The effect of pH on glucoamylase production, glycosylation and chemostat evolution of Aspergillus niger

Wallis, Gregg; Swift, Richard J.; Atterbury, Robert J.; Trappe, Susanne; Rinas, Ursula; Hemming, Frank W.; Wiebe, Marilyn G.; Trinci, Anthony P. J.; Peberdy, John F.

doi:10.1016/s0304-4165(01)00145-3

Cited by 14 publications

(12 citation statements)

References 42 publications

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“…A similar effect of pH on production and secretion of glucoamylase (GA) by A. niger was described by Wallis et al (22). They demonstrated that GA production was higher at pH 4.0 than at pH 5.5.…”

Section: Effect Of Carbon Source Media Initial Ph and Temperature Ofsupporting

confidence: 65%

Production of thermostable glucoamylase by newly isolated Aspergillus flavus A 1.1 and Thermomyces lanuginosus A 13.37

Gomes¹,

Souza²,

Grandi³

et al. 2005

Braz. J. Microbiol.

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Thirteen thermophilic fungal strains were isolated from agricultural soil, tubers and compost samples in tropical Brazil. Two strains were selected based on of their ability to produce considerable glucoamylase activity while growing in liquid medium at 45ºC with starch as the only carbon source. They were identified as Aspergillus flavus A1.1 and Thermomyces lanuginosus A 13.37 Tsiklinsky. The experiment to evaluate the effect of carbon source, temperature and initial pH of the medium on enzyme production was developed in a full factorial design (2x2x3). Enzyme productivity was influenced by the type of starch used as carbon source. Cassava starch showed to be a better substrate than corn starch for glucoamylase production by A. flavus but for T. lanuginosus the difference was not significant. Enzyme activities were determined using as substrates 0.3% soluble starch, 0.3% maltose or 0.3% of starch plus 0.1% maltose. The enzymes from A. flavus A1.1 hydrolyzed soluble starch preferentially but also exhibited a significant maltase activity. Moreover higher quantities of glucose were released when the substrate used was a mixture of starch and maltose, suggesting that this fungus produced two types of enzyme. In the case T. lanuginosus A 13.37, the substrate specificity test indicated that the enzyme released also hydrolyzed starch more efficiently than maltose, but there was no increase in the liberation of glucose when a mixture of starch and maltose was used as substrate, suggesting that only one type of enzyme was secreted. Glucoamylases produced from A. flavus A1.1 and T. lanuginous A.13-37 have high optimum temperature (65ºC and 70ºC) and good thermostability in the absence of substrate (maintaining 50% of activity for 5 and 8 hours, respectively, at 60ºC) and are stable over in a wide pH range. These new strains offer an attractive alternative source of enzymes for industrial starch processing.

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Section: Effect Of Carbon Source Media Initial Ph and Temperature Ofsupporting

confidence: 65%

Production of thermostable glucoamylase by newly isolated Aspergillus flavus A 1.1 and Thermomyces lanuginosus A 13.37

Gomes¹,

Souza²,

Grandi³

et al. 2005

Braz. J. Microbiol.

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“…For Aspergillus glaA, which encodes glucoamylase (43), or exlA, which encodes exoxylanase (7), glucose and fructose (the preferred fungal carbon sources) must be absent or present at limiting concentrations, and specific inducers, such as starch, xylan, cellulose, or derivatives of these compounds, must be supplied. Similarly, the regulation and activities of extracellular proteases which are of major importance in yield optimization are both carbon and nitrogen source sensitive (19) and dependent on ambient pH (44). These constraints on medium composition often prevent the use of cost-effective raw materials in commercial fermentations.…”

Section: Discussionmentioning

confidence: 99%

Metabolically Independent and Accurately AdjustableAspergillussp. Expression System

Pachlinger

Mitterbauer

Adam

et al. 2005

Appl Environ Microbiol

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Filamentous fungi are well-established expression hosts often used to produce extracellular proteins of use in the food and pharmaceutical industries. The expression systems presently used in Aspergillus species rely on either strong constitutive promoters, e.g., that for glyceraldehyde-3-phosphate dehydrogenase, or inducible systems derived from metabolic pathways, e.g., glaA (glucoamylase) or alc (alcohol dehydrogenase). We describe for Aspergillus nidulans and Aspergillus niger a novel expression system that utilizes the transcriptional activation of the human estrogen receptor by estrogenic substances. The system functions independently from metabolic signals and therefore can be used with low-cost, complex media. A combination of positive and negative regulatory elements in the promoter drives the expression of a reporter gene, yielding a linear dose response to the inducer. The off status is completely tight, yet the system responds within minutes to induction and reaches a level of expression of up to 15% of total cell protein after 8 h. Both Aspergillus species are very sensitive to estrogenic substances, and low-cost inducers function in the picomolar concentration range, at which estrogenic substances also can be found in the environment. Given this high sensitivity to estrogens, Aspergillus cells carrying estrogen-responsive units could be used to detect xenoestrogens in food or in the environment.In their natural environment, fungi use extracellular enzymes to gain access to complex, often water-insoluble carbon and nitrogen sources. Extracellular protein production by filamentous fungi usually is very efficient for homologous proteins, for which levels of grams per liter can be obtained. Aspergillus and Trichoderma strains have been reported to secrete up to 30 g of homologous proteins per liter in fermentation processes (e.g., glucoamylases) (11). The production of recombinant proteins of mammalian origin can be up to 4 orders of magnitude lower even when the same expression signals are used.The reason for this difference is not completely understood (2). Along with mRNA stability, codon usage, and translational efficiency, overloading the secretory machinery with a protein that has a "foreign" structure appears to be a critical bottleneck (16,34). High concentrations of such heterologous proteins can result in incorrect folding-leading to translational feedback inhibition by phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 (23) and subsequent intracellular degradation of the misfolded protein. Thus, strong but highly regulatable expression systems are needed to control the flow of proteins through the secretory pathway.The use of strong promoters derived from housekeeping genes, such as those of the various fungal glyceraldehyde-3-phosphate dehydrogenase genes, has the disadvantage of continuous, growth-related expression levels (31). This problem may reduce yields of proteins susceptible to degradation or lead to cell death if the overexpressed foreign proteins are toxic to the ...

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“…The role of some extracellular polysaccharides of fungi may be to reserve nutrient sources of carbon. During C limitation, GM and other galf epitopes might be broken down by the enzyme exo-␤-D-galactofuranosidase to produce galactose, which can then be used as a secondary C source, as suggested for Aspergillus niger (57) and Penicillium fellutanum (40). The production of this enzyme seemed to be medium dependent, with glucose as a repressor (10).…”

Section: Vol 44 2006 Release Of Surrogate Markers By Aspergillus Fumentioning

confidence: 99%

In Vitro Release by Aspergillus fumigatus of Galactofuranose Antigens, 1,3-β- d -Glucan, and DNA, Surrogate Markers Used for Diagnosis of Invasive Aspergillosis

et al. 2006

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Aspergillus markers are becoming increasingly important for the early diagnosis of invasive aspergillosis. The kinetics of release of these surrogate markers, however, is largely unknown. We investigated the release of ␤-(1-5)-galactofuranosyl (galf) antigens (Platelia Aspergillus), 1,3-␤-D-glucan (BG) (Fungitell), and DNA (PCR) in an in vitro model of Aspergillus fumigatus. The results showed that release is correlated to the growth phase of the fungus, which depends on available nutrients. Whereas galf antigens and BG are released during logarithmic growth, DNA is released only after mycelium breakdown. During early logarithmic growth, galf antigens seem to be released somewhat earlier than BG. Furthermore, galf antigen concentrations of more than 120,000 times the serum cutoff value (0.5 ng/ml) can be measured, while BG concentrations reach a value only 978 times the serum cutoff value (60 pg/ml). During lytical growth, release of galf antigens further increased to a maximum level, which depended on pH. After that, the concentration of galf antigens stayed high (pH 7.4) or decreased to zero within 4 days (pH 5.0). In contrast to galf antigens, BG concentration decreased after 1 day of growth. The decrease of galf components seems to be due to the enzyme ␤-galactofuranosidase, which is able to destroy galf epitopes and whose activity fluctuates in the culture filtrates in parallel with galf antigen concentration. Fungal DNA seems to be released only due to autolysis caused by nutrient limitation. In conclusion, several factors clearly influence the release of surrogate markers in vitro. These same factors might also play a role at the infection site of Aspergillus disease in humans.Invasive aspergillosis (IA) has become a leading cause of death among immunocompromised hosts, including transplant patients, those treated for hematological malignancy, and those treated with high-dose corticosteroids (41, 49). In addition, IA is increasingly observed in the nonneutropenic phase after hematopoietic stem cell transplantation and in nonclassic settings, such as intensive care units with critically ill patients (13, 16). The high mortality is due partly to the difficulty in establishing a diagnosis at an early stage of infection, since presenting symptoms are nonspecific and sensitivity of cultures is low. Techniques to improve timely diagnosis have focused on the detection of circulating surrogate markers released by the fungus (43,45,50). With the development of nonculture-based methods, such as PCR and antigen detection, circulating markers can be detected at an early stage of infection in patients with invasive disease (12, 27, 28).The commercially available sandwich enzyme-linked immunosorbent assay (ELISA) (Platelia Aspergillus [PA]; Bio-Rad, Marnes-la-Coquette, France) is based on the rat immunoglobulin M monoclonal antibody EB-A2, which binds the ␤-(1-5)-galactofuranosyl (galf) side chains of the Aspergillus galactomannan (GM) molecule (24,50,51). In addition to GM, fungal glycoproteins also react with the EB-...

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The effect of pH on glucoamylase production, glycosylation and chemostat evolution of Aspergillus niger

Cited by 14 publications

References 42 publications

Production of thermostable glucoamylase by newly isolated Aspergillus flavus A 1.1 and Thermomyces lanuginosus A 13.37

Production of thermostable glucoamylase by newly isolated Aspergillus flavus A 1.1 and Thermomyces lanuginosus A 13.37

Metabolically Independent and Accurately AdjustableAspergillussp. Expression System

In Vitro Release by Aspergillus fumigatus of Galactofuranose Antigens, 1,3-β- d -Glucan, and DNA, Surrogate Markers Used for Diagnosis of Invasive Aspergillosis

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