**7. Soybean as animal feed – what urease has got to do with it?**

It is estimated that soybean meal accounts for around 67% of all protein sources used in ani‐ mal feeds around the world [96], due mainly to its high protein concentration (44 to 48%) [97]. Nevertheless, soybean contains an unusually large number of bioactive compounds with antinutritional and/or toxic properties, which have a negative effect on body metabo‐ lism of animals [98]. Urease is one of these factors. Urease content was not evidently differ‐ ent among 11 soybean cultivars tested [99]. In contrast, urease content was found very variable among several other soybean cultivars [100-102], and the levels of urease correlated positively with antinutritional effects in rats [101].

The negative effects of using urease-containing meals as animal feed are reported in the lit‐ erature. Urea is frequently added to animal feed and, when unprocessed soybeans are mixed with urea, ammonia will be released by the action of urease, which is an undesired effect in a mixed feed [103]. In ruminants, ammonia rapidly enters the blood and can cause adverse affects ranging from depressed feed intake and animal performance, to death from ammonia toxicity [104]. In dairy cows, the liver, responsible for removing potentially toxic ammonia from circulation, was able to remove ammonia added to portal blood until the supply reached 182 mg/min but, at higher infusion rates, peripheral blood ammonia concen‐ trations increased, supporting the assessment that a rapid hydrolysis of dietary urea can ex‐ ceed the liver's capacity to remove it [105]. In chickens, it was demonstrated that soybean meals from one particular source consistently produced a high incidence of tibial dyschon‐ droplasia (TD) and the most striking difference between the meals was the high antitrypsin and urease values in those that induced the disease [106]. The incidence of TD was demon‐ strated previously to be increased in broilers when ammonium chloride (1.5 or 30%) was added to the diet [107], but not when calcium chloride was used [108]. These may be indica‐ tions that the release of ammonia by urease could play a role in the incidence of TD in soy‐ bean meal fed chickens.

In order to allow the addition of supplemental nitrogen to the animal feed, while protecting the animals against the production of toxic levels of ammonia, pre-treatment of the soybean meal is necessary. Heat treatment is the main method used to abolish or decrease the effects of the antinutritional and/or toxic factors in soybean, including urease [109, 110], but these treatments should be kept to a minimum, due to the possibility of destroying important seed constituents [98]. To abolish urease activity, several treatments are effective, including steam-heating at 102 °C for 40 min or at 120 °C for 7.5 min [109], boiling at 92 °C for 60 min [101], and dry-heating at 100 °C and 2 kgf/cm2 for 60 min [111]. All those treatments abolish‐ ed urease activity, along with a decrease of several antinutritional factors.

The best way to evaluate the adequacy of the processing and final quality of soybean meals is conducting biological tests. However, the cost, time requirement and complexity of these tests impair their use. Since the 1940's, the urease test is used as an indirect way to evaluate the adequacy of heat processing of soybeans due to its rapidity, low skill and minimum amount of laboratory equipment requirements. One research study [109] showed a high cor‐ relation among the activities of trypsin inhibitors, urease and lectins, indicating that the ade‐ quacy of soybean processing can be estimated to a considerable extent by these analytical criteria. Over the years, many protocols were developed to facilitate the measurement of urease activity. These protocols quantify the released ammonia directly or indirectly. One of the first to be developed, in the Caskey-Knapp method [112] the meal is incubated with urea in a buffered solution and then phenol red is added. After incubation, insufficiently process‐ ed meals will cause an increase in the pH of the solution, indicated by a change in color (from red-orange to pink), while adequately processed meals produce little or no color change. One study [113] proposed an alternative method, with the potential to differentiate between meals with low levels of urease activity, based on the incubation of the meal with urea in a buffered solution and the colorimetric determination of the residual urea with *p*dimethylaminobenzaldehyde. A method for direct titration of ammonia as a measure of ure‐ ase activity was proposed [114] and adapted [115], in which the incubation of urea and the meal is performed, maintaining the pH of the solution by slowly adding HCl. The system is then titrated with NaOH. The difference in titration between a control (urease inactivated) and the sample is taken as the urease activity of the meal. Two other methods were devel‐ oped to determine ammonia directly, based on the phenol-hypochlorite reaction [116, 117].

Several modifications and adaptations of these methods were developed during time. But, regardless of the method chosen, urease activity is a very good indicator of under process‐ ing of soybean meals. It is worth noting, however, that this activity is not a good indicator of over processing of soybean meals.

### **8. The biotechnological potential of soybean ureases**

The questions of why are ureases so large, and why are they multimeric have been raised, and a possible explanation is that a "primordial" enzyme could have acquired other "traits" under the evolutionary pressure of competition in an increasingly complex biosphere [73]. In the view of these "extra traits" discovered for ureases, some biotechnological applications can now be proposed.

Soybean can be attacked by many different organisms, including fungi, insects, virus and nematodes. These pathogens and pests can cause damage in seeds, roots, leaves, stems and pods, and usually are tissue-specific [118]. And, despite control measures, pests reduce worldwide soybean production by almost 28% [119]. The development of new technologies to control these pests is urgent, and exploring natural plant compounds is a major strategy.

Plant ureases and their derived peptides have a great biotechnological potential. Ureases are abundant in many edible sources, including legumes and potatoes, and even eaten raw in cucumbers, or fruits such as melon and watermelon [73]. Thus, possible biosafety issues could be more manageable. Since JBU, es-SBU and the derived peptide Jaburetox seem non toxic to mammals [75, 90], the entomotoxic and fungitoxic properties of these molecules are relevant when considering biotechnological strategies aiming to protect commercially-rele‐ vant crops against natural enemies. The evidences of an *in vivo* effect of soybean urease in protecting the plant from fungi [93] are exciting. The possibility of selecting soybean culti‐ vars with higher urease content or increasing the production of these proteins in the plant through genetic manipulation, in order to increase the resistance against insects and fungi, is very promising. Also, the premise of using plant naturally occurring proteins to improve re‐ sistance seems much more appealing to the general public than the alternative of inserting foreign genes (from microorganisms or animals) into crops.

As pointed out a long time ago [15, 120], soybeans with a high content of ureases could also be agronomically valuable, regardless of the defense role, for permitting more efficient as‐ similation of urea fertilizer by the plant. Also, considering the wide use of soybean meal as animal feed (as discussed above) and the potential of being a protein source for humans, a higher urease content in soybean could be interesting for improving soybean nutritional quality, after the appropriated processing, since urease is richer in methionine than many others soybean seed proteins. Soybean has a limited amount of sulfur aminoacids, almost half of which are considered ideal for animal feed. Although this problem can be overcome by feed supplementation with free methionine, there are problems associated with the sup‐ plementation, such as leaching of methionine during processing and bacterial degradation leading to formation of undesirable volatile sulfides [121]. Improving the content of methio‐ nine in soybean through the increase of the biosynthesis of endogenous proteins, such as ureases, is a very interesting approach.

## **9. Conclusion**

In order to allow the addition of supplemental nitrogen to the animal feed, while protecting the animals against the production of toxic levels of ammonia, pre-treatment of the soybean meal is necessary. Heat treatment is the main method used to abolish or decrease the effects of the antinutritional and/or toxic factors in soybean, including urease [109, 110], but these treatments should be kept to a minimum, due to the possibility of destroying important seed constituents [98]. To abolish urease activity, several treatments are effective, including steam-heating at 102 °C for 40 min or at 120 °C for 7.5 min [109], boiling at 92 °C for 60 min [101], and dry-heating at 100 °C and 2 kgf/cm2 for 60 min [111]. All those treatments abolish‐

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

The best way to evaluate the adequacy of the processing and final quality of soybean meals is conducting biological tests. However, the cost, time requirement and complexity of these tests impair their use. Since the 1940's, the urease test is used as an indirect way to evaluate the adequacy of heat processing of soybeans due to its rapidity, low skill and minimum amount of laboratory equipment requirements. One research study [109] showed a high cor‐ relation among the activities of trypsin inhibitors, urease and lectins, indicating that the ade‐ quacy of soybean processing can be estimated to a considerable extent by these analytical criteria. Over the years, many protocols were developed to facilitate the measurement of urease activity. These protocols quantify the released ammonia directly or indirectly. One of the first to be developed, in the Caskey-Knapp method [112] the meal is incubated with urea in a buffered solution and then phenol red is added. After incubation, insufficiently process‐ ed meals will cause an increase in the pH of the solution, indicated by a change in color (from red-orange to pink), while adequately processed meals produce little or no color change. One study [113] proposed an alternative method, with the potential to differentiate between meals with low levels of urease activity, based on the incubation of the meal with urea in a buffered solution and the colorimetric determination of the residual urea with *p*dimethylaminobenzaldehyde. A method for direct titration of ammonia as a measure of ure‐ ase activity was proposed [114] and adapted [115], in which the incubation of urea and the meal is performed, maintaining the pH of the solution by slowly adding HCl. The system is then titrated with NaOH. The difference in titration between a control (urease inactivated) and the sample is taken as the urease activity of the meal. Two other methods were devel‐ oped to determine ammonia directly, based on the phenol-hypochlorite reaction [116, 117].

Several modifications and adaptations of these methods were developed during time. But, regardless of the method chosen, urease activity is a very good indicator of under process‐ ing of soybean meals. It is worth noting, however, that this activity is not a good indicator of

The questions of why are ureases so large, and why are they multimeric have been raised, and a possible explanation is that a "primordial" enzyme could have acquired other "traits" under the evolutionary pressure of competition in an increasingly complex biosphere [73].

**8. The biotechnological potential of soybean ureases**

over processing of soybean meals.

Relationships

328

ed urease activity, along with a decrease of several antinutritional factors.

Soybean ureases were undoubtedly landmarks in science, being the subject of investigations since the beginning of the 1900's. But, despite the more than one century of studies, we still have a long way until fully understanding the complexity of such a striking molecule. The many properties of these proteins revealed that ureases are much more than urea hydrolyz‐ ing enzymes, and present a vast array of interesting biotechnological applications. Exploring

the toxic properties of plant ureases can be of great interest for the development of alterna‐ tive strategies to protect agricultural relevant crops against several natural enemies.
