稻瘟病菌一个假想几丁质酶的表达分析

为研究稻瘟病菌几丁质酶在稻瘟病菌形态建成和致病过程中的作用,对稻瘟病菌MGG_08054.6编码的一个假想几丁质酶进行生物信息学分析、基因表达及其融合蛋白分泌特性研究.结果显示:稻瘟病菌基因MGG_08054.6编码的蛋白MoChil与里氏木霉、桔绿木霉、烟曲霉、禾布氏白粉菌已知的18家族几丁质酶(EC 3.2.1.14)蛋白序列同源性大于40%,并具有几丁质酶催化区域的两个保守基序GGW和DG-D-DWE及部分几丁质结合区的活性位点,该蛋白序列经程序SignalP 3.0分析含有信号肽,在16T-17S和26Q-27S处有两个可能的剪切位点;将MGG 08054.6基因在稻瘟病菌Guy11进行过量表达,获得分泌蛋白,证明了该蛋白的分泌特性.半定量RT-PCR分析表明,该基因在不同生长发育阶段及侵染水稻阶段均有表达,其中菌丝阶段表达量最高.     

A Broad-Specificity Chitinase from Penicillium oxalicum k10 Exhibits Antifungal Activity and Biodegradation Properties of Chitin

Penicillium oxalicum k10 isolated from soil revealed the hydrolyzing ability of shrimp chitin and antifungal activity against Sclerotinia sclerotiorum. The k10 chitinase was produced from a powder chitin-containing medium and purified by ammonium sulfate precipitation and column chromatography. The purified chitinase showed maximal activity toward colloidal chitin at pH 5 and 40 °C. The enzymatic activity was enhanced by potassium and zinc, and it was inhibited by silver, iron, and copper. The chitinase could convert colloidal chitin to N-acetylglucosamine (GlcNAc), (GlcNAc)2, and (GlcNAc)3, showing that this enzyme had endocleavage and exocleavage activities. In addition, the chitinase prevented the mycelial growth of the phytopathogenic fungi S. sclerotiorum and Mucor circinelloides. These results indicate that k10 is a potential candidate for producing chitinase that could be useful for generating chitooligosaccharides from chitinous waste and functions as a fungicide.     

Identification and Characterization of Three Chitinases with Potential in Direct Conversion of Crystalline Chitin into N,N′-diacetylchitobiose

Chitooligosaccharides (COSs) have been widely used in agriculture, medicine, cosmetics, and foods, which are commonly prepared from chitin with chitinases. So far, while most COSs are prepared from colloidal chitin, chitinases used in preparing COSs directly from natural crystalline chitin are less reported. Here, we characterize three chitinases, which were identified from the marine bacterium Pseudoalteromonas flavipulchra DSM 14401^T, with an ability to degrade crystalline chitin into (GlcNAc)₂ (N,N’-diacetylchitobiose). Strain DSM 14401 can degrade the crystalline α-chitin in the medium to provide nutrients for growth. Genome and secretome analyses indicate that this strain secretes six chitinolytic enzymes, among which chitinases Chia4287, Chib0431, and Chib0434 have higher abundance than the others, suggesting their importance in crystalline α-chitin degradation. These three chitinases were heterologously expressed, purified, and characterized. They are all active on crystalline α-chitin, with temperature optima of 45–50 °C and pH optima of 7.0–7.5. They are all stable at 40 °C and in the pH range of 5.0–11.0. Moreover, they all have excellent salt tolerance, retaining more than 92% activity after incubation in 5 M NaCl for 10 h at 4 °C. When acting on crystalline α-chitin, the main products of the three chitinases are all (GlcNAc)₂, which suggests that chitinases Chia4287, Chib0431, and Chib0434 likely have potential in direct conversion of crystalline chitin into (GlcNAc)₂.     

Isolation,Characterization, Kinetics,and Enzymatic and Nonenzymatic Microbicidal Activities of a Novel c-Type Lysozyme from Plasma of Schistocerca gregaria (Orthoptera: Acrididae)

A protein, designated as Sgl, showing a muramidase lytic activity to the cell wall of the Gram-positive bacterium Micrococcus lysodeikticus was isolated for the first time from plasma of Escherichia coli-immunized fifth instar Schistocerca gregaria. The isolated Sgl was detected as a single protein band, on both native- and SDS-PAGE, has a molecular weight of ∼15.7 kDa and an isoelectric point (pI) of ca 9.3 and its antiserum has specifically recognized its isolated form. Fifty-nine percentage of Sgl lytic activity was recovered in the isolated fractions and yielded ca 126-fold increase in specific activity than that of the crude. The partial N-terminal amino acid sequence of the Sgl has 55 and 40% maximum identity with Bombyx mori and Gallus gallus c-type lysozymes, respectively. The antibacterial activity against the Gram-positive and the Gram-negative bacteria were comparatively stronger than that of the hen egg white lysozyme (HEWL). The detected Sgl poration to the inner membrane that reach a maximum ability after 3 h was suggested to operate as a nonenzymatic mechanism for Gram-negative bacterial cell lysis, as tested in a permease-deficient E. coli, ML-35 strain. Sgl showed a maximal muramidase activity at pH 6.2, 30–50°C, and 0.05 M Ca²⁺ or Mg²⁺; and has a K_m of 0.5 μg/ml and a V_max of 0.518 with M. lysodeikticus as a substrate. The Sgl displayed a chitinase activity against chitin with a K_m of 0.93 mg/ml and a V_max of 1.63.     

茶树几丁质酶基因的克隆及其在干旱胁迫下的表达分析在干旱胁迫下的表达分析

以铁观音茶树叶片为材料,利用逆转录PCR及RACE法,克隆了茶树几丁质酶基因CsChi(GenBank登录号为KR078345).CsChi基因的cDNA全长为1 192 bp,包含972 bp的开放阅读框(ORF),编码323个氨基酸.生物信息学分析结果表明,CsChi蛋白的分子量为34.33 ku;理论等电点pI为8.44;原子组成为C1519H2285N413O464S18,总原子数为4 699;蛋白质结构分析显示该蛋白有6个蛋白的跨膜区域,属于跨膜蛋白;存在于细胞外;没有卷曲螺旋结构存在;CsChi基因编码的蛋白属于糖苷水解酶19家族,含有保守的ChtBD1结构域,与溶菌酶的保守结构域类似,可能兼具几丁质酶活性和溶菌酶活性,qPCR定量分析结果显示在不同干旱胁迫处理下茶树的CsChi基因的表达量,与对照组相比有所增加.推测CsChi基因在茶树干旱等逆境胁迫中起重要作用.     

Overexpression and Characterization of a Novel Thermostable β-Agarase YM01-3, from Marine Bacterium Catenovulum agarivorans YM01T

Genome sequencing of Catenovulum agarivorans YM01^T reveals 15 open-reading frames (ORFs) encoding various agarases. In this study, extracellular proteins of YM01^T were precipitated by ammonium sulfate and separated by one-dimensional gel electrophoresis. The results of in-gel agarase activity assay and mass spectrometry analysis revealed that the protein, YM01-3, was an agarase with the most evident agarolytic activity. Agarase YM01-3, encoded by the YM01-3 gene, consisted of 420 amino acids with a calculated molecular mass of 46.9 kDa and contained a glycoside hydrolase family 16 β-agarase module followed by a RICIN superfamily in the C-terminal region. The YM01-3 gene was cloned and expressed in Escherichia coli. The recombinant agarase, YM01-3, showed optimum activity at pH 6.0 and 60 °C and had a K_m of 3.78 mg mL⁻¹ for agarose and a V_max of 1.14 × 10⁴ U mg⁻¹. YM01-3 hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Notably, YM01-3 was stable below 50 °C and retained 13% activity after incubation at 80 °C for 1 h, characteristics much different from other agarases. The present study highlights a thermostable agarase with great potential application value in industrial production.     

Preliminary Characterization,Antioxidant Properties and Production of Chrysolaminarin from Marine Diatom Odontella aurita

A new chrysolaminarin, named CL2, with a molecular mass of 7.75 kDa, was purified from the marine diatom, Odontella aurita, using DEAE-52 cellulose anion-exchange chromatography and Sephadex G-200 gel-filtration chromatography. The monosaccharide and structural analysis revealed that CL2 was a glucan mainly composed of glucose, which was linked by the β-d-(1→3) (main chain) and β-d-(1→6) (side chain) glycosidic bond, demonstrated by infrared spectroscopy (IR) and nuclear magnetic resonance (NMR). The antioxidant activity tests revealed that the CL2 presented stronger hydroxyl radical scavenging activity with increasing concentrations, but less was effective on reducing power analysis and scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The influences of nitrogen concentration and light intensity on chrysolaminarin production of O. aurita were further investigated in a glass column photobioreactor, and a record high chrysolaminarin productivity of 306 mg L⁻¹ day⁻¹ was achieved. In conclusion, the chrysolaminarin CL2 from O. aurita may be explored as a natural antioxidant agent for application in aquaculture, food and pharmaceutical areas.     

Mycemycins A–E,New Dibenzoxazepinones Isolated from Two Different Streptomycetes

Five new dibenzoxazepinone derivatives, mycemycins A–E (1–5), were isolated from the ethanol extracts of mycelia of two different streptomycetes. 1 and 2 were isolated from an acidic red soil-derived strain, Streptomyces sp. FXJ1.235, and 3–5 from a gntR gene-disrupted deep-sea strain named Streptomyces olivaceus FXJ8.012Δ1741. The structures of mycemycins were elucidated by a combination of spectroscopic analyses, including 1D- and 2D-NMR techniques.     

Structural and Immunochemical Studies of the Lipopolysaccharide from the Fish Pathogen,Aeromonas bestiarum Strain K296, Serotype O18

Chemical analyses and mass spectrometry were used to study the structure of the lipopolysaccharide (LPS) isolated from Aeromonas bestiarum strain K296, serotype O18. ESI-MS revealed that the most abundant A. bestiarum LPS glycoforms have a hexa-acylated or tetra-acylated lipid A with conserved architecture of the backbone, consisting of a 1,4′-bisphosphorylated β-(1→6)-linked d-GlcN disaccharide with an AraN residue as a non-stoichiometric substituent and a core oligosaccharide composed of Kdo₁Hep₆Hex₁HexN₁P₁. 1D and 2D NMR spectroscopy revealed that the O-specific polysaccharide (OPS) of A. bestiarum K296 consists of a branched tetrasaccharide repeating unit containing two 6-deoxy-l-talose (6dTalp), one Manp and one GalpNAc residues; thus, it is similar to that of the OPS of A. hydrophila AH-3 (serotype O34) in both the sugar composition and the glycosylation pattern. Moreover, 3-substituted 6dTalp was 2-O-acetylated and additional O-acetyl groups were identified at O-2 and O-4 (or O-3) positions of the terminal 6dTalp. Western blots with polyclonal rabbit sera showed that serotypes O18 and O34 share some epitopes in the LPS. The very weak reaction of the anti-O34 serum with the O-deacylated LPS of A. bestiarum K296 might have been due to the different O-acetylation pattern of the terminal 6dTalp. The latter suggestion was further confirmed by NMR.     

Chitin Degradation Machinery and Secondary Metabolite Profiles in the Marine Bacterium Pseudoalteromonas rubra S4059

Genome mining of pigmented Pseudoalteromonas has revealed a large potential for the production of bioactive compounds and hydrolytic enzymes. The purpose of the present study was to explore this bioactivity potential in a potent antibiotic and enzyme producer, Pseudoalteromonas rubra strain S4059. Proteomic analyses (data are available via ProteomeXchange with identifier PXD023249) indicated that a highly efficient chitin degradation machinery was present in the red-pigmented P. rubra S4059 when grown on chitin. Four GH18 chitinases and two GH20 hexosaminidases were significantly upregulated under these conditions. GH19 chitinases, which are not common in bacteria, are consistently found in pigmented Pseudoalteromonas, and in S4059, GH19 was only detected when the bacterium was grown on chitin. To explore the possible role of GH19 in pigmented Pseudoalteromonas, we developed a protocol for genetic manipulation of S4059 and deleted the GH19 chitinase, and compared phenotypes of the mutant and wild type. However, none of the chitin degrading ability, secondary metabolite profile, or biofilm-forming capacity was affected by GH19 deletion. In conclusion, we developed a genetic manipulation protocol that can be used to unravel the bioactive potential of pigmented pseudoalteromonads. An efficient chitinolytic enzyme cocktail was identified in S4059, suggesting that this strain could be a candidate with industrial potential.     

Assignment of the CD Cotton Effect to the Chiral Center in Pseurotins,and the Stereochemical Revision of Pseurotin A2

Pseurotins A₁ (1) and A₂ (2) were isolated from a culture broth of the fungal strain Aspergillus fumigatus WFZ-25 as stereoisomers of pseurotin A (3) in 2011. We also isolated 1 and 2 together with 3 from A. fumigatus OUPS-T106B-5 separated from the marine fish Mugil cephalus. In this study, we re-examined the stereochemistry of 1 and 2 using chemical transformation and the CD spectra, and found the relationship between the CD Cotton effect and the absolute configurations of 1 and 2, which led us to revise the stereostructure of pseurotin A₂.     

The Glycolytic Enzymes Activity in the Midgut of Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) adult and their Seasonal Changes

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) is an important pest of maize. The diet of the D. virgifera imago is rich in starch and other polysaccharides present in cereals such as maize. Therefore, knowledge about enzymes involved in digestion of such specific food of this pest seems to be important. The paper shows, for the first time, the activities of main glycolytic enzymes in the midgut of D. virgifera imago: endoglycosidases (α-amylase, cellulase, chitinase, licheninase, laminarinase); exoglycosidases (α- and β-glucosidases, α- and β-galactosidases) and disaccharidases (maltase, isomaltase, sucrase, trehalase, lactase, and cellobiase). Activities of α-amylase, α-glucosidase, and maltase were the highest among assayed endoglycosidases, exoglycosidases, and disaccharidases, respectively. This indicates that in the midgut of D. virgifera imago α-amylase, α-glucosidase and maltase are important enzymes in starch hydrolysis and products of its digestion. These results lead to conclusion that inhibition of most active glycolytic enzymes of D. virgifera imago may be another promising method for chemical control of this pest of maize.     

Characterization of an Alginate Lyase,FlAlyA, from Flavobacterium sp. Strain UMI-01 and Its Expression in Escherichia coli

A major alginate lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the polysaccharide-lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I—Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.     

Purification and Characterization of a Fucoidanase (FNase S) from a Marine Bacterium Sphingomonas paucimobilis PF-1

The Search for enzyme activities that efficiently degrade marine polysaccharides is becoming an increasingly important area for both structural analysis and production of lower-molecular weight oligosaccharides. In this study, an endo-acting fucoidanase that degrades Miyeokgui fucoidan (MF), a sulfated galactofucan isolated from the sporophyll (called Miyeokgui in Korean) of Undaria pinnatifida, into smaller-sized galactofuco-oligosaccharides (1000–4000 Da) was purified from a marine bacterium, Sphingomonas paucimobilis PF-1, by ammonium sulfate precipitation, diethylaminoethyl (DEAE)-Sepharose column chromatography, and chromatofocusing. The specific activity of this enzyme was approximately 112-fold higher than that of the crude enzyme, and its molecular weight was approximately 130 kDa (FNase S), as determined by native gel electrophoresis and 130 (S1), 70 (S2) and 60 (S3) kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of FNase S were pH 6.0–7.0 and 40–45 °C, respectively. FNase S activity was enhanced by Mn²⁺ and Na⁺ (115.7% and 131.2%), but it was inhibited by Ca²⁺, K⁺, Ba²⁺, Cu²⁺ (96%, 83.7%, 84.3%, and 89.3%, respectively), each at 1 mM. The K_m, V_max and K_cat values of FNase S on MF were 1.7 mM, 0.62 mg·min⁻¹, and 0.38·S⁻¹, respectively. This enzyme could be a valuable tool for the structural analysis of fucoidans and production of bioactive fuco-oligosaccharides.     

乙烯利和乙烯刺激对橡胶树胶乳中几丁质酶活性和胶乳产量的影响

以巴西橡胶树无性系PR107,RRIM600,GT1为试验材料,研究乙烯利和乙烯刺激对胶乳黄色体中几丁质酶活性和胶乳产量的影响。结果表明:在乙烯利和乙烯的有效刺激浓度范围内,几丁质酶活性随乙烯利和乙烯浓度增加而增加;乙烯和乙烯利持续刺激一段时间后,几丁质酶活性达到最大值;3种不同排胶特性品系橡胶树在施用乙烯利刺激后胶乳中几丁质酶活性和胶乳产量较施用乙烯利刺激前均有所增加,其增加强度存在着品系差异,表现为:PR107>GT1>RRIM600。      

玉米螟虫生真菌球孢白僵菌几丁酶基因的克隆与分析

球孢白僵菌是最常见的昆虫病原真菌之一,已成功应用于玉米螟和松毛虫等害虫的生物防治。球孢白僵菌主要通过分泌多种胞外水解酶降解昆虫体壁侵入寄主,其分泌的水解酶主要包括蛋白酶、几丁质酶、脂酶等。本研究从玉米螟虫生真菌球孢白僵菌中分离到一个几丁质酶基因BbChitI,该基因的编码区包含1047bp,编码了348个氨基酸,氨基酸序列与来源于小菜蛾虫生真菌的白僵菌几丁质酶的同源性高达99%,与来源于木霉菌的几丁质酶的同源性达到82%。     

Vibrio vulnificus MO6-24/O Lipopolysaccharide Stimulates Superoxide Anion,Thromboxane B2, Matrix Metalloproteinase-9, Cytokine and Chemokine Release by Rat Brain Microglia in Vitro

Although human exposure to Gram-negative Vibrio vulnificus (V. vulnificus) lipopolysaccharide (LPS) has been reported to result in septic shock, its impact on the central nervous system’s innate immunity remains undetermined. The purpose of this study was to determine whether V. vulnificus MO6-24/O LPS might activate rat microglia in vitro and stimulate the release of superoxide anion (O₂⁻), a reactive oxygen species known to cause oxidative stress and neuronal injury in vivo. Brain microglia were isolated from neonatal rats, and then treated with either V. vulnificus MO6-24/O LPS or Escherichia coli O26:B6 LPS for 17 hours in vitro. O₂⁻ was determined by cytochrome C reduction, and matrix metalloproteinase-2 (MMP-2) and MMP-9 by gelatinase zymography. Generation of cytokines tumor necrosis factor alpha (TNF-α), interleukin-1 alpha (IL-1α), IL-6, and transforming growth factor-beta 1 (TGF-β1), chemokines macrophage inflammatory protein (MIP-1α)/chemokine (C-C motif) ligand 3 (CCL3), MIP-2/chemokine (C-X-C motif) ligand 2 (CXCL2), monocyte chemotactic protein-1 (MCP-1)/CCL2, and cytokine-induced neutrophil chemoattractant-2alpha/beta (CINC-2α/β)/CXCL3, and brain-derived neurotrophic factor (BDNF), were determined by specific immunoassays. Priming of rat microglia by V. vulnificus MO6-24/O LPS in vitro yielded a bell-shaped dose-response curve for PMA (phorbol 12-myristate 13-acetate)-stimulated O₂⁻ generation: (1) 0.1–1 ng/mL V. vulnificus LPS enhanced O₂⁻ generation significantly but with limited inflammatory mediator generation; (2) 10–100 ng/mL V. vulnificus LPS maximized O₂⁻ generation with concomitant release of thromboxane B₂ (TXB₂), matrix metalloproteinase-9 (MMP-9), and several cytokines and chemokines; (3) 1000–100,000 ng/mL V. vulnificus LPS, with the exception of TXB₂, yielded both attenuated O₂⁻ production, and a progressive decrease in MMP-9, cytokines and chemokines investigated. Thus concentration-dependent treatment of neonatal brain microglia with V. vulnificus MO6-24/O LPS resulted in a significant rise in O₂⁻ production, followed by a progressive decrease in O₂⁻ release, with concomitant release of lactic dehydrogenase (LDH), and generation of TXB₂, MMP-9, cytokines and chemokines. We hypothesize that the inflammatory mediators investigated may be cytotoxic to microglia in vitro, by an as yet undetermined autocrine mechanism. Although V. vulnificus LPS was less potent than E. coli LPS in vitro, inflammatory mediator release by the former was clearly more efficacious. Finally, we hypothesize that should V. vulnificus LPS gain entry into the CNS, it would be possible that microglia might become activated, resulting in high levels of O₂⁻ as well as neuroinflammatory TXB₂, MMP-9, cytokines and chemokines.     

Purification,Characterization and in vitro Anti-Tumor Activity of Proteins from Arca subcrenata Lischke

Two purified proteins G-6 and G-4-2 were obtained from Arca subcrenata Lischke using the homogenization, salting-out with ammonium sulfate, ion-exchange chromatography and gel filtration chromatography techniques. The purity of G-6 and G-4-2 was over 96%, as measured by RP-HPLC. G-6 and G-4-2 were measured by SDS-PAGE and IEF-PAGE to have molecular weights of 8.2 kDa and 16.0 kDa, and isoelectric points of 6.6 and 6.1, respectively. The amino acid constituents of G-6 and G-4-2 were also determined. The existence of saccharides in G-6 was demonstrated by the phenol-sulfuric acid method. G-6 and G-4-2 inhibited the proliferation of human tumor cells in vitro. By MTT assay, the IC₅₀ values of G-4-2 were 22.9 μg/mL, 46.1 μg/mL and 57.7 μg/mL against Hela, HL-60 and KB cell lines, respectively, and the IC₅₀ value of G-6 against HL-60 cell line was measured to be 123.2 μg/mL.     

Identification and Function Analysis of enolase Gene NlEno1 from Nilaparvata lugens (St?l) (Hemiptera:Delphacidae)

The enolase [EC 4.2.1.11] is an essential enzyme in the glycolytic pathway catalyzing the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP). In this study, a full-length cDNA encoding α-enolase was cloned from rice brown planthopper (Nilaparvata lugens) and is provisionally designated as NlEno1. The cDNA sequence of NlEno1 was 1,851 bp with an open reading frame (ORF) of 1,305 bp and encoding 434 amino acids. The deduced protein shares high identity of 80–87% with ENO1-like protein from Hemiptera, Diptera, and Lepidoptera speices. The NlEno1 showed the highest mRNA expression level in hemolymph, followed by fat body, salivary gland, ovaries and egg, and showed trace mRNA levels in testis. The mRNA of NlEno1 showed up-regulated level in virulent N. lugens population Mudgo, IR56 and IR42 when compared with TN1 population. Injection of double-stranded RNA (dsRNA) of NlEno1 into the adults significantly down-regulated the NlEno1 mRNA level along with decreased eggs and offspring. Moreover, injection of NlEno1-dsRNA decreased mRNA level of Vitellogenin (Vg) gene. These results showed that the NlEno1, as a key glycolytic enzyme, may play roles in regulation of fecundity and adaptation of N. lugens to resistant rice varieties.