Urease isolation

Urease activity in soils has been found to reflect the bacterial count and content of organic matter. The urease isolated from an Australian forest soil (87) was crystallized and found to have a specific activity of 75 Sumner units (S.U.) per mg. The molecular weight species were estimated (sedimentation velocity) to be 42, 131, and 217 X 103. That urease activity persists in soils is shown by the finding that enzymic activities, including urease, could be demonstrated in soil samples over 8000 years old (88). 

It has not been possible so far to establish that Cr is an essential element required by plants, however, addition of Cr to soils deficient in the element has been shown to increase growth rates and yields of potatoes, maize, rye, wheat or oats (Scharrer and Schropp, 1935 Huffman and Allaway, 1973 Bertrand and De Wolf, 1986). Nickel appears to be an essential element for plants (Farago and Cole, 1988). Zerner and coworkers (Dixon et al., 1975) demonstrated that urease isolated from jack bean (Canavalia ensiformis) was a nickel enzyme. Eskew et al. (1983) have shown that Ni is an essential micronutrient for legumes. Most plants contain nickel in the range 1 - 6 mg kg-1 (Vanselow, 1966 Hutchinson, 1981). The uptake of Ni is enhanced by low pH values, and available nickel increases at pH less than 6.5 as a consequence of the breakdown of Ni complexes in the soil with Fe and Mn oxides. Uptake of nickel by plants and questions of toxicity and tolerance have been reviewed by Farago and Cole (1988). Nickel toxicity toward plants has been reviewed by Vanselow (1966) and Hutchinson (1981). 

Roughly 30% of enzymes are metalloenzymes or require metal ions for activity and the present chapter will concentrate on the chemisty and structure of the plant metalloenzymes. As analytical methods have improved it has been possible to establish a metal ion requirement for a variety of enzymes which were initially considered to be pure proteins. A dramatic example is provided by the enzyme urease isolated from Jack beans and first crystallised by Sumner (1926) (the first enzyme to be crystallised). Sumner defined an enzyme as a pure protein with catalytic activity, however, Zerner and his coworkers (Dixon et al., 1975) established that urease is in fact a nickel metalloenzyme. Jack bean urease contains two moles of nickel(II) per mole of active sites and at least one of these metal ions is implicated in its mechanism of action. 

The X-ray crystallographic studies of the enzyme isolated from Klebsiella aerogenes reveals an active site composed of a dinickel center with 3.5-A Ni- Ni separation. A schematic view of the active site of urease isolated from K. aerogenes is... 

Figure 2 A schematic drawing of the active site of urease isolated from K. aerogenes ...

Urea undergoes hydrolysis in the presence of acids, bases, or the enzyme urease (isolable from jack beans generated by many bacteria, such as Micrococcus ureqey... 

Until the discovery in 1975 of nickel in jack bean urease (which, 50 years previously, had been the first enzyme to be isolated in crystalline form and was thought to be metal-free) no biological role for nickel was known. Ureases occur in a wide variety of bacteria and plants, catalyzing the hydrolysis of urea,... 

Hevesy first used a radioisotope as a tracer. Warburg. Importance of iron pigments in oxidation. Keilin rediscovered cytochromes. Sumner crystallized urease. The Eggletons and Fiske and SubbaRow isolated phospho-creatine. 

McGee DJ, May CA, Garner RM, et al. 1999. Isolation of Helicobacter pylori genes that modulate urease activity. J Bacteriol 181 2477-84. 

The protein nature of enzymes was established through the seminal work of James Sumner. In 1926, Sumner succeeded in isolating the enzyme urease in crystalline form from jack bean meal. This was the first time in history that an enzyme had been obtained in crystalline, though not completely pure, form. Subsequently, Sumner established that the crystalline enzyme was a protein. Urease is an enzyme that degrades one of the human end products of nitrogen metabolism, urea, to ammonia and carbon dioxide ... 

Phenotypical isolates from purified water shall be characterized. Biochemical testing (such as oxidase test, urease test, catalase test, citrate test, coagulase test, and indole test) and commercial test kits (such as API tests) and reagents may be used for conformation of some unique isolates. 

The isolation and crystallization of urease by James Sumner in 1926 provided a breakthrough in early enzyme studies. Sumner found that urease crystals consisted entirely of protein, and he postulated that all enzymes are proteins. In the absence of other examples, this idea remained controversial for some time. Only in the 1930s was Sumner s conclusion widely accepted, after John Northrop and Moses Kunitz crystallized pepsin, trypsin, and other digestive enzymes and found them also to be proteins. During this period,... 

Urease, which was first isolated from the jack bean has a special place in biochemical history as the first enzyme to be crystallized. This was accomplished by J. B. Sumner in 1926, and although Sumner eventually... 

Nickel has long been suspected to be an essential trace element for living organisms, but the identification of its functions in molecular terms is relatively recent. The first nickel protein to be identified was urease (urea ammonia hydrolase) (i). This was demonstrated 49 years after the original isolation and crystallization of the enzyme by Sumner (2). This enzyme is of widespread occurrence, and the specific requirement for nickel explains many of the effects of nickel deficiency in plants (3, 4). 

The enzyme urease was not discovered until 1926 (by Sumner). It was the first enzyme to be isolated as a crystalline protein. Sumner s accomplishment confirmed the then growing belief that enzymes, the... 

In view of the isolation of the crystalline enzyme from this source (chosen because of the high concentration) 45 years ago, it might be supposed that a discussion of preparative procedures would be unnecessary. On the contrary, vigorous contentions concerning the purity and activity of crystalline urease have been a hallmark of its literature right to the present. Several factors contribute to this situation. The urease content of jack bean meal varies according to the origin and... 

A method for the isolation of urease from hydrated jack beans developed by Sehgal and Naylor (15) employs DEAE-cellulose as a final purification step. A method intended for purification of urease from mammalian sources (16) was developed using jack bean meal as a standard source. It employed no acetone but is a combination of (NH4)2S04 precipitation and Sephadex column separations. [Note Fish-... 

Much uncertainty reigned over the nature of proteins, the best known of which were hemoglobin, the digestive enzymes, and later, insulin. Properties of individual amino acids and the peptide bond were studied early in this century, but it was not until urease was crystallized by Sumner1 in 1926, followed by the isolation of other pure enzymes, that it was finally accepted in the 1930s that enzymes were proteins and that their catalytic properties were not the function of some adsorbed low molecular weight entity. Somewhat later, towards the end of the 1930s, coenzymes were isolated and their roles established. 

Several other proteins show a low, but significant amino acid identity with atzA (Table 22.4). All of these, urease-alpha subunit (urea amidohydrolase), cytosine deaminase, and imidazolone-5-propionate hydrolase, catalyze hydrolytic reactions with substrates involved in the metabolism of nitrogenous compounds (Sadowsky et al 1998). A Rhizobium strain capable of atrazine dechlorination has been isolated from a soil that was previously treated with atrazine (Bouqard et al., 1997). This bacterium could not mineralize atrazine, and it accumulated hydroxyatrazine as the sole metabolite after long-term incubations. Interestingly, 22 of the 24 identified amino acids at the N-terminus of the atrazine halidohydrolase from Rhizobium were identical with atzA from Pseudomonas strain ADP. 

The proteins from the jack bean (Canavala ensiformis) were first studied over 60 years ago by Jones and Johns(6). Several years later, Sumner(7), while studying urease (also from the jack bean), isolated three other proteins, two of which could be crystallized, concanavalin A and B. It should be noted that this report of crystalline Con A by Sumner appeared about seven years before he reported the first crystallization of an enzyme, urease(8). 

D. E. Koshland and J. T. Edsall have recorded their own experiences in early protein research. Koshland stresses the value of his induced fit theory,192 while Edsall describes his long career at Harvard after two years with Hopkins at Cambridge.193 Fruton has also described the work of T. B. Osborne (1859-1928), noted for his analyses of amino-acids from seed proteins.194 The idea that enzymes might be proteins was a matter of heated debate among chemists from about 1915, including arguments between Willstatter and James Sumner, who in 1926 isolated the enzyme urease and showed it to be a protein.195 However, while it is most important to emphasize protein chemistry, the contributions made to protein science by physics... 

The chemical nature of enzyme was controversial for a long time, until Buchner succeeded in isolating an enzyme system (zymase) from yeast in a cell-free extract in 1897.2) Urease was then crystallized by Sumner in 1926,3) followed by crystallization of several proteolytic enzymes by Northlop and his colleagues. At present the chemical nature of enzyme is defined as a protein with catalytic activity based on the specific activaiton of its substrate. However, this definition has been somewhat open to debate since a catalytic RNA, ribozyme, was discoved in 1982. 

Sumner JB (1926) The isolation of the enzyme urease preliminary paper. J Biol Chem... 

Several alternatives are used to reduce ammonia elimination. Applied in relatively small quantities, urease inhibitors such as A-(n-butyl) thiophosphoric acid triamide reduce the rate of microbial hydrolysis of urea and increase its efficiency as a fertilizer (Manahan, 2005). Ammonia volatilization could also be reduced using a mixture of urea with tropical peat soil or free humic substances, such as humic and fulvic acids, isolated from peat soils (Bernard et al., 2009). Another application of green technologies is the use of thermal polyaspartate, a product formed by the condensation and base treatment of a natural compound, aspartic acid. This has been found to be effective in stimulating plant uptake of fertilizer thus reducing the amount of fertilizer required (Manahan, 2005). 

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