 2.5 غ من البكتين في الوسط وتركيز المعلق الفطري من الأبواغ 107بوغة/ مل، وحجم لقاح (معلق فطري) 1.5 مل، وباستخدام بكتين البرتقال كركيزة للوسط الصلب ومصدر للكربون، وكبريتات الأمونيوم كمصدر للآزوت.

A Decade of Climate-Smart Agriculture in Major Agri-Food Systems: Earthworm Abundance and Soil Physico-Biochemical Properties

Earthworms (EWs) could be a viable indicator of soil biology and agri-food system management. The influence of climate-smart agriculture (CSA)-based sustainable intensification practices (zero tillage, crop rotations, crop residue retention, and precision water and nutrients application) on earthworms’ (EWs) populations and soil physico-biochemical properties of rice-wheat cropping system in the Indo-Gangetic plains of South Asia was investigated. This study investigates the effect of 10-years adoption of various CSA practices on the abundance of earthworms and physical and biochemical properties of the soil and EWs’ casts (EWC). Five scenarios (Sc) were included: conventionally managed rice-wheat system (farmers’ practices, Sc1), CSA-based rice-wheat-mungbean system with flood irrigation (FI) (Sc2) and subsurface drip irrigation (SDI) (Sc3), CSA-based maize-wheat-mungbean system with FI (Sc4), and SDI (Sc5). Results revealed that EWs were absent under Sc1, while the 10-year adoption of CSA-based scenarios (mean of Sc2–5) increased EWs’ density and biomass to be 257.7 no. m−2 and 36.05 g m−2, respectively. CSA-based maize scenarios (Sc4 and Sc5) attained higher EWs’ density and biomass over rice-based CSA scenarios (Sc2 and Sc4). Also, SDI-based scenarios (Sc3 and Sc5) recorded higher EWs’ density and biomass over FI (Sc2 and Sc4). Maize-based CSA with SDI recorded the highest EWs’ density and EWs’ biomass. The higher total organic carbon in EWC (1.91%) than in the bulk soil of CSA-based scenarios (0.98%) and farmers’ practices (0.65%) suggests the shift of crop residue to a stable SOC (in EWC). EWC contained significant amounts of C and available NPK under CSA practices, which were nil under Sc1. All CSA-based scenarios attained higher enzymes activities over Sc1. CSA-based scenarios, in particular, maize-based scenarios using SDI, improved EWs’ proliferation, SOC, and nutrients storage (in soil and EWC) and showed a better choice for the IGP farmers with respect to C sequestration, soil quality, and nutrient availability.

Vaginal bleeding as a sign of Crimean\u2013Congo hemorrhagic fever infection: a case report

BackgroundCrimean–Congo hemorrhagic fever is a severe vector-borne viral hemorrhagic fever with considerable mortality in humans. This disease is endemic in Afghanistan, and its incidence rate has rapidly increased in recent years. This infection can cause a broad range of hemorrhage manifestations including epistaxis, petechial or purpuric rashes, hematemesis, and melena; however, vaginal bleeding is also reported as a rare manifestation.Case presentationWe report the case of a previously healthy 30-year-old Afghan female of shepherding occupation, with a sudden onset of fever, generalized body pain, epistaxis, and vaginal bleeding. She was admitted to the hospital after 7 days of symptom manifestation, with predominant signs being high fever, vaginal bleeding, and elevated liver enzymes. The serological test result for Crimean–Congo hemorrhagic fever was positive. She was treated with oral ribavirin and discharged with normal parameters.ConclusionsPeople in high-risk professions in endemic areas should be informed that vaginal bleeding is a serious symptom and requires immediate action and, therefore, might be attributed to nongynecologic disorders.

Valorization of Agro-industrial Discards in Fermentation for the Production of Cellulase Enzyme

Cellulases are commercially important enzymes with application in various industries such as biofuel, detergent, food processing, brewery, pulp and paper. To make its production cost-effective, a preferred method is to use solid-state fermentation and with use of inexpensive substrates. Solid-state fermentation is an alternative culturing method and yields higher enzymes compared to submerged fermentation. In the current study, Aspergillus niger was isolated and further developed as inoculum for solid-state fermentation. Agroindustrial discards like banana pseudostem, jackfruit waste were used as the substrates. The substrates were pretreated by acid and were characterized by FTIR analysis to confirm the presence of cellulosic content. Different concentrations of the substrates were attempted for fermentation and the yield of the enzyme was compared. The solid-state fermentation was stable for enzyme production as well as microbial growth. The cellulase activity per gram of the substrate (U/g) was obtained maximum for jackfruit waste-based media (17±1.1 U/g). Both the lignocellulosic substrates were potential substrates for the production of cellulase enzyme. With further optimization and scale-up, this could be a cheap and sustainable process. This study has validated agro-industrial waste’s bioconversion into value-added products that have a remarkable environmental and economic advantage.

Bundling the removal of emerging contaminants with the production of ligninolytic enzymes from residual streams

Enzymes offer interesting features as biological catalysts for industry: high specificity, activity under mild conditions, accessibility, and environmental friendliness. Being able to produce enzymes in large quantities and having them available in a stable and reusable form reduces the production costs of any enzyme-based process. Agricultural residues have recently demonstrated their potential as substrates to produce ligninolytic enzymes by different white rot fungi. In this study, the biotechnological production of a manganese peroxidase (MnP) by Irpex lacteus was conducted through solid-state fermentation (SSF) with wheat straw as substrate and submerged fermentation (SmF) employing wheat straw extract (WSE). The obtained enzyme cocktail also showed manganese-independent activity (MiP), related to the presence of a short MnP and a dye-decolorizing peroxidase (DyP) which was confirmed by shotgun proteomic analyses. In view of the enhanced production of ligninolytic enzymes in SmF, different parameters such as WSE concentration and nitrogen source were evaluated. The highest enzyme titers were obtained with a medium formulated with glucose and peptone (339 U/L MnP and 15 U/L MiP). The scale-up to a 30 L reactor achieved similar activities, demonstrating the feasibility of enzyme production from the residual substrate at different production scales. Degradation of five emerging pollutants was performed to demonstrate the high oxidative capacity of the enzyme. Complete removal of hormones and bisphenol A was achieved in less than 1 h, whereas almost 30% degradation of carbamazepine was achieved in 24 h, which is a significant improvement compared to previous enzymatic treatments of this compound.Key points• Wheat straw extract is suitable for the growth of I. lacteus.• The enzyme cocktail obtained allows the degradation of emerging contaminants.• Mn-dependent and Mn-independent activities increases the catalytic potential.Graphical abstract

Production, characterization, and antitumor efficiency of l-glutaminase from halophilic bacteria

BackgroundHalophiles are an excellent source of enzymes that are not only salt stable, but also can withstand and carry out reaction efficiently under extreme conditions. l-glutaminase has attracted much attention with respect to proposed applications in several fields such as pharmaceuticals and food industries. The aim of the present study was to investigate the anticancer activity of l-glutaminase produced by halophilic bacteria. Various halophilic bacterial strains were screened for extracellular l-glutaminase production. An attempt was made to study the optimization, purification, and characterization of l-glutaminase from Bacillus sp. DV2-37. The antitumor activity of the produced enzyme was also investigated.ResultsThe potentiality of 15 halophilic bacterial strains isolated from the marine environment that produced extracellular l-glutaminase was investigated. Bacillus sp. DV2-37 was selected as the most potent strain and optimized for enzyme production. The optimization of fermentation process revealed that the highest enzyme activity (47.12 U/ml) was observed in a medium supplemented with 1% (w/v) glucose as a carbon source, 1% (w/v) peptone as a nitrogen source, 5% (w/v) NaCl, the initial pH was 7.0, at 37 °C, using 20% (v/v) inoculum size after 96 h of incubation. The produced crude enzyme was partially purified by ammonium sulfate precipitation and dialysis. Of the various parameters tested, pH 7, 40 °C, and 5% NaCl were found to be the best for l-glutaminase activity. The enzyme also exhibited high salt and temperature stability. The antitumor effect against human breast (MCF-7), hepatocellular (HepG-2), and colon (HCT-116) carcinoma cell lines revealed that l-glutaminase produced by Bacillus sp. DV2-37 showed potent cytotoxic activity of all the tested cell lines in a dose-dependent manner with an IC50 value of 3.5, 3.4, and 3.8 µg/ml, respectively.ConclusionsThe present study proved that l-glutaminase produced by marine bacteria holds proper features and it has a high potential to be useful for many therapeutic applications.

Composition of Zingiber officinale Roscoe (Ginger), Soil Properties and Soil Enzyme Activities Grown in Different Concentration of Mineral Fertilizers

Ginger is rich in different chemical compounds such as phenolic compounds, terpenes, polysaccharides, lipids, organic acids, minerals, and vitamins. The present study investigated the effect of mineral fertilizers on the content of mineral elements in the rhizomes of Zingiber officinale Roscoe, soil enzymes activity, and soil properties in Surkhandarya Region, Uzbekistan. To the best of our knowledge, the present study is the first in Uzbekistan to investigate the mineral elements of ginger rhizome inhabiting Termez district, Surkhandarya region. A Field experiment was conducted at the Surkhandarya experimental station research Institute. Four treatments have been studied (Control with no fertilizers (T-1), N75P50K50 kg/ha (T-2), N125P100K100 kg/ha (T-3) and N100P75K75 + B3Zn6Fe6 kg/ha (T-4)). Results showed that T-4 treatment significantly increased ginger rhizome K, Ca, P, Mg, Fe, Na, Mn, Zn, Si, Li, and V content as compared to all other treatments and control. T-3 treatment significantly increased Mo, Ga, and Ag content in comparison to other treatments. Soil enzymes showed a significant increase for all treatments against control, while T-4 treatment has recorded the highest enzyme activity in comparison to all other treatments in urease, invertase, and catalase content. Soil chemical properties have significantly changed for all treatments against the non-cultivated soil and the zero fertilizers plantation with variation among different treatments. Results showed that ginger root is rich in minerals and can be used as a great potential for nutritional supplements and soil enrichment. This study suggest that combination of macro-microelements have the potential to increase the content of mineral elements in the rhizomes of ginger in field conditions.