Abstract: Yeast isolates GSP 101, 102 and 103 capable of producing both pectin lyase and pectate lyase were isolated from rotten apple. Among this, isolate GSP 101 exhibited higher levels of pectin lyase and pectate lyase. Based on 26S rDNA D1/D2 sequence analysis, the isolate was identified as Debaryomyces nepalensis (99.8% sequence identity). The isolate produced maximum 3.2 U mL-1 of pectin lyase and 2.3 U mL-1 of pectate lyase when grown in basal medium supplemented with pectin (5 g L-1). The presence of glucose in medium did not show any negative effect on production of pectic lyases suggesting that there is no catabolite repression. Among the tested naturally available pectic substances, lemon peel was best inducer and carbon source for the production of PL and PGL. This is the first report on production of pectin lyase and pectate lyase by Debaryomyces nepalensis.
Introduction
Pectic substances are naturally occurring heterogeneous macromolecular polyuronides widely distributed in plant tissues. The principle constituent of all pectic substance is rhamnogalacturonan. The primary chain in the polymer consists of α-D-galacturonate units linked through α-1, 4-glycosidic linkages. The side chains of pectin molecule consist of rhamnose, arabinose, galactose and xylose. In most plants, about 70% of the galacturonate units are esterified with methanol. Based on the degree of esterification, pectic substances are classified into three main types: protopectin, highly esterified parent pectic substance, which is insoluble in water; pectin where the degree of polymerization varies between 70-90% and polygalacturonic acid (unesterified) (Whitaker, 1991). The degradation of pectic substances involves the combined action of different pectinases, viz., esterases and depolymerases (hydrolases and lyases).
Pectic transeliminases or pectic lyases are one among the pectinases, which degrade pectic substances by β-elimination mechanism yielding 4:5 unsaturated oligogalacturonates. Pectin Lyase (PL) acting on pectin and polygalacturonate lyase or pectate lyase (PGL) acting on polygalacturonic acid are two important transeliminases acting on pectic substances. Fungal strains are mainly found to produce PL and bacterial strains were used for the production of PGL (Gummadi and Kumar, 2005). PL and PGL can be differentiated by their substrate requirement and the absolute requirement of calcium for PGL activity (Henrissat et al., 1995). Pectic lyases are extensively used in extraction and clarification of fruit juices, degumming of ramie and jute fibers, scouring of crude cotton fibers, pretreatment of wastewater from food processing industries (Bruhlmann, 1994; Naidu and Panda, 1998; Tanabe et al., 1998; Kashyap et al., 2001; Hoondal et al., 2002; Gummadi and Panda, 2003; Gummadi and Kumar, 2005). Since applications of pectic lyases are in an increasing trend, new strains capable of producing pectic lyases (both PL and PGL) are needed. The strain Debaryomyces nepalensis isolated from rotten apple was found to produce both PL and PGL. In this study we report for the first time the production of pectic lyases from Debaryomyces nepalensis. The isolate was able to use lemon peel as carbon source and inducer for production of PL and PGL.
Materials and Methods
Chemicals
Apple pectin (Sigma) was used as the substrate for PL and polygalacturonic
acid (Sigma) was substrate for PGL. The other chemicals were of analytical grade
procured in India.
Media
The screening medium had the following composition (g L-1): CaCl2,
0.05; KH2PO4, 0.2; MgS04.7H20, 0.1;
(NH4)2SO4, 1.0; pectin, 0.5; agar, 15. The
pH of the medium was maintained at 6.4. The Basal Medium (BM) had the following
composition (g L-1): MgS04.7H20, 0.1; NH4Cl,
2; Na2HPO4, 6; K2HPO4, 3; NaCl,
5. The initial pH was adjusted to 7.0. The minimal medium (MM) had the same
composition of BM with glucose (8 g L-1). The isolates were maintained
in YEPD agar medium. YEPD medium has the following composition (g L-1):
yeast extract, 10; peptone, 20; dextrose, 20. For preparation of solid medium,
YEPD medium was supplemented with agar (15 g L-1).
Screening and Identification of Strains Producing PL and PGL
Samples (soil, rotten orange, apple and guava and decaying wood) were used
to screen for pectin degrading microorganisms. Each test sample (1 g) was mixed
with 10 mL of sterile saline and incubated for 15 min, 100 μL of this was
added to 900 μL of sterile saline and a serial dilution (10 -1
to 10-6) was prepared, of which 100 μL was added and distributed
on an screening medium. The plates were incubated at 30°C for two days.
Isolates capable of utilizing the pectin as sole source of carbon was picked
and sub-cultured to single colony. Pure isolates were further screened based
on their ability to produce PL and PGL by submerged fermentation in BM supplemented
with 0.5 g L-1 pectin in test tubes. Pectinase producing cultures
from National collection of industrial microorganisms (NCIM, Pune) and Institute
of Microbial Type Culture Collection (IMTECH, Chandigarh) were also screened
for PL and PGL production. Among all the tested strains, the best strain producing
PL and PGL was further characterized by 26S rDNA D1/D2 sequence analysis, which
was performed at National collection of yeast cultures (NCYC), Norwich, UK.
Production of PL and PGL by Submerged Fermentation
Prior to cultivation of isolated strains on production medium a loop of
the strain from YEPD agar plates was transferred to 5 mL sterile YEPD medium
and incubated on rotary shaker at 180 rpm and 30°C. After 12 h, 2% (v/v)
of the seed culture was transferred into a 100 mL Erlenmeyer flask containing
25 mL of medium and incubated at 180 rpm and 30°C. Pectic lyases production
was studied in BM and MM with and without pectin and polygalacturonic acid (5
g L-1).
Effect of Different Naturally Available Pectic Substances on PL and PGL
Production
The effect of different naturally available pectic sources (corn, peels
of orange, lemon and banana, sugarcane bagasse and sugar beet) on the production
of PL and PGL was studied. These natural sources were cut into small pieces,
dried in oven at 70°C for two days and ground to fine powder (approximately
200 μm) and stored in airtight bags. The naturally available pectic substance
used in this study act as an inducer and carbon source and was added to BM at
5 g L-1.
Enzyme Assay
Supernatant was used as the source for enzyme assay. PL activity was assayed
by measuring the formation of unsaturated oligogalacturonates at 235 nm (Albersheim,
1966). The reaction mixture contained 0.19% pectin in 100 mM citrate phosphate
buffer (pH 5.2) and 100 μL of enzyme. The assay mixture was incubated for
3 min at 30°C and the increase in absorbance at 235 nm was measured using
Perkin Elmer UV-Visible spectrophotometer. To test PGL activity pectin was replaced
by 0.15% (w/v) polygalacturonic acid in 75 mM Tris-HCl buffer (pH-8.0) with
1 mM CaCl2. One unit of enzyme activity was defined as an increase
of 1.0 unit of absorbance at 235 nm of the reaction mixture per minute (Nakagawa
et al., 2000).
Analytical Methods
Cell growth was monitored by measuring the absorbance at 600 nm (OD600
of 0.5 corresponds to 0.56 g dry weight L-1). Protein was measured
by the method of Lowry with bovine serum albumin as the standard. All the experiments
were performed in triplicates and the activity values reported are mean values
with ±5 to ±8% error.
Results and Discussion
Screening and Identification of Microorganism
Eleven isolates (2 fungal, 3 yeast and 6 bacterial isolates) were obtained
from natural sources capable of degrading pectin when pectin was used as the
sole source of carbon. In addition, five pure cultures from culture banks capable
of producing pectinases were selected for screening of pectic lyases production.
All the 16 isolates were tested for production of PL and PGL in submerged medium
and their activities were shown in Table 1. All the fungal
isolates produced only PL, whereas the bacterial isolates B4, B5, B8 produced
only PGL. However, three yeast strains GSP 101, 102 and 103 produced both PL
and PGL. Of this, isolate GSP 101 produced higher levels of PL and PGL. Hence,
this isolate was used for further studies. The isolate GSP 101 was a Gram-positive
with budding cells, showed creamy white smooth colonies on sabourand agar and
negative for Germ tube test, suggesting that isolate GSP 101 was non-pathogenic
yeast. The isolate was further characterized based on 26S rDNA analyses and
the sequence was compared using EMBL database. The D1/D2 sequence of the strain
shows 99.8% sequence identity for both Candida sp. BG02-6-6-2-1 and Debaryomyces
nepalensis. However, the strain was found to sporulate on corn meal agar
(CMA) after 17 days of incubation at 20°C, thus confirming its identity
as Debaryomyces nepalensis. The 26 S rDNA D1/D2 sequence is given in
the 5’ to 3’ direction (Fig. 1).
Many microorganisms have been reported to produce pectic lyases but most of them produce either PL or PGL. PL has been primarily produced by fungi (Alana et al., 1990; Manachini et al., 1998; Panda and Naidu, 2000; Piccoli-Valle et al., 2003) where as PGL by bacteria (Dave and Vaughn, 1971; McCarthy et al., 1985; Hayashi et al., 1997; Singh et al., 1999).
| Table 1: | Production of PL and PGL by different isolates and strains obtained from culture banks |
| Table 2: | Comparison of PL and PGL activities with and without calcium for crude and partially purified enzyme |
However, very few reports were available where both the pectic lyases being produced by single microorganism (Nakagawa et al., 2000; Soriano et al., 2005). This is the first report on production of both pectic transeliminases by Debaryomyces nepalensis.
Effect of Different Media on PL and PGL Production
The effect of different media on the growth and production of PL and PGL
by Debaryomyces nepalensis was studied. Maximum growth was observed when
glucose was used as the carbon source (MM) followed by MM supplemented with
pectin and polygalacturonic acid. The isolate was also able to grow in medium
containing pectin and polygalacturonic acid as the sole carbon source (Fig.
2a). PL and PGL activities were detected in BM and MM suggesting that the
enzymes are constitutive in nature. The production of pectic lyases was induced
by the presence of pectic substances in the medium. The strain showed highest
pectic lyases production when grown on basal medium supplemented with pectin
than compared to polygalacturonic acid (Fig. 2b and c).
The presence of glucose in the medium did not show any pronounced negative effect
on the production of pectic lyases, suggesting that pectic lyases production
in the isolate was not repressed by glucose. However, it has been reported that
PL and PGL production by Candida boidinii and Paenibacillus sp.
was repressed by the glucose in the medium (Nakagawa et al., 2000; Soriano
et al., 2005). In all the media, maximum PL and PGL were produced within
24-36 h of fermentation and the production was growth associated (Fig.
2a-2c). Maximum activities of PL and PGL by Debaryomyces
nepalensis obtained in this study were 3.2 and 2.3 U mL-1 respectively
in 24-36 h of fermentation.
| Fig. 1: | The 26 S rDNA D1/D2 sequence of the isolated strain Debaryomyces nepalensis |
These results were higher to those reported for pectic lyases production by different strains such as, 0.009 U mL-1 of PL by Aspergillus niger NCIM 548 (Panda and Naidu, 2000), 0.06 U mL-1 of PL by Penicillium griseoroseum CCT 6421(McCarthy et al., 1985), 0.07 U mL-1 of PL by Penicillium italicum CECT 2294 (Alana et al., 1990), 0.4 U mL-1 of PGL by Bacillus licheniformis (Singh et al., 1999), 0.96 U mL-1 of PGL by Pseudomonas marginalis (Hayashi et al., 1997) and 1.4 U mL-1 of PGL by Bacillus pumilis (Dave and Vaughn, 1971). However, Paenibacillus sp. (47.2 U mL-1 of PL and 23.4 U mL-1 of PGL) and Bacillus sp. (53 U mL-1 of PL) showed higher activities than reported in the present study (Kashyap et al., 2000; Soriano et al., 2005). The specific activities of PL (4.3 U mg-1) and PGL (3.3 U mg-1) reported in this report were much higher than reported for Candida boidinii (0.673 U mg-1 of PL and PGL) (Nakagawa et al., 2000). Production of pectic lyases in shorter fermentation time is advantageous and alternative to fungal production.
PL and PGL Production Using Naturally Available Pectic Substances
The ability of the isolate to produce PL and PGL using naturally available
pectic substances was studied to reduce the overall cost of fermentation (using
nutrient medium with pectin). For this purpose, corn, sugar beet and wastes
containing pectic substances such as orange, banana and lemon peels and sugar
cane bagasse (5 g L-1) was used as the sole carbon source and inducer
for production of PL and PGL. The isolate Debaryomyces nepalensis showed
maximum PL production in lemon peel medium, which is equal to that obtained
in BM, supplemented with pectin (Fig. 3a). The production
of PGL was same when BM was supplemented with lemon peel and orange peel but
lower than compared to BM supplemented with pectin (Fig. 3b).
From this result, it is clear that lemon peel was the best carbon source and
inducer for production of both PL and PGL among the tested pectic substances.
Hence, pectin in the medium can be replaced by lemon peel.
Characterization of Lyase Activities
In order to confirm whether the pectinase activity obtained in this study
was due to pectic lyases, the enzymatic reaction was performed as discussed
in materials and methods. The product of reaction was scanned from 200 nm to
500 nm using UV-Visible spectrophotometer. The results clearly showed that the
product of enzymatic reaction has maximum absorbance around 235 nm for both
PL and PGL (result not shown), which is due to the formation of unsaturated
oligogalacturonates by transelimination mechanism (Albersheim, 1966). It is
know that PGL has absolute requirement of calcium for its activity whereas PL
is independent of calcium. To study the effect of Ca2+ on PL and
PGL activity, the enzymatic reaction was performed with (1 mM) and without Ca2+
for PL and PGL activity. It was found that Ca2+ has no effect
on PL activity in the crude culture broth.
| Fig. 2: | Effect of different media on growth and production of PL and PGL by Debaryomyces nepalensis (a) Growth profile (b) PL activity profile © PGL activity profile. PGA: polygalacturonic acid |
But the absence of Ca2+ in the assay mixture slightly reduced the PGL activity. However, PGL activity was not completely abolished in the absence of Ca2+. Calcium is found abundantly in the cell walls of plants which is required for the in vivo activity of PGL in plants (Hepler and Wayne, 1985). The calcium ions present in the cell walls of lemon peel might have interfered with the PGL activity and hence enzyme assay were performed with both crude and partially purified enzyme (90% ammonium sulfate saturation and elute of anion exchange column).
| Fig. 3: | Effect of naturally available pectic substances on production of PL and PGL by Debaryomyces nepalensis in BM (a) PL activity profile (b) PGL activity profile |
It was found that activity of PL was not affected in the presence and absence of Ca2+ in crude and partially purified fractions. However, ammonium sulfate fraction shows reduction of PGL activity drastically by almost less than half without Ca2+ whereas in elute of Q6 PGL activity is completely abolished (Table 2). These results clearly indicate that presence of Ca2+ is absolutely necessary for PGL activity whereas calcium is not required for PL.
Acknowledgements
This study was supported by research grant from Department of Science and Technology, India. Authors thank National Collection of Yeast Cultures, Norwich, UK for their help in strain identification.