Extraction and purification of amyloglucosidase produced by solid state fermentation with Aspergillus niger

Summary Aqueous two-phase extraction was employed, as a new approach, for the recovery of amyloglucosidase (AMG) after solid state fermentation. In an aqueous two-phase system of PEG 6000 and potassium phosphate, 95% of the AMG was recovered in the bottom salt phase, with all visible particles from solid fungal bran in the interface, giving a purification factor of 11. Affinity purification of AMG was also done by adsorption onto crosslinked starch, in the presence of PEG or salt. Both were found to enhance the adsorption. In the presence of PEG 6000 (10% w/w), two fold increase in AMG adsorption, 85% of AMG recovery and 6.0 fold of purification were achieved/The purified AMG was found homogeneous by SDS polyacrylamide gel electrophoresis.     

Partitioning of amylase produced by Aspergillus niger in solid state fermentation using aqueous two-phase systems

Equilibrium data of aqueous two-phase systems composed of polyethylene glycol (4000 g mol⁻¹ or 6000 g mol⁻¹) and Li₂SO_4, (NH₄)₂SO₄ or Na₂SO₄ at pH 6.5 and 25 °C were obtained. The efficiency of these in the partition of amylases derived from Aspergillus niger was determined. The experimental data of binodal curves and tie lines were used to estimate the group interaction parameters using the UNIFAC model. Additionally, the influence of phases on the activity of the enzymes was investigated. The results indicate that the polymer molar mass did not influence the biphasic region size. However, the cations under study presented differences in induction to phase formation. It was verified that the systems formed with the Na⁺ presented a larger biphasic region. The increase in the molar mass of the polymer caused the increase in the exclusion volume from 3970.732 g mol⁻¹ to 5700.873 g mol⁻¹. The transfer Gibbs free energy of enzymes presented values between −1296.30 kJ mol⁻¹ and −2867.70 kJ mol⁻¹, that is, the process was spontaneous for all systems studied. The systems formed by (NH₄)₂SO₄ and PEG 4000 g mol⁻¹ presented the best K_e result (3.421) and theoretical recovery of 80.35 %.     

黑曲霉固态发酵生产单宁酶的条件优化

研究采用响应面法优化黑曲霉固态发酵生产单宁酶的培养条件。应用Plackett—Burman试验筛选出重要影响因子：五倍子粉含量、（NH4）2SO4浓度以及接种孢子量,最陡爬坡试验逼近最大响应区域。应用Box．Behnken响应面试验对重要影响因子进一步优化。得到最佳培养条件：每250mL三角瓶中装入1．0g五倍子粉、4．4g稻壳和0．5g麸皮、液固比（mL／g）2：1且营养盐溶液组成为（NH4）2s0421g/L、MgSO4·7H2O1g／L、NaCl1g／L,培养基pH自然,接种5．7×10^7个孢子后在30℃温度下培养4d。在此条件下,单宁酶产量从40U／g提高到114U／g,3次重复验证性试验平均值为115U／g,验证了模型的可靠性。     

Protease-conidia relationships of Aspergillus niger grown in solid state fermentation

Protease activity of Aspergillus niger growing on solid substrate correlated well with conidia formation (R: 0.91–0.96) for initial moisture contents of 38–48% (wet basis), initial pH 5.4 and 6 and temperature (29–37 °C ). However, conidia/protease ratio varied with most of these conditions and by NaCl addition indicating only a partial association between them.     

Purification and characterization of polygalacturonases produced by the hyphal fungus Aspergillus niger

Five endo-polygalacturonases (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) and one exo-polygalacturonase (poly(1,4-alpha-D-galacturonide) galacturonohydrolase, EC 3.2.1.67) were isolated from a commercial pectinase preparation derived from Aspergillus niger. All five endo-enzymes could be purified to homogeneity by affinity chromatography on cross-linked alginate, ion-exchange chromatography, chromatofocusing, and gel permeation chromatography. The exo-polygalacturonase was only partially purified but free from endo-polygalacturonase activity. The two most abundant endo-polygalacturonases (endo-I and endo-II), with molecular masses of 55 and 38 kDa, respectively, are quite different with respect to their isoelectric point, specific activity, mode of action on oligomeric substrates, and amino acid composition. The physicochemical properties of the other three endo-polygalacturonases (endo-IIIA, endo-IIIB, and endo-IV), present in low amounts, are quite similar to those of the endo-I type. The pH optima of all these endo-polygalacturonases are in the range of 4.3-4.9.     

Air pressure pulsation solid state fermentation of feruloyl esterase by Aspergillus niger

Air pressure pulsation solid state fermentation (APP-SSF) was applied to produce feruloyl esterase (FAE) by Aspergillus niger. With the optimization of some variables by orthogonal design, the optimal condition obtained was 0.2 MPa (gauge pressure) of high pressure intensity, 30 min of low pressure duration and 20s of high pressure duration. Based on the optimized condition, the APP-SSF achieved the reasonable enzyme yield of 881 mU/g at 48 h, which was 58% more than that by static solid state fermentation (static SSF) at 72 h. By comparison of two fermentation methods in temperature, O(2) and CO(2) concentration, and respiration intensity, it was concluded that APP-SSF enhanced heat and mass transfer of fermentation system and strengthened the metabolism of microorganisms. The APP-SSF had a greatly positive effect on FAE production by A. niger, by enhancing mass and heat transfer and activating growth and metabolism.     

Recovery of pectolytic enzymes produced by solid state culture of Aspergillus niger

Pectinases are enzymes with a wide range of applications in the food and drink industries. In the present work, the extraction of pectinases produced by Aspergillus niger in a solid state fermentation system was investigated. The purpose was to reduce enzyme losses in the fermented solids and at the same time obtain a crude extract as concentrated as possible. Initially the performances of stirred tank and fixed bed extractors were compared. Polygalacturonase activity and viscosity reducing capacity obtained in the stirred tank system were 105% and 15% superior, respectively. Repeated extractions and multiple stage countercurrent extraction were studied, employing stirred tanks. It was possible to observe that three stages were enough for total recovery of the enzymes contained in the solids. The final enzyme extract obtained by counter-current extraction with three stages showed a polygalacturonase activity 81% higher than the one obtained by one-stage extraction.     

Production and characterization of a highly active cellobiase from Aspergillus niger grown in solid state fermentation

Summary Aspergillus niger produced extracellular cellobiase when grown on different lignocellulosic substrates in solid state fermentation. The enzyme activity and yield were variable according to the carbon source. In Vogel’s medium, the cellobiase productivity was significantly higher on wheat bran, followed by Leptochloa fusca (kallar grass) straw augmented with corn steep liquor. Maximum yield of cellobiase/g wheat bran was significantly higher than the values reported on other potent fungi, bacteria and recombinants, harboring heterologous gene for cellobiase. This enzyme in the presence and absence of Trichoderma reesei and celluloclast, saccharified the biomass and the percentage saccharification as well as glucose yield from lignocellulosic biomass was doubled in its presence. The partially purified enzyme was thermotolerant as evidenced by melting temperature, activation energy demand for active catalysis, enthalpy and entropy of activation for reversible or irreversible thermal inactivation.     

Purification and characterization of acid-stable protopectinase produced by Aspergillus awamori in solid-state fermentation

Aspergillus awamori IFO 4033 produced an acid-stable protopectinase in solid-state fermentation using wheat bran as the medium. The enzyme was purified to a homogeneous preparation with anion-exchange, hydrophobic, and size-exclusion chromatography. The enzyme was a monomeric protein of 52 kDa, by SDS-PAGE analysis, with an isoelectric point of pH 3.7. The optimum pH for enzyme activity was 2.0, and it was most active at 50 degrees C (at pH 2.0) and was stable up to 50 degrees C (at pH 2.0). The enzyme showed pectin-releasing activity toward protopectins from various origins, especially on lemon protopectin. An outstanding characteristic of the enzyme was its extreme stability in acidic conditions: the enzyme activity was not lost after incubating at pH 2.0 and 37 degrees C for 24 h.     

Water and water activity in the solid state fermentation of cassava starch by Aspergillus niger

Summary During the solid state fermentation (SSF) of cassava starch by Aspergillus niger estimations were made of total water, consumed water and the residual water remaining in small quantities after 23 h. A theoretical calculation based on the Ross equation showed that the water activity (a _w) of the substrate decreased to 0.85 towards the end of the culture. Such low values were assumed to be inhibitory to growth. The a _w of the substrate was increased when sugarcane bagasse was used as a high water retention capacity support. Higher growth rates and substrate conversion to biomass were obtained with this system, confirming that water availability is a critical factor in the SSF of starch substrates.Abbreviations A, B Experimental constants - a _w Water activity - H₂O_c Consumed water - H₂O_R Residual water - H₂O_T Total water - IDW Initial dry weight - IMC Initial moisture content - OUR Oxygen uptake rate - S Substrate dry weight - Sc Substrate conversion: consumed substrate/initial substrate - S _H Amount of sugars hydrolysed - SSF Solid state fermentation - X Biomass dry weight - W ^* Amount of solids/g of water     

Xylanase production in solid state fermentation by Aspergillus niger mutant using statistical experimental designs

The initial moisture content, cultivation time, inoculum size and concentration of basal medium were optimized in solid state fermentation (SSF) for the production of xylanase by an Aspergillus niger mutant using statistical experimental designs. The cultivation time and concentration of basal medium were the most important factors affecting xylanase activity. An inoculum size of 5 x 10(5) spores/g, initial moisture content of 65%, cultivation time of 5 days and 10 times concentration of basal medium containing 50 times concentration of corn steep liquor were optimum for xylanase production in SSF. Under the optimized conditions, the activity and productivity of xylanase obtained after 5 days of fermentation were 5,071 IU/g of rice straw and 14,790 IU l(-1) h(-1), respectively. The xylanase activity predicted by a polynomial model was 5,484 IU/g of rice straw.     

Production of pectinases by Aspergillus niger in solid state fermentation at high initial glucose concentrations

Exopectinase (exo-p) and endopectinase (endo-p) production by Aspergillus niger CH4 in solid state culture was studied at initial glucose concentrations of 100, 250, 350 and 450 g/l. The highest activity of exo-p (35 U/g) was produced at 72 and 120 h in the medium containing 100 and 250 g glucose/l, respectively. The maximum endo-p activity (9 U/g) was produced at 72 h in the medium with 250 g glucose/l. The reduction in pectinase production at 350 and 450 g/l initial glucose concentration was due neither to repression of the synthesis of the enzyme nor to the glucose consumption rate of the strain but due to a drastic drop in pH of the medium.S. Solis-Pereyra, E. Favela-Torres, M. Gutiérrez-Rojas, G. Saucedo-Castañeda and G. Viniegra-González are with the Departamento de Biotecnologia, Universidad Autónoma Metropolitana, A.P. 55-535, C.P. 09340, México D.F., México; S. Roussos is with the Laboratoire de Biotechnologie, ORSTOM, B.P. 5045, 34032, Montpellier Cedex, France, and P. Gunasekaran is with the Department of Microbial Technology, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625-021, India.     

Gluconic acid production by Aspergillus niger mutant ORS-4.410 in submerged and solid state surface fermentation

Aspergillus niger ORS-4.410, a mutant of Aspergillus niger ORS-4 was produced by repeated irradiation with UV rays. Treatments with chemical mutagnes also resulted into mutant strains. The mutants differed from the parent strain morphologically and in gluconic acid production. The relationship between UV treatment dosage, conidial survival and frequency of mutation showed the maximum frequency of positive mutants (25%) was obtained along with a conidial survival of 59% after second stage of UV irradiation. Comparison of gluconic acid production of the parent and mutant ORS-4.410 strain showed a significant increase in gluconic acid production that was 87% higher than the wild type strain. ORS-4.410 strain when transferred every 15 days and monitored for gluconic acid levels for a total period of ten months appeared stable. Mutant ORS-4.410 at 12% substrate concentration resulted into significantly higher i.e. 85-87 and 94-97% yields of gluconic acid under submerged and solid state surface conditions respectively. Further increase in substrate concentration appeared inhibitory. Maximum yield of gluconic acid was obtained after 6 days under submerged condition and decreased on further cultivation. Solid state surface culture condition on the other hand resulted into higher yield after 12 days of cultivation and similar levels of yields continued thereafter.     

Influence of carbon, nitrogen and phosphorous sources on glucoamylase production by Aspergillus awamori in solid state fermentation

It was the objective of the present study to increase the production of glucoamylase by Aspergillus awamori through solid state fermentation, using wheat bran as the main carbon source and (NH4)2SO4, urea, KH2PO4, glucose, maltose and starch as additional nitrogen, phosphorus, and carbon sources. The production of glucoamylase is strongly influenced by N and C sources. A 100% increase was observed when the (NH4)2SO4 was replaced by urea, with C/N = 4.8, using maltose as the additional carbon source. C/P ratios in a range of 5.1 to 28.7 did not induce glucoamylase production under the studied conditions.     

嗜热拟青霉固体发酵产木聚糖酶的纯化和性质

研究一株新的嗜热拟青霉J18的固体发酵产木聚糖酶的纯化和性质。固体发酵的粗酶液经硫酸铵沉淀、凝胶过滤层析和离子交换层析得到了一种分子量约为26 kDa的电泳纯木聚糖酶,酶活力回收率为33.5%,纯化了5.27倍。该木聚糖酶具有很好的温度和pH稳定性,在pH7.0~pH 9.0下,60℃处理24 h,酶活力能保存80%以上。该酶水解玉米芯木聚糖生成以木二糖、木三糖和木四糖为主的低聚木糖,薄层层析分析表明不含木糖,适合生产低聚木糖。     

Production and extraction of pectinases obtained by solid state fermentation of agroindustrial residues with Aspergillus niger

In this work soy and wheat bran were employed as raw materials for the production of pectinases by Aspergillus niger through solid-state fermentation. Several fermentation and recovery parameters were studied. The kinetics of enzyme synthesis was investigated in the range from 13 to 96 h with moisture contents varying from 25% to 70% (w/w). A medium moisture content of 40% and a fermentation time of 22 h were selected, as these conditions resulted in high pectolytic activity and enhanced polygalacturonase productivity. In order to optimise the recovery step, the best combination of temperature of extraction, contact time and solvent type was investigated. Acetate buffer (pH 4.4), 35°C and 30 min provided the best recovery. The present results show that optimising the extraction conditions is a simple way of obtaining more concentrated enzyme extracts and could be a useful instrument to extract more selectively a desired biomolecule from fermented solids.     

Optimization of extraction and purification of glucoamylase produced by Aspergillus awamori in solid-state fermentation

Various parameters such as solvent selection, concentration, soaking time, and temperature were tested in a single bioreactor in order to determine optimum extraction conditions of glucoamylase, when produced simultaneously with protease by Aspergillus awamari nakazawa MTCC 6652. Optimum conditions were achieved in a 10% glycerol solution soaked for 2 h at 40°C, followed by concentration of extracted glucoamylase (9,157 U/gds) by acetone precipitation (1:2, v/v), which yielded 51.9% recovery. Ion exchange chromatography and gel filtration showed specific activities of 270.5 and 337.5 U/mg, respectively, while SDS-PAGE and zymogram analysis of glucoamylase indicated the presence of three starch-hydrolyzing isoforms with molecular weights of approximately 109.6, 87.1, and 59.4 kDa, respectively     

Production of high activity thermostable phytase from thermotolerant Aspergillus niger in solid state fermentation

The thermotolerant fungus, Aspergillus niger NCIM 563, was used for production of extracellular phytase on agricultural residues: wheat bran, mustard cake, cowpea meal, groundnut cake, coconut cake, cotton cake and black bean flour in solid state fermentation (SSF). Maximum enzyme activity (108 U g⁻¹ dry mouldy bran, DMB) was obtained with cowpea meal. During the fermentation phytic acid was hydrolysed completely with a corresponding increase in biomass and phytase activity within 7 days. Phosphate in the form of KH₂PO₄ (10 mg per 100 g of agriculture residue) increased phytase activity. Among various surfactants added to SSF, Trition X-100 (0.5%) exhibited a 30% increase in phytase activity. The optimum pH and temperature of the crude enzyme were 5.0 and 50°C respectively. Phytase activity (86%) was retained in buffer of pH 3.5 for 24 h. The enzyme retained 75% of its activity on incubation at 55°C for 1 h. In the presence of 1 mM K⁺ and Zn²⁺, 95% and 55% of the activity were retained. Scanning electron microscopy showed a high density growth of fungal mycelia on wheat bran particles during SSF. Journal of Industrial Microbiology & Biotechnology (2000) 24, 237–243. Received 07 June 1999/ Accepted in revised form 18 December 1999     

Biomass estimation of Aspergillus niger growing on real and model supports in solid state fermentation

Summary Direct hydrolysis of Aspergillus niger mycelium growth on amberlite IRA-900 or sugar cane bagasse on solid state fermentation followed by the analysis of soluble protein by the dye binding method was carried out. Hydrolysis with phosphoric acid 0.25M during 7 min allowed maxima protein extraction available to be measured. Color interference of medium components was not observed, allowing the use of this method for biomass estimation when amberlite IRA-900 or sugar cane bagasse are used as support in solid state fermentation processes.     

Purification and characterization of invertase from Aspergillus niger

Invertase produced by a strain of Aspergillus niger showed the following main characteristics: maximum activity at 60°C, pH 5.0; K _m with sucrose as substrate, 0.0625mm; V _max 0.013 mol/min; and free energy 9132 cal/mol. The metal ions and p-chloromercuribenzoate (PCMB) acted as inhibitors respectively.