Abstract: Penicillium species grew in a synthetic medium with pectin as the sole carbon source producing proteins which expressed alkaline pectin lyase activity. The enzyme was purified by a combination of ammonium sulphate precipitation, dialysis, gel filtration and ion-exchange chromatography. Optimum activity of the enzyme was at 35°C and at pH 80. The enzyme was stimulated by K+, Na+, Ca++ and Mg++ but inhibited by EDTA and Hg++. The enzyme was able to degrade pectin with optimum activity expressed at 16 mg mL‾1.
INTRODUCTION
Pectin lyase, also known as pectin transeliminase or pectinase is involved in the degradation of pectic substances (Gummadi and Kumar, 2005). The enzyme is a virulent factor, which degrade the pectic components of the plant cell wall (Mayans et al., 1997). The enzyme acts to cleave certain activated glycosidic linkages present in acidic polysaccharides. It acts through an eliminase mechanism rather than through hydrolysis, resulting in unsaturated oligosaccharide products (Linhardt et al., 1986).
Microorganisms have been reported to produce pectin lyase during plant and fruit infection (Schink et al., 1981; Sakiyama et al., 2001).
This study was designed to confirm whether an isolated species of Penicillium is capable of producing pectin lyase in a synthetic growth medium with pectin as sole carbon source. Attempts were made to purify the enzyme and determine some of its properties.
MATERIALS AND METHODS
Organism and Culture Conditions
An isolate of a Penicillium species used for this study was obtained
from the culture collection of Professor P.O. Olutiola, Department of Microbiology,
Obafemi Awolowo University, Ile-Ife. Osun state Nigeria. It was grown and maintained
on potato dextrose agar plates and slants.
The isolate was allowed to grow in a defined medium made up of K2HPO4, KH2PO4, MgSO4. 7H2O, pectin, thiamine, biotin, FeSO4 and KNO3. The carbon source, which is pectin, was autoclaved separately and aseptically added to the basal medium to give a final concentration of 1%. The growth medium (50 mL) in conical flasks was inoculated with 1 mL of spore suspension (5χ107 spores mL‾1) of the culture organism. The inoculated growth medium was incubated at 25°C and analysed daily for protein content (Lowry et al., 1951) and pectin lyase activity (Olutiola and Akintunde, 1979; Alana et al., 1990).
Extraction of Enzyme
The crude enzyme in each flask was clarified by filtering through filter
paper (Whatman G/A) and analysed for pectin lyase activity.
Ammonium Sulphate Precipitation and Dialysis
The proteins of the extracted crude filtrate was precipitated using ammonium
sulphate (analytical grade) to 90% saturation. Precipitation during salt saturation
was at 4°C for 24 h. The precipitate was there after redissolved in 0.02
M citrate phosphate buffer pH 6.0. Analysis for pectin lyase activity was carried
out.
Dialysis was carried out over night with three changes to 0.02 M citrate buffer pH 6.0.
Fractionation on Sephadex G-100 Column
Ten milliliter of the dialysed enzyme was applied to a column (2.5χ70
cm) of Sephadex G-100 which had been calibrated earlier with proteins of known
molecular weights (Andrews, 1964). Fractions (5 mL/tube) were collected and
optical density, measured at 280 nm and analysed for pectin lyase activity.
Fractions which showed appreciable activity were pooled and applied to a column
of Sephadex C-50.
Fractionation on Sephadex C-50 Column
Ten milliliter of pooled fractions from Sephadex G-100 column which showed
appreciable pectin lyase activity was applied to a column (2.5χ40 cm) of
Sephadex C-50. Fractions (5 mL/tube) were collected. Optical density of the
fractions was measured at 280 nm and analysed for pectin lyase activity.
Pectin Lyase Assay
Pectin lyase activity was assayed by the pectin degradation method using
the thiobarbituric acid technique of Olutiola and Akintunde (1979). The substrate
1.2% (w/v) pectin (Sigma) was prepared in 0.2 M Tris-HCL buffer (pH 8.5). The
reaction mixture was 2 mL of enzyme added to 3 mL of substrate. Incubation was
at 35°C for 1 h. The reaction mixture was terminated by adding 3 mL of 0.04
M Thiobarbituric Acid (TBA) and 1.5 mL of 1 N HCl. Controls which initially
contained only 3 mL of substrate were incubated with the experimental tubes
at 35°C for 1 h. Three milliliters thiobarbituric acid was added to the
contents of each control tube followed by 2 mL of enzyme. Both experimental
and control tubes with contents were boiled in a water bath for 20 min. Optical
density measurements were taken at 540 nm.
One unit of pectin lyase activity was arbitrarily defined as the amount of enzyme in the reaction mixture, which caused an 0.01 increase in absorbance in 1 h under assay conditions.
RESULTS
Penicillium species grew in a synthetic growth medium with pectin as sole carbon source at 25°C within ten days of incubation. Pectin lyase activity was detected in the growth medium. Uninoculated medium lacked pectin lyase activity. The enzyme, purified by a combination of ammonium sulphate precipitation, dialysis, gel filtration (Sephadex G-100) and ion- exchange chromatography (Sephadex C-50) (Table 1), expressed optimum activity at 35°C within a temperature range of 10-60°C (Fig. 1). Within a pH range to 5.0 to 9.0, optimum activity was observed at 8.0 (Fig. 2).
| Table 1: | Purification of pectin lyase obtained from Penicillium species |
| Fig. 1: | Effect of temperature on the activity of partially purified (SP C-50 fraction) pectin lyase obtained from Penicillium species |
| Fig. 2: | Effect of pH on the activity of partially purified (SP C-50 fraction) pectin lyase obtained from Penicillium species |
Activity of the enzyme was stimulated with increasing concentrations (within a range of 0-30 mM) of K+, Na+, Ca++ and Mg++. Optimum activities were observed at 15 mM concentration of Ca++ 20 mM concentration of K+ and 25 mM concentration of Na+ and Mg++. There after, there were declines for Ca++, K+ and Mg++. Na+ was stable at 30 mM concentration. The activity of the enzyme was inhibited by Hg++ and EDTA within a range of 0-10 mM concentrations. Activity was completely lost at 6 mM concentration of Hg++ .Within different concentrations of pectin as substrate (ranges of 5-20 mg mL‾1), optimum activity was observed at 16 mg mL‾1 (Fig. 5).
| Fig. 3: | Effect of cations on the activity of partially purified (SP C-50 fraction) pectin lyase obtained from Penicillium species |
| Fig. 4: | Effect of chemicals on the activity of partially purified (SP C-50 fraction) pectin lyase obtained from Penicillium species |
| Fig. 5: | Effect of substrate concentration on the activity of partially purified (SP C-50 fraction) pectin lyase obtained from Penicillium species |
DISCUSSION
Penicillium species produced pectin lyase in a synthetic growth medium with pectin as sole carbon source. According to Gonzalez-Candelas (1992), Fusarium solani f.sp. pisi is capable of producing pectin lyase in growth medium with pectin as sole carbon source. Nakagawa et al. (2005) reported that the methylotropic yeast Pichia methalonica is able to grow on pectin compounds, pectin and polygalacturonate as sole carbon sources.
Temperature ranges affected pectin lyase activity from Penicillium species. Optimum activity was at 35°C. An almost similar temperature of 50°C for optimum activity of pectin lyase produced by Bacillus subtilis has been reported by Soriano (2000). Optimum activity of pectin lyase by Penicillium species was observed at an alkaline pH of 8.0 Penicillium italicum has been found exhibit optimum pectin lyase activity at alkaline pH range of 6.0-7.0 (Alana et al., 1990).
K+, Na+, Ca++ and Mg++ enhanced activity of the Pectin lyase produced by Penicillium species with optimum activity at different concentrations. Mayans et al. (1997) observed that a species of Aspergillus is able to produce a Calcium- dependent pectin lyase. EDTA and Hg++ were inhibitory to the activity of pectin lyase produced by Penicillium species. According to Soriano (2000), Ba++ is inhibitory to a similar enzyme produced by Bacillus subtilis. Alana et al. (1989) reported that glycerol inhibits pectin lyase produced by Penicillium italicum.
Penicillium species was able to degrade pectin as substrate with optimum activity at 16 mg mL‾1 concentration. Strains of psychrophilic yeasts with taxonomic affiliations similar to Cyrptococcus cylindricus, Mrakia frigida and Cystofilobasidium capitatum are able to grow on and degrade pectin at about 5°C (Nakagawa et al., 2004). According to Alana et al. (1990), Penicillium italicum produces an extracellular pectin lyase in a liquid medium with Km of 3.2 mg mL‾1 for pectin. Silva et al. (1993) reported that Penicillium expansum produces a monomeric pectin lyase with molecular mass of 36.5 kDa capable of degrading citrus and apple pectin, with a Km for citrus pectin at 9 mg mL‾1.
Alkaline pectinases are among the most important industrial enzymes and are of great significance in textile processing, degumming of plant bast fibers, treatment of waste waters, paper making, coffee and tea fermentation (Hoondai et al., 2002).