Characterization of a Hyaluronic Acid Utilization Locus and Identification of Two Hyaluronate Lyases in a Marine Bacterium Vibrio alginolyticus LWW-9

Hyaluronic acid (HA) is a negatively charged and linear polysaccharide existing in the tissues and body fluids of all vertebrates. Some pathogenic bacteria target hyaluronic acid for adhesion and/or infection to host cells. Vibrio alginolyticus is an opportunistic pathogen related to infections of humans and marine animals, and the hyaluronic acid-degrading potential of Vibrio spp. has been well-demonstrated. However, little is known about how Vibrio spp. utilize hyaluronic acid. In this study, a marine bacterium V. alginolyticus LWW-9 capable of degrading hyaluronic acid has been isolated. Genetic and bioinformatic analysis showed that V. alginolyticus LWW-9 harbors a gene cluster involved in the degradation, transport, and metabolism of hyaluronic acid. Two novel PL8 family hyaluronate lyases, VaHly8A and VaHly8B, are the key enzymes for the degradation of hyaluronic acid. VaHly8A and VaHly8B have distinct biochemical properties, reflecting the adaptation of the strain to the changing parameters of the aquatic habitats and hosts. Based on genomic and functional analysis, we propose a model for the complete degradation of hyaluronic acid by V. alginolyticus LWW-9. Overall, our study expands our knowledge of the HA utilization paradigm within the Proteobacteria, and the two novel hyaluronate lyases are excellent candidates for industrial applications

Formation of sclerotia in Sclerotinia ginseng and composition of the sclerotial exudate

Background Sclerotinia ginseng is a major devastating soil-borne pathogen of ginseng that can cause irreparable damage and large economic losses. This pathogen produces sclerotia, which are among the most persistent resting structures produced by filamentous fungi. The production of an exudate is a common feature of sclerotial development. Methods S. ginseng was cultured on 10 different media and the following parameters were measured: mycelial growth rate (mm/day), initial formation time of exudate droplets, total quantity of exudate, number of sclerotia per dish, and sclerotial fresh/dry weight. The composition of the sclerotial exudate was analyzed using four methods (high performance liquid chromatography, gas chromatography-mass spectrometry, flame atomic absorption spectrometry, and Nessler’s reagent spectrophotometry). Results We found that PDA was the optimal medium for exudate production, while SDA medium resulted in the highest mycelial growth rate. The earliest emergence of exudate droplets from sclerotia was on OA-YE and V8 media. The largest amount of sclerotia and the smallest sclerotia were produced on V8 medium. The maximum and minimum dry/fresh weight were obtained on MEA medium and V8 medium, respectively. The exudate contained organic acids (oxalic acid, gallic acid, ferulic acid, vanillic acid, caffeic acid, and tannic acid), carbohydrates (inositol, glucose, and trehalose), various ions (potassium, sodium, and magnesium), and ammonia. Discussion The functions of the identified compounds are discussed within the context of pathogenicity, sclerotial development, and antimicrobial activity. Our findings provide information about the production of sclerotia and the composition of sclerotial exudate that may be useful to develop strategies to control this disease

Effect of Clay Mineral Composition on Low-Salinity Water Flooding

Low-salinity water (LSW) flooding technology has obvious operational and economic advantages, so it is applied to practice in many oilfields. However, there are differences in the oil recovery efficiencies in different oilfields, the reasons for which need to be further studied and discussed. This paper studies the effect of different clay mineral compositions on low-salinity water flooding. For this purpose, three groups of core displacement experiments were designed with cores containing different clay mineral compositions for comparison. In the process of formation water and low-salinity water driving, the oil recovery and produced-water properties were measured. By comparing the two types of water flooding, it was found that the cores with the highest montmorillonite content had the best effect (5.7%) on low-salinity water flooding and the cores with the highest kaolinite content had the least effect (1.9%). This phenomenon is closely related to the difference in ion exchange capacity of the clay minerals. Moreover, after switching to low-salinity water flooding, the interfacial tension and wetting angle of the produced-water increased and the value of pH decreased, which are important mechanisms for enhancing oil recovery by low-salinity water flooding. This study reveals the influence of clay mineral composition on low-salinity water flooding and can provide more guidance for conventional and unconventional oilfield application of low-salinity water flooding technology

YsHyl8A, an Alkalophilic Cold-Adapted Glycosaminoglycan Lyase Cloned from Pathogenic Yersinia sp. 298

A high enzyme-yield strain Yersinia sp. 298 was screened from marine bacteria harvested from the coastal water. The screening conditions were extensive, utilizing hyaluronic acid (HA)/chondroitin sulfate (CS) as the carbon source. A coding gene yshyl8A of the family 8 polysaccharide lyase (PL8) was cloned from the genome of Yersinia sp. 298 and subjected to recombinant expression. The specific activity of the recombinase YsHyl8A was 11.19 U/mg, with an optimal reaction temperature of 40 °C and 50% of its specific activity remaining after thermal incubation at 30 °C for 1 h. In addition, its optimal reaction pH was 7.5, and while it was most stable at pH 6.0 in Na2HPO4-citric acid buffer, it remained highly stable at pH 6.0–11.0. Further, its enzymatic activity was increased five-fold with 0.1 M NaCl. YsHyl8A, as an endo-lyase, can degrade both HA and CS, producing disaccharide end-products. These properties suggested that YsHyl8A possessed both significant alkalophilic and cold-adapted features while being dependent on NaCl, likely resulting from its marine source. Yersinia is a typical fish pathogen, with glycosaminoglycan lyase (GAG lyase) as a potential pathogenic factor, exhibiting strong hyaluronidase and chondroitinase activity. Further research on the pathogenic mechanism of GAG lyase may benefit the prevention and treatment of related diseases

A new permeability calculation method using nuclear magnetic resonance logging based on pore sizes: A case study of bioclastic limestone reservoirs in the A oilfield of the Mid-East

It is difficult to accurately obtain the permeability of complex lithologic reservoirs using conventional methods because they have diverse pore structures and complex seepage mechanisms. Based on in-depth analysis of the limitation of classical nuclear magnetic resonance (NMR) permeability calculation models, and the understanding that the pore structure and porosity are the main controlling factors of permeability, this study provides a new permeability calculation method involving classifying pore sizes by using NMR T2 spectrum first and then calculating permeability of different sizes of pores. Based on this idea, taking the bioclastic limestone reservoir in the A oilfield of Mid-East as an example, the classification criterion of four kinds of pore sizes: coarse, medium, fine and micro throat, was established and transformed into NMR T2 standard based on shapes and turning points of mercury intrusion capillary pressure curves. Then the proportions of the four kinds of pore sizes were obtained precisely based on the NMR logging data. A new NMR permeability calculation model of multicomponent pores combinations was established based on the contributions of pores in different sizes. The new method has been used in different blocks. The results show that the new method is more accurate than the traditional ones. Key words: reservoir, pore structure, nuclear magnetic resonance, permeability, bioclastic limestone, well loggin

Site-Specific Covalent Immobilization of <i>Methylobacterium extorquens</i> Non-Blue Laccse Melac13220 on Fe₃O₄ Nanoparticles by Aldehyde Tag

In the present study, the non-blue laccase Melac13220 from Methylobacterium extorquens was immobilized using three methods to overcome problems related to the stability and reusability of the free enzyme: entrapment of the enzyme with sodium alginate, crosslinking of the enzyme with glutaraldehyde and chitosan-, and site-specific covalent immobilization of the enzyme on Fe3O4 nanoparticles by an aldehyde tag. The site-specific covalent immobilization method showed the highest immobilization efficiency and vitality recovery. The optimum temperature of Melac13220 was increased from 65 °C to 80 °C. Immobilized Melac13220 showed significant tolerance to some organic solvents and maintained approximately 80% activity after 10 cycles of use. Differential scanning calorimetry (DSC) indicated that the melting temperature of the enzyme was increased (from 57 °C to 79 °C). Immobilization of Melac13220 also led to improvement in dye decolorization such that Congo Red was completely decolorized within 10 h. The immobilized enzyme can be easily prepared without purification, demonstrating the advantages of using the aldehyde tag strategy and providing a reference for the practical application of different immobilized laccase methods in the industrial field

Identification and Biochemical Characterization of a Surfactant-Tolerant Chondroitinase VhChlABC from Vibrio hyugaensis LWW-1

Chondroitinases, catalyzing the degradation of chondroitin sulfate (CS) into oligosaccharides, not only play a crucial role in understanding the structure and function of CS, but also have been reported as a potential candidate drug for the treatment of high CS-related diseases. Here, a marine bacterium Vibrio hyugaensis LWW-1 was isolated, and its genome was sequenced and annotated. A chondroitinase, VhChlABC, was found to belong to the second subfamily of polysaccharide lyase (PL) family 8. VhChlABC was recombinant expressed and characterized. It could specifically degrade CS-A, CS-B, and CS-C, and reached the maximum activity at pH 7.0 and 40 °C in the presence of 0.25 M NaCl. VhChlABC showed high stability within 8 h under 37 °C and within 2 h under 40 °C. VhChlABC was stable in a wide range of pH (5.0~10.6) at 4 °C. Unlike most chondroitinases, VhChlABC showed high surfactant tolerance, which might provide a good tool for removing extracellular CS proteoglycans (CSPGs) of lung cancer under the stress of pulmonary surfactant. VhChlABC completely degraded CS to disaccharide by the exolytic mode. This research expanded the research and application system of chondroitinases

Superoxide radical-mediated photocatalytic oxidation of phenolic compounds over Ag+/TiO2: Influence of electron donating and withdrawing substituents

A comparative study was constructed to correlate the electronic property of the substituents with the degradation rates of phenolic compounds and their oxidation pathways under UV with Ag+/TiO2 suspensions. It was verified that a weak electron withdrawing substituent benefited photocatalytic oxidation the most, while an adverse impact appeared when a substituent was present with stronger electron donating or withdrawing ability. The addition of p-benzoquinone dramatically blocked the degradation, confirming superoxide radicals (O-center dot(2)-) as the dominant photooxidant, rather than hydroxyl radicals, singlet oxygen or positive holes, which was also independent of the substituent. Hammett relationship was established based on pseudo-first-order reaction kinetics, and it revealed two disparate reaction patterns between center dot O-2(-) and phenolic compounds, which was further verified by the quantum chemical computation on the frontier molecular orbitals and Mulliken charge distributions of center dot O-2(-) and phenolic compounds. It was found that electron donating group (EDG) substituted phenols were more likely nucleophilically attacked by center dot O-2(-), while center dot O-2(-) preferred to electrophilically assault electron withdrawing group (EWG) substituted phenols. Exceptionally, electrophilic and nucleophilic attack by center dot O-2(-) could simultaneously occur in p-chlorophenol degradation, consequently leading to its highest rate constant. Possible reactive positions on the phenolic compounds were also detailedly uncovered. (C) 2015 Elsevier B.V. All rights reserved

Optimizing the Incorporated Amount of Chinese Milk Vetch (<i>Astragalus sinicus</i> L.) to Improve Rice Productivity without Increasing CH₄ and N₂O Emissions

Chinese milk vetch (CMV) is a leguminous green manure that is commonly cultivated in paddy fields and can partially substitute synthetic nitrogen fertilizer. However, the impacts of incorporating CMV on CH4 and N2O emissions are still a subject of controversy. Therefore, we conducted a field experiment over three years to investigate emissions under different substitution ratios: urea only (CF); incorporating a traditional amount of CMV (MV); and with incorporation ratios of 1/3 (MV1/3), 2/3 (MV2/3), and 4/3 (MV4/3) of MV for partial urea substitution. Compared with CF, MV2/3, MV, and MV 4/3 resulted in increased yields. MV and MV4/3 reduced N2O emissions but increased CH4 emissions by 28.61% and 85.60% (2019), 32.38% and 103.19% (2020), and 28.86% and 102.98% (2021), respectively, resulting in an overall increase in total global warming potential (except for MV in 2021). MV2/3 exhibited a low greenhouse gas intensity value ranging from 0.46 to 0.47. Partial least-squares-path model results showed that CH4 and N2O emissions were influenced by substitution ratios, which indirectly regulated the gene abundances of mcrA and nosZ. Overall, the impact of CMV on CH4 and N2O emissions was determined by substitution ratios. MV2/3, which involved partial substitution of synthetic N fertilizer with 15.0 t ha−1 of CMV, resulted in improved rice productivity without increasing CH4 and N2O emissions, making it a recommended approach in the study area