Characterization of Nariginase Obtained from Aspergillus niger by Submerged Fermentation Using Naringin Extracted from Lemon Peels

Naringin was extracted from lemon peels with a yield of 0.25%. The activity of crude naringinase was 157.7μmol/min and 493.64μmol/ min using naringin disappearance and reducing sugars appearance assay methods respectively. Naringinase was precipitated at 50% ammonium sulphate saturation. After gel filtration, a purification fold of 32.84 with specific activity of 7279.37μmol/min/mg was observed while using naringin disappearance assay method and a 20.48 purification fold with specific activity of 14463.57μmol/min/mg with reducing sugars appearance method of assay. Percentage yields of 11.63% and 7.38% were observed using naringin disappearance and reducing sugars appearance methods of assay respectively. The enzyme pH and temperature optima were found to be 3.50 and 50 °C with a Km and Vmax obtained as 5.8mg/ml and 1111.11μmol/ min respectively. This investigation suggests that lemon peels could be a good source of naringin which could be used as a carbon source in submerged fermentation for naringinase production using Aspergillus niger.


Introduction
Bitterness is the major limiting factor for commercial acceptance of processed citrus fruit such as juice, wine, and vinegar [1]. The bitterness in citrus fruit is affected by limonin and naringin which are flavonoids generally recognized for making juice bitter [2]. Naringin which was first found in grapefruit is abundant in immature fruit with its concentration decreasing as fruit ripens [3]. Naringin has sugar complexes (α-L-rhamnose and β-D-glucose) and an aglycone (Naringenin) part. Naringinase is a bienzyme consisting of a-L-rhamnosidase (EC 3. 2.1.40) and flavonoid-bglycosidase (EC 3.2.1.21). b-L-Rhamnosidase hydrolyses naringin into the flavonoid prunin, which is converted to naringenin (4,5,7trihydroxyflavonone) by b-D-glucosidase. It has been observed that Naringinase catalyzes the hydrolysis of naringin to naringenin, glucose, and rhamnose. Fruit Juice industries process fruits at the lowest possible cost while maintaining the organoleptic quality and stability of the finished products, with increased consumer acceptability [4].
Numerous techniques used in naringin reduction including adsorptive debittering and chemical methods have limitations by altering nutrient composition by either chemical reactions or removal of nutrients, flavor and color [3]. A suitable debittering is achieved by treating the juice with naringinase. The enzyme naringinase hydrolyses naringin and produces a tasteless compound naringenin by two step reactions [5]. The reduction in bitterness as a result of the enzymatic process controls the quality and increase the acceptance by the consumers. Therefore, debittering with naringinase will not reduce the health promoting effects of fruit juices [6]. Naringinase has been produced from plants [7][8][9]. Ilamathi et al. [10] observed that the debittering process could be more cost effective and economically viable if naringinase production is achieved industrially using microorganisms.
In recent times, naringinase has been produced from several bacteria such as; Penicillium decumbens [11], Candida tropicalis [12], Aspergillus niger [13,14], Aspergillus Oryzae JMU 316 [1], and Penicillium purpurogenum [5], using different methods. Up till now there is a dearth of information on production of nariginase from Aspergillus niger using submerged fermentation. Kanokpan et al. [13] have only carried out preliminary enzyme characterization and optimization of culture parameters for production of Naringinase isolated from Aspergillius niger. This work is aimed at extraction of naringin and production of naringinase from Aspergillus niger in submerged fermentation system using the extracted naringin as the only carbon source.

By monitoring the magnitude of naringin disappearance
Naringinase activity was assayed using the method described by Ribeiro & Rabaca [5] with the following modifications. 0.3ml 0.1M sodium acetate buffer pH 4.0 was added to 1ml of 5% naringin. Thereafter, 0.2ml of the enzyme was added. This was incubated at 50 °C for 1hr. 0.1ml of the incubated reaction mixture was added to 5ml of 90% diethylene glycol containing 0.1ml of 4M NaOH and allowed to stand for 10min at room temperature. The residual naringin was then measured at 420nm using a JENWAY 6405 UV/VIS spectrophotometer (Beckman/Instruments, Inc., Huston Texas). One unit (U) of naringinase activity is defined as the amount of enzyme that hydrolyzes 1µmol of naringin per minute under the assay conditions.

By monitoring the appearance of reducing sugar
The reducing sugars were quantified by the 2, 4-dinitrosalicyclic acid (DNS) method of Millers (1959) with the following modifications; 0.3ml of sodium acetate buffer (0.1M pH 4.0) was added to 1ml of 5% naringin followed by the addition of 0.2ml of the enzyme. This was allowed to stand for 1hr at 50 °C. 1ml of DNS was added. The mixture was boiled for 10min using a water bath. 1ml of sodium potassium tartarate was added and the mixture was brought to 0 °C under ice blocks. The absorbance was then read at 540nm using a JENWAY 6405 UV/VIS spectrophotometer (Beckman/Instruments, Inc., Huston Texas). One unit of naringinase activity is defined as the amount of enzyme that catalyzes the release of 1µmole of glucose and rhamnose per minute under the assay conditions.

Effect of incubation time on naringinase activity
Different test tubes (pyrex) containing 1ml of substrate, 0.3ml of sodium acetate buffer (0.1M pH 4.0) and 0.2ml crude enzyme were incubated at 50 °C for varying period of time (min) ranging from 0-120. At the end of each incubation period, the reaction mixture was assayed for naringinase activity as earlier described.

Enzyme purification
Partial purification of the naringinase: Naringinase was purified by a modification of the method of Chen et al. [17]. The crude enzyme was brought to 50% (NH 4 ) 2 SO 4 saturation with solid (NH 4 ) 2 SO 4 . This was kept at 4 °C for 30hr, thereafter it was centrifuged at 4000rmp for 30min using a Cole-palmer VS-13000 micro centrifuge. The precipitate was collected and redisolved in 0.1M acetate buffer pH 4.0. 10ml of the redissolved protein was introduced onto a sephadex G-200 gel chromatographic column (50x2.5cm) for gel filtration. The column was pre equilibrated with 0.1M sodium acetate buffer pH 4.0. Fractions were collected at a flow rate of 5ml/9min. The protein concentration of the eluents was monitored at 280nm using a JENWAY 6405 UV/VIS spectrophotometer (Beckman/Instruments, Inc., Huston Texas). The naringinase activity of the collected fractions was assayed as earlier described and the active fractions were pooled and stored at -10 °C.
Effect of pH on naringinase activity: Naringinase activity was determined by a modification of the method of Ghada et al. [18]. In the following assay buffer systems using the assay methods earlier described; 0.1M sodium acetate buffer pH 3.5-5.5, 0.1M sodium phosphate buffer pH 6.0-7.5 and 0.1M Tris-HCl buffer 8.0-9.0.
Effect of temperature on naringinase activity: Naringinase activity was determined at various temperatures; 30, 40, 50, 60, 70 and 80 °C using a modification of the method of Ghada et al. [18]. The lag phase was minimized by preincubating the reaction cocktail in a circulating water bath at the desired temperature for 10min followed by the introduction of 0.1ml of the enzyme solution which was rapidly mixed and assayed as earlier described.

Effect of Substrate Concentration on naringinase activity:
The kinetics of Naringinase reaction (K m and V max ) was determined in duplicate by a modification of the method of Ribeira & Rabaca [19]. The enzyme was incubated with 5, 10, 15, 20, 25 and 30mg/ ml of lemon naringin at pH 3.5 and 50 °C. Naringin was extracted from lemon peels with a yield of 0.25%. Naringinase was produced by A. niger in a submerged fermentation system using naringin as the only carbon source. Pilot study was carried out to monitor the day of highest naringinase activity and production. The highest naringinase concentration was obtained on day five while the highest naringinase activity was obtained on day four of fermentation ( Figure 1 & 2). Different periods of fermentation (days) have been reported for optimum naringinase activity. Chen et al. [20] reported day five while Radhakrishnan et al. [21] observed highest activity on day seven. Saranya et al. [12] obtained maximum activity on day five. The effect of period of incubation on the activity of naringinase using naringin as the only carbon source was investigated. 55min of incubation time yielded the highest Naringinase activity of 27039.84µmol using the reducing sugar production method of assay ( Figure 3).

Results and Discussion
Crude naringinase was partially purified on a two-step purification step of ammonium sulphate precipitation and gel filtration. 50% ammonium sulphate saturation yielded naringinase with a specific activity of 335.40U/mg using naringin hydrolysis method of assay and 846.60U/mg using reducing sugars production assay method (Table 1 & 2). Saranya et al. [12] reported a specific activity of 0.061U/mg using naringin hydrolysis assay method upon 65% ammonium sulphate precipitation. Figure 4 & 5 show the elution profiles of naringinase on gel filtration with sephadex G-200 using the two methods of assay. A 32.84 and 20.48 purification folds were observed with specific activities of 7279.37µmol/min/ mg and 14463.57µmol/min/mg upon gel filtration using naringin disappearance assay method and reducing sugars appearance assay methods respectively (Table 1 & 2). The percentage yields were found to be 11.63% using naringin disappearance assay method and 7.38% using reducing sugars production assay methods. Saranya et al. [12] reported specific activity of 1.307U/ mg using reducing sugar production assay method which is very low compared to the one obtained in this work.       Figure 6 shows an optimum pH of 3.5. Nourouzian et al. [22] reported a pH optimum of 4.0 using Pencillium decumbens while Puri et al. [23] reported a pH of 4.0 using Aspergillus niger. The observed pH was comparatively lower than the report of the above authors but comparatively higher than 3.0 reported by Saranya et al. [12] using Candida tropicalis. In Figure 7, as temperature increased from 25 °C to 50 °C, the naringinase activity increased. Further, increase in temperature beyond of 50 °C decreased naringinase activity making 50 °C the optimum temperature. The decrease in naringinase activity at higher temperature may be due to enzyme denaturation or inactivation. Hence the optimum temperature 50 °C was used for further studies. The optimum temperature is comparatively high when compared to 25 °C obtained using Candida tropicalis but lower when compared to 55 °C obtained using Penicillium decumbens and higher compared to 45 °C obtained using Aspergillus niger [21][22][23].  The K m and V max obtained from Linewaever-Burk plot of initial velocity data at different concentrations of substrates ( Figure  8) were found to be 7.1mM and 1000µmol/min. This shows that partially purified naringinase obtained in this work has high affinity for naringin from grape peel and could be of industrial use. Saranya et al. [12] reported K m and V max of 0.19mM and 0.1µmole/ min, using Candida tropicalis which were lower than the K m and V max