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1.
The growth and the enzymatic production of two microbial fungal associations were studied: Aspergillus niger and Fusarium moniliforme and Trametes versicolor and Aspergillus niger. The synergistic interrelations between the species of the first mixed culture increased the biosynthesis of α-amylase and
pectinase. T. versicolor and A. niger proved to be compatible partners in the overproduction of the enzyme laccase, whose synthesis surpassed 8.4 times the enzymatic
level in the monoculture, with both of the mixed microbial populations cocultivation facilitating the amplified synthesis
of enzymes rather than their growth acceleration. A further proof of the presence of synergism established by the cultures
was the enzyme volumetric productivities in both of the mixed microbial cultures, which increased parallel to the rise in
the combined biomass synthesis. The competent selection of compatible partners can adjust the desired enzymatic levels and
compositions in mixed fungal systems aimed at a number of specified designations. Thus, a very high level of laccase production
(97,600 IU/g dry weight) was achieved. The chosen fungal strains produce a variety of different enzymes, but first microbial
association produces mainly amylase and pectinase, necessary for their growth, and second association produces mainly laccase
and pectinase. 相似文献
2.
We have investigated transformation of eight industrial dyes by a whiterot fungus, Trametes versicolor. The fungus was found to decolorize Reactive Golden Yellow R, Procion Red, Reactive Violet 5, Reactive Blue 28, and Ponceau
Red 4R at an initial dye concentration of 80 ppm within 72 h of incubation, whereas it took 5 d to completely decolorize Reactive
Black 5 (40 ppm). However, it did not significantly decolorize Reactive Red 152 and Novatic Blue BC S/D. During decolorization
in liquid medium, laccase and manganese-independent peroxidase (MiP) activities were detected in culture filtrate of T. versicolor. Dye-decolorizing activity of the culture was found to be associated with H 2O 2-dependent activity of the culture filtrate. Furthermore, dye-decolorizing activity of the culture filtrate was not influenced
by Mn 2+ or veratryl alcohol, thus suggesting a role of extracellular MiP in decolorization of synthetic dyes by T. versicolor. 相似文献
3.
A new process involving the filamentous fungi Aspergillus niger and Pycnoporus cinnabarinus has been designed for the release of ferulic acid by enzymic degradation of a cheap and natural agricultural byproduct (autoclaved
maize bran) and its biotransformation into vanillic acid and/or vanillin with a limited number of steps. On the one hand,
the potentialities of A. niger I-1472 to produce high levels of polysaccharide-degrading enzymes including feruloyl esterases and to transform ferulic acid
into vanillic acid were successfully combined for the release of free ferulic acid from autoclaved maize bran. Then vanillic
acid was recovered and efficiently transformed into vanillin by P. cinnabarinus MUCL 39533, since 767 mg/L of biotechnologic vanillin could be produced in the presence of cellobiose and XAD-2 resin. On
the other hand, 3-d-old high-density cultures of P. cinnabarinus MUCL39533 could be fed with the autoclaved fraction of maize bran as a ferulic acid source and a. niger I-1472 culture filtrate as an extracellular enzyme source. Under these conditions, P. cinnabarinus MUCL39533 was shown to directly biotransform free ferulic acid released from the autoclaved maize bran by A. niger I-1472 enzymes into 584 mg/L of vanillin. These processes, involving physical, enzymic, and fungal treatments, permitted
us to produce crystallin vanillin from autoclaved maize bran without any purification step. 相似文献
4.
Laccase production by solid-state fermentation (SSF) using an indigenously isolated white rot basidiomycete Ganoderma sp. was studied. Among the various agricultural wastes tested, wheat bran was found to be the best substrate for laccase
production. Solid-state fermentation parameters such as optimum substrate, initial moisture content, and inoculum size were
optimized using the one-factor-at-a-time method. A maximum laccase yield of 2,400 U/g dry substrate (U/gds) was obtained using
wheat bran as substrate with 70% initial moisture content at 25°C and the seven agar plugs as the inoculum. Further enhancement
in laccase production was achieved by supplementing the solid-state medium with additional carbon and nitrogen source such
as starch and yeast extract. This medium was optimized by response surface methodology, and a fourfold increase in laccase
activity (10,050 U/g dry substrate) was achieved. Thus, the indigenous isolate seems to be a potential laccase producer using
SSF. The process also promises economic utilization and value addition of agro-residues. 相似文献
5.
Production of xylanolytic enzymes by an Aspergillus niger CCMI 850 isolate was investigated in batch cultures. The effect of the composition of a fermentation medium that did not
include chemical inducers, on β-xylanase, β-xylosidase, α- l-arabinofuranosidase, and total cellulase activity was studied. With 4% xylan as the carbon source, about 65 U/mL of β-xylanase
was obtained, whereas the total cellulase activity was undetectable, under the specified conditions. This β-xylanase activity
represents the highest reported for a wild-type strain of A. niger. The effect of pH and temperature on the activity of β-xylanase was studied. Partial characterization of the β-xylanase showed
that with insoluble birchwood as substrate the K
m
and V
max were 0.3 m M and 19 μmol/min, respectively. Aspects of using the crude β-xylanase preparation for applications in the pulp and paper industry
were discussed. 相似文献
6.
The hydrolytic activity of fungal originated β-glucosidase is exploited in several biotechnological processes to increase
the rate and extent of saccharification of several cellulosic materials by hydrolyzing the cellobiose which inhibits cellulases.
In a previous presentation, we reported the screening and liquid fermentation with Aspergillus niger, strain C-6 for β-glucosidase production at shake flask cultures in a basal culture medium with mineral salts, corn syrup liquor, and
different waste lignocellulosic materials as the sole carbon source obtaining the maximum enzymatic activity after 5–6 d of
8.5 IU/mL using native sugar cane bagasse. In this work we describe the evaluation of fermentation conditions: growth temperature,
medium composition, and pH, also the agitation and aeration effects for β-glucosidase production under submerged culture using
a culture media with corn syrup liquor (CSL) and native sugar cane bagasse pith as the sole carbon source in a laboratory
fermenter. The maximum enzyme titer of 7.2 IU/mL was obtained within 3 d of fermentation. This indicates that β-glucosidase
productivity by Aspergillus niger
C-6 is function of culture conditions, principally temperature, pH, culture medium conditions, and the oxygen supply given in
the bioreactor. Results obtained suggest that this strain is a potential microorganism that can reach a major level of enzyme
production and also for enzyme characterization. 相似文献
7.
Laccases are very interesting biocatalysts for several industrial applications. Its production by different white-rot fungi
can be stimulated by a variety of inducing substrates, and the use of lignocellulosic wastes or industrial by-products is
one of the possible approaches to reduce production costs. In this work, various industrial wastes were tested for laccase
production by Trametes versicolor MZKI G-99. Solid waste from chemomechanical treatment facility of a paper manufacturing plant showed the highest potential
for laccase production. Enzyme production during submerged cultivation of T. versicolor on the chosen industrial waste has been further improved by medium optimization using genetic algorithm. Concentrations of
five components in the medium were optimized within 60 shake-flasks experiments, where the highest laccase activity of 2,378 U dm −3 was achieved. Waste from the paper industry containing microparticles of CaCO 3 was found to stimulate the formation of freely dispersed mycelium and laccase production during submerged cultivation of
T. versicolor. It was proven to be a safe and inexpensive substrate for commercial production of laccase and might be more widely applicable
for metabolite production by filamentous fungi. 相似文献
8.
The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by
lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such
as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production
by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch
fermentation process for rMnP production in P. pastoris αMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase ( mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation
of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake
flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at
pH less than 5.5, although cell growth rates were similar from pH 4–7. Investigations of the cause of low rMnP production
at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during
the fermentation that are active against rMnP at pH less than 5.5. 相似文献
9.
Horticultural waste collected from a landscape company in Singapore was utilized as the substrate for the production of laccase
under solid-state fermentation by Trametes versicolor. The effects of substrate particle size, types of inducers, incubation temperature and time, initial medium pH value, and
moisture content on laccase production were investigated. The optimum productivity of laccase (8.6 U/g substrate) was achieved
by employing horticultural waste of particle size greater than 500 μm and using veratryl alcohol as the inducer. The culture
was at 30 °C for 7 days at moisture content of solid substrate of 85% and initial pH 7.0. The decolorization was also investigated
in order to assess the degrading capability of the ligninolytic laccase obtained in the above-mentioned cultures. The decolorization
degree of a model dye, phenol red, was around 41.79% in 72 h of incubation. By far, this is the first report on the optimization
of laccase production by T. versicolor under solid-state fermentation using horticultural waste as the substrate. 相似文献
10.
The enzyme laccase was produced by the white-rot fungus Trametes versicolor in repeated batches cultures with immobilized mycelium. Two different culture conditions were used. Enzymes produced were
evaluated regarding their stability a thigh temperatures (55°C and 65°C) and at alkaline conditions (pH 7.0 and pH 8.0) having
in view the application of these enzymes in biobleaching of hardwood Kraft pulp.
Biobleaching experiments were divided in two parts, enzymatic prebleaching followed by chemical bleaching. In the enzymatic
prebleaching the enzyme laccase was used at two conditions of pH and temperature, whereas the reaction time was fixed at 1h
in all pretreatments. In the chemical bleaching the DEDED and DEpDED sequences were used.
The enzyme action was evaluated by Kappa number, viscosity, and brightness at the end of bleaching sequences. There were obtained
values of Kappa numbers lower than control assays, viscosities compatible with industrial pulps, and brightness higher than
controls, when pulps were pretreated for 1 h with laccase at pH 8.0 and 55°C. 相似文献
11.
This work aims to evaluate cell recycle of a recombinant strain of Pichia pastoris GS115 on the Xylanase A (XynA) production of Thermomyces lanuginosus IOC-4145 in submerged fermentation. Fed-batch processes were carried out with methanol feeding at each 12h and recycling
cell at 24, 48, and 72 h. Additionally, the influence of the initial cell concentration was investigated. XynA production
was not decreased with the recycling time, during four cell recycles, using an initial cell concentration of 2.5 g/L. The
maximum activity was 14,050 U/L obtained in 24h of expression. However, when the initial cell concentration of 0.25 g/L was
investigated, the enzymatic activity was reduced by 30 and 75% after the third and fourth cycles, respectively. Finally, it
could be concluded that the initial cell concentration influenced the process performance and the interval of cell recycle
affected enzymatic production. 相似文献
12.
Aspergillus niger NRRL330 produces extracellular β-fructofuranosidase (Ffase), and its production is subject to repression by hexoses in the
medium. After ultraviolet mutagenization and selection, seven derepressed mutants resistant to 2-deoxyglucose (2-DG) were
isolated on Czapek’s minimal medium containing glycerol. One of the mutants, designated DGRA-1, produced higher levels of
Ffase. A considerable difference occurred in the mutants with reference to hexokinase and intracellular acid phosphatase activities.
The hexokinase activity of the mutant DGRA-1 (0.69 U/mg) was 1.8-fold higher than the wild type (0.38U/mg). Intracellular
acid phosphatase activity of the mutant DGRA-1 (0.83 U/g of mycelia) was twofold higher than that of the wild type (0.42U/g
of mycelia), suggesting that phosphorylation and dephosphorylation steps could attribute to the 2-DG resistance of A. niger. However, additional mutations could account for the increased production of Ffase in the mutant DGRA-1. 相似文献
14.
Laccase activity was detected in a soil bacterium Stenotrophomonas maltophilia AAP56 identified by biochemical and molecular methods. It was produced in cells at the stationary growth phase in Luria Bertani
(LB) medium added by 0.4 mM copper sulfate. The addition of CuSO 4 in culture medium improved production of laccase activity. However, one laccase enzyme was detected by native polyacrylamide
gel electrophoresis. The enzyme showed syringaldazine ( K
m = 53 μM), 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ( K
m = 700 μM), and pyrocatechol ( K
m = 25 μM) oxidase activity and was activated by addition of 0.1% ( v/ v) Triton-X-100 in the reaction mixture. Moreover, the laccase activity was increased 2.6-fold by the addition of 10 mM copper
sulfate; the enzyme was totally inhibited by ethylenediaminetetraacetic acid (5 mM), suggesting that this laccase is a metal-dependant
one. Decolorization activity of some synthetic dyes (methylene blue, methyl green, toluidine blue, Congo red, methyl orange,
and pink) and the industrial effluent (SITEX Black) was achieved by the bacteria S. maltophilia AAP56 in the LB growth medium under shaking conditions. 相似文献
15.
Aspergillus niger ORS-4.410, a mutant of A. niger ORS-4, was generated by repeated ultraviolet (UV) irradiation. Analysis of the UV treatment dose on wild-type (WT) A. niger ORS-4, conidial survival, and frequency of mutation showed that the maximum frequency of positive mutants (25.5%) was obtained
with a 57% conidial survival rate after the second stage of UV irradiation. The level of glucose oxidase (GOX) production
from mutant A. niger ORS-4.410 thus obtained was 149% higher than that for WT strain A. niger ORS-4 under liquid culture conditions using hexacyanoferrate (HCF)-treated sugarcane molasses (TM) as a cheaper carbohydrate
source. When subcultured monthly for 24 mo, the mutant strain had consistent levels of GOX production (2.62±0.51 U/mL). Mutant
A. niger ORS-4.410 was markedly different from the parent strain morphologically and was found to grow abundantly on sugarcane molasses.
The mutant strain showed 3.43-fold increases in GOX levels (2.62±0.51 U/mL) using HCF-TM compared with the crude form of cane
molasses (0.762±0.158 U/mL).
The results reported herein were obtained while the author was working at the Department of Biotechnology, Indian Institute
of Technology, Roorkee-247667, India. 相似文献
16.
The filamentous fungi Trichoderma reesei and Penicillium funiculosum produce highly effective enzyme mixtures that degrade the cellulose and hemicellulose components of plant cell walls. Many
fungal species produce a glycoside hydrolase family 7 (Cel7A) cellobiohydrolase, a class of enzymes that catalytically process
from the reducing end of cellulose. A direct amino acid comparison of these two enzymes shows that they not only have high
amino acid homology, but also contain analogous N-linked glycosylation sites on the catalytic domain. We have previously shown (Jeoh et al. in Biotechnol Biofuels, 1:10,
2008) that expression of T. reesei cellobiohydrolase I in a commonly used industrial expression host, Aspergillus niger var. awamori, results in an increase in the amount of N-linked glycosylation of the enzyme, which negatively affects crystalline cellulose degradation activity as well as thermal
stability. This complementary study examines the significance of individual N-linked glycans on the surface of the catalytic domain of Cel7A cellobiohydrolases from T. reesei and P. funiculosum by genetically adding or removing N-linked glycosylation motifs using site directed mutagenesis. Modified enzymes, expressed in A. niger var. awamori, were tested for activity and thermal stability. It was concluded that N-linked glycans in peptide loops that form part of the active site tunnel have the greatest impact on both thermal stability
and enzymatic activity on crystalline cellulose for both the T. reesei and P. funiculosum Cel7A enzymes. Specifically, for the Cel7A T. reesei enzyme expressed in A. niger var. awamori, removal of the N384 glycosylation site yields a mutant with 70% greater activity after 120 h compared to the heterologously
expressed wild type T. reesei enzyme. In addition, similar activity improvements were found to be associated with the addition of a new glycosylation motif
at N194 in P. funiculosum. This mutant also exhibits 70% greater activity after 120 h compared to the wild type P. funiculosum enzyme expressed in A. niger var. awamori. Overall, this study demonstrates that “tuning” enzyme glycosylation for expression from heterologous expression hosts is
essential for generating engineered enzymes with optimal stability and activity. 相似文献
17.
Aspergillus niger NRRL3 was cultivated in a moist wheat bran and ground corncob solid medium supplemented with inorganic minerals for the production
of cellobiase (β-1,4-glucosidase, EC 3.2.1.21). With this method, A. niger NRRL3 was able to produce a high concentration of cellobiase (215 IU/gofsolid substrate) after 96 h of incubation. Temperature
and moisture content affected final cellobiase titers. The best conditions for cell obiase production from solid substrate
by A. niger NRRL3 were determined to be 70% moisture and 35°C. 相似文献
18.
An extracellular laccase (Lacc10) was discovered in submerged cultures of Pleurotus ostreatus var. florida bleaching ß-carotene effectively without the addition of a mediator (650 mU/L, pH 4). Heterologous expression in P. pastoris confirmed the activity and structural analyses revealed a carotenoid-binding domain, which formed the substrate-binding pocket and is reported here for the first time. In order to increase activity, 106 basidiospore-derived monokaryons and crosses of compatible progenies were generated. These showed high intraspecific variability in growth rate and enzyme formation. Seventy-two homokaryons exhibited a higher activity-to-growth-rate-relation than the parental dikaryon, and one isolate produced a very high activity (1800 mU/L), while most of the dikaryotic hybrids showed lower activity. The analysis of the laccase gene of the monokaryons revealed two sequences differing in three amino acids, but the primary sequences gave no clue for the diversity of activity. The enzyme production in submerged cultures of monokaryons was stable over seven sub-cultivation cycles. 相似文献
19.
The Pichia pastoris clone producing streptokinase (SK) was optimized for its nutritional requirements to improve intracellular expression using
statistical experimental designs and response surface methodology. The skc gene was ligated downstream of the native glyceraldehyde 3-phosphate dehydrogenase promoter and cloned in P. pastoris. Toxicity to the host was not observed by SK expression using YPD medium. The transformant producing SK at level of 1,120 IU/ml
was selected, and the medium composition was investigated with the aim of achieving high expression levels. The effect of
various carbon and nitrogen sources on SK production was tested by using Plackett–Burman statistical design and it was found
that dextrose and peptone are the effective carbon and nitrogen sources among all the tested. The optimum conditions of selected
production medium parameters were predicted using response surface methodology and the maximum predicted SK production of
2,136.23 IU/ml could be achieved with the production medium conditions of dextrose ( x1), 2.90%; peptone ( x2), 2.49%; pH, 7.2 ( x3), and temperature, 30.4 ( x4). Validation studies showed a 95% increase in SK production as compared to that before optimization at 2,089 IU/ml. SK produced
by constitutive expression was found to be functionally active by plasminogen activation assay and fibrin clot lysis assay.
The current recombinant expression system and medium composition may enable maximum production of recombinant streptokinase
at bioreactor level. 相似文献
20.
Enhanced production of laccases from Streptomyces psammoticus in solid-state fermentation was carried out using two different strategies: laccase inducers and scale-up process. Laccase
yield was enhanced by a wide range of aromatic inducers. The best inducer was pyrogallol, which yielded 116 U/g as compared
to the control (55.4 U/g). Scale-up studies in packed bed bioreactor was performed at different aeration rates. Aeration at
1.5 vvm was identified as the optimum condition for laccase production (75.4 U/g) in the column bioreactor. The enzyme yield
was enhanced further by combining the best conditions from the first two experiments. Fermentation was carried out in bioreactors
in the presence of 1 mM pyrogallol, which resulted in 3.9-fold increase in laccase yield (215.6 U/g). The role of laccase
in azo dye decolorization was evaluated in the presence of four different laccase mediators, at different concentrations.
1-Hydroxybenzotriazole (HOBT) proved to be the best mediator for S. psammoticus laccase and decolorized the azo dyes efficiently. Acid orange, Methyl orange, and Bismarck brown were decolorized at the
rates of 86%, 71%, and 75% respectively, by HOBT. 相似文献
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