Urease from Vigna umbellata seeds: isolation, partial purification, characterization and antifungal activity

Urease (EC 3.5.1.5) hydrolyzes urea to produce ammonia and carbamate. Urease can be synthesized by plants, fungi and bacteria. In agriculture, urease is needed efficiently to play a major role in the urea cycle as source of plant nitrogen in the soil. Rice beans (Vigna umbellata) are leguminous plants from the Fabaceae family that contain high urease activity. This study isolated urease from the seed coat and without the seed coat of rice beans. The urease activity in rice beans without seed coat was higher than that of the activity with seed coat. The amount of ammonia produced with the addition of 1 µL of urease enzyme of rice beans without seed coat was 13 µg which was higher than the amino produced from the seed coat sample (8.8 µg). The concentration of urease enzyme from rice beans was obtained at 7.238 mg/mL. Analyses of gel electrophoresis indicated that urease from rice beans are composed of four polypeptide chains with molecular weights of about 52, 33, 22 and 10 kDa. Then rice beans urease exhibited no antifungal activity against Aspergillus niger, Aspergillus fumigatus, Microsporum canis, Fusarium oxysporum subsp. lini, Trichophyton rubrum, Candida glabrata and Candida albicans.


Introduction
Urease (EC 3.5.1.5)is an enzyme that catalyzes the hydrolysis of urea into ammonia and carbamate as source of nitrogen for plants which can be synthesized from plants, fungi and bacteria [1].Urease is found in several plant tissues, especially in abundance in t h e germination of seeds from the Fabaceae and Cucurbitaceae family [2].The main role of urease in plants is to contribute to nitrogen availability, participate in defense mechanisms and provide energy for plants to use urea or hydroxyurea as a nitrogen source [3].Urease is found in large quantities in peas, beans and plant seeds.Urease was isolated and purified from black-eyed (Vigna unguiculata) peas [2], Momordica charantia seeds [4], cotton (Gossypium hirsutum) seeds [5], Pisum sativum seeds [6] and pigeon pea (Cajanus cajan L.) seeds [7].
There are many benefits of urease such as its use for industrial waste treatment, hemodialysis, medical diagnosis and measurement of urea content in human blood [2].Urease is also involved in the human body because many diseases of the urinary tract, gastroduodenal and some cancers are related to this [6].Some ureases have antifungal properties, especially urease produced from leguminous plants (jack beans and soybeans) and Helicobacter pylori bacteria, sub-micromolar concentrations of IOP Publishing doi:10.1088/1755-1315/1302/1/012091 2 antifungal activity can inhibit spore germination and mycelial growth of several filamentous fungi [5].
Rice beans (V.umbellata) also known as small red beans or uci beans (local language in Indonesia), are annual legumes from the Fabaceae family that are grown mainly in South and Southeast Asian countries such as India, Nepal, Bhutan, Myanmar, China, Vietnam, Thailand and India [8].Rice beans as a legume can fix nitrogen and increase soil fertility for most of the surrounding plants because they have a high protein content of around 17.26 to 21.42% [9].Rice beans have high adaptability and are abundant in Indonesia.Rice beans are not popular in Indonesia and are only used as food complement and animal feed.The high protein content and ability of rice beans to increase the fertility of surrounding plants means that rice beans also contain high levels of urease.The urease found in rice beans can be an additional income for rice bean farmers in the economic sector.Through this study, the urease enzyme, which is usually purchased from several companies, can now be produced by farmers in an easy way and from readily available sources, such as rice bean seeds.

Isolation of Urease from Rice Beans
Fifty grams of rice beans soaked in deionized water 200 mL for 6 hours.Then rice beans are drained and put into a net basin.The net basin was covered with wet muslin cloth and germinated at room temperature for 4 days.Germination was carried out twice, first for germination of rice beans with seed coat and the second for germination without seed coat.This treatment was carried out to observe comparison of urease produced between rice beans with seed coat and without seed coat.
Germinated rice beans were mashed using mortar and pestle, then suspended in 400 mL phosphate buffer (0.2 M; pH 7) at temperature of 4 o C and stirred in magnetic stirrer.Two layers of filtrate and suspension produced were then separated by muslin cloth.The filtrate was centrifuged at 14600 rcf for 30 minutes at 4 ℃.The supernatant obtained was centrifuged by protein concentrator at 4500 rpm for 10 minutes at 4 ℃ to remove water from the extract.The extract was centrifuged until 5 mL of crude extract was obtained.The same treatment to rice beans without seed coat where previously the rice beans skin was peeled and then the rice beans are carried out by the extraction process.

Gel Filtration Chromatography
The crude extract of rice bean urease was purified by chromatography gel filtration using Sephadex G-150 column (1 x 25 cm) and phosphate buffer (0.2 M; pH 7) for elution.The fractions each of about 2 mL were collected to determine purified enzyme molecular weight using SDS-PAGE Electrophoresis.The fractions that demonstrated the purest enzyme were centrifuged by protein concentrator at 4500 rpm for 10 minutes at 4 ℃ until the volume was 500 µL.Then the fractions with the purest enzyme are used for another purification step by gel filtration against t h e Sephadex G-150 column.The fractions of the second purification step were used to measure molecular weight using SDS-PAGE Electrophoresis.The fractions that show t h e purest enzyme of second purification were pooled together and centrifuged by protein concentrator at 4500 rpm for 10 minutes at 4 ℃ until the volume was 500 µL.Then the final fraction was used to determine urease activity by UV-visible spectrophotometry at OD620 nm and the enzyme concentration was measured by nanodrop spectrophotometer.

Urease Assay
The urease assay in purified enzyme of rice beans was carried out using reagent A and reagent B. Reagent A contained 200 mg phenol and 6 mg sodium nitroprusside dissolved in 20 mL DI water, and reagent B consisted of 200 mg sodium hydroxide and 150 µL sodium hypochlorite dissolved in 50 mL DI water.Urease assay was carried out using 0.2 M phosphate buffer (pH 7).For the substrate solution, 5 mg of urea was dissolved in 5 mL of phosphate buffer.Jack bean urease 2.92 mg was dissolved in 2.5 mL phosphate buffer by gently shaking.A 96-well plate was used and each well contained 200 µL of reaction volume.The composition of reaction urease assay is described in Table 1.The sample mixture was incubated at 37 ℃ for 30 minutes.Following incubation, reagents A and B were added.Then the sample mixture was incubated at 37 ℃ for 10 minutes.All reactions were analyzed the absorbance by UV-visible spectrophotometry at OD620.The standard graph was prepared to see the production of ammonium sulfate in units of µg and then the results of the enzyme activity obtained will be compared with how much ammonium sulfate is produced.

Quantification of Protein Concentration
The protein concentration of urease was quantified spectrophotometrically using a Nanodrop 2000 spectrophotometer.Two microliters of purified urease were used for analysis and 2 µL of nucleasefree water was used as blank.Absorbance measurements taken at 280 nm were used to calculate concentration.

SDS-PAGE Electrophoresis
Thirty microliters of purified urease were analyzed by SDS-PAGE (Sodium dodecyl sulfatepolyacrylamide gel electrophoresis) Bis-Tris gel 4-12% polyacrylamide concentration size 1.5 mm.The protein band was detected using 10 µL Coomassie brilliant blue dye, then shaken purified urease with blue dye using thermal mixer at 98 ℃ for 10 minutes.Blue protein marker was used for the reference of protein molecular weight.

Antifungal Test
The yeast Candida albicans and Candida glabrata, and filamentous fungi Aspergillus niger, Aspergillus fumigatus, Microsporum canis, Trycophyton rubrum and Fusarium oxysporum subsp.line were from fungal collections maintained at the molecular bank of the International Center for Chemical and Biology Sciences (ICCBS), University of Karachi, Pakistan.The yeasts and fungi were maintained on sabouraud dextrose agar (SDA) (1% peptone, 2% glucose and 1.7% agar) that dissolved DMSO (Dimethyl Sulfoxide).
Yeast growth media contains SDA dissolved in distilled water.Then, 66.66 µL of the media was removed and replaced with urease crude extract which had been mixed with distilled water.The agar media was mixed with the extract and the media was allowed to polymerize.After polymerizing, the yeast cells C. albicans and C. glabrata were inoculated.Then yeast cells were incubated for 48 hours at 27 ℃.The same sequence was also for filamentous fungi, but incubation was carried out for 7 days.

Results and discussion
The germination was carried out twice with the first treatment where the rice beans were isolated using the seed coat and the second treatment where the rice beans were isolated without the seed coat.The germination of rice beans (V.umbellata) was done for 1, 2, 3 and 4 days.The best germination occurred on day 4, more than that there was fungal growth (Data is not shown).The germinated rice beans were weighed, the weight with the seed coat was 163.36 g and the weight without the seed coat was 133.61 g.Urease was isolated from germinating plants because during the germination process, the plant releases much urea hydrolyzed by urease.Urease was found to be very abundant when seeds germinated, but in vegetative tissues, urease was only found in smaller amounts [10].Increased urease content and ureolytic activity play an important role in the early stages of plant development.In the early stages of plant development, plants produce large amounts of nitrogen, this is due to the catalytic activity and tissue distribution in the plant [11].Furthermore, the germination rice beans extraction process was carried out by crushing the germinated rice beans with a mortar then dissolved in 400 mL phosphate buffer (0.2 M; pH 7) then stirred for 3 days at 4 o C. The solution was filtered using a muslin cloth, then centrifuged with a protein concentrator to remove water and collected 5 mL of crude extract.The crude extract was used to measure enzyme activity.
The urease from rice beans was done to purity of crude extract.The purification was done using chromatography gel filtration by Sephadex G-150 column (1 x 25 cm).Then the enzyme activity was determined by the absorbance value of urease with UV-visible spectrophotometry at OD620.Table 2 shows the results of purified urease activity with different treatments on the seed coat.Based on the data obtained, it can be observed that there is an increase in enzyme activity in unpeeled rice bean seeds and peeled seed coat rice bean seeds.The urease activity values obtained at different concentrations produced different activities, indicating that enzyme concentration can affect enzyme activity.The higher urease activity values are accompanied by increasing enzyme concentration.
Urase activity increases with substrate concentration, reaches an optimum value and decreases with increasing concentration.The three-dimensional structure of a protein in an enzyme influences the activity of the resulting enzyme.The protein's primary structure and the enzyme environment regulate changes in enzyme activity.Environmental factors change the enzyme's shape and block substrate access to the active site, thereby affecting enzyme activity [12].Figure 1.shows how the ratio of urease activity is produced during the treatment before the seed coat of rice bean is peeled and after it is peeled.This shows that the rice seed coat contains many additional compounds that cause enzyme work to be less than optimal.The skin of rice beans also causes urease production by the seeds to be disrupted and limited.The seeds of legume plants contain a high protein content of around 20-40% which is stored in globulin [13].Most of the phenolic compounds were concentrated in the seed coats of legumes from the Fabaceae family.The Compounds found in the seed coat are phenolic acids, flavonoids, phydroxybenzoic acid and tannins.This compound functions as important bioactive compound that can bind free radicals and interact with proteins [14].The assay activity of urease in hydrolyzing urea substrate to ammonium and carbon dioxide was carried out by comparing the absorbance of urease activity in hydrolyzing urea with the standard graph of ammonium sulfate.
Based on the standard graph of ammonium sulfate, the amount of ammonia present in the given V. umbellata urease without seed coat is 13 µg of ammonium (NH3) formed / 1 µL of enzyme / 15 minutes and the amount of ammonia present in the given urease enzyme of V. umbellata with seed coat is 8.8 µg of ammonium (NH3) formed / 1 µL of enzyme / 15 minutes.The concentration of urease from rice beans obtained from the Nanodrop spectrophotometer test was 7.238 mg/mL, with the molecular weight setting being 83.93 kDa and the extinction coefficients being 42.455 mg/mL Urease from rice beans was purified using chromatography and then determined by molecular weight using SDS-PAGE (Figure 2).The result shows urease from rice beans is thought to have four polypeptides of molecular weights of about 52, 33, 22 and 10 kDa.This shows that urease isolated from rice beans has similarities with the urease isolated from black-eyed pea (Vigna unguiculata ssp unguiculata L.) and consists of four polypeptides of molecular weight about 55, 35, 17 and 15 kDa [2].
Antifungal test was carried out to see the effect and activity of the urease extracted from rice bean (V.umbellata) seeds.After the incubation period, there was no antifungal activity of urease isolated from rice bean seeds was detected.All tested fungi grew normally as shown in Figure 3. From the results obtained urease extracted from rice bean seeds (V.umbellata) does not have antifungal ability.This indicates that not all enzymes extracted from plants have benefits as antifungals.However, Menegassi et al. urease from cotton seeds poses antifungal activity [5].Urease which has antifungal capabilities usually contains lectin protein in its structure, the seeds of members of the Leguminosae are generally rich sources of urease and lectin [15].Urease from jack bean seeds contains concanavalin A, a glucose-mannose-specific lectin [16] and jaburetox (JBTX) which is a peptide that can be used as an antifungal [17].Therefore, urease extracted from sword beans may have antifungal capabilities, in contrast to urease extracted from peas which may not contain protein that can be used as an antifungal.

Conclusions
Rice beans (V.umbellata) are legume plants containing high protein.The beans have relatively high urease activity.In agriculture, urease is needed efficiently to play a major role in the urea cycle as source of plant nitrogen in the soil.Urease (EC 3.5.1.5),the enzyme that catalyzes the hydrolysis of urea into ammonia and carbon dioxide was isolated and partially purified and characterized from rice beans.The urease activity was higher in rice beans without seed coat than that of from the seed coat sample.Urease enzyme from V. umbellata did not have antifungal activity as it is reported by some researchers.The enzyme consisted of four different polypeptide chains.

Figure 1 .
Figure 1.Comparison of urease activity in rice beans with seed coat and without seed coat.

Figure 2 .
Figure 2. SDS-PAGE profile of urease from Rice beans.

Table 1 .
The composition of reaction urease assay wells.

Table 2 .
Urease assay activity on optical density at 620nm.