Bioinformatics Analysis of QoD1PO Gene of Mangrove Endophytic Fungus Aspergillus terreus

[Objectives]To analyze the gene structure of the protein that predicts 6-hydroxymellein synthase of Aspergillus terreus and predict the characteristics and functions of the protein structure encoded by the gene.[Methods]Various information analysis tools in NCBI,CBS and ExPASy websites were adopted.[Results]The QODIPO gene had a full length of 2954 bp,with 952 amino acids in the coding area,and QODIPO had the highest homology with the hypothetical protein ATETN484_0003008800.The molecular weight of QOD1PO protein was 105040.56,the theoretical isoelectric point(pl)was 5.69 and the grand average of hydropathieity was-0.242.It was speculated that QODIPO was an unstable and non-secretory hydrophilic protein located in cytoplasm without transmembrane domain or signal peptide.It could be predicted that the secondary structure of QODIPO encoding protein consisted mainly of random coil,α-helix and a PKS-DH anhydrase domain.[Conclusions]The results will lay a theoretical foundation for cloning and expression of 6-hydroxymellein synthase and further understanding of its activity and function.

头花蓼内生真菌Aspergillus terreus油脂类代谢物的鉴定及其抗多药耐药菌和抗炎作用研究

目的:鉴定头花蓼内生真菌Aspergillus terreus油脂类代谢物并考察其抗多药耐药菌和抗炎作用。方法:采用气相色谱-质谱联用技术(GC-MS)分析、鉴定A.terreus的油脂类代谢物;采用96孔板微量稀释法测试油脂类代谢物和主要成分对10株多药耐药菌(肺炎克雷伯杆菌、普通变形杆菌、表皮葡萄球菌、大肠埃希菌、金黄色葡萄球菌、阴沟肠杆菌、铜绿假单胞菌、奇异变形杆菌、屎肠球菌和鲍曼不动杆菌)的最低抑菌浓度(MIC),用涂布平板法测定各样品对细菌的最低杀菌浓度(MBC)。以脂多糖诱导体外RAW264.7细胞炎症模型,考察不同质量浓度(50、100、200μg/m L)油脂类代谢物作用24 h后对细胞中一氧化氮(NO)、肿瘤坏死因子α(TNF-α)释放的影响。结果:从A.terreus的油脂类代谢物中共鉴定了13个化合物,其中棕榈酸、硬脂酸、亚油酸和油酸为主要成分,其相对百分含量依次为29.35%、10.87%、21.94%、34.85%。油脂类代谢物对大肠埃希菌和肺炎克雷伯杆菌表现出较好的抗菌活性,MIC分别为12.5、50 mg/m L,MBC分别为25、100 mg/m L;4个主要成分均具有抑制大肠埃希菌生长的作用,MIC范围为0.5~1 mg/m L,其中棕榈酸显示出较为广泛的抗菌活性,且对屎肠球菌活性最强(MIC为0.25 mg/m L)。50、100μg/m L的油脂类代谢物可显著抑制RAW264.7细胞中炎症因子NO和TNF-α的释放。结论:头花蓼内生真菌A.terreus的油脂类代谢物具有抗多药耐药菌和抗炎作用。

内生菌Aspergillus terreus PC-038次生代谢产物中抗菌化学成分研究

研究内生菌Aspergillus terreus PC-038次生代谢物中抗菌活性成分。采用现代分离技术对A.terreus次生代谢物中活性组分进行分离纯化,利用现代质谱和核磁等波谱技术鉴定其结构,采用96孔板二倍稀释法测试单体化合物的抗菌作用。从A.terreus代谢物的活性组分中分离得到17个化合物。化合物3~7和10~14均为从内生菌A.terreus代谢物中首次分离,其中烟曲霉酸为主要成分。抗菌结果显示1,2-二氢烟曲酶酸和烟曲霉酸对Staphylococcus aureus ATCC25923有显著的抑制作用,MIC分别为8μg/mL和4μg/mL。研究丰富了A.terreus次生代谢物的化合物库,明确了1,2-二氢烟曲酶酸和烟曲霉酸为A.terreus PC-038的抗菌主要活性成分,为新型抗生素的研发奠定了基础。

一株太子参内生真菌Aspergillus terreus TZS-201607中抗肿瘤活性代谢产物研究

为研究太子参内生真菌Aspergillus terreus TZS-201607所产的次级代谢产物,利用硅胶柱层析、Sephadex LH-20凝胶柱层析、反相柱层析及半制备高效液相色谱等技术,从其PDB培养基发酵产物的乙酸乙酯萃取物中分离纯化得到16个单体化合物。利用波谱学方法结合文献数据分析分别鉴定为柄曲霉素(1)、5-甲氧基柄曲霉素(2)、variecoxanthone A(3)、chryxanthone A(4)、6,8-di-O-methylaverufin(5)、6,8-di-O-methylnidurufin(6)、6,8,1′-tri-O-methyl averantin(7)、(22E,24R)-ergosta-7,9(11),22-trien-3β-ol(8)、(22E,24R)-ergosta-4,6,8(14),22-trtraen-3-one(9)、(22E,24R)-3β,5α-dihydroxy-ergosta-7,22-dien-6-one(10)、(22E,24R)-3β,5α,9α-trihydroxy-ergosta-7,22-diene-6-one(11)、(22E,24R)-3α-ureido-ergosta-4,6,8(14),22-tetraene(12)、(22E,24R)-ergosta-7,22-dien-3β,5α,6β-triol(13)、(22E,24R)-5α,8α-epidioxyergosta-6,22-dien-3β-ol(14)、demethylincisterol A3(15)和(17R)-17-methylincisterol(16)。化合物1~7、12、14~16为首次从A.terreus中分离得到。体外抗肿瘤活性测试显示,化合物6、7和14对人肿瘤细胞株A549、BT-549、HeLa和THP-1表现出较强的细胞毒活性(IC50<10μM)。

Production of Cellulase and Xylanase by Aspergillus terreus KJ829487 Using Cassava Peels as Subtrates

Cassava (Manihot esculenta, Crantz) is one of the most important food plants in West Africa. Its peels are made up of cellulose, hemicellulose and lignin. This lignocellulolytic biomass can be converted using microbial enzymes to fermentable sugars which have wide range of biotechnological relevance in many fermentation processes. The aim of this study is to screen filamentous fungi from decaying cassava peels that are good producers of xylanases and cellulases. Decaying parts of cassava peels were obtained and brought to the laboratory for further work. Fungi were isolated, identified and screened for cellulase and xylanase production. Isolate with highest frequency of occurrence and enzyme production was identified using phenotypic and molecular method. Optimisation of growth conditions for enzymes production was monitored using the DNSA method, also saccharification of cassava peel were carried out using the enzymes obtained from the isolate. Aspergillus terreus KJ829487 was the predominant fungus. It produces cellulases and xylanases optimally at 40°C, pH 6 and 8, utilising carboxymethylcellulose (CMC) or xylose and yeast extracts as its carbon and nitrogen sources respectively. Saccharification of the peels yielded 584 mg/L glucose, 78 mg/L xylose and 66 mg/L rhamnose. Aspergillus terreus KJ829487 obtained from cassava peels have the ability to produce high concentration cellulases and xylanases which effectively hydrolysed the lignocelluloses’ biomass to fermentable sugars.

Purification and Biochemical Characterization of Native and Pegylated Form of L-Asparaginase from Aspergillus terreus and Evaluation of Its Antiproliferative Activity

L-asparaginase is a chemotherapeutic drug used in the treatment of lymphoblastic leukemia. In the present study, the extracellular L-asparaginase produced by strain (PC-1.7A) of Aspergillus terreus was purified, characterized, and modified with polyethylene glycol. Moreover, its antiproliferative activity was evaluated. The apparent molecular weight of the enzyme was found to be 136 kDa. The optimal pH and temperature for the enzyme were 9.0℃ and 40℃, respectively. The enzyme retained 100% of the activity at 40℃ for 120 min. Pegylated L-asparaginase was more thermostable and more resistant to trypsin than native enzyme. Native L-asparaginase against human normal cells did not show cytotoxicity. However, in the leukemia cell lines RS4;11 and HL60 the antiproliferative effects of native L-asparaginase were observed after 96 and 72 h of incubation, respectively. For the first time, an L-asparaginase from fungus was evaluated as an antitumor agent in human cells lines and further investigations should be conducted to improve the knowledge about this enzyme.

Directional Breeding of High Itaconic Acid Yielding Strain of <i>Aspergillus terreus</i>with a New Plate Technique

Itaconic acid is commercially produced by the cultivation of Aspergillus terreus using starch hydrolysate as carbon source. The degree of hydrolysis had a great influence on itaconic acid production which was suitable when corn starch was saccharified at 35 DE. The α-amylase was sufficient to drive the starch hydrolysis to the degree. The agar plate assay with LiCl treatment provided a rapid, simple and unequivocal method for screening large numbers of colonies for itaconic acid producing strains. It was learned by experience that the strains on the plates with thick hyphae and light-colored spores often accompanied high itaconic acid production. A strain, designated Ast165, producing itaconic acid with a high yield, was successfully obtained by directional breeding of metabolic end products resistant strains. The itaconic acid concentration produced by Ast165 was 53.8 g/l from 100 g/l of starch hydrolysate in shake flasks. The conversion rate was 61.3%, which was the highest value found in tests.

Genetic variability of Aspergillus terreus from dried grapes

RAPD was used to examine the genetic variability among five isolates of Aspergillus terreus spp.. Two random primers were selected for the RAPD assay PG01–5’ CAGGTGTTGC 3’ and PG02–5’ CTGGACAGAC 3’ (Progen Technologies). The characterization of Aspergillus terreus species have been mostly applied on the basis of morphology, phenotype and physiology. DNA Polymorphisms are based on differences in DNA sequences and have advantages over protein polymorphisms. But morphological characterization besides molecular tools will remain a basic and powerful key in the identification of Aspergillus terreus species. The objective of the present study was to isolate the fungal contaminants from dried grapes and compare the genomic profile of the Aspergillus terreus speices isolated from the dried grapes, through RAPD analysis. In the present study with primer PG 01 four different discriminations was there among the A. terreus isolates. There was a homology of genotype between the isolates 1 & 3. And with primer PG 02 four different discriminations were there and there was a homology between 1 & 3. The predominant type was type I in primer I & II. The other isolates belonged to 2, 3 and 4. No similarity was detected for isolates 3, 4 and 5 indicating great genomic diversity of A. terreus.

通过红曲霉-土曲霉属间原生质体融合提高Monacolin K产量

为获得高产Monacolin K的红曲霉菌株，将高产Monacolin K的土曲霉Aspergillus terreus CA99与低产Monacolin K的红曲霉Monascus anka M-3进行原生质体融合．生长24h的A．terrus CA99和M．anka M-3菌株用0．5％溶壁酶、0．3％蜗牛酶和0．3％纤维素酶混合酶液分别在34℃和30℃下处理5h和3．5h，原生质体的形成率分别为1．76×10^7／mI。和1．68×10^7／mL．两种原生质体分别置于30W的紫外灯下，距离30cm照射3min灭活，等浓度混合后用浓度为30％的PEG 6000融合15min．再生平板上共选取363株融合子，从中筛选得到多株Monacolin K产量高于出发菌株的高产融合子，其中有10株产量的提高幅度超过60％，有2株融合子（F49和F104）产量提高了将近一倍，分别为460μg／mL。和457μg／mL。对F49的发酵培养基进行优化，Monacolin K发酵单位达到了1216μg／mL。

Crystal Structure of 2-(2-Carboxy-4-hydroxy-6-methoxyphenoxy)-3,5-dichloro-6-hydroxy-4-methylbenzoic Acid 1-Methyl Ester

The title compound, neogeodin hydrate （C17H14C1208, CAS： 94540-50-8）, was derived from marine fungus Aspergilhts terreus CRIM301. It was unequivocally characterized by IR, NMR spectroscopies, and single-crystal X-ray crystallography and tested for various biological activities. Neogeodin hydrate crystallizes in the triclinic space group P1 with a = 8.1159（5） A, b = 8.2472（4） A, c= 14.1278（7） A, a = 81.448（2）°, β = 84.860（2）°, γ= 70.400（2）°, V = 880.13（8） A3; Z = 2. It comprises a diphenyl ether, asterric acid skeleton and dichloro substituents. The methoxyphenoxy rings of the inversely related molecules form a ribbon-like structure that is stabilized by O-H...O hydrogen bonds through the doubly disordered carboxyl groups and by C-H...O interactions, generating the same R22（8） ring motif. The chlorinated methylbenzoate rings, making mostly a right angle, link the parallel upper and lower ribbons via bifurcated O-H...O and C-H...O hydrogen bonds, yielding endless channels. The channels formed are further sustained by C-H...O and π...π interactions Neogeodin hydrate exhibits inhibition against superoxide anion radical formation in the xanthine/xanthine oxidase （XXO） assay, but has no aromatase inhibitory activity.

Computer Image Analysis as a Tool for Microbial Viability Assessment: Examples of Use and Prospects

Application of the computer image analysis for improving microbial viability assessment by plate count and fluorescence microscopy was investigated. Yeast cells were used as a model microorganism. The application of the improved methods for the viability assessment of yeast cells after preservation by freezing and freeze-drying was demonstrated.

Solid state fermentation of pomegranate seed for lovastatin production: a bioprocessing approach

This study reports lovastatin production by solid state fermentation using pomegranate seeds as a substrate. Six different fungal strains and several agro-industrial wastes were selected and screened. Various physico-chemical parameters were optimized to improve lovastatin. Moreover, chemical mutation was systematically employed to enhance lovastatin yield on selected strains. Productivity of 3 ± 0.06 mg lovastatin/gm dfm was obtained prior to optimization. One factor a time followed by Response Surface Methodology (RSM) gave 4.2 ± 0.10 mg lovastatin/gm dfm yield in an optimized setup with pomegranate seed powder (5 gms), KH2PO4 (0.1% w/v), glucose (5% w/v), moisture (60% w/w), pH 5 in a 15 days fermentation cycle. The production was further increased to 6.5 ± 0.08 mg lovastatin/gm dfm through chemical mutation of the strain. This process is simple and reproducible for the production of lovastatin using pomegranate seed as an agro-industrial waste.

1株南沙群岛柳珊瑚来源真菌Aspergillus terreus中化合物及mPTPB酶抑制活性研究

目的从1株南沙群岛柳珊瑚来源真菌Aspergillus terreus(NS02-09)中分离鉴定海洋天然产物,对所得化合物进行结核分枝杆菌酪氨酸磷酸激酶(mPTPB)抑制活性评价。方法运用多种色谱手段分离纯化化合物,利用NMR、CD等现代波谱分析方法,对化合物进行结构鉴定,通过衍生物制备获得2个乙酰化衍生物(2a和2b);并对化合物2及其衍生物2a和2b进行mPTPB酶抑制活性测试。结果鉴定了1个土曲霉酮(1)和1个丁烯酸内酯(2)的结构;2具有较强的mPTPB酶抑制活性,而其乙酰化产物(2a和2b)的mPTPB酶抑制活性显著降低。运用Sybyl X 1.3软件,对2与mPTPB酶的模拟对接计算发现,丁烯酸内酯环及环上的羟基是化合物2发挥酶抑制活性的重要作用基团。结论从柳珊瑚来源真菌A.terreus(NS02-09)中发现了具有mPTPB酶抑制活性的丁烯酸内酯类化合物,并对其作用机制进行了计算研究,该类化合物的相关研究对抗结核药物先导化合物发现具有借鉴作用。

1株海兔来源真菌Aspergillus terreus丁内酯类化合物及其生物活性研究

目的在生物活性和化学方法指导下,从1株海兔来源真菌Aspergillus terreus（RA29-5）中分离鉴定海洋天然产物,并对其进行生物活性评价。方法运用硅胶柱层析、Sephadex LH-20凝胶柱层析和HPLC等方法分离纯化化合物,利用NMR等现代波谱分析方法,对化合物进行结构鉴定,并对化合物进行抗菌、卤虫致死和斑马鱼胚胎毒性活性评价。结果鉴定了9个丁内酯类化合物（1～9）的结构;抗菌活性表明,除化合物6和9外,所有化合物对4株致病细菌白色葡萄球菌Staphylococcus epidermidis、金黄色葡萄球菌Staphylococcus aureus、蜡状芽孢杆菌Bacillus subtilis和四联球菌Tetragenococcus halophilus都显示一定的抗菌活性;化合物1～4和7均对真菌芒果叶枯菌Pestalotia mangiferae显示抗菌活性;特别的,化合物7对测试的13株致病菌都具有广谱地抗菌活性,其MIC值在1.11～8.88μmol/L之间;此外,还对化合物的抗菌活性构效关系进行了初步探讨。结论海兔来源真菌A.terreus（RA29-5）可产生结构多样的丁内酯类化合物,该类化合物具有开发成抗菌剂的潜力。

蓝花黄芩内生真菌Aspergillus terreus HQ100X-1次级代谢产物研究

从蓝花黄芩内生真菌土曲霉(Aspergillusterreus)HQ100X-1发酵产物中分离得到2个新化合物[terrustone(1)和asperteretone G (2)], 10个已知化合物(3~12), terrustone (1)为具有4个连续手性中心的三羟基环戊酮类化合物,它们的结构以及绝对构型是通过1D/2D NMR结合电子圆二色谱(ECD)计算来确定.抗菌实验表明,化合物rbrolide R (4)对金黄色葡萄球菌有一定的抑菌活性,其最低抑菌浓度(MIC)值为2.5μg/mL.

Fumigaclavine I, a new alkaloid isolated from endophyte Aspergillus terreus

The present study was designed to isolate and purify chemical constituents from solid culture of endophyte Aspergillus terreus LQ, using silica gel column chromatography, gel filtration with Sephadex LH-20, and HPLC. Fumigaclavine I(1), a new alkaloid, was obtained, along with seven known compounds, including fumigaclavine C(2), rhizoctonic acid(3), monomethylsulochrin(4), chaetominine(5), spirotryprostatin A(6), asperfumoid(7), and lumichrome(8). The structure of compound 1 was elucidated by various spectroscopic analyses(UV, MS, 1D and 2D NMR). The in vitro cytotoxicity of compound 1 was determined by MTT assay in human hepatocarcinoma cell line SMMC-7721, showing weaker cytotoxicity, compared with cisplatin, a clinically used cancer chemotherapeutic agent.

Peptides and polyketides isolated from the marine sponge-derived fungus Aspergillus terreus SCSIO 41008

Two new isomeric modified tripeptides,aspergillamides C and D(compounds 1 and 2),together with fifteen known compounds(compounds 3-17),were obtained from the marine sponge-derived fungus Aspergillus terreus SCSIO 41008.The structures of the new compounds,including absolute configurations,were determined by extensive analyses of spectroscopic data(NMR,MS,UV,and IR)and comparisons between the calculated and experimental electronic circular dichroism(ECD)spectra.Butyrolactone I(compound 11)exhibited strong inhibitory effects against Mycobacterium tuberculosis protein tyrosine phosphatase B(MptpB)with the IC_(50) being 5.11±0.53μmol·L^(–1),and acted as a noncompetitive inhibitor based on kinetic analysis.

Asperterzine,a symmetric aromatized derivative of epipolythiodioxopiperazine,from the endophytic fungus Aspergillus terreus PR-P-2

A new epipolythiodioxopiperazine （ETP）, asperterzine （1）, along with two known analogs, bisdethiobis （methylthio）-acetylaranotin （2） and bisdethiobis（methylthio）-acetylapoaranotin （3）, was isolated from the plant endophytic fungus Aspergillus terreus PR-P-2. The structure elucidation of 1 was accomplished by a combination of spectral methods and electronic circular dichroism （ECD） spectrum. Asperterzine （1） was a symmetric aromatized ETP found as a natural product for the first time. Compounds 2 and 3 showed strong cytotoxicity against HL-60 cell line. The putative biosynthetic pathway of 1 was also detailed in the text.

海洋真菌来源化合物(＋)-terrein的发酵优化及其生物活性

目的对海洋来源真菌Aspergillus terreus中的(+)-terrein代谢产物进行发酵优化,以期获得足够的样品进行药理活性评价。方法采用不同发酵条件对微生物进行发酵优化,运用柱色谱层析技术对目标产物进行分离纯化,对化合物进行细胞毒及抗细菌活性评价。结果研究表明,最优发酵条件为土豆液体发酵培养基,发酵时间为28d,在该条件下目标产物单一,易分离纯化。用上述最优发酵条件,摇床发酵时间12d的产量与静置发酵28d的产量相当。细胞毒活性测试结果显示,(+)-terrein具有强的细胞毒活性,对NCIH460、A549和BT474肿瘤细胞株的IC50值分别为3.12、3.32和3.45μg/mL。结论在优化条件下,静置发酵28d,(+)-terrein的产量可达0.254g·L-1,而摇床发酵12d其产量为0.263g·L^(-1),明显缩短了发酵时间。此外,该化合物对NCI-H460、A549和BT474肿瘤细胞具有较好的细胞毒活性,具有开发为抗癌药物的潜在价值。

烟草内生真菌土曲霉中一个新的苯乙酸类化合物

目的研究分离自烟草中内生真菌土曲霉(Aspergillus terreus)的次生代谢产物。方法运用硅胶柱色谱、C18反相硅胶柱色谱、Sephadex LH-20凝胶柱色谱和薄层制备等色谱技术对菌株A.terreus的发酵液粗提物进行分离纯化,采用波谱方法对化合物进行结构鉴定。结果从烟草内生真菌A.terreus的发酵粗提物中分离得到10个化合物,分别鉴定为(2′R)-westerdijkin B(1)、(2′R)-westerdijkin A(2)、4-羟基苯乙酸甲酯(3)、ampelopyrone(4)、sporogen-AO 1(5)、(S)-(+)-11-dehydrosydonic acid(6)、sydowic acid(7)、asperdemin(8)、asperversin E(9)和asperversin G(10)。其中,化合物1为新化合物,化合物2~5为首次从菌株A.terreus中分离获得。结论在抑菌活性测试中,化合物1对大肠杆菌具有明显的抑制活性,其MIC值为4 mg·L^(-1)。化合物5具有显著的抗植物病原真菌活性,对苹果黑腐皮壳菌的MIC为4 mg·L^(-1)。