Function of Urease in Plants with Reference to Legumes: A Review

Abstract

Urease (urea amidohydrolase, EC 3.5.1.5) is a nickel-containing enzyme produced by plants, fungi, and bacteria that catalyzes the hydrolysis of urea into ammonia and carbamate. Plant (especially legumes) ureases hold a special place in science history, participating on some important landmarks of biochemistry as it was the first enzyme ever to be crystallized in 1926. Finding nickel in urease’s active site in 1975 was the first indication of a biological role for this metal. Despite the abundance of urease in tissues and seeds of some members of Legumes families, and its ubiquity in virtually all plants little has been revealed of the roles of urease. This review will explore many faces of these ureases from legumes and other plants, their roles, nutritional relationship between plants and the commensal bacteria with which they associate. In addition, we will explore the possibility that bacteria participate in turnover of the “plant” urea pool. Plant ureases possess insecticidal and fungitoxic properties independent of its ureolytic activity. Altogether, with this review we wanted to invite the readers to take a second look at ureases from versatile plants especially legumes for various biotechnological applications.

1. Introduction

Ureases (urea amidohydrolase, EC 3.5.1.5) are ubiquitous enzymes produced by plants, bacteria and fungi, animals do not produce these metalloenzymes. They are found to be the most proficient known enzymes to date, the enzyme catalyzes the hydrolysis of urea to form carbamate and ammonia; the carbamate then decomposes to form carbon dioxide and another molecule of ammonia, enabling the reaction rate to be faster by at least a factor of 10¹⁴ when it is compared to the decomposition of urea by elimination reaction [1, 2, 3, 4]. The proficiency of urease Computational modeling brought about a proposal of a value equivalent to 10³² multiplied by the theoretical rate of uncatalyzed hydrolysis of urea [5]. But in solution, it can be debated upon that the value obtained is not visible based on some limitation imposed due to the substrate diffusion in water. Ureases from Plants maintain a special position in the history of science, involving in some relevant events in biochemistry. For example, Urease contributed about three landmarks in the history of Biochemistry. One, the Canavalia ensiformis (jack bean) seeds urease isolated and crystallized by a scientist called James B. Sumner, who in 1926 demonstrated the enzymes’ proteinaceous nature [6], this findings in 1946 was laureated with the Nobel Prize in Chemistry. Two, the biological importance of nickel (N²⁺) was in 1975 recognized as obligatory for urease’ catalytic activity after studies conducted by Zerner’s and colleagues who reveal the existence of nickel ions in jack bean urease’ active site [7]. Three, the identification of a toxin in plant as a urease in the year 2001 may be regarded as another breakthrough that has to do with ureases, which brought about the discovery of properties of these enzymes that are non-catalytic [8]. This discovery increased our knowledge on the functions carried out by these proteins, asides their function in metabolism of nitrogen [9]. This urease enzymes belongs to the super family of phosphotriesterases and amidohydrolases, that possesses in their active sight two catalytic Nickel metal(s) with a few reported exceptions [8, 10]. Takeuchi’s findings as stated in Real-Guerra et al. make all this possible after he observed that Glycine max (soybean) seeds’ crude extracts shows high amounts of urease activity [11]. As at that time, research on urease only focus on microorganisms and in algae. Takeuchi’s discovery was the first report that shows the existence of ureases in higher plants. The advantage of this finding is that it gave researchers knowledge on the large availability of urease globally, thereafter, so many other researches on ureases were carried out, taking advantage of the leguminous plant urease having the aim to understand functions of the enzyme. Urease from G. max was among the main focus on enzymology development, involving several researches associated with this enzyme which brought about hypothesis that were important to Michaelis and Menten’s observation on the reaction rate of enzymes and the substrates they catalyzed [12]. Till date, after those first experiments more than a century ago, urease from legumes continued to generate attention by researchers globally, in various fields such as biochemistry, physiology and genetic.

A leguminous plant belongs to the family Leguminosae otherwise known as Fabaceae. They produce their seeds around a pod [13, 14]. This plant family is large with more than 18,000 species shrubs, climbers, trees and herbs whereby only few has been studied for urease extraction and utilization. Common legumes that have been used for the extraction of urease include mung bean, Pisumsativum seeds, peas, some beans species, peanuts, lentils, soybeans, upins, lotus, green beans and sprouts are known as food or grain legumes. Different legumes are shown in Figure 1.

Figure 1.

Some species of legumes.

In this review, we shall focus on ureases from legumes, providing information generated over time and exposes some areas that need to be focused by researchers.

2. Metabolic origins of urea in plants

Mammals synthesize urea via the Krebs-Henseleit cycle (also called the arginine, ornithine, or urea cycle) as nontoxic form of jettisoned ammonia [15]. Plants usually have the opposite problem, i.e., how to conserve nitrogen, which after carbon, is the most limiting element in plant nutrition [16]. This contention is consistent with the presence of urease in plants and in most bacteria and fungi [17] and its absence in mammals. Whereas in the latter, urea is a nontoxic “waste” form of ammonia in excess, in the former, ureolytic activity is necessary to recycle urea nitrogen (urea is 47% nitrogen). We discuss in this section the metabolic and tissue origins of plant urea.

2.1 The precursor of urea

Arginine, by the action of arginase, is the immediate precursor of urea in the mammalian arginine (urea) cycle. Although an active functional arginine cycle in plants has been debated [18], it is clear that arginase (EC 3.5.3.1) is widely spread in the plant world. Plant arginases resemble the animal enzyme in their high pH optima (approximately 9.7) and Mn²⁺ requirement [18, 19, 20, 21, 22]. As arginase is widely spread, so is its substrate abundant: arginine is a main nitrogen transport and plants’ storage compound. It is the nitrogen main transport compound of deciduous [23] and coniferous [24] trees and a major component of underground storage organs (bulbs, roots, tubers [25]). It was shown to be among the amino acids in the seeds of 379 angiosperms that is predominant [26]. It was recalculated (in mol %) the reported average seed amino acid composition of these 379 species. Arginine accounted for 7.7% of seed amino acids and its “N-weighted” contribution was 21.1% of total amino acid nitrogen, the highest contribution of any amino acid, with glutamine a close second (18.6% based on the assumption that half of the glutamate in protein hydrolyses came from glutamine) [26]. In Glycine max, arginine contains 18% of seed protein-bound nitrogen [27]. At least 50% of free amino acid N in developing seeds of pea [28] and soybean [27] is in the arginine pool. In addition, many legume seeds are exceedingly rich in free canavanine [29], an arginine analog. Half of arginine nitrogen (and two-thirds of canavanine nitrogen), in the guanidino moiety, is convertible to urea by arginase kanavinase action.

3. Repercussion of urease activity elimination in plants

In higher plants it appears that urea can be assimilated only by urease action. Urease-negative plants and cultures, induced genetically [30] with urease inhibitor [31, 32] or by nickel deprivation [33, 34, 35, 36], have been observed either to accumulate urea or to be blocked in the ability to employ urea as a nitrogen source. All plant [37] and bacterial [3] ureases are probably nickel metalloenzymes. Seed ureases from jackbean [7, 38] and soybean [39] have been shown to contain nickel. Duckweed plants [40] and callus of soybean, rice, and tobacco [34, 37] are dependent on nickel for maximal growth with urea as sole nitrogen source. Urease appears to be the only nickel-requiring enzyme in plants since, as indicated below, nickel-deprived soybean plants have the same phenotype as those genetically blocked in urease synthesis [41]. Thus, higher plants appear to lack the ATP-dependent urea amidohydrolase reported in algae and yeast [42, 43, 44, 45]. This biotin-containing carboxylase/hydrolase appears not to be a nickel metalloenzyme and has a urea-assimilatory function (e.g., [46] in these urease-negative lower eukaryotes. In an interesting example of the potential of urease to provide nitrogen for the plant, [47] developed transgenic tobacco plants engineered for resistance to cyanamide. The resistance gene, from soil fungus Myrothecium verrucaria, codes a cyanamide hydratase that converts cyanamide to urea. Although urea levels are raised in such plants, the endogenous urease apparently hydrolyzes much of the liberated urea.

3.1 Effect urease on protein deposition and embryo development

Given that urease is the plant’s only means of assimilating urea, the next question is the metabolic version of “If you are so smart how come you are not rich?” When applied to urease it would read, “If you’re so important how come the plant survives without you?” Indeed, completely urease negative mutant soybean plants develop to maturity and produce a relatively good yield of seeds that germinate at normal frequency to propagate another generation. However, if the role of urease is to recycle urea nitrogen generated from arginine (and possibly ureide) degradation, then a protein-rich plant such as soybean may provide a “suppressing” background for a urease defect. Soybean has indeed been intensively bred for large and protein-rich seeds. However, even in soybean we question the dispensability of urease. Urease-negative mutant plants [41] and nickel-deprived wild type [35, 36] exhibit necrotic leaf tips, apparently due tourea “burn.” Similar observations were made in nickel-deprived tomato [48, 49]. More important, perhaps, than the deleterious effects of leaf burn is the loss of significant quantities of nitrogen in a urea dead-end, lost nitrogen that could have a significant negative impact on seed protein deposition during pod fill. The assessment of the agronomic impact of a urease-negative phenotype on soybean performance requires extensive field testing of isogenic or nearly isogenic paired urease-positive and urease-negative lines, preferably in multiple environments. To obtain these lines we are currently engaged in the long process of backcrossing, generation advance, selection for uniformity in maturity group and plant architecture, etc., and amplification of seed stocks [50]. Our prediction that total seed protein of plant will decrease as suggested by [36], who reported a correlation between seed yield and nickel content per seed. However, there was too much variation in their material to obtain a statistically significant difference. It was observed that at the time of flowering completely urease-negative soybean mutants accumulated approximately 100 dry wt of total leaf (green plus necrotic tip) [41]. Assuming that 10% of the leaf dry weight is protein (16% nitrogen), much of which is destined to provide amino acids during pod fill, accumulated urea (47% nitrogen) represents 18% of the nitrogen in leaf protein. The developing soybean embryo does not generate urea [41]. Thus, other than recycling maternally derived urea, urease appears to play no direct role in embryo metabolism. However, it is possible that in monocots a urease role is more critical in embryo development. It has been reported that nickel is essential for development of viable barley embryos [51]. Post dormancy grains could not be rescued by nickel, whereas developing grains could be rescued by the feeding of nickel to the maternal plant. The role of nickel in barley embryo development is not known and may be unrelated to urease. (Urease is the only nickel metalloenzyme yet identified in plants). However, it is possible that a loss of urease activity under nickel-deprivation conditions leads either to urea poisoning or to nitrogen starvation of the embyro. It would be informative to study barley embryo development in vitro, where both nickel availability and nitrogen source can be manipulated.

3.2 Effects of urease on germination

What is the consequence of blocking urease action during germination when large of amounts of arginine and ureides are mobilized and generate urea? In soybean we observed a 7–8 h delay in germination of its protein-rich seeds imbibed in the potent urease inhibitor [52, 53, 54] phenylphosphorodiamidate (PPD) [55]. Protein-poor Arabidopsis thaliana seeds imbibed in water with 50 PPD did not germinate at all. That PPD acts specifically on urease and brings about inhibition of germination by nitrogen limitation is indicated by parallel dose-response curves for germination and urease inhibition, and by reversal of inhibition of germination with added nitrogen sources, NH, NO, (5 mM), or casein hydrolysate (1 mg/ml) [55]. Similar respose was observed for a cyclotriphosphazatriene urease inhibitor [56]. Within A. thaliana, large seeds germinated in distilled water tend to produce seedlings that survive longer than those from small seeds [57]. Phenyl phosphorodiamidate may effectively reduce seed size by depriving the seedling of that portion of its nitrogenous reserves catabolized to urea. Hydrolyzed A. thaliana seed meal contains 5.5 g arginine per 16 g total nitrogen [26]. Thus, 1.76 g or 11% of seed nitrogen (free plus that bound in protein and nucleic acids) is in arginine and, potentially, 5.5% (or more, if arginine is proportionally higher in “reserves”) seed nitrogen can be converted to urea by arginase. Seed nitrogen in nucleic acids is another generator of urea; greater than 40% of urea generated in 6-day Arabidopsis seedlings is eliminated by allopurinol [55]. In other species, e.g., Lupinus texensis [58] and wild radish [59], larger seeds tend to germinate at a higher frequency than small seeds. We have observed a crude correlation, across species, between seed size and resistance to PPD inhibition of germination and will extend studies of relative PPD sensitivity to large seeded soybeans vs. selected small-seeded sister lines [60], when these lines are made available to the public.

3.3 Loss of chemical protection

As described in the next section, G. max contains two different isozymes of urease: the enzyme which is ubiquitous is made in all the examined tissues, more so, embryo-specific synthesis of urease is dependent to the embryo that is developing and it is conserved in the seed that is mature where its specific activity is about 1000-fold higher than that of the ubiquitous urease in any tissue [61, 62]. Since mutants that lacks the embryo-specific urease do not display any of the abnormalities related with loss of the ubiquitous enzyme necrotic leaf tips, accumulation of urea in leaves or seeds, retarded germination [41] it was concluded that this enzyme has no important physiological use. In vitro culture of developing cotyledons of pea [63] and G. max [41, 64] indicates that ureases play little or no role in embryo nutrition since urea was an extremely poor source of nitrogen. Indeed, urea is not normally generated within the developing glycine max cotyledon in vivo [41], in agreement with the lack of ureide delivery to the legume embryo from maternal tissues [65, 66]. The obvious question from the observations of the previous paragraph is why would the developing soybean embryo and those of jackbean, watermelon, and many other members of Leguminosae and Cucurbitaceae [67] invest in a very active ureolytic activity when it never “sees” urea. Although much urea is generated upon germination [41] and although much of the embryo-specific urease is retained in seedling cotyledons and roots [68], the loss of the embryo-specific urease causes no discernible increase in seedling urea levels over those of wild type [41]. It is suggested, therefore, that the embryo-specific urease plays no urea assimilatory role but rather that of chemical defense in seeds. To draw two parallels, at least, with the pathogenic effects of bacterial urease on vertebrates, active seed urease could cause either hepatic coma by subversion of the urea cycle or peptic ulceration by localized increases in NH⁺ and OH-ions (urea + 3H₂O + 2NH₄⁺ + HCO⁻ + OH⁻). A micro aerophilic, bacterium, Helicobacter pylori, can colonize gastric epithelium because its active urease creates a more basic micro environment in this acidic milieu [69, 70]. Arguments summarized by [71] link urease from bacteria to ulceration of the gastric mucosa either by cytotoxicity of ammonia directly or by its prevention of proton flux from gastric glands to the gastric lumen leading in a back-diffusion of protons. Hepatic coma results when intestinally derived nitrogenous compounds, e.g., ammonia, bypass the liver and get to the brain. Administration of urease inhibitors has proven effective in reducing hyperammonemia [72, 73]. It is easy to visualize an active seed urease mimicking these bacterial effects (the bacterial and plant seed ureases have >50% amino acid identity) [74], especially urease aided by other cytotoxic components in the seed i.e. protease inhibitors, lectins that disrupt intestinal brush borders [75], etc. Another postulated chemical defense for seed urease is its induction of a hostile environment upon microbial and perhaps insect attack. With this second model, wounding or infection of the immature embryo will lead to the release of arginase from mitochondria that is ruptured. Cytoplasmic arginase would generate urea from the large pool of arginine which is at least 50% of free amino acid nitrogen [27, 28] and cytoplasmic urease would rapidly convert urea to ammonia. It was observed that cultured cotyledons containing the embryo specific urease commit suicide (probably by the combined effects of ammonia toxicity and medium alkalinization) in the presence of urea (20 mM), whereas those containing only the ubiquitous urease isozyme survive and utilize this urea, albeit poorly [41]. The assessment of pest resistance and herbivore avoidance of soybean cultivars lacking the embryo-specific urease requires extensive field testing of isogenic lines exposed to a variety of pathogens and pests. In addition to the abundant storage proteins, seeds contain several moderate- to high-abundance proteins with enzymatic or other biological activity. It is easy to invoke a plant protection role for many of these proteins: phytohemagglutinins (lectins) [75], lipoxygenases [76], ribosomal inactivators [77] and inhibitors of amylase [78], and animal proteases [79]. The lack of an essential physiological role for many of these proteins in plant is suggested by the relatively high frequency of cultivars and varieties lacking one of the urease [61], lipoxygenases [80, 81, 82], etc. Lack of an essential physiological role for the seed (embryo specific) urease is further suggested by the high prevalence of seed urease nulls in Japanese populations of Glycine soja [83], a sexually compatible close relative of cultivated soybean (Glycine max).

4. Physiology of leguminous soybean ureases and their functions

Carbon followed by nitrogen is the major limiting element for the performance of plant [84], and a regular demand for utilization of nitrogen targeting the development of mechanism that are efficient for the uptake of nitrogen and metabolic pathways for remobilization if nitrogen in plants [85, 86]. A pressure like this eventually led to nitrogen content reduction of proteins from plants [87]. Since urea is known to be a major source of nitrogen in plants, arginase’ activity is found to be the only known pathway involved in the in vivo generation of urea; it may be generated also due to ureides and purines degradation [88], even though this pathway happens to attract so much debate. The only way urea can be assimilated is after it has been hydrolyzed by urease into carbon dioxide and ammonia [89], this happens to be the major physiological function attributed to plant ureases [85]. Re-assimilation of ammonia will then be performed by an enzyme called glutamine synthetase utilizing glutamate as substrate [64]. The activity of urease is found in almost all species of plant and is found to be ubiquitous in all tissues of plants [62, 68, 90], this shows the great advantage of its physiological function to the entire plant. For ages, the importance of urease-mediated metabolism of urea in plant was not regarded because, there was an assumption that plants have difficulty in taking up urea, otherwise they are broken down by soil microorganisms leading to the absorption of nitrate and ammonia. Presently it has been established that plant can absorb urea from the soil actively, via the activity of a committed transporters urea [91] and can also process urea imported from the soil efficiently, even though the concentrations is high. These discoveries focuses at urease as an area for research on improving plant nitrogen metabolism urea that is urea based, the widely utilized fertilizer globally [92]. G. max happens to be an exciting model for research based on the physiological function of urease in import plants, because it has so far become the only plant that the genome have been sequenced which shows above one isoform of the enzyme. Soy bean urease ub-SBU has been identified to be the isoform accountable for recycling every urea that is derived metabolically [93, 94, 95]. This has been shown because mutants with noes-SBU never assemble urea in any tissue and do not have any deterioration on urea utilization as the major source of nitrogen, although ub-SBU is available at levels only 0.1–0.3% that of es-SBU [61, 93]. G. max mutants that have no activity of ub-SBU show a distinguishing feature phenotype consisting of necrosis of the tips of leaf, because of urea “burn”, and accumulation of urea in many tissues [41, 96]. Urease is the only Nickel dependent enzyme discovered in plants and the same phenotype as mentioned earlier, has been studied for plants grown under the deprivation of Nickel [35].

It is interesting to note that there is no physiological function, either assimilatory or that of any other characteristics, could possibly be seen due to bountiful es-SBU. In reality, types of cultured cotyledons that are wild were not able to grow due to urea presence, because of a sudden increase in pH as a result released ammonia that was not controlled. A similar effect was not seen on mutants having ub-SBU only [41]. It was concluded that es-SBU could possibly be associated in protection against predators in plant. For the case of microbes or attack of insect, protection of chemical was postulated. Using a model like this, immature embryo that is infected would bring about the release of arginase release from mitochondria that is ruptured which will generate urea in abundance from the pool of arginine and urease from the cytoplasm would transform urea to ammonia rapidly [96]. This hypothesis still waiting for demonstration to be carried out, meanwhile a report has been published stating that mutants that lack the activity of urease were highly susceptible to be infected by microbes [97].

5. Biotechnological potential functions of urease from legumes

Many questions on why ureases are ubiquitous and multimeric has been asked, a possible answer is that the “earliest” enzyme may have gotten other form of “traits” during the evolutionary pressure of a biosphere that is complex which led to increase in competition [97]. Due to these “extra traits” findings on ureases, some applications of biotechnology can be suggested. Legumes such as G. max may be targeted and attacked by several organisms, which may include virus, fungi, nematodes, and insects. These pests and pathogens may cause serious damage in pods, seeds, stems, roots and leaves, and most times are specific to tissues [98]. Even though measures have been taken for control, pests decrease the G. max production globally by about 28% [99], there is need for urgent development of new technology for the control of these pests; also exploring natural compounds of plant is an important strategy. There are so many biotechnological potentials in respect to plant ureases and their derived peptides. Many sources that are edible are found to be in abundant in ureases, which include legumes and potatoes, they are even eaten in their raw form e.g. cucumbers, or some fruits like watermelon and melon [97]. Hence, issues of bio-safety could possibly be managed better since es-SBU, JBU and Jaburetox’ derived peptide seems to be non-toxic to mammalians [100, 101], the fungi toxic and entomotoxic characteristics of these molecules are very important when putting into consideration the strategies biotechnology which aims to provide security to crops that are relevant commercially against natural enemies. The proof of an in vivo effect of G. max urease in protecting the plant against fungi [102] are creating excitement. The chances of choosing G. max cultivars having higher content of urease or to increase the production of these proteins found in the plant via genetic manipulations, so as to improve the resistance to fungi and insects, is encouraging. In addition, the premise of utilizing naturally occurring proteins in plant to increase resistance seems to be more appealing to the public more than the option of foreign genes insertion (from animals or microorganisms) into crops. As stated some time back that G. max having a high ureases content could be also agronomically valuable, not regarding the defense function, for allowing more thorough absorption of fertilizer containing urea by the plant [33, 39]. More so, considering the fact that G. max meal is widely used as feed for animals and the potential of becoming a source of protein to humans, a higher content of urease in G. max could be interesting for increasing nutritional quality of G. max, after processing it appropriately, because urease has higher methionine content more than many other G. max seed proteins. G. max has a very low amount of amino acids containing sulfur, about half of which are regarded as best as feed for animal. Even though this issue can be dealt with by supplementing the feed with free methionine, the supplementation are associated with problems, including leaching of the methionine when processing the degradation of bacteria leading to formation of volatile sulfides that are not desirable [103]. Improving methionine content in G. max by increasing the biosynthesis of endogenous proteins, like ureases, is an approach that is so interesting.

6. Some effects of legumes containing urease used as animal feed

It has been estimated that G. max meal accounts for about 67% of the total sources of protein utilized in feeds of animal around the globe [104], because of majorly its high concentration of protein which is around 44–48% [105]. Nonetheless, G. max possesses an unusual high amounts of bioactive compounds having antinutritional or/and properties that are toxic, that possesses on the body of animals’ metabolism a negative effect [106]. The content of urease was not different evidently among 11 tested soybean cultivars [107]. Contrarily, the content of urease was found to vary among several other soybean cultivars [108, 109, 110], and the urease levels positively correlated with antinutritional effects in rats [109]. The negative effects of utilizing meals containing urease as feed for animals are reported. Urea is regularly mixed with the feed of animals and, when G. max that are not processed are mixed with urea, the activity of urease will enabled the release of ammonia, which is an effect that is not desired in a feed that are mixed [111]. In ruminant animals, ammonia quickly enters the blood which can cause affects that is adverse ranging from depressed feed intake and performance of the animal, to Mortality due to toxicity of ammonia [112]. In dairy cows, the liver whose responsibility is remove potentially toxic ammonia from circulation, was able to remove ammonia added to portal blood until the supply was up to 182 mg/min but, at infusion rates that is high, peripheral blood concentrations of ammonia increased, supporting the assessment that a rapid hydrolysis of dietary urea can rise above the capacity of the liver to remove it [113]. In chickens, it was shown that meals of G. max from a particular source regularly produced a high incidence of tibialdyschondroplasia (TD) and the major striking difference between the meals was the high antitrypsin and values of urease in those that induced the disease [114]. TD incidence was previously shown in broilers to increase when 1.5 or 30% ammonium chloride was added to the feed [115], meanwhile it was not shown when calcium chloride was added [116]. These could indicate that the ammonia released by urease may a function in TD incidence on meals of G. max fed on chickens. For addition of nitrogen supplement to be allowed in the feed of animals, so as to protect the animals from the production of high ammonia level which is toxic, there is need for pre-treatment of the G. max meal. The major method used is the treatment by heat to do away or reduce the anti-nutritional effects and/or factors that are toxic in G. max, which include urease [103, 117], meanwhile these treatments should be kept at a low level, because it may be possible to destroy some important constituents of the seed [106]. To do away with the activity of urease, many effective treatments are required, which includes steam-heating at 120°C for 7.5 min or at102°C for 40 min [103], boiling for 60 min. at 92°C [109], and dry-heating at 100°C and 2 kgf/cm² for 60 min [117]. All these treatments do away with the activity of urease, coupled with a reduction of many anti-nutritional factors. The best method to evaluate the processing adequacy and final quality of the G. max meals is by conducting biological tests. Nevertheless, the required time, complexity and cost of these tests have negative effect their use. From 1940s, the use of urease test has been in existence as an indirect method of evaluating the adequacy processing G. max using heat because it is fast, require minimum skill and minimum amount of laboratory equipment. A discovery revealed a correlation that is high among trypsin inhibitors activities, lectins and urease, which indicate that the G. max processing can be estimated adequately by these analytical criteria to a considerable extent. In the past years, several guidelines were developed to facilitate the measurement of urease activity [103]. These guidelines are used to quantify directly or indirectly the amount of ammonia released. The one that was first developed is the method by Caskey-Knapp [118] in a buffer solution the meal is incubated with urea and then the addition of phenol red. Meals that are not properly processed will lead to an increase in the pH of the solution after incubation, which will be observed by a change in color of the solution from red-orange to pink, but meals that are well processed will show a little or no color change. A study suggested another alternative method, having the potential to distinguish meals with low levels of the activity urease, based on the meal incubation with urea in a buffered solution and using colorimeter to determine the urea residual with pdimethylaminobenzaldehyde [119]. Also, a method was propose d for direct titration of ammonia as a measure of urease activity [120] and adapted [121], whereby urea incubation with the meal is carried out, maintaining the pH of the solution by adding HCl slowly. The system is therefore titrated with NaOH. The difference in titration between a urease inactivated (control) and the sample is considered as the urease activity of the meal. Other two methods were developed for determination of ammonia directly, which is based on phenol-hypochlorite reaction [122, 123]. So many adaptations and modifications of these methods were developed as time passes by. Meanwhile, regardless of the method used, the activity of urease is a very good indicator of G. max meals that are not properly processed. This method could also be applied to other meals suspected to contain urease. However, this activity is not a good indicator G. max meals that are over processed.

7. Mechanism of action for insecticidal property in leguminous plant

Ureases from plants such as G. max (soy bean) and jack bean (JBU and canatoxin) are discovered to exhibit insecticidal activity in insects with cathepsin B-based (cysteine protease) and cathepsin D-based (aspartyl protease) digestive system. But no effect was observed in insects that relay on digestive enzymes which are trypsin-like [124, 125]. It is interesting to note that, no change was seen in the insecticidal effects of canatoxin and G. max ureases was after the enzyme was treated with an irreversible inhibitor of ureolytic activity, which indicate that ureolytic and insecticidal activities are not related [100]. In canatoxin, it was shown that its entomotoxic effect depends on an internal 10-kDa peptide (called pepcanatox), which is released by canatoxinhydrolysis by cathepsins in the digestive system of insects that are susceptible. Nonetheless, a 13-kDa recombinant peptide named jaburetox-2Ec, analog to pepcanatox, was cloned in Escherichia coli and was discovered to have insecticidal activity [126]. But with ureases from plant, there was no insecticidal activity seen in bacterial (Bacillus pasteurii) urease [100] and it was proposed that the entomotoxic peptide released from canatoxin by insect cathepsins is not present in ureases from microbes due to the subunit structure which is made up of two or three chains [100] and peptides that links and connect the different chains exhibit the insecticidal activity. Canatoxins has been studied extensively in several applications including industrial, agricultural, and medical [127]. Insect pest are the main cause of damage in agriculture having negative effect on commercially important crops globally. Having transgenic crops with intrinsic resistance to pest propose an alternative that is promising for chemical pesticides to prevent the crop losses [128]. Since plant ureases exhibit insecticidal property, to know their three-dimensional structure and the structural basis of their mechanism of its endomotoxic peptide could be used effectively for the development of transgenic plants that are resistant to insects. From the industrial point of view, a large number of research papers have been published four decades ago in legumes urease immobilization studies. Legumes urease structural integrity and important resistance to chemical and thermal deactivation have been studied extensively for immobilization studies. Based on these facts, the present study reveals some details of leguminous canatoxin urease, which may be utilized by researchers to design better carriers for enzyme immobilization and areas by utilizing these plants.

8. Summary and prospects for research on urease from legumes

In summary there are several indications that urease is important for efficient nitrogen assimilation. The urease substrate urea is derived from ureides and arginine. Arginine, is the richest nitrogen repository among the amino acids of legumes and plant seeds storage proteins. Urease are significant during the fixation of nitrogen in “tropical” legumes for example soybean and other plants. Urease-negative plants accumulate substantial, non-utilizable urea in both maternal and embryonic tissue. During germination of urease-negative seeds, further urea accumulates as a dead end in nitrogen metabolism. Although this accumulation may not be a lethal defect for large protein-rich legume seeds like soybean, small or protein-poor seeds, such as Arubidopsis, may be severely retarded or blocked in germination by the lack of an active urease. The better known, abundant legume seed ureases, for example, canatoxin urease, may perform a chemical defense function, similar to those postulated for some ureases of pathogenic bacteria. The ureases characterized to date, both from plants and bacteria, resemble each other in primary structure and in their requirement for accessory genes. Although the functions of the accessory genes have not yet been fully characterized they are undoubtedly involved in insertion of a nickel cofactor at the urease active site. A study of urease has revealed what may be a universal phenomenon in plant biochemistry: that enzyme profiles may be a composite of both plant and microbial activities. Indeed, the microbial contributions may be physiologically significant to the plant; in legume such as soybean it has been observed that bacteria are responsible for generating urea from ureides and mitigating urea accumulation in some urease negative mutants. In the future, there is a real need to quantify nitrogen fluxes through urea and to assess in the field and greenhouse the effects of urease inactivation on germination, seedling vigor, protein deposition, etc. Such studies are best performed in isogenic plant lines differing in the presence or absence of urease. Leguminous plants such as Soybean is so far the best experimental subject in light of its battery of urease mutants, its importance as a protein crop, and knowledge of its physiology. The ability to eliminate the abundant embryo specific urease, exclusively, will allow us to focus on its possible defense roles, especially when the seed urease-negative trait is combined with those eliminating lectins [82], protease inhibitors [129], and other putative defense factors. Obviously, improved understanding of the extent of plant-bacterium interdependence requires that we successfully cure plants of the bacterium. It is not clear whether this has been achieved, even in plants regenerated from cell culture. Finally, we need the knowledge and understanding of the nature of ureases’ control and also to find out if urease is participate in the ensemble of N assimilatory enzymes whose expression is coordinately controlled.

9. Conclusion

Plant ureases especially those from legumes are without any iota of doubt important events in the history of science, which has been an area of research as early as 1900s. Nevertheless, despite several researches, there are still lots of work that need to be carried out to completely understanding this complex molecule. The several characteristics exhibited by these enzyme shows that ureases are not just enzymes for the hydrolysis of urea, it also presents a wide array of biotechnological applications which are so interesting.

10. Future perspective of urease from legumes

Intensive study of the toxic properties of ureases from plants can be of great interest in developing an alternative strategy in agriculture as biosecurity which will be very important to crops against so many natural enemies. Urease from legumes can also be isolated, purified, characterized and immobilized for the diagnosis of urea level of patients in the medical laboratories.