Acta Physiologiae Plantarum

, Volume 34, Issue 2, pp 657–667

Oxalic acid and oxalate oxidase enzyme in Costus pictus D. Don

Authors

    • Department of Crop PhysiologyTamil Nadu Agricultural University
  • Antoney Augustin
    • Centre for Plant Biotechnology and Molecular BiologyCollege of Horticulture
Original Paper

DOI: 10.1007/s11738-011-0866-x

Cite this article as:
Sathishraj, R. & Augustin, A. Acta Physiol Plant (2012) 34: 657. doi:10.1007/s11738-011-0866-x
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Abstract

The leaves of Costus pictus are sour in taste due to the presence of oxalic acid in the leaves. Different stages of leaves were collected and the samples were designated as stage one, stage two and stage three. It was found that oxalate content and oxalate oxidase activity were maximum in second leaf stage followed by first leaf stage and third leaf stage. Drying causes substantial loss of oxalate content and complete loss of oxalate oxidase activity. With various solvents water recovered more oxalate followed by methanol and ethanol while oxalate oxidase activity was maximum in ethanol followed by methanol and water. The ethanol or methanol extract of second leaf stage of C.pictus can be used for isolating active principles. The oxalate oxidase from C.pictus can be used as a cheap source of oxalate oxidase enzyme which is used in oxalate determination in biological fluids. Moreover, the sensitivity of oxalate determination employing oxalate oxidase from C.pictus will be more as oxalate oxidase in C.pictus has Km 20 times lesser than the oxalate oxidase enzyme from barley seedling.

Keywords

Costus pictusOxalic acidOxalate oxidase enzyme

Introduction

Costus pictus D. Don is native of Mexico and known as Mexican cane or spiral ginger. It is commonly known as insulin plant in Kerala. The hypoglycemic property of C. pictus was well established. “Preparation process and a regenerative method and technique for prevention, treatment and glycemic control of diabetic mellitus using Costus pictus extract” was patented by Merina Benny in 2007. Administration of C. pictus extract about 500–2,000 mg day−1 brings down the blood glucose level to normal in diabetic patients (Benny 2007). The leaves of C. pictus are sour in taste due to the presence of oxalic acid in the leaves (Benny 2006). Excess oxalate intake through oxalate rich foods leads to nephrolithiasis, a condition in which oxalate crystallises to form stones in the kidney, bladder and urethra (Siener et al. 2005). But C.pictus is used to treat kidney problems even though it contains substantial quantity of oxalic acid (Camargo et al. 2006; Moron et al. 2007). Hence, the present study was conducted to investigate the oxalic acid and oxalate oxidase enzyme in C.pictus for better utilisation of its medicinal properties.

Materials and methods

Sample plants of C. pictus were maintained in the department field. Fully opened leaves from top 1st to 3rd, 4th to 6th and 7th to 9th were collected for analysis of oxalic acid and oxalate oxidase enzyme. The samples were designated as stage one, stage two and stage three, respectively. The typical characteristics of the C.pictus used for the present analysis is given in Fig. 1. Chemicals and dialysing tube for dialysis of enzyme sample were obtained from Sigma-Aldrich.
https://static-content.springer.com/image/art%3A10.1007%2Fs11738-011-0866-x/MediaObjects/11738_2011_866_Fig1_HTML.jpg
Fig. 1

Morphology of Costuspictus

Oxalate content and oxalate oxidase enzyme activity were determined in all the three leaf stages (for both fresh and dry samples). Partial purification was done for the leaf stage which showed higher enzyme activity. The partially purified enzyme is used to study the effect of pH, temperature and substrate concentration on enzyme activity in contrast to crude enzyme.

Oxalate

Oxalate from C.pictus was isolated by Burrows method (1950) and Baker method (1952) with slight modifications. The method of estimating oxalate content was adopted from Burrows (1950) with slight modifications.

Oxalate isolation

In Burrows method, oxalate was isolated as total oxalate but in Baker method the oxalate was isolated as soluble and total oxalate.

Burrows method

The method of isolating total oxalate from fresh and dried leaf sample of C.pictus was adopted from Burrows (1950) with slight modifications.

Reagents:

Citric acid reagent. Citric acid reagent was prepared by dissolving 2 g of citric acid and 5 g of anhydrous calcium chloride in minimum quantity of distilled water and made up to 100 ml. It was heated to boiling. Saturated ammonium oxalate solution was added drop by drop until a permanent turbidity was formed then it was boiled for 10 min and kept overnight. It was filtered through Whatman No. 42 filter paper before use.

HCl 0.4 N. Hydrochloric acid 0.4 N was prepared by dissolving 3.5 ml of concentrated hydrochloric acid (min. assay: 35%; sp.gr.: 1.18 kg) in minimum quantity of distilled water and made up to 100 ml with distilled water.

Sample preparation: In fresh sample, 5 g of leaf sample was ground with 10 ml of distilled water and 10 ml of citric acid reagent in a pestle and mortar at room temperature. In dry sample, a 10 ml of distilled water and 10 ml of citric acid reagent were added to 1-g dried leaf sample in a 100 ml volumetric flask. Correspondingly, the extract was filtered by Whatman No. 42 filter paper. The precipitate in the filter paper was dissolved in 50 ml of 0.4 N HCl. The dissolved precipitate was again filtered by Whatman No. 42 filter paper. The filtrate was saved and used for the estimation of total oxalate.

Baker method

The method for isolating soluble and total oxalate from fresh and dried leaf sample was adopted from Baker (1952) with slight modifications. The difference between total and water-soluble oxalate gives insoluble oxalate content.

Reagents:

Ammonium hydroxide. Concentrated ammonium solution with specific gravity 0.880 was dissolved in distilled water at 1:1 v/v ratio.

Diluted hydrochloric acid. Concentrated hydrochloric acid (min. assay: 35%; sp.gr: 1.18 kg) was dissolved in distilled water in the ratio of 1:1 v/v.

Phosphoric-tungstate reagent. Sodium tungstate 2.4 g was dissolved in 4 ml of orthophosphoric acid and made up to 100 ml with distilled water.

Calcium chloride buffer. Anhydrous calcium chloride was dissolved in 50 ml of 50% v/v glacial acetic acid and this solution was added to the solution of 66 g of sodium acetate in minimum quantity of distilled water and it was made up to 100 ml with distilled water.

Wash solution. Acetic acid solution 5% v/v kept over calcium oxalate at room temperature for saturation and used as a wash solution. The solution was stirred periodically and filtered before use for complete saturation.

Sample preparation: In the isolation of oxalate by Baker method, there was a difference in the protocols for extracting total and water-soluble oxalate. Total oxalate is isolated with strong-acid solution leading to the dissolution of crystalline calcium oxalate. Isolation of tissues with water removes free oxalic acid as well as potassium oxalates and sodium oxalates but does not remove calcium oxalate.

Five gram of fresh leaf sample/one gram of dried leaf sample was homogenised with 25 ml of diluted hydrochloric acid (1 + 1) or distilled water in pestle and mortar at room temperature for total/water soluble oxalate. The homogenate was transferred to a 50 ml beaker and was boiled for 10 min and then 10 ml of diluted hydrochloric acid (1 + 1) was added and kept aside overnight at room temperature. The extract was filtered through Whatman No. 42 filter paper. Phosphoric-tungstate reagent was added to the filtrate and mixed by vortexing and kept aside for 5 h at room temperature. It was centrifuged at 5,000 rpm for 10 min at 4°C. The supernatant was transferred to a fresh tube and added ammonium hydroxide solution drop by drop until a slight precipitate was formed. Then 5 ml calcium chloride buffer was added and mixed by vortexing and kept aside overnight at 4°C. It was centrifuged at 5,000 rpm for 10 min at 4°C. The supernatant was discarded and the precipitate was dissolved in 20 ml of wash solution and centrifuged at 5,000 rpm for 10 min at 4°C. The supernatant was discarded and the precipitate was dissolved in 10 ml of 0.4 N HCl and used for the estimation of total/water soluble oxalate.

Solvent extraction

To study the effect of solvents for the isolation of oxalate content in fresh and dry leaf samples, water, ethanol and methanol were used. Five gram of fresh/one gram of dry leaf sample was ground in 25 ml of corresponding solvent and centrifuged at 15,000×g for 20 min. The supernatant was collected and stored at 4°C and used for the estimation of oxalate content.

Oxalate estimation

Estimation of oxalate was done by iron ferron method of Burrows (1950). In iron ferron method, the fading effect of oxalate on the colour produced by iron with ferron reagent was used for the estimation of oxalate. The fading of colour was directly proportional to the concentration of ferron reagent and oxalic acid. The concentration of oxalic acid is determined by keeping the concentration of ferron reagent constant. The fading of colour was read at 540 nm.

Reagents:

Iron ferron stock solution. Iron ferron reagent was prepared by dissolving 0.4 g of ferron in 50 ml of hot distilled water containing 0.1 g of ferric chloride, 30 ml of 2 N HCl and 6.8 g of sodium acetate, and the mixture was allowed to cool to room temperature and then made up to 100 ml.

Iron ferron working solution. Working solution was prepared by diluting 10 ml of the iron ferron stock solution to 100 ml with distilled water and it was used for oxalate estimation.

Standard stock solution. Stock solution was prepared by dissolving 1 g of commercially available calcium oxalate crystals in 100 ml of 0.4 N HCl.

Standard working solution. Working standard solution was prepared by dissolving 1 ml of stock in 10 ml of 0.4 N HCl. The solution was shook well before drawing the aliquot for calibration curve preparation.

Sample reading: 2 ml of extract was added to 3 ml of iron ferron reagent and the fading of colour was read at 540 nm against zero setting blank of 0.4 N HCl. 3 ml iron ferron reagent with 2 ml 0.4 N HCl was used as a sample blank.

Standard curve was prepared using commercially available calcium oxalate dissolved in 0.4 N HCl. The concentration of oxalate in the sample was determined by extrapolating the standard curve prepared using calcium oxalate in the range of 0.1–0.5 mg.

The concentration of oxalate in different solvents was determined by extrapolating the standard curve prepared by dissolving the oxalic acid in the corresponding solvent in the concentration range of 0.1–0.5 mg.

Oxalate oxidase enzyme

Method for isolation and assay of oxalate oxidase was adopted from Singh et al. (2006) with slight modifications.

Isolation and assay of oxalate oxidase

Oxalate oxidase was isolated with 0.1 M phosphate buffer pH 7.0 at 4°C. Oxalate oxidase was assayed by 4-aminophenazone method. The hydrogen peroxide produced by the oxidation of oxalate by oxalate oxidase enzyme combines with 4-aminophenazone and phenol in the presence of peroxidase to form quinoneimine dye. The concentration of quinoneimine dye is directly proportional to the concentration of hydrogen peroxide which in turn proportional to the activity of oxalate oxidase enzyme. The colour produced due to quinoneimine dye formation was read at 520 nm.
$$ {\text{Oxalate}} + {\text{O}}_{2} \mathop{\longrightarrow} \limits^{{{\text{oxalate}}\,\,{\text{oxidase}}}}{\text{H}}_{2} {\text{O}}_{2} + 2{\text{CO}}_{2} $$
$$ 2{\text{H}}_{2} {\text{O}}_{2} + {\text{Phenol}} + 4{\text{-}} {\text{aminoantipyrine}}\mathop{\longrightarrow} \limits^{\text{peroxidase}}{\text{quinoneimine\ dye}} + 4{\text{H}}_{2} {\text{O}} $$

Reagents:

Phosphate buffer. Phosphate buffer 0.1 M pH 7.0 and phosphate buffer 0.4 M pH 7.0 were used.

Succinate buffer. Succinate buffer 0.05 M, pH 5.0 was prepared by dissolving 5.9045 g of succinic acid (MW: 118.09 g mole−1) in 500 ml of distilled water and adjusting the pH to 5.0 with 1 N NaOH and made up to 1,000 ml with distilled water.

Copper sulphate. Copper sulphate 0.01 M was prepared by dissolving 0.25 g of copper sulphate (CuSO4·5H2O, MW: 294.68 g mole−1) in 100 ml of distilled water.

Oxalic acid. Oxalic acid 0.01 M was prepared by dissolving 0.216 g of oxalic acid (MW: 126.07 g mole−1) in 100 ml of distilled water.

Colour reagent. The colour reagent was prepared by dissolving 50 mg 4-aminophenazone, 0.1 g solid phenol and 1 mg HOPD in 100 ml of 0.4 M sodium phosphate buffer (pH 7.0). It was stored in amber coloured bottles at 4°C and prepared for every 7 days.

Sample preparation: 5 g of fresh leaf sample/1 g of dried leaf sample was homogenised with 25 ml of 0.1 M phosphate buffer pH 7.0 and/or corresponding solvents in ice-cold sterile pestle and mortar at 4°C. The homogenate was centrifuged at 15,000×g for 20 min. Supernatant was collected as crude enzyme.

Oxalate oxidase assay

The assay of oxalate oxidase was carried out in tubes wrapped with aluminium foil. To each tube 1.7 ml succinate buffer (0.05 M, pH 5.0), 0.1 ml CuSO4 (0.01 M) and 0.1 ml extract were added. The reaction mixture was pre-incubated at 40°C for 2 min. The reaction was started by adding 0.1 ml oxalic acid (10 mM) to each tube. After incubating it at 40°C for 5 min, 1.0 ml colour reagent was added to each tube and kept at room temperature for 30 min in dark to develop the colour. Absorbance was read at 520 nm against the zero setting blank of 0.05 M succinate buffer pH 5.0. A sample blank was maintained which contains all the reagents except the enzyme extract. The protein in the sample was estimated by Bradford method. One unit of oxalate oxidase was defined as the amount of enzyme required to produce 1 nmol of H2O2 per 5 min under standard assay conditions.

Standard curve was prepared using commercially available hydrogen peroxide solution dissolved in succinate buffer 0.05 M, pH 5.0. The amount of hydrogen peroxide produced from the sample due to oxalate oxidase activity on oxalic acid was determined by extrapolating the standard curve prepared using hydrogen peroxide in the range of 0.01–0.30 μmol.

Protein estimation

The soluble protein in the C.pictus leaf extract was determined by Bradford (1976) with slight modification. This method is based on the principle that protein binds to coomassie brilliant blue G-250 in acid solution and forms a blue complex whose extension coefficient (λmax = 595 nm) is much greater than the free dye (λmax = 465 nm) itself. The dye binds strongly to positively charged groups of proteins and also hydrophobic regions in proteins. As a result, a blue colour is formed with a λmax at 595 nm (on binding to proteins, the λmax is shifted from 465 to 595 nm).

Reagents:

Bradford dye solution. Bradford dye solution was prepared by dissolving 100 mg of coomassie brilliant blue G 250 in 50 ml methanol (95% v/v) and 100 ml of concentrated orthophosphoric acid and the volume was made up to 1,000 ml with distilled water and stored at 4°C.

Standard stock solution. Standard stock solution was prepared by dissolving 100 mg of bovine serum albumin (BSA) fraction V in 100 ml of 0.1 N NaOH.

Standard working solution. The stock solution was diluted to get a working solution containing BSA of 100 μg ml−1.

Protein assay

Protein concentration in the enzyme extract was determined by pipetting out 0.2 ml of the extract into a tube containing 0.8 ml of NaOH and 5.0 ml of Bradford dye solution shook well and allowed to stand for 5 min. Absorbance was measured at 595 nm against the zero setting blank of 0.1 N NaOH. A sample blank was maintained essentially except adding the enzyme extract. The concentration of protein was determined by extrapolating the standard curve prepared using BSA fraction V in the concentration ranging from 10 to 100 μg.

Partial purification

The method for partial purification of the enzyme was adopted from Goyal et al. (1999) with slight modification. All the steps were carried at 4°C.

Preparation of sample

Ten gram leaf sample was collected (4th to 6th leaf from top) and ground in ice-cold sterile pestle and mortar with 50 ml of 0.1 M phosphate buffer at 4°C. The extract was centrifuged at 15,000×g for 20 min. The supernatant was collected and 25 ml of extract was used for partial purification and the excess is stored at 4°C.

Precipitation and dialysis

Hundred millimeters of 100% saturated ice-cold ammonium sulphate solution pH 7.0 was added drop by drop to the 25 ml of crude enzyme extract at 4°C. The mixture was kept at 4°C overnight for precipitation of proteins. On the next day, it was centrifuged at 10,000×g for 30 min. The precipitates were collected and dissolved in 15 ml of 0.1 M phosphate buffer pH 7.0 and dialysed against the same buffer (0.05 M, pH 7.0) at 4°C with four changes of buffer at an interval of 6 h for each change.

Effect of pH, temperature and substrate concentration

To find out the optimum pH for the enzymatic activity, 0.05 M succinate buffer of various pH from 3 to 8 was prepared with increment of 1 pH units and subsequently the increment was reduced to 0.1 pH units for the pH range 4–6.

To find out the optimum temperature of the enzyme activity, the reaction mixture containing 1.7 ml succinate buffer (0.05 M, pH 5.0), 0.1 ml CuSO4 (0.01 M), 0.1 ml oxalic acid (10 mM) and 0.1 ml of enzyme extract was incubated at various temperatures from 10 to 100°C with the increment of 10°C and 5°C.

To find out the effect of substrate concentration on the enzyme activity, the enzyme is assayed with increasing concentration of substrate from 0.1 to 20 mM under standard assay conditions.

Statistical analysis

Data are the mean of three independent replicates. Each replicate represents the mean of five samples. The analyses of variance were computed and statistically significant differences (P < 0.05) determined based on the appropriate F tests. The mean differences were compared utilising Latin square design (LSD). All the data were statistically significant at 0.05%.

Result and discussion

Oxalate

The oxalate content was high in second stage followed by first and third stage in fresh and dry samples (Table 1). Hoover and Karunaratnam (1965) reported that in plant sample, oxalate was stable at 1.5 N HCl for 12 h above which the oxalic acid slowly degrades. But in Baker method the oxalate was isolated using (1 + 1) diluted HCl with boiling for 15 min as a result reduction in oxalic acid content occurred. Moreover, a series of steps were involved in Baker method which contributed to the loss of oxalate during isolation. In Burrows method, simple procedure was followed which reduced the loss of oxalate during isolation. Hence, oxalate content was high in Burrows method compared to that of Baker method. Oxalate was less in dry sample compared to fresh sample due to the chemical degradation of oxalate during drying (Baker 1952). The oxalate loss was about 4.53 mg/g during the sun drying for 2 weeks.
Table 1

Oxalate content in different leaf stages of Costuspictus

Stage

Burrows method

Baker method

Total oxalate (mg g−1)

Soluble oxalate (mg g−1)

Insoluble oxalate (mg g−1)

Total oxalate (mg g−1)

Fresh

Dry

Fresh

Dry

Fresh

Dry

Fresh

Dry

First

16.41

12.10

10.75

6.09

0.02

0.25

10.77

6.34

Second

16.50

12.21

10.83

6.19

0.23

0.28

11.06

6.47

Third

15.82

11.47

9.67

5.06

0.50

0.20

10.18

5.26

Mean

16.24

11.93

10.42

5.78

0.25

0.24

10.67

6.02

SD

0.0211

0.0334

0.0257

0.0320

0.0115

0.0110

0.0645

0.0545

CD (0.05)

0.0460

0.0727

0.0560

0.0698

0.0252

0.0239

0.1405

0.1187

CV (%)

0.21

0.44

0.39

0.88

7.30

7.12

0.96

1.43

The oxalate content in C.pictus was moderately high compared to oxalate rich plants like spinach, rhubarb, rumex, etc. Rhubrarb contains 18–216 mg g−1 of oxalate in the petiole in fresh sample. Spinach contains 10.14–13.7 mg g−1 of oxalate in leaf. In spinach, greater variability of oxalate occurs between species. The highest oxalate was recorded in New Zealand spinach leaf (Tetragonia tetragonioldes) which has 117 mg g−1 (Hui et al. 2001). The plant which we use regularly in our daily life also contains considerable amount of oxalate. For example, curry leaf contains 13.52 mg g−1 of oxalate; Piperbetel (betel leaf) contains 13.5 mg g−1 of oxalate; Coriandersativum (coriander) leaf contains 12.68 mg g−1 of oxalate (Hui et al. 2001).

Forms of oxalate in Costuspictus

Depending upon the plant species, oxalate accumulates primarily as soluble oxalate, insoluble oxalate or a combination of these forms. In C. pictus, the oxalate accumulates primarily as soluble oxalate which is also observed in spinach in which 98% oxalate accumulates as soluble oxalates (Siener et al. 2006). In C. pictus, the soluble oxalate decreases with the maturity. In first leaf stage, 99.8% oxalate accumulated as soluble oxalates. In second leaf stage, 97.9% oxalate accumulated as water soluble oxalates. In third leaf stage, 94.9% oxalate accumulated as soluble oxalate. In other words, the insoluble oxalate formation increased with maturity of leaf. The insoluble salt formed primarily as magnesium or iron oxalate because calcium oxalate crystals are absent in Costaceae (Tomlinson 1969).

Even though the quantity of oxalate varied at different stages of maturity, the maximum quantity recorded was 16.5 mg g−1 in fresh leaves. The lethal dose of oxalic acid for human varies from 2 to 30 g (Hui et al.2001). According to these data, 0.12–1.82 kg of C.pictus should be taken to reach the lethal dose which is practically impossible. Hence, the oxalate in C.pictus will not cause harmful effect.

Effect of solvents on the recovery of oxalate content

Oxalate was isolated with water, ethanol and methanol from three different stages of fresh and dry samples. The fresh sample recorded more oxalate content than dry sample which was due to degradation of oxalic acid during drying. Water extract contains more oxalate in fresh as well as in dry sample followed by methanol and ethanol. The oxalate content of different solvents were low, compared to the oxalate content isolated by standard methods but the trend of oxalate variation at different stages of maturity were same (Table 2). The second stages expressed more oxalate content followed by first and third leaf stage. The reduction in oxalate content in solvents was due to poor solubility of oxalate in the solvents (Benjamin 2005). Extraction with ethanol followed by methanol for active principles can eliminate the oxalate content in C.pictus.
Table 2

Effect of solvents on oxalate content in different leaf stages of Costuspictus

Stage

Water

Ethanol

Methanol

Total oxalate (mg g−1)

Total oxalate (mg g−1)

Total oxalate (mg g−1)

Fresh

Dry

Fresh

Dry

Fresh

Dry

First

8.53

3.73

3.81

2.90

4.17

1.11

Second

8.70

3.87

4.29

3.72

5.01

1.76

Third

8.35

3.61

3.69

2.64

3.52

0.56

Mean

8.53

3.74

3.93

3.08

4.23

1.14

SD

0.0288

0.0254

0.0181

0.0161

0.0198

0.0171

CD (0.05)

0.0626

0.0554

0.0394

0.0351

0.0432

0.0373

CV (%)

0.53

1.08

0.73

0.83

0.74

2.37

Oxalate oxidase enzyme activity at different stages of leaf maturity

In fresh leaf sample oxalate oxidase activity was 93.17, 125.58 and 84.81 U in first, second and third leaf stages, respectively. Dry samples showed no oxalate oxidase activity. Table 3 shows the activity of oxalate oxidase enzyme at different leaf stages.
Table 3

Oxalate oxidase activity in crude extract of three different stages of fresh and dry leaf samples of Costuspictus

Stage

Protein (mg g−1)

Oxalate oxidase activity (U)

Specific activity (U mg−1)

Fresh

Dry

Fresh

Dry

Fresh

Dry

First

0.93

0.69

93.17

0

100.18

0

Second

0.67

0.51

125.58

0

187.43

0

Third

0.52

0.41

84.81

0

163.10

0

Mean

0.71

0.54

101.19

0

150.24

0

SD

0.0239

0.0113

0.0701

0

0.1900

0

CD (0.05)

0.0522

0.0245

0.1527

0

0.4140

0

CV (%)

5.36

3.32

0.11

0

0.20

0

1 Unit nmol H2O2 g−1 5 min−1

In C.pictus, oxalate content was high in the second leaf stage followed by first and third leaf stage (Table 1). The activity of oxalate oxidase was also high in the second leaf stage followed by first and third leaf stage (Table 3). This indicates that the oxalate and oxalate oxidase enzyme exist in an equilibrium and the oxalate oxidase enzyme regulates the oxalate content.

The specific activity was high in the second leaf stage (187.43 U mg−1) followed by third leaf stage (163.10 U mg−1) and first leaf stage (100.18 U mg−1). The trend in the specific activity indicates that oxalate oxidase helps in the lignification of cell wall in the immature stage and in the full maturity it was a source of free radical which causes senescence, because the protein content in the third leaf stage was half of that in the first leaf stage. Even though the protein content was less in the third leaf stage, the specific activity was more which indicated that the oxalate oxidase was high with in the low protein content. It is in agreement with the report of Deunff et al. (2004).

The high oxalate oxidase activity at early stage of development in C. pictus revealed the close association of the enzyme with cell wall development. The high rate of oxalate oxidase enzyme activity at early stage and high specific activity at later stage throw light to the different roles of the oxalate oxidase enzyme. The activity of the enzyme at early stage is much concerned with the developmental process and defense against fungal pathogens. The high specific activity at later stage was more concerned with senescence process and defense against fungal pathogen. Expression of germin oxalate oxidase after wounding or during senescence confers protection against fungal pathogens (Donaldson et al. 2001; Liang et al. 2001; Ramputh et al. 2002). Hydrogen peroxide is considered as an alarming signal involved in both constitutive defence (senescence) and adaptative defence (wounding) against pathogens (Deunff et al. 2004). Hence, it can be very well presumed that defense against fungal pathogens seems to be common function of oxalate oxidase enzyme at all the stages of development of C. pictus.

Effects of solvents on the oxalate oxidase enzyme activity

Oxalate oxidase was isolated with water, ethanol and methanol from fresh and dried leaf samples. The enzyme activity was in the order of ethanol > methanol > water. But the trend in the oxalate oxidase activity at different stages was same in all solvents. High activity was recorded in the second leaf stage followed by first stage and third stage (Table 4).
Table 4

Effect of solvents on oxalate oxidase enzyme activity in crude extract of three different leaf stages of Costuspictus

Stage

Water

Ethanol

Methanol

Protein (mg g−1)

Oxo activity (U)

SA (U mg−1)

Protein (mg g−1)

Oxo activity (U)

SA (U mg−1)

Protein (mg g−1)

Oxo activity (U)

SA (U mg−1)

Fresh

Dry

Fresh

Dry

Fresh

Dry

Fresh

Dry

Fresh

Dry

Fresh

Dry

Fresh

Dry

Fresh

Dry

Fresh

Dry

First

0.47

0.37

89.19

0

189.77

0

1.24

0.90

453.97

0

366.10

0

0.56

0.44

80.66

0

144.04

0

Second

0.44

0.35

96.75

0

219.89

0

1.18

0.85

549.30

0

465.51

0

0.39

0.32

101.02

0

259.03

0

Third

0.35

0.29

74.44

0

212.29

0

1.04

0.76

396.38

0

381.13

0

0.29

0.25

53.17

0

183.34

0

Mean

0.42

0.34

86.79

0

207.32

0

1.15

0.84

466.55

0

404.25

0

0.41

0.34

78.28

0

195.47

0

SD

0.0121

0.0077

0.0224

0

0.0314

0

0.0093

0.0110

0.0210

0

0.0261

0

0.0134

0.0121

0.0653

0

0.1129

0

CD (0.05)

0.0264

0.0169

0.0487

0

0.0684

0

0.0203

0.0239

0.0457

0

0.0568

0

0.0292

0.0264

0.1423

0

0.2460

0

CV (%)

4.56

3.64

0.04

0

0.02

0

1.28

2.07

0.01

0

0.01

0

5.13

5.69

0.13

0

0.09

0

Oxo oxalate oxidase enzyme, SA specific activity, 1 U nmol H2O2 g −1 5 min−1

Ethanol was extracting only selected proteins. Oxalate oxidase enzyme was one of the protein in which active site was exposed to substrate. So ethanol increases the sensitivity of oxalate oxidase (Zhang et al. 1996). Hence, the activity was high in the ethanol extract compared to methanol and water extract.

Protein content and oxalate oxidase activity

Among the three leaf stages first leaf stage expressed high quantity of protein followed by the second and third stage. Even though the protein content was high in the first leaf stage, oxalate oxidase activity was lower than the second leaf stage (Table 3). It was an indication of the presence of other enzymes/proteins contributing to the growth and development of the plant.

The partially purified sample with heat treatment at 80°C for 3 min recorded substantial loss of protein content. The protein available after heating showed oxalate oxidase activity, in which specific activity was higher than the recorded specific activity of other samples (Tables 3, 5). The protein content was reduced due to degradation of proteins to simple biomolecules and secondary products. The presence of oxalate oxidase enzyme even after heating recorded the stability of the enzyme under stress condition.
Table 5

Oxalate oxidase enzyme activity in crude and partially purified sample of second leaf stage of Costuspictus

 

Protein (mg g−1)

Oxalate oxidase activity (U)

Specific activity (U mg−1)

Crude

0.81

34.69

42.83

Partial purification

 Without heat treatment

0.744

155.52

209.03

 With heat treatment

0.48

110.03

229.23

Mean

0.6767

100.0800

160.3633

SD

0.0139

0.0153

0.0265

CD (0.05)

0.0303

0.0333

0.0576

CV (%)

3.25

0.02

0.03

1 U nmol H2O2 g−1 5 min−1

Partial purification of oxalate oxidase

The crude extract of C. pictus was partially purified by ammonium sulphate precipitation (80%) with and with out heat treatment.

Purification of oxalate oxidase employing heat treatment and ammonium sulphate precipitation was done in barley seedlings (Chiriboga 1966; Kotsira and Clonis 1998; Requena and Bornemann 1999), sorghum (Pundir 1991a) and wheat (Hu and Guo 2009). Purification of oxalate oxidase employing ammonium sulphate precipitation with out heat treatment was done in Amaranthus (Mongkolisirikieat and Srisawan 1987; Goyal et al. 1999), maize (Vuletic and Šukalovic 2000), barley (Thakur et al. 2000) and sorghum leaves (Singh et al. 2006).

Partial purification yielded 4.5-fold increased purification compared to crude extract of C.pictus (Table 5). The heat treated sample shows decrease in activity (29%) compared to heat untreated ammonium sulphate precipitated sample. But the specific activity was 1.1 times more compared to heat untreated ammonium sulphate precipitated sample, which indicates that even though there was decrease in activity (29%) after heat treatment, the enzyme was stable.

Partial purification along with heat treatment reduced the protein content by 40.7% compared to crude but in partial purified without heat treated sample the protein content was reduced only by 8.1% compared to crude. Specific activity of oxalate oxidase in partial purified heat treated sample is 1.1 times higher than the partial purified without heat treated sample and 5.4 times higher than the crude.

The activity of partial purified enzyme with out heat treatment was 155.52 U which is 4.5 times higher than crude. The specific activity of partial purified enzyme with out heat treatment is 209.03 U mg−1 which is 4.9 times higher than the crude. The protein content of partial purified sample with out heat treatment was 0.744 mg g−1. The protein content in the partial purified sample with heat treatment was 0.48 mg g−1.

Effect of pH on oxalate oxidase enzyme activity

The oxalate oxidase was assayed at varying pH from 3 to 8. The enzyme showed maximum activity at pH 5.2 for crude extract and 5.8 for partially purified extract when incubated at 40°C (Fig. 2).
https://static-content.springer.com/image/art%3A10.1007%2Fs11738-011-0866-x/MediaObjects/11738_2011_866_Fig2_HTML.gif
Fig. 2

Effect of pH on oxalate oxidase enzyme activity. a Effect of pH on oxalate oxidase enzyme activity in crude and partially purified sample of second leaf stage of C. pictus with the increment of 1.0 pH unit from pH 3 to 8. b Effect of pH on oxalate oxidase enzyme activity in crude and partially purified sample of second leaf stage of C. pictus with the increment of 0.1 pH unit from pH 4 to 6 for crude and 5 to 7 for partially purified sample

The pH optima in the acidic range has been reported for oxalate oxidase from various sources such as sorghum roots pH 5.0 (Pundir 1991b; Pundir and Kuchhal 1989), sorghum leaves (Pundir and Nath 1984; Pundir and Satyapal 1998) and banana peel pH 5.2 (Inamdar et al. 1986). The increase in pH from 5.2 (crude) to 5.8 (partially purified) was due to the availability of organic acids and other biomolecules which contribute to the pH at acidic range in the crude extract.

Effect of temperature on oxalate oxidase enzyme activity

The enzyme showed maximum activity at 45°C for crude and partially purified above which it showed a rapid decline due to the denaturation of the enzyme (Fig. 3). The temperature for maximum activity at 45°C was reported for Amaranthus leaves (Goyal et al. 1999). The oxalate oxidase enzyme from C.pictus retained 6.4% of its maximum activity on heating for 5 min at 80°C in 0.05 M succinate buffer, pH 5.0, whereas barley enzyme has been reported to be more thermostable, since it retained 60% activity on heating at 80°C for 5 min at its optimal pH (Kotsira and Clonis 1997).
https://static-content.springer.com/image/art%3A10.1007%2Fs11738-011-0866-x/MediaObjects/11738_2011_866_Fig3_HTML.gif
Fig. 3

Effect of temperature on oxalate oxidase enzyme activity. a Effect of temperature on oxalate oxidase enzyme activity in crude and partially purified sample of second leaf stage of C. pictus with the increment of 10°C from 10 to 100°C. b Effect of temperature on oxalate oxidase enzyme activity in crude and partially purified sample second leaf stage of C. pictus with the increment of 5°C from 40 to 60°C

Effect of substrate concentration on oxalate oxidase enzyme activity

The activities of the crude, partially purified enzyme were measured with increasing concentration of oxalic acid from 0.1 to 20 mM. The activity of the crude extract was steadily decreased with increase in substrate concentration. The activity of the partially purified enzyme showed a hyperbolic relationship with oxalic acid concentration only up to 0.8 mM above which the enzyme showed decrease in activity due to substrate inhibition (Fig. 4).
https://static-content.springer.com/image/art%3A10.1007%2Fs11738-011-0866-x/MediaObjects/11738_2011_866_Fig4_HTML.gif
Fig. 4

Effect of substrate concentration on oxalate oxidase enzyme activity

Earlier hyperbolic relationship between the initial velocity and the substrate concentration was reported up to 4 × 10−3 M for grain sorghum leaf (Satyapal and Pundir 1993), barley seedling (Sugiura et al. 1979) and barley root (Kotsira and Clonis 1997). A Lineweaver–Burk plot of 1/S versus 1/V revealed an Km of 0.065 mM and Vmax of 427.5 nmol of H2O2 g−1 5 min−1 at 40°C, pH 5.0 (Fig. 5). Lower the Km value higher the affinity towards the substrate. The affinity of oxalate oxidase enzyme for oxalic acid can be exploited for clinical purposes.
https://static-content.springer.com/image/art%3A10.1007%2Fs11738-011-0866-x/MediaObjects/11738_2011_866_Fig5_HTML.gif
Fig. 5

Lineweaver–Burk plot

Substrate inhibition of oxalate oxidase is also reported in various plants. Barley root oxalate oxidase showed substrate inhibition when oxalic acid reached to 4 mM and had Km of 0.27 (Chiriboga 1966) and 0.42 mM (Kotsira and Clonis 1997). In contrast, oxalate oxidase from barley seedling did not exhibit substrate inhibition until oxalic acid was up to 200 mM and has a Km of 1.3 mM (Requena and Bornemann 1999), 0.42 mM (Sugiura et al. 1979). The oxalate oxidase from C.pictus had the Km of 0.065 mM which is 20 (Km 1.3 mM) and 6.5 (Km 0.42 mM) times lesser than barley seedling. This comparison leads to speculation that the use of oxalate oxidase from C.pictus in enzymatic determination of oxalate in biological fluids could enhance the sensitivity of the method over the barley seedling enzyme.

Conclusion

Oxalate in C.pictus accumulated primarily as a water soluble oxalate. This indicates that the oxalate and oxalate oxidase enzyme exist in an equilibrium and the oxalate oxidase enzyme regulates the oxalate content. Recovery of oxalate was more in water than the methanol and ethanol. The fresh leaf showed oxalate oxidase whereas the dry sample shows no oxalate oxidase activity. The second leaf stage showed maximum oxalate oxidase activity as well as specific activity compared to other two leaf stages. Ethanol extract showed maximum oxalate oxidase activity than the methanol and water extract. Partial purification of oxalate oxidase yielded 4.5 times increase in activity compared to crude extract. The partially purified oxalate oxidase showed hyperbolic relationship with substrate concentration up to 0.8 mM above which it showed substrate inhibition. The Lineweaver–Burk plots showed Vmax of 427.2/5 min and Km of 0.065 mM. The sensitivity of the oxalate determination in biological fluids employing oxalate oxidase from C.pictus will be more as it had Km value 20 times lesser than the oxalate oxidase from barley seedling. C. pictus can be safely used for reducing blood glucose level in diabetic patients without risk of kidney stone formation and the oxalate oxidase enzyme from C. pictus can be commercially exploited for determining oxalic acid in biological fluids.

Acknowledgments

We thank Dr. Bhabesh Dutta, Post Doctoral Associate, Department of Plant Pathology, Coastal Plain Research Station, University of Georgia, Tifton, for providing the valuable references for this study. We also thank Dr. C. Vijayalakshmi, Professor and Head, Department of Crop Physiology, Tamil Nadu Agricultural University, Coimbatore, for giving valuable suggestions in manuscript preparation.

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2011