Production of β-glucosidase by Aspergillus niger using carbon sources derived from agricultural wastes

The effects of various medium carbon sources and salt solutions on the production of β-glucosidase (βG, EC 3.2.1.21) by Aspergillus niger have been studied. β-Glucosidase productivity was found to be 50 times greater than that reported previously.¹ This higher productivity was achieved by employing a mutant strain of the organism, readily available and inexpensive carbon sources, such as cellulose and orange peel, and a simple nutrient salt solution.     

Biotransformation of isoflavones by Aspergillus niger as biocatalyst

Biotransformation of the isoflavones, 6,7,4′‐trimethoxyisoflavone ( 1 ) and 5,7,4′‐trimethoxyisoflavone ( 2 ) by Aspergillus niger was investigated. Compound 1 was transformed to 4′‐hydroxy‐6,7‐dimethoxyisoflavone ( 3 ) and 2 to 4′‐hydroxy‐5,7‐dimethoxyisoflavone ( 4 ). This suggested that 1 and 2 were demethylated at the C‐4′ position with regioselectivity by Aspergillus niger. Copyright © 2006 Society of Chemical Industry     

Use of Aspergillus niger to improve filtration of olive mill waste-waters

The high levels of dry matter (70–200 g dm^?3) and total suspended solids (TSS) of up to 50 g dm^?3 which are present in olive mill waste-waters (OMW) render these very difficult to filter. During filtration of the OMW viscous solution, a cake is formed by the TSS which decreases the kinetics of the process. Addition of pellets of the fungus Aspergillus niger modifies the cake porosity and thus improves the OMW filtration process. Improvement to the filtration is directly related to the quantity of A, niger added and is attributable to the fermentation of OMW by A. niger since the TSS are trapped by the fungal pellets. The degradation of pectin by A. niger pectinases may also be partly responsible for the increase in the filtration capacity.     

Production of ellagic acid from degradation of valonea tannins by Aspergillus niger and Candida utilis

Two fungal strains, effective in converting valonea (Quercus aegilops) tannins into ellagic acid (EA), were isolated from soil contaminated by valonea tannins, and were tentatively identified as Aspergillus niger and Candida utilis. Properties of EA accumulation by the two isolates from valonea tannins' fermentation were studied. Both the strains preferred sucrose to glucose as the additional carbon and energy source. The most suitable concentrations of valonea tannins in fermentation media for EA accumulation by A niger and C utilis were 5.0 and 9.0 g dm^?3 with EA yields of 12.1 and 8.9% (w/w), respectively. The optimal temperature for the two strains was 28 °C, while the preferred pH values of the fermentation media were 4.5–5.0 for A niger and 4.8–5.2 for C utilis. The tannin tolerances of A niger and C utilis were adapted to 20 and 25 g dm^?3 by gradually increasing the concentrations of valonea tannins in the culture media. The adapted strain of A niger was able to completely degrade 20 g dm^?3 valonea tannins with an EA yield of 14.3% in 9 days. Meanwhile, the adapted strain of C utilis decreased the valonea tannins' level from 25 to 9.1 g dm^?3 with an EA yield of 11.48%. The degradation ability of A niger came from tannase whose activity in the medium was 63 U cm^?3 at the ninth day of fermentation, and that of C utilis was due to both tannase and polyphenol oxidase (PPO) whose activities were 32 U cm^?3 and 29 U cm^?3 at the ninth day. The coculture of both the adapted strains could completely degrade 25 g dm^?3 valonea tannins in only 7 days with a remarkably increased EA yield (21%). The activities of tannase and PPO of the coculture at the seventh day were 66 U cm^?3 and 47 U cm^?3 respectively, which proved the synergistic effect of the two enzymes on valonea tannins' degradation and EA accumulation. Copyright © 2005 Society of Chemical Industry     

Production,thermal stability and immobilisation of inulinase from Fmarium oxysporum

Fusarium oxysporum produced maximum extracellular inulinase after 9 days of its growth at 25°C on a medium (pH 5.5) containing 3% fructan and 0.2% sodium nitrate. The level of this enzyme decreased on the addition of either glucose, fructose, galactose or sucrose to F. oxysporum already growing on a fructan-containing medium. A significant increase in invertase production which resulted in an increase of the invertase/inulinase (S/I) ratio, was observed on addition of inulin to this fungus growing on other carbon sources. Glycerol (10%) gave better protection to inulinase against thermal denaturation at 50°C compared to ethylene glycol and sorbitol. Inulinase immobilised in polyacrylamide gel retained 45% of its original activity. The immobilised enzyme showed a higher optimum temperature (45°C) compared to free enzyme (37°C). The immobilised enzyme after storage at 25°C for 96 h showed 58% activity. Thermal stability of entrapped inulinase increased in the presence of inulin.     

The antifungal activity and biotransformation of diisophorone by the fungus Aspergillus niger

The microbial transformation of racemic diisophorone was investigated using the plant pathogen Aspergillus niger as a biocatalyst. Incubation of diisophorone with Aspergillus niger gave 8α‐hydroxy‐diisophorone, 10‐hydroxydiisophorone and 17‐hydroxydiisophorone on the basis of their spectroscopic data including two‐dimensional NMR analysis, nOe and an X‐ray crystallographic study. The antifungal activity of diisophorone against Aspergillus niger was also examined. Copyright © 2004 Society of Chemical Industry     

黑曲霉实时荧光PCR检测方法的建立

目的建立黑曲霉实时荧光PCR检测方法。方法对6种主要病原曲霉(黑曲霉、烟曲霉、杂色曲霉、构巢曲霉、土曲霉及黄曲霉)的GAPDH基因序列进行比对分析,选择黑曲霉特异位点设计引物和探针,对黑曲霉进行实时荧光PCR扩增,并检测该方法的灵敏度及特异性。结果该方法可检出2.78×10-10μg/ml的黑曲霉基因组DNA;对亲源关系较近的11株不同种曲霉及4株其他属临床常见的病原真菌进行实时荧光PCR检测,未发现有交叉反应。结论已建立了灵敏度高、特异性好的快速检测黑曲霉的实时荧光PCR方法。     

Fructooligosacharides production in aqueous medium with inulinase from Aspergillus niger and Kluyveromyces marxianus NRRL Y-7571 immobilized and treated in pressurized CO2

This work investigated the influence of compressed CO₂ treatment on the enzymatic activity of immobilized inulinases, and the production of fructooligosacharides in aqueous medium using these enzymes. The effects of system pressure, exposure time and depressurization rate on the enzymatic activity were evaluated through central composite designs (CCD) 2³. Inulinase from Kluyveromyces marxianus NRRL Y-7571 presented an increase of 104% in the residual activity using CO₂ at 275 bar submitted to 6 h treatment, at a depressurization rate of 10 kg m^?3 min^?1. For Aspergillus niger commercial inulinase, a decrease in enzyme activity was observed (residual activity of 39%) using CO₂ treatment at 75 bar for 6 h exposure at the highest depressurization rate (200 kg m^?3 min^?1). Enzymatic activities changed significantly depending on the enzyme source and the experimental treatment conditions investigated. The values of FOS obtained using inulinases from A. niger were 30.64% of GF2; 13.90% of GF3 and 2.88% of GF4 in the medium containing inulin as substrate. Results demonstrate that the use of compressed CO₂ might be of technological importance as a preceding, preparation step, to improve enzyme activity, hence helping the development of new biotransformation processes.     

半纤维素酶的细胞固定化研究

用海藻酸钙凝胶将黑曲霉six－1孢子包埋，在含有0．5g葡萄糖和0．075g蛋白胨的培养基中摇床培养，生长状态丰满，产生的木聚精酶活力可达441．7IU／mL，并可维持10天以上。     

Production,purification and immobilisation of inulinase from Kluyveromyces fragilis

Kluyveromyces fragilis (NCIM 3217), Kluyveromyces marxianus (NCIM 3231), Hansenula polymorpha (NCIM 3377), Pichia fermentan (NCIM 3408), Pichia polymorpha (NCIM 3419) and Debaryomyces castellii (NCIM 3446) were grown on an inulin-based growth medium. Only K. fragilis produced extracellular inulinase with a maximum after 36 h of growth at 25–27°C. Sucrose and fructose were weak inducers of inulinase as compared to inulin whereas with glucose the inulinase level was minimal. An aqueous extract of chicory roots containing 1% fructan was a better carbon source than inulin and peptone was the best nitrogen source for the production of inulinase. The maximum yield of inulinase was about 7 units cm^?3 of medium. The invertase to inulinase ratio of 10 in the culture filtrate was reduced to 1·6 on purifying inulinase by ethanol precipitation followed by chromatography on Sephadex G-200, DEAE-cellulose and CM-cellulose columns. Using this purification procedure, inulinase was purified 26-fold. With inulin as substrate, the shape of the velocity curve was nearer to a sigmoidal pattern whereas with sucrose the curve was hyperbolic. The molecular weight of inulinase was determined as 250 ± 10 kDa. The crude and purified inulinase preparations did not release sucrose or oligosaccharides from inulin, indicating that the enzyme has primarily exo-inulinase activity. Using the metal-link chelation method, 40% of inulinase was immobilised on cellulose. Maximum activity of crude, purified and immobilised inulinase preparations was observed at 55°C. The half-life of immobilised inulinase at 25°C was 5 days.     

Bubble column fermentation of olive mill wastewaters by Aspergillus niger

A bubble column (tower) fermenter was used to study the growth of Aspergillus niger on olive mill wastewaters (OMW). The gas hold-up, mixing time and oxygen transfer coefficient values were studied in terms of superficial air velocity. These characteristics were clearly affected by OMW. The growth of A. niger resulted in a filamentous suspension with no pellet formation and the specific growth rate was 0–2 h^?1. The growth rate was limited by the oxygen transfer rate at 90 mmol dm^?3 h^?1 when pO₂ was lower than 2%.     

Thermally treated low density polyethylene biodegradation by Penicillium pinophilum and Aspergillus niger

Differential scanning calorimetry (DSC), X‐ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) were used to determine morphological, structural and surface changes (biodegradation) on thermo‐oxidized (80°C, 15 days) low‐density polyethylene (TO‐LDPE) incubated with Aspergillus niger and Penicillium pinophilum fungi, with and without ethanol as cosubstrate for 31 months. TO‐LDPE mineralization by fungi was also evaluated. Significantly morphological and structural final changes on biologically treated TO‐LDPE samples were observed. Decreases to three units on crystallinity and crystalline lamellar thickness (0.4–1.8 Å), and increases in small‐crystals content (up to 3.2%) and mean crystallite size (8.4–14 Å) were registered. An oxidation decrease (almost twice) on samples without ethanol with respect to the control was observed, while in those with ethanol it was increased (up to 2.5 times). Double bond index increased more than twice from 21 to 31 months. The higher TO‐LDPE changes and fungi‐LDPE interaction was observed in samples with ethanol, suggesting that ethanol favors the TO‐LDPE biodegradation, at least in case of P. pinophilum, probably by means of a cometabolic process. Mineralization of 0.50 % and 0.57 % for A. niger, and of 0.64 % and 0.37 % for P. pinophilum were obtained, for samples with and without ethanol, respectively. A model to explain morphological and structural changes on biologically treated TO‐LDPE is also proposed. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 83: 305–314, 2002     

黑曲霉作为分泌蛋白细胞工厂的研究进展

黑曲霉具有极强的分泌蛋白的能力,其分泌的木质纤维素降解酶、淀粉酶、蛋白酶、脂肪酶等广泛应用于食品、饲料和生物技术等相关工业中。本文围绕黑曲霉生产同源和异源分泌蛋白,阐述黑曲霉作为分泌蛋白细胞工厂的巨大潜力。首先,通过总结黑曲霉表达系统的优越性,确定了黑曲霉作为分泌蛋白细胞工厂的开发前景。随后,介绍了初步构建黑曲霉分泌蛋白细胞工厂的一般流程。接着,总结了近十年来黑曲霉作为同源分泌酶细胞工厂的研究进展,提出在黑曲霉中定向表达分泌酶是深入研究黑曲霉自身分泌酶性质、功能和结构的理想策略。最后,总结了近年来黑曲霉作为异源分泌蛋白细胞工厂的研究进展,并从异源蛋白表达中遇到的难点出发介绍了完善黑曲霉细胞工厂来提高异源蛋白产量的对策。     

黑曲霉β-葡萄糖苷酶的研究进展

系统介绍了近年来黑曲霉β-葡萄糖苷酶(EC3.2.1.2)的酶解特征、酶学性质、催化机制、活性中心、生物学功能及应用技术等方面的研究进展,并展望了其应用前景。     

四株黑曲霉对高磷铁矿脱磷的研究

研究了四株不同来源的黑曲霉对高磷铁矿的脱磷作用,结果表明,在NBRIP培养基中,四株黑曲霉都能有效脱除高磷铁矿中的磷,但脱磷效率存在一定的差异,脱磷率从高到低依次为WIT-4>WIT-3>WIT-2>WIT-1。分析了四株黑曲霉对高磷铁矿的脱磷机制,发现黑曲霉生长代谢产生有机酸使体系可滴定酸浓度增加及pH下降是其脱磷的主要原因。     

Permeabilization of Aspergillus niger by Reverse Micellar Solutions and Simultaneous Purification of Catalase

Abstract

Reverse micellar solutions(RMS) of sodium bis‐ (2‐ethylhexyl)‐sulfosuccinate (AOT) in aliphatic organic solvents were used for permeabilization and protein removal directly from Aspergillus niger cells. Most of the cell wall proteins (～95–100%) were solubilized into the reverse micelles solutions. A significant fraction of intracellular catalase (26–30%) permeates out of the cells and remains on the cell surface and is recovered in a cell wash. Because of it's size the catalase in not solubilized in the RM water pools and thus is not detected in the stripping solution used to recover other proteins from the organic reverse micellar solutions. The remaining amount of catalase is recovered by breaking the cells in purer form. Multiple extractions were used for the extraction of the cell wall proteins followed by ultrasonication of the cells to recover intracellular catalase in a purified form. Therefore, catalase with 5 fold purification was recovered in 88% yield from the RMS‐ treated cells.     

黑曲霉负载银纳米颗粒的制备及其抗菌性能

采用非酶还原法,以黑曲霉菌原位还原银氨离子制备一种新型银纳米颗粒（AgNPs）/菌体复合抗菌材料,着重考察了反应温度与pH值对还原过程和所得复合材料的抗菌性能及稳定性的影响。结果表明,在温度为30℃、60℃和pH 9.5、11.5条件下,能够合成出粒径为6.9～8.2 nm的近球形AgNPs。该AgNPs均匀地分布在菌体表面上,对E.coli显示出高的抗菌性能：最小抑菌浓度(MIC)为217～434 mg·L^-1（以菌粉总质量表示）或8～20 mg Ag·L^-1（以银含量表示）。提高反应温度有利于提高菌体银负载量,但AgNPs粒径增大,抗菌性能有所下降;提高反应pH值有利于提高还原速率,而对抗菌性能影响不显著。复合材料中AgNPs与菌体结合牢固,单位质量复合材料释出的Ag⁺含量为1.7～6.8 mg·g^-1,提高反应温度和pH值后Ag⁺的释出均减少。     

高碱性铜尾矿的黑曲霉生物浸出

以湖南某高碱性铜尾矿为研究对象,进行了黑曲霉摇瓶浸出实验,研究了浸出时间和PSA培养基成分、含量对铜浸出的影响. 结果表明,浸出时间为7 d时,铜浸出率最高,随浸出时间延长,铜浸出率显著下降. PSA培养基中的马铃薯和蔗糖含量对铜浸出有明显影响,马铃薯含量为200 g/L、蔗糖含量为20 g/L时,铜浸出率最高. 在接种量0.02%(j)、矿浆浓度50 g/L、温度30℃、转速200 r/min、浸出时间7 d、马铃薯和蔗糖含量分别为200 g/L和20 g/L条件下,铜浸出率可达79.03%.     

Effect of Ozone on Aspergillus niger Causing Black Rot Disease in Onion

The effect of ozone on Aspergillus niger causing black rot disease in onion was studied in culture. Ozone induced the spore germination in all treatments and few spores showed rapid swelling, resulting in the production of 2-3 germ tubes per spore compared to control. Although all the ozone treated spores germinated, all of them did not produce uniform colony morphology. Some colonies which developed from ozone treated spores failed to produce spores and such colonies appeared as grey patches of mycelia without spores amidst surrounding black sporulating colonies. Further work is in progress to study the mechanism involved in formation of sterile mycelia by ozone.