Kinetic,thermodynamic parameters and in vitro digestion of tannase from Aspergillus tamarii URM 7115

Tannase is an enzyme used in various industries and produced by a large number of microorganisms. The aim of this study was to evaluate tannase production to determine the biochemical, kinetic, and thermodynamic properties and to simulate tannase in vitro digestion. The tannase-producing fungal strain was isolated from “jamun” leaves and identified as Aspergillus tamarii. Temperature at 26°C for 67?h was the best combination for maximum tannase activity (6.35-fold; initial activity in Plackett–Burman design—15.53?U/mL and average final activity in Doehlert design—98.68?U/mL). The crude extract of tannase was optimally active at 40°C, pH 5.5 and 6.5. Moreover, tannase was stimulated by Na⁺, Ca²⁺, Mg²⁺, and Mn²⁺. The half-life at 40°C lasted 247.55?min. The free energy of Gibbs, enthalpy, and entropy, at 40°C, was 81.47, 16.85, and ?0.21?kJ/mol?·?K, respectively. After total digestion, 123.95% of the original activity was retained. Results suggested that tannase from A. tamarii URM 7115 is an enzyme of interest for industrial applications, such as gallic acid production, additive for feed industry, and for beverage manufacturing, due to its catalytic and thermodynamic properties.     

Adaptive relationships of epoxide hydrolase in herbivorous arthropods

Epoxide hydrolase catalyzes a simple hydrolysis of reactive cyclic ethers that may otherwise alkylate and impair critical proteins and nucleic acids required for life. Although much less studied than the cytochrome P-450 monooxygenases that produce epoxides, differences in subcellular, tissue, pH, substrate, and inhibitor specificities argue for at least three forms of insect epoxide hydrolase. Increasing numbers of epoxides are being identified as plant allelochemicals, antifeedants, and essential hormones or precursors for herbivorous arthropods, and in many cases an associated alkene to diol pathway of metabolism is found. A role for epoxide hydrolase in arthropod-plant interactions is strongly supported by species comparisons and by age-activity and induction studies. Two major limitations for study in biochemical ecology of epoxide hydrolase are the lack of an effective in vivo inhibitor and a range of commercially available radiolabeled substrates for the enzymes.     

有机磷水解酶生物传感器载体固定化酶的研究

有机磷水解酶（OPH）传感器作为检测农产品中农药残留的新型检测装置,其酶的固定化对OPH传感器的灵敏度和稳定性有重要的影响。研究了几种酶固定化载体、孔径大小、固定方式、固定方法（试剂组成）对传感器pH值的影响。结果显示：采用孔径为0.45μm的硝酸纤维素膜制备固定化酶片的pH值要大于其余几种;采用浸泡方式制备固定化酶片的pH值明显大于传统的滴定法;采用牛血清白蛋白（BSA）、戊二醛交联固定的效果优于酶直接吸附法和BSA固定法,且当戊二醛体积分数为2.5％,BSA为10％时,酶固定化效果最好。     

Insights into the Structure of the Highly Glycosylated Ffase from Rhodotorula dairenensis Enhance Its Biotechnological Potential

Rhodotorula dairenensis β-fructofuranosidase is a highly glycosylated enzyme with broad substrate specificity that catalyzes the synthesis of 6-kestose and a mixture of the three series of fructooligosaccharides (FOS), fructosylating a variety of carbohydrates and other molecules as alditols. We report here its three-dimensional structure, showing the expected bimodular arrangement and also a unique long elongation at its N-terminus containing extensive O-glycosylation sites that form a peculiar arrangement with a protruding loop within the dimer. This region is not required for activity but could provide a molecular tool to target the dimeric protein to its receptor cellular compartment in the yeast. A truncated inactivated form was used to obtain complexes with fructose, sucrose and raffinose, and a Bis-Tris molecule was trapped, mimicking a putative acceptor substrate. The crystal structure of the complexes reveals the major traits of the active site, with Asn387 controlling the substrate binding mode. Relevant residues were selected for mutagenesis, the variants being biochemically characterized through their hydrolytic and transfructosylating activity. All changes decrease the hydrolytic efficiency against sucrose, proving their key role in the activity. Moreover, some of the generated variants exhibit redesigned transfructosylating specificity, which may be used for biotechnological purposes to produce novel fructosyl-derivatives.     

A Bacillus licheniformis Glycoside Hydrolase 43 Protein Is Recognized as a MAMP

Glycoside hydrolases from pathogens have often been reported as inducers of immune responses. However, the roles of glycoside hydrolase from plant-growth-promoting rhizobacteria (PGPR) in the resistance of plants against pathogens is not well studied. In this study, we identified a glycoside hydrolase 43 protein, H1AD43, produced by Bacillus licheniformis BL06 that can trigger defense responses, including cell death. Ion-exchange and size-exclusion chromatography were used for separation, and the amino acid sequence was identified by mass spectrometry. The recombinant protein generated by prokaryotic expression was able to elicit a hypersensitive response (HR) in Nicotiana benthamiana and trigger early defense responses, including reactive oxygen species (ROS) burst, callose accumulation, and the induction of defense genes. In addition, the protein could induce resistance in N. benthamiana, in which it inhibited infection by Phytophthora capsici Leonian and tobacco mosaic virus-green fluorescent protein (TMV-GFP) expression. H1AD43 thus represents a microbe-associated molecular pattern (MAMP) of PGPR that induces plant disease resistance and may provide a new method for the biological control of plant disease.     

Comprehensive Analysis of Copy Number Variations on Glycoside Hydrolase 45 Genes among Different Bursaphelenchus xylophilus Strains

Bursaphelenchus xylophilus is considered the most dangerous quarantine pest in China. It causes enormous economic and ecological losses in many countries from Asia and Europe. The glycoside hydrolase 45 gene family has been demonstrated in early studies to contribute to the cell wall degradation ability of B. xylophilus during its infection. However, the copy number variation (CNV) of the GH45 gene and its association with B. xylophilus pathogenicity were not fully elucidated. In this study, we found that the GH45 gene with two copies is the most predominant type among 259 B. xylophilus strains collected from China and Japan. Additionally, 18 strains are identified as GH45 genes with a single copy, and only two strains are verified to have three copies. Subsequent expression analysis and inoculation test suggest that the copy numbers of the GH45 gene are correlated with gene expression as well as the B. xylophilus pathogenicity. B. xylophilus strains with more copies of the GH45 gene usually exhibit more abundant expression and cause more severe wilt symptoms on pine trees. The aforementioned results indicated the potential regulatory effects of CNV in B. xylophilus and provided novel information to better understand the molecular pathogenesis of this devastating pest.     

GH-16 Type β-1,3-Glucanase from Lysobacter sp. MK9-1 Enhances Antifungal Activity of GH-19 Type Chitinase,and Its Glucan-binding Domain Binds to Fungal Cell-wall

The GH-16 type β-1,3-glucanase (BgluC16MK) gene of Lysobacter sp. MK9-1 was cloned to study its antifungal activities. BgluC16MK displays amino acid sequence similarity with GluC from L. enzymogenes strain N4-7. BgluC16MK includes a signal sequence, a catalytic domain and carbohydrate-binding module family 6-type β-glucan binding domain (B-GBD). The expression of the BgluC16MK gene in Escherichia coli without the signal sequence resulted in antifungal activity at a dose of 0.6-0.8 nmol/disk. However, BgluC16MK displayed antifungal activity at a dose of 0.025 nmol/disk in combination with Chi19MK. Substrate-specific assay revealed that purified BgluC16MK hydrolyzed insoluble curdlan more readily than the soluble substrate. Furthermore, to explore the binding selectivity of B-GBD of BgluC16MK, we constructed a fusion protein (B-GBD-GFP) using the B-GBD and green fluorescent protein. The activity of the fusion protein against various substrates indicates that B-GBD was selective for glucans with β-1,3-linkages. An additional study demonstrated the binding ability of B-GBD-GFP to the cell-wall of living fungi, such as T. reesei and Aspergillus oryzae. These findings suggest that BgluC16MK can be utilized to generate antifungal enzyme preparations and that the fusion protein B-GBD-GFP can be used to identify the fungal cell surface structure using β-glucans.     

Anti-Inflammatory Activity of Soluble Epoxide Hydrolase Inhibitors Based on Selenoureas Bearing an Adamantane Moiety

The inhibitory potency of the series of inhibitors of the soluble epoxide hydrolase (sEH) based on the selenourea moiety and containing adamantane and aromatic lipophilic groups ranges from 34.3 nM to 1.2 μM. The most active compound 5d possesses aliphatic spacers between the selenourea group and lipophilic fragments. Synthesized compounds were tested against the LPS-induced activation of primary murine macrophages. The most prominent anti-inflammatory activity, defined as a suppression of nitric oxide synthesis by LPS-stimulated macrophages, was demonstrated for compounds 4a and 5b. The cytotoxicity of the obtained substances was studied using human neuroblastoma and fibroblast cell cultures. Using these cell assays, the cytotoxic concentration for 4a was 4.7–18.4 times higher than the effective anti-inflammatory concentration. The genotoxicity and the ability to induce oxidative stress was studied using bacterial lux-biosensors. Substance 4a does not exhibit genotoxic properties, but it can cause oxidative stress at concentrations above 50 µM. Put together, the data showed the efficacy and safety of compound 4a.     

水杨酸处理对库尔勒香梨果实贮藏期细胞壁组分和水解酶活性变化的影响

本研究探讨水杨酸处理对库尔勒香梨贮藏期果实质地的影响。通过对库尔勒香梨进行水杨酸处理后,定期检测库尔勒香梨果实贮藏期细胞壁含量、细胞壁组成物质含量、果胶酶和纤维素酶活性等指标。结果表明水杨酸处理较好地保持香梨果实细胞壁含量,在贮期结束时达19.91 mg/g左右,并显著抑制了香梨果实贮藏期果胶酶和纤维素酶活性,减缓了水溶性和离子型果胶含量上升和共价结合型果胶含量及纤维素和半纤维素含量的下降。水杨酸处理对保持香梨果实细胞壁组分,延缓库尔勒香梨贮藏期果实衰老有积极作用。