Alcohol oxidase of Aspergillus flavipes grown on hexadecanol

Abstract: The activity of alcohol oxidase in Aspergillus flavipes was induced by growth on hexadecanol, though highest activities were obtained using a mixture of hexadecanol and olive oil. The enzyme showed a wide range of substrate specificity towards aliphatic primary alcohols from C₈ to C₁₈. The preferred substrate was decanol. The enzyme had an optimum pH of 9.5. It also used cis -unsaturated alcohols better than the trans -isomers. ω-Hydroxy fatty acids and α,ω-diols were not attacked.     

Production and characterization of alpha-galactosidase from Aspergillus flavipes

An extracellular alpha-galactosidase from the culture filtrate of Aspergillus flavipes grown on melibiose as a carbon source was partially purified by hydroxylapatite and diethylaminoethylcellulose chromatographies. Electrophoretic analysis showed protein bands corresponding to alpha-galactosidase and invertase activities. The optimum pH and temperature were determined as 4.5-5.0 and 45 degrees C, respectively. The Km value for p-nitrophenyl-alpha-d-galactopyranoside was found to be 1.89 mm. The results reported in this study indicate that Aspergillus flavipes is indeed an active source of extracellular alpha-galactosidase.     

Analysis of polysaccharide hydrolases secreted by Aspergillus flavipes FP-500 on corn cobs and wheat bran as complex carbon sources

Abstract

Aspergillus flavipes FP-500 is a Mexican native strain that has been reported as a good producer of xylanases and pectinases; therefore, it promises a strong impact on biotechnology. To provide an overview of protein secretion by A. flavipes, we carried out a comparative proteome analysis of extracellular proteins in liquid cultures with two heterogeneous agro-industrial residues; corn cob (CC) and wheat bran (WB), as carbon sources. Extracellular proteins obtained from both cultures were identified using MS/MS spectrometry. We identified 134 proteins, which were classified into four groups: glycosyl hydrolases (GH), esterases/proteases, miscellaneous proteins, and unidentified proteins. Around 50% of the total proteins identified were GH such as xylanases, β-xylosidases, β-galactosidases, cellulolytic enzymes like β-glucosidase, endoglucanases, and cellobiohydrolases. From this family, a core of 22 (16%) of the proteins identified were found in both substrates, CC and WB, whereas 30% and 54% were unique for CC and WB, respectively. In the esterases/proteases group, proteases, lipases and esterases like feruloylesterases, and acetyl-xylanesterase were identified. Proteins with diverse functions such as monophosphate dehydrogenase or N-acetylglucosaminidase were present. Here, we present strong evidences indicating that the composition and heterogeneity of the used carbon source determine the specific set of protein secreted by the fungus.     

Isolation and Characterization of Anti-Inflammatory Sorbicillinoids from the Mangrove-Derived Fungus Penicillium sp. DM815

Marine derived fungus has gained increasing ground in the discovery of novel lead compounds with potent biological activities including anti-inflammation. Here, we first report the characterization of one new sorbicillinoid ( 1 ) and fourteen known compounds ( 2 – 15 ) from the ethyl acetate (AcOEt) extract of a cultured mangrove derived fungus Penicillium sp. DM815 by UV, IR, HR ESI-Q-TOF MS, and NMR spectra. We then evaluated the anti-inflammatory effects of eleven sorbicillinoids ( 1 – 11 ) using cultured macrophage RAW264.7 cells. The results show that compound 9 , and to a lesser degree compound 5 , significantly inhibited the Gram-negative bacteria lipopolysaccharide (LPS)-induced upregulation of the inducible nitric oxide synthase (iNOS). Consistently, compounds 5 and 9 significantly reduced the level of nitric oxide (NO), the product of iNOS, induced by LPS. We further show that these two compounds dose-dependently inhibited LPS-triggered iNOS expression and NO production, but had no effect on proliferation of RAW264.7 cells in the presence of LPS. In conclusion, our study identifies novel and known sorbicillinoids as potent anti-inflammatory agents, holding the promise of developing novel anti-inflammation treatment in the future.     

Two New Phenol Derivatives from the Cold Seep-Derived Fungus Aspergillus insuetus SD-512

Two new phenol derivatives, namely insphenol A ( 1 ) and acetylpeniciphenol ( 2 ), along with seven known analogs ( 3 – 9 ), were isolated from the deep-sea cold seep-derived fungus, Aspergillus insuetus SD-512. The structures of 1 and 2 were established by extensive interpretation of NMR and mass spectroscopic data. The absolute configuration of 1 was determined by the combination of coupling constant analysis and acid hydrolysis. Among the isolated compounds, insphenol A ( 1 ) represents the first example of isopentenyl phenol derivative with a unique 1-glycosylation from the species Aspergillus insuetus. The isolated new compounds were evaluated for antibacterial activities against six human or aquatic pathogens, while compound 2 exhibited inhibitory effect against Edwardsiella tarda, Vibrio alginolyticus, and V. vulnificus, with MIC values of 4, 8, and 8 μg/mL, respectively.     

Aspergillus fumigatus catalases: cloning of an Aspergillus nidulans catalase B homologue and evidence for at least three catalases

The presence of catalases in the water soluble fractions of three Aspergillus fumigatus strains was investigated using non-denaturing and denaturing polyacrylamide gel electrophoresis and Western analysis. Using non-denaturing polyacrylamide gel electrophoresis and staining for catalase activity, three separate catalases were identified. An A. fumigatus catalase gene (catB) was cloned from genomic DNA using the Aspergillus niger catR gene as a probe. Polyclonal antibodies were raised to a glutathione S-transferase-CatB fusion product expressed in Escherichia coli. Western analysis indicated that, under denaturing conditions, the polyclonal antibody recognised a 90-kDa band and under non-denaturing conditions, two separate bands were identified. These results indicate that A. fumigatus in addition to CatB, produces at least two other catalases, one of which is similar in size to CatB. The polyclonal antibody was also used to observe catalase expression in mice, experimentally infected with A. fumigatus. Staining was observed heterogeneously throughout the fungal hyphae. This result indicates that catalase is produced by A. fumigatus during invasive aspergillosis.     

Glycoengineering of Aspergillus nidulans to produce precursors for humanized N-glycan structures

Filamentous fungi secrete protein with a very high efficiency, and this potential can be exploited advantageously to produce therapeutic proteins at low costs. A significant barrier to this goal is posed by the fact that fungal N-glycosylation varies substantially from that of humans. Inappropriate N-glycosylation of therapeutics results in reduced product quality, including poor efficacy, decreased serum half-life, and undesirable immune reactions. One solution to this problem is to reprogram the glycosylation pathway of filamentous fungi to decorate proteins with glycans that match, or can be remodeled into, those that are accepted by humans. In yeast, deletion of ALG3 leads to the accumulation of Man₅GlcNAc₂ glycan structures that can act as a precursor for remodeling. However, in Aspergilli, deletion of the ALG3 homolog algC leads to an N-glycan pool where the majority of the structures contain more hexose residues than the Man_3-5GlcNAc₂ species that can serve as substrates for humanized glycan structures. Hence, additional strain optimization is required. In this report, we have used gene deletions in combination with enzymatic and chemical glycan treatments to investigate N-glycosylation in the model fungus Aspergillus nidulans. In vitro analyses showed that only some of the N-glycan structures produced by a mutant A. nidulans strain, which is devoid of any of the known ER mannose transferases, can be trimmed into desirable Man₃GlcNAc₂ glycan structures, as substantial amounts of glycan structures appear to be capped by glucose residues. In agreement with this view, deletion of the ALG6 homolog algF, which encodes the putative α-1,3- glucosyltransferase that adds the first glucose residue to the growing ER glycan structure, dramatically reduces the amounts of Hex_6-7HexNAc₂ structures. Similarly, these structures are also sensitive to overexpression of the genes encoding the heterodimeric α-glucosidase II complex. Without the glucose caps, a new set of large N-glycan structures was formed. Formation of this set is mostly, perhaps entirely, due to mannosylation, as overexpression of the gene encoding mannosidase activity led to their elimination. Based on our new insights into the N-glycan processing in A. nidulans, an A. nidulans mutant strain was constructed in which more than 70% of the glycoforms appear to be Man_3-5GlcNAc₂ species, which may serve as precursors for further engineering in order to create more complex human-like N-glycan structures.     

秸杆降解菌株抗药性标记的筛选

两株菌株原生质体融合技术中,为了将融合子与双亲本分离出来,要对其中的至少一个亲本进行标记。该文从降解秸杆的两种不同的真菌出发,在两株菌株原生质融合前进行抗药性标记的筛选,为两种菌株融合后融合子的筛选和分离提供依据。本实验采用10种药物,对双亲本进行抗药性初筛,然后对临界药剂用量筛选,通过对比和重复实验,镜检和酶活测定,获得两种对白腐真菌抗药而康宁木霉非抗性的药品,氟康唑和十一烯酸真菌清,剂量分别为400μg/mg和500μg/mg。     

Development of enzyme technology for Aspergillus oryzae,A. sojae,and A. luchuensis,the national microorganisms of Japan

This paper describes the modern enzymology in Japanese bioindustries. The invention of Takadiastase by Jokiti Takamine in 1894 has revolutionized the world of industrial enzyme production by fermentation. In 1949, a new γ-amylase (glucan 1,4-α-glucosidase, EC 3.2.1.3) from A. luchuensis (formerly designated as A. awamori), was found by Kitahara. RNase T₁ (guanyloribonuclease, EC 3.1.27.3) was discovered by Sato and Egami. Ando discovered Aspergillus nuclease S₁ (single-stranded nucleate endonuclease, EC 3.1.30.1). Aspergillopepsin I (EC 3.4.23.18) from A. tubingensis (formerly designated as A. saitoi) activates trypsinogen to trypsin. Shintani et al. demonstrated Asp76 of aspergillopepsin I as the binding site for the basic substrate, trypsinogen. The new oligosaccharide moieties Man₁₀GlcNAc₂ and Man₁₁GlcNAc₂ were identified with α-1,2-mannosidase (EC 3.2.1.113) from A. tubingensis. A yeast mutant compatible of producing Man₅GlcNAc₂ human compatible sugar chains on glycoproteins was constructed. The acid activation of protyrosinase from A. oryzae at pH 3.0 was resolved. The hyper-protein production system of glucoamylase was established in a submerged culture.     

过表达LaeA激活海洋土曲霉中isoflavipucine类化合物的合成及其抗弧菌活性研究

丝状真菌土曲霉因产生结构独特且药理活性强烈的次级代谢产物而受到真菌学家和药学家的广泛关注。然而,在丝状真菌中,大部分次级代谢基因在常规实验室培养条件下表达量较低甚至沉默。本研究通过过表达全局性调控因子LaeA试图激活一株海洋来源土曲霉中的沉默基因簇以发现新次级代谢产物。结果表明,laeA基因过表达突变中未知产物基因簇被激活,指纹图谱分析发现了2个在野生菌株不存在的化合物吸收峰。质谱和核磁共振波谱分析确证了这2个化合物为dihydroisoflavipucine类小分子,特别是化合物1的产量在突变体中高达183 mg/L。此外,抗菌活性测试发现,这2个化合物对4种病原弧菌显示了强烈的活性,特别是化合物1抗弧菌活性更强,MIC低至16μg/mL。本研究提供了一条大量合成dihydroisoflavipucine类抗生素的新路线,并证明了次级代谢调控因子LaeA在丝状真菌中的功能是高度保守的,过表达LaeA是激活丝状真菌中沉默基因簇的有效手段。     

Purification and characterization of laccase II of Aspergillus nidulans

Sexual development in Aspergillus nidulans is a morphogenetic differentiation process triggered by internal and environmental signals. As a first step in analyzing the developmental pathway at the molecular level, laccase II (EC 1.10.3.2), which is specifically expressed in early stages of fruitbodies, was isolated. The enzyme was purified to apparent homogeneity from a mutant strain (SMS1) in which the sexual cycle dominates and the number of cleistothecia is increased tenfold. Laccase II was enriched 560-fold to a specific activity of 892 U (mg protein)^–1. The apparent molecular mass was determined to be 80 kDa under denaturing conditions and to be 100–120 kDa under native conditions. The internal peptide sequences gained from the protein will allow the isolation of the corresponding gene as a first step in determining the key regulators of sexual development. Received: 8 January 1998 / Accepted: 14 April 1998     

AoAtg26, a putative sterol glucosyltransferase,is required for autophagic degradation of peroxisomes,mitochondria, and nuclei in the filamentous fungus Aspergillus oryzae

Autophagy is a conserved process in eukaryotic cells for degradation of cellular proteins and organelles. In filamentous fungi, autophagic degradation of organelles such as peroxisomes, mitochondria, and nuclei occurs in basal cells after the prolonged culture, but its mechanism is not well understood. Here, we functionally analyzed the filamentous fungus Aspergillus oryzae AoAtg26, an ortholog of the sterol glucosyltransferase PpAtg26 involved in pexophagy in the yeast Pichia pastoris. Deletion of Aoatg26 caused a severe decrease in conidiation and aerial hyphae formation, which is typically observed in the autophagy-deficient A. oryzae strains. In addition, cup-shaped AoAtg8-positive membrane structures were accumulated in the Aoatg26 deletion strain, indicating that autophagic process is impaired. Indeed, the Aoatg26 deletion strain was defective in the degradation of peroxisomes, mitochondria, and nuclei. Taken together, AoAtg26 plays an important role for autophagic degradation of organelles in A. oryzae, which may physiologically contribute to the differentiation in filamentous fungi.     

海带渣微生物药肥对土壤中产毒真菌寄生曲霉的抑制作用研究

研究了施用多功能生防芽孢杆菌Hitwh-BA2菌株发酵生产的海带渣微生物药肥对栽培土壤中产毒真菌寄生曲霉的生态防治效果。结果表明：施用海带渣微生物药肥能极显著的降低栽培土壤中寄生曲霉的数量水平,有效降低寄生曲霉占总真菌数的百分含量,其作用效益优于海带渣;施用海带渣微生物药肥能极显著提高栽培土壤中细菌的数量水平,作用效果极显著优于海带渣。研究结果期望为土壤黄曲霉毒素产毒真菌的防控提供参考。     

来自红海榄根际土壤的曲霉属真菌F7菌株及其生物活性分析

基于菌落和菌体的形态特征以及ITS序列分析结果,将分离自中国海南省海口市东寨港红树林保护区红海榄根际土壤的菌株F7鉴定为曲霉属黄绿组的一种真菌。在确定和优化菌株F7培养基的基础上,初步确定了适合其分泌活性代谢产物的改良查氏培养基组成:3%乳糖,1%蛋白胨,0.3%NaNO3,0.05%KCl,0.05%MgSO4·7H2O,0.001%FeSO4,0.1%K2HPO4,pH7.0。将菌株F7接种于该培养基中,28℃下160r/min振荡培养7d后收获发酵液,经乙酸乙酯和正丁醇萃取后获得了乙酸乙酯萃取物(EAE)、正丁醇萃取物(BE)以及水萃取物(WSE),其中EAE和BE对金黄色葡萄球菌、藤黄八叠球菌和枯草芽孢杆菌的生长显示出明显的抑制活性,最低抑菌浓度(MIC)介于0.625-5.0mg/mL之间。同时,上述3种萃取物对人肝癌细胞株HepG2的增殖也表现出一定的抑制活性,IC50约为120μg/mL左右。     

Synchrotron radiation-based microcomputed tomography for three-dimensional growth analysis of Aspergillus niger pellets

Filamentous fungi produce a wide range of relevant biotechnological compounds. The close relationship between fungal morphology and productivity has led to a variety of analytical methods to quantify their macromorphology. Nevertheless, only a µ-computed tomography (µ-CT) based method allows a detailed analysis of the 3D micromorphology of fungal pellets. However, the low sample throughput of a laboratory µ-CT limits the tracking of the micromorphological evolution of a statistically representative number of submerged cultivated fungal pellets over time. To meet this challenge, we applied synchrotron radiation-based X-ray microtomography at the Deutsches Elektronen-Synchrotron [German Electron Synchrotron Research Center], resulting in 19,940 3D analyzed individual fungal pellets that were obtained from 26 sampling points during a 48 h Aspergillus niger submerged batch cultivation. For each of the pellets, we were able to determine micromorphological properties such as number and density of spores, tips, branching points, and hyphae. The computed data allowed us to monitor the growth of submerged cultivated fungal pellets in highly resolved 3D for the first time. The generated morphological database from synchrotron measurements can be used to understand, describe, and model the growth of filamentous fungal cultivations.     

Utilization of AFLP markers for PCR-based identification of Aspergillus carbonarius and indication of its presence in green coffee samples

AIMS: The objective of this work was to test whether ochratoxin A (OTA) production of Aspergillus niger and A. carbonarius is linked to a certain genotype and to identify marker sequences with diagnostic value aiding identification of A. carbonarius, a fungus of major concern regarding OTA production in food and food raw materials. METHODS AND RESULTS: Aspergillus niger and A. carbonarius were isolated mainly from Brazilian coffee sources. The ability of isolates to produce OTA was tested by thin layer chromatography (TLC). Strains were genetically characterized by AFLP fingerprinting and compared with each other and with reference strains. Cluster analysis of fingerprints showed clear separation of A. niger from A. carbonarius strains. To obtain marker sequences, AFLP fragments were isolated from silver stained polyacrylamide gels, cloned and sequenced. Sequences obtained were used to develop species- specific PCR primers for the identification of A. carbonarius in pure culture and in artificially and naturally infected samples of green coffee. CONCLUSIONS: No clear correlation between genetic similarity of the strains studied and their potential to produce OTA was found. The PCR assays designed are a useful and specific tool for identification and highly sensitive detection of A. carbonarius. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assays allow specific and sensitive detection and identification of A. carbonarius, a fungus considered to be one of the major causative agents for OTA in coffee and grape-derived products. Assays may provide powerful tools to improve quality control and consumer safety in the food processing industry.     

Development of VNTR markers for three Aspergillus species used in brewing

Using a bioinformatics approach, we developed 18 variable number of tandem repeat markers for Aspergillus oryzae for use in population genetic studies. Repeat sequences in the genome sequences of A. oryzae were identified by a tandem repeat finding program. Length polymorphisms at 18 loci were examined in 41 strains of A. oryzae. The total number of alleles per locus ranged from two to 20. Investigation of cross-species amplifications with A. sojae and A. tamarii showed success. The variable number of tandem repeat markers will be used to determine the population structure of these three Aspergillus species used in brewing.     

荧光定量PCR方法检测白酒发酵过程中Aspergillus tubingensis生物量

【目的】为了更好地分析霉菌在白酒发酵过程中的作用,需要快速准确地测定发酵过程中霉菌生物量的变化,本实验以白酒酿造中常用的塔宾曲霉(Aspergillus tubingensis)为例,建立一套快速准确定量塔宾曲霉生物量的方法。【方法】优化从酒醅中提取基因组的方法,设计和验证专一性引物,建立实时荧光定量PCR(Real-time quantitative PCR)方法,验证方法的有效性并应用于白酒发酵过程中塔宾曲霉生物量的检测。【结果】用原位机械破碎法提取酒醅中总基因组,其DNA的浓度能够达到1.060×105 ng/g酒醅;同时建立了一套快速准确测定固态基质中霉菌生物量的方法,并应用于白酒生产(制曲、堆积发酵和窖池发酵过程)中塔宾曲霉生物量的定量。【结论】实时荧光定量PCR方法能够快速准确地测定固态基质中霉菌的生物量,且检测限较低,对今后的相关研究具有借鉴意义。