World

Journal

of Microbiology

& Biofechnology

12. 239-242

Rapid and simple assay for feruloyl p-coumaroyl esterases F.H. O’Neill,

L.P. Christov,*

P.J. Botes

and

and B.A. Prior

A rapid, simple and sensitive method for detection of ferulic and p-coumaric acids using HPLC has been developed which can be used to determine the respective phenolic acid esterase activities of microorganisms. Prior concentration, purification or derivatization of the samples are not required. As little as 0.5 mg ferulic or pcoumaric acid/l could be detected and estimated in < 1 h. The method is specific for the two phenolic acids since no interference by other components was observed. Key words: p-coumaric

acid, ferulic acid, HPLC, phenolic acid esterase assay.

Xylan is one of the major constituents of hemicellulose in plants. It is composed of a linear chain of P-l&linked xylose residues that are substituted at the C2 and/or C3 positions with arabinose, acetic acid and J-O-methylglucuronic acid (Fengel & Wegener 1984). For instance, arabinose can be bound to the xylan backbone at the C3 position. On the other hand, the same arabinose molecule may be esterified at the C5 position by phenolic acids such as ferulic acid and p-coumaric acid to form crosslinks between xylan and lignin (Hartley & Ford 1989). These links provide the fibre with mechanical strength and increase its resistance towards chemical or biological degradation. If a process of xylanase-aided conversion or bleaching of the plant material is desired, the presence of xylan-lignin complexes in the cell wall may be one of the limiting factors for the effectiveness of the enzymatic hydrolysis. Accessibility to the branched xylan polymer can be improved by using xylanases in conjunction with ferulic and p-coumaric acid esterases (Christov & Prior 199j). The latter can break down the ester bonds between the arabinose side groups of xylan and the phenolic acids (ferulic and p-coumaric) of lignin, thereby enhancing the penetration of xylanase into the cell wall. A number of methods employing various techniques currently exists for determining the activity of feruloyl and p-coumaroyl esterases. These assay methods use various

The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State (UOFS). PO Box 339, Bloemfontein 9300, South Africa; fax: 27 51 448 2004. ‘Corresponding author.

@ 1996 Rapid

Science

natural as well as synthetic substrates. The natural substrates include wheat bran (MacKenzie et al. 19877, wheat straw (Tenkanen et al. 1991), Coastal Bermuda grass (Bomeman ef aI. 1991), and Italian ryegrass (Castanares et al. 1992). Well-characterized definitive compounds isolated from natural substrates have also been used (Bomeman et al. 1990a, 1991). The main drawback of using synthetic substrates such as methyl ferulate and p-coumarate (Bomeman et al. 199Ob; McDermit et al. 1990) or p-nitrophenyl butyrate (Donnelly & Crawford 1988) is that they are dissimilar to the naturally occurring substrates in terms of accessibility and the type of the ester bonds to be cleaved. The use of artificial substrates cannot, therefore, reveal the actual activity of the enzymes against natural substrates. The cinnamic-based acids released during the assay procedure have been quantified by a number of different methods: HPLC (Hatfield et al. 1991; Castanares et al. 1992; McCrae et al. 1994); spectroscopy (McDermit et al. 1990); TLC (Tenkanen et al. 1991); and GC (Bomeman et al. 1990b). The most common method to quantify the liberated phenolic acids is HPLC analysis, with variations in the actual assay, the parameters for the analysis and the eluants used. However, most of the techniques used involve timeconsuming procedures for preparation of the substrate and preliminary manipulation of the samples (Bomeman et al. 199oa; Hatfield et al. 1991; Tenkanen et al. 1991). The aim of the present study was to develop a rapid, simple and sensitive method for detection of phenolic acids, which can be used for the determination of microbial feruloyl and p-coumaroyl esterase activities.

Publishers World Journal

of Mimobiology

& Biotechnology.

I/o1 12. 1996

239

0’Neill et al.

F.H.

0

2

4

6

6

10

Time

12

14

16

18

20

(min)

Figure 1. HPLC chromatogram of a standard (25 mg/l) and p-coumaric acid (25 mg/l).

solution

of ferulic

160oool/

Results

Concentration

(mg/l)

Figure 2. Calibration curve for phenolic acid esterase assay using ferulic acid (m) and p-coumaric acid (A) as standards.

Materials

supematant) and incubating for 20 min at 50°C. the reaction was terminated by boiling the samples for 3 min. The HPLC analysis of the supematant (obtained by centrifugation at 10 000 x g for 2 min) was conducted on a Waters HPLC system (Millipore) equipped with a Spherisorb S5-ODSZ column (Phase Sep, Queensferry, UK). The mobile phase consisted of water/formic acid/ acetonitrile (7:1:2, by vol) at a flow rate of 0.5 ml/min and at ambient temperature. Samples of 20 ~1 were injected for analysis. A variable-wavelength U.V. detector (Waters UV Lambda Max, Model 640) set at 300 nm was used to determine the absorbance of the eluant. Peaks were recorded with the aid of a computer program and then integrated manually. One unit of activity was defined as the amount of enzyme that catalyses the release of 1 pmol product (ferulic or p-coumaric acid)/min.

and Methods

Subsfrate Preparation The wheat bran used as substrate for the determination of the phenolic acid esterase activities was de-starched by incubation in 0.25% (w/v) potassium acetate for 10 min at 95°C (MacKenzie et al. 1987). The treated bran was then washed with water and air dried prior to use. Enzyme Production and Preparation Atrreobasidium plcllulans NRRL Y-2311-1, Thermomyces lanuginosis (DSM 5826 and a local isolate) and unidentified species (local isolates) of Aspergih and Ftrsariwn were tested as sources of phenolic acid esterases. The organisms were maintained on yeast extract/malt (YM) slants and streaked out on YM agar plates prior to use. For the assay, the organisms were grown on a rotary shaker at 30°C for a period of 5 days. The growth medium contained (%, w/v): oat spelts xylan, 0.5; yeast nitrogen base, 0.67; asparagine, 0.2; and K,HPO,, 0.5; at pH 5.0. After growth, the cells were harvested by centrifugation (10 000 x g for 10 min) and the supematant stored at 4°C until use. Enzyme Assay For the assay of phenolic esterase activity, 100 mg de-starched wheat bran were suspended in 1 ml 100 mM MOPS buffer at pH 6.0 (MacKenzie et al. 1987). After adding the enzyme (1 ml

and Discussion

Standard solutions of fen&c acid and p-coumaric acid (up to 100 mg/l) were injected into the HPLC to analyse the effectivity of the column and buffer combination used. A satisfactory separation of the two phenolic acids was obtained with all concentrations tested and retention times for p-coumaric acid and ferulic acid of 15 and 17 min, respectively (Figure I). The linearity of the plot of peak area versus phenolic acid concentration was confirmed after HPLC analysis (Figure 2). The minimum detectable amount of phenolic acid was in the range of 0.5 mg/l (0.01 pg) or 2.6 pmol fen&c acid/l and 3.0 pmol p-coumaric acid/l, respectively. Statistical analysis was performed on the values obtained from the HPLC. The efficiency value (IV), which gives an indication of the effectiveness of the total system, was 2437. The resolution value (R,) of 1.8 and the selectivity value (a) of 1.3 are each above the minimum accepted value of I, indicating good separation between the two phenolic acids. The standard deviation for ferulic and p-coumaric acid was 0.26% and 0.36%, respectively. For comparison, the standard deviation for ferulic acid using amperometric detection was 1.1% (Madigan et al. 1994). The yields (99.7% and 99.6%) and r2 values (0.9999 and 0.9998) obtained for the two phenolic acids provided further indication of the effectiveness of the method. The adsorption of phenolic acids by wheat bran was studied by incubating standard solutions of ferulic (50 mg/l) and p-coumaric acid (20 mg/l) or their mixture (25 mg/l and 20 mg/l, respectively) with de-starched wheat bran under assay conditions. The substrate did adsorb a small percentage (4%) of ferulic acid and a relatively large percentage (25%) of p-coumaric acid. Therefore, for determination of the actual esterase activity using wheat bran as substrate, corrections should be made for the loss of phenolic acids due to their partial adsorption to the substrate under the particular assay conditions used. No interference of ferulic and p-coumaric acid from any other components was detected when analysing the

Phenolic acid esterase assay

I

Ferulic

p-Coumaric

Time Figure crude

enzymes

0.7

at 50°C

II

(min)

3. HPLC chromatogram wheat bran after

de-starched

acid

acid

of the products incubation with

released

Au.

from

pu//u/ans

for 20 min.

a 1

0.1. OI 0

5

10

15

20

25

30

35

Time (min) 35 1 30

b

Using this assay and detection method, a number of microorganisms were tested for esterase activities. Aureobasidium pullulans grown on oat spelts xylan had 163.3 mU feruloyl and 5.6 mU p-coumaroyl e&erase/ml. Aspergillus sp. produced 9.6 mU feruloyl and 5.6 mU p-coumaroyl esteraseiml, whereas T. lanuginosis (local isolate) and Fusarium sp. produced only feruloyl esterase activity, of 2.6 and 2.1 mu/ml, respectively. The sensitivity of detecting, separating and quantifying the product was satisfactory and comparable with that of other methods described. Thus, as little as 0.01 pg (0.5 mg/l) of phenolic acid can be detected and estimated. Hartley & Buchan (x979), using the same detection system and water/acetic acid/n-butanol as eluant, reported a limit of detection of 0.1 pg of ferulic or p-coumaric acid. However, the sensitivity can be further increased (0.02 mg/l) by applying a very sensitive electrochemical (amperometric) detection (Madigan ef al. 1994). In conclusion, the present HPLC detection method proved to be rapid, with results available in C 1 h, which compares well with other similar methods (Madigan ef al. 1994). An inexpensive and readily available substrate (wheat bran) was used to determine both esterase activities simultaneously, avoiding their separate assay. Simple techniques without prior concentration, purification or derivatization of the samples were applied; other methods require pre-treatment of the samples (Castanares et al. 1992). Since no other components released from the substrate during the assay had retention times close to that of either ferulic acid or p-coumaric acid, the method would be specific for these two phenolic acids.

Acknowledgements We thank the Foundation for Research Development and SAPPI SAICCOR (Pty) Ltd for financial support.

(FRD)

References

Figure de-starched enzymes undiluted

4. Release

of p-coumaric

wheat from (A).

Au.

bran

acid

incubated puhlans diluted

W.S., Hartley, R.D., Himmelsbach, D.S. & Ljungdahl, L.G. 1990a Assay for trans-p-coumaroyl esterase using a specific substrate from plant cell walls. Analytical Biochemistry 190,

Bomeman,

Time (min) (a) or ferulic

acid

(b) from

at 50°C with the crude 4-(H) or 2-fold (0) or

129-133. Bomeman,

dahl,

W.S., Hartley, R.D., Morrison, W.H., Akin, D.E. & LjungL.G.

1990b

Feruloyl

and

p-coumaroyl

esterase

from

anaerobic fungi in relation to plant cell wall degradation. Applied hydrolysates of wheat bran digested with the Au. pullulans enzymes (Figure 3). When different dilutions of the Au. pullulans enzymes were applied to de-starched wheat bran, the reaction rate proved to be linear over a 20-min period for both of the phenolic esterases (Figure 4). However, the rate of release of the phenolic acid from the substrate was not proportional to the change in the enzyme concentrations applied, indicating that enzyme activities will be dependent upon the protein concentration used.

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and Biotechnology

33, 345-35

I.

W.S., Ljungdahl, L.S., Hartley, R.D. & Akin, D.E. 1991 Isolation and characterization of p-coumaroyl esterase from

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of Microbmlogy

6 Biotechnology.

Vol 12, 1996

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et al.

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3 November

1995;

accepted

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1995)