Isolation and characterization of urease from Aspergillus niger

Pt, Smith; Ad, King; N, Goodman

doi:10.1099/00221287-139-5-957

Cited by 43 publications

(22 citation statements)

References 26 publications

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“…As none of these products appeared in the respective culture media, the transient accumulation of urea (and not ornithine or GABA) suggests that A. niger prefers the prior catabolism of amino products over urea. However, aspergilli do possess a functional urease, and the A. niger enzyme has a reasonable K m for its substrate (3.0 mM) (46). Deferred urea utilization could be due to the paucity of urease activity per se under those growth conditions.…”

Section: Resultsmentioning

confidence: 99%

Novel Route for Agmatine Catabolism in Aspergillus niger Involves 4-Guanidinobutyrase

Kumar¹,

Saragadam²,

Punekar³

2015

Appl Environ Microbiol

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Agmatine, a significant polyamine in bacteria and plants, mostly arises from the decarboxylation of arginine. The functional importance of agmatine in fungi is poorly understood. The metabolism of agmatine and related guanidinium group-containing compounds in Aspergillus niger was explored through growth, metabolite, and enzyme studies. The fungus was able to metabolize and grow on L-arginine, agmatine, or 4-guanidinobutyrate as the sole nitrogen source. Whereas arginase defined the only route for arginine catabolism, biochemical and bioinformatics approaches suggested the absence of arginine decarboxylase in A. niger. Efficient utilization by the parent strain and also by its arginase knockout implied an arginase-independent catabolic route for agmatine. Urea and 4-guanidinobutyrate were detected in the spent medium during growth on agmatine. The agmatinegrown A. niger mycelia contained significant levels of amine oxidase, 4-guanidinobutyraldehyde dehydrogenase, 4-guanidinobutyrase (GBase), and succinic semialdehyde dehydrogenase, but no agmatinase activity was detected. Taken together, the results support a novel route for agmatine utilization in A. niger. The catabolism of agmatine by way of 4-guanidinobutyrate to 4-aminobutyrate into the Krebs cycle is the first report of such a pathway in any organism. A. niger GBase peptide fragments were identified by tandem mass spectrometry analysis. The corresponding open reading frame from the A. niger NCIM 565 genome was located and cloned. Subsequent expression of GBase in both Escherichia coli and A. niger along with its disruption in A. niger functionally defined the GBase locus (gbu) in the A. niger genome.

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Section: Resultsmentioning

confidence: 99%

Novel Route for Agmatine Catabolism in Aspergillus niger Involves 4-Guanidinobutyrase

Kumar¹,

Saragadam²,

Punekar³

2015

Appl Environ Microbiol

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“…500 μl of a 2% alkaline hypochlorite solution and 150 μl water were added, the mixture was then shaken, and the colour complex was allowed to develop for a minimum of 10 min at room temperature. Ammonia concentrations generated from the urease reaction, represented by the colour complex, were determined at 625 nm and compared to a standard curve made with NH 4 Cl 33 . Experiments were performed at a final concentration of enzyme c e = 10 nM.…”

Section: Methodsmentioning

confidence: 99%

The heat released during catalytic turnover enhances the diffusion of an enzyme

Riedel

Gabizon

Wilson

et al. 2014

Nature

221

380

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Recent studies have shown that the diffusivity of enzymes increases in a substrate-dependent manner during catalysis1,2. Although this observation has been reported and characterized for several different systems3–10, the precise origin of this phenomenon is unknown. Calorimetric methods are often used to determine enthalpies from enzyme-catalysed reactions and can therefore provide important insight into their reaction mechanisms11,12. The ensemble averages involved in traditional bulk calorimetry cannot probe the transient effects that the energy exchanged in a reaction may have on the catalyst. Here we obtain single-molecule fluorescence correlation spectroscopy data and analyse them within the framework of a stochastic theory to demonstrate a mechanistic link between the enhanced diffusion of a single enzyme molecule and the heat released in the reaction. We propose that the heat released during catalysis generates an asymmetric pressure wave that results in a differential stress at the protein–solvent interface that transiently displaces the centre-of-mass of the enzyme (chemoacoustic effect). This novel perspective on how enzymes respond to the energy released during catalysis suggests a possible effect of the heat of reaction on the structural integrity and internal degrees of freedom of the enzyme.

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“…The ureases of eukaryotic microorganisms, such as Schizosaccharomyces pombe, Aspergillus nidulans, and A. niger had masses of 212 kDa, 19) 240 kDa, 20) and 250 kDa, 21) respectively. The early studies on ureases from prokaryotic microorganisms suggested that they had molecular weights of 200 kDa, 224 kDa, 360 kDa, and 230 kDa for Brevibacterium anmmoniagenes, Klebsiella aerogenes, Selenomonas ruminantium, and Sporosarcina (formerly Bacillus) pasteurii, respectively.…”

Section: Discussionmentioning

confidence: 99%

Ureases of Extreme Halophiles of the GenusHaloarculawith a Unique Structure of Gene Cluster

Mizuki

Kamekura

DasSarma

et al. 2004

Bioscience, Biotechnology, and Biochemistry

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We searched for urease activities in 71 strains of extreme halophiles by a urea-phenol red-agar plate method. Positive strains were further investigated by measuring the ammonia released from urea in cell-free extracts. Only 4 strains of the genus Haloarcula, Har. aidinensis, Har. hispanica, Har. japonica, and Har. marismortui were finally shown as the urease producers. A partially purified urease from Har. hispanica was a typical halophilic enzyme in that it showed maximum activity at 18-23% NaCl and lost the activity irreversibly in the absence of NaCl. Partial genes (1596 bp) of the urease encoding from upstream of the subunit down to the N-terminal 139 amino acids of the subunit, were PCR amplified from the four strains, as well as from five urease-negative Haloarcula strains. Strains of other genera, which were urease-negative, did not yield PCR products. The deduced amino acid sequences of the subunit and partial subunit were similar to each other (92-100% similarities) and to those from other organisms. Analysis of the draft genome sequence of Har. marismortui, however, suggested that the order of the genes encoding the three subunits (with the total number of amino acids of 834) and four accessory proteins was ---UreG-UreD-UreE-UreF. This order is quite unique, since in other microorganisms the order is ---UreE-UreF-UreG-UreD in most cases. No open reading frames were detected in the PCR-amplified upstream of the subunit, suggesting that all Haloarcula species have the same unique structure of the urease gene cluster.

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Isolation and characterization of urease from Aspergillus niger

Cited by 43 publications

References 26 publications

Novel Route for Agmatine Catabolism in Aspergillus niger Involves 4-Guanidinobutyrase

Novel Route for Agmatine Catabolism in Aspergillus niger Involves 4-Guanidinobutyrase

The heat released during catalytic turnover enhances the diffusion of an enzyme

Ureases of Extreme Halophiles of the GenusHaloarculawith a Unique Structure of Gene Cluster

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