Screening of Yeast Strains for Pectinolytic Activity: Effects of Different Carbon and Nitrogen Sources in Submerged Fermentations

Correspondence Address: Mustafa Oskay Department of Biology, Faculty of Arts and Sciences, Section of Basic and Industrial Microbiology, Celal Bayar University, Manisa, Turkey Tel: +90 236 201 3265 Fax: +90 236 201 3040 E-mail: mustafa.oskay@cbu.edu.tr Abstract: Twenty yeast strains were screened for pectinolytic activity, among the strains, only nine were positive for pectinase production. The best strain was Kluyveromyces marxianus NRRL-Y-1109 which gave high quantities of pectinase activity, by submerged fermentation. Different parameters such as incubation time, pH (3.5-7), temperature (20-45°C), nitrogen and carbon source, were optimized. The optimal incubation time, temperature and pH for pectinase production were found to be 48 h, 30°C and 6, respectively. Studies were conducted on the production of pectinase in submerged fermentation using agro-industrial residues such as wheat bran, grape waste, brewer's malt, beet molasses and corncob at a concentration of 1.5% (wt./vol.). Under optimized fermentation conditions, maximal enzyme were produced when citrus pectin (4.8 U/mL) was used as the carbon source, but high enzyme production was also obtained on wheat bran (2.2 U/mL) and grape waste (1.8 U/mL) in shaking conditions (120 rpm) for 48 h. Peptone and yeast extract used together as nitrogen source gave best enzyme production. The effects of temperature (30-70°C), pH (3.5-8) and salt concentration (1, 2, 5 and 10%) on the pectinase activity were determined. The optimum activity was obtained when temperature 45°C and pH at 5.5. The enzyme was stable at 45°C for more than 50 min, suggesting that it is relatively thermostable and being of interests for food processing.


Introduction
The production of enzymes has evolved rapidly and nowadays, enzymes are the most important products obtained for human needs through microbial sources. Enzymes have application in a variety of areas including food biotechnology, environment, animal feed, pharmaceutical, textile, paper and others technical and chemical industries. Due to the large industrial application and significant cost, there is a necessity to develop processes able to minimize the production costs. In this sense, the production of enzymes by a variety of agro-industrial wastes may be used as support material, carbon and nitrogen sources that would allow obtaining more economical fermentation processes avoiding the use of expensive chemical components in the media formulation (Mussatto et al., 2012).
Pectinases comprise several enzymes that promote the natural degradation of pectins, which are high molecular weight acid polysaccharides, present in plant cell walls, primarily made up of α-(1→4) linked D-galacturonic acid residues with a small number of rhamnose residues in the main chain and arabinose, galactose and xylose in the side chain (Blanco et al., 1999;Kashyap et al., 2001;Pedrolli et al., 2009). The presence of pectic substances causes problems in the elaboration of fruit juices, particularly during the clarification and filtration steps (Gummadi and Panda, 2003;Serrat et al., 2011). Microbial pectinolytic enzymes are known to play a commercially important role in the current biotechnological area widely used in industry, with applications such as the extraction and clarification of fruit juices, wastewater treatment, tea and coffee fermentations, bleaching of paper, the production of fermentable sugars from plant biomass, oil and pigment extraction and processing of textile fibers (Favela-Torres et al., 2006;Sharma et al., 2012). They are also used in formulations of in poultry feed additives, contributing to better nutrient assimilation. In the industrial production of fruit juices, it is necessary to eliminate the pectin released during fruit processing in order to reduce the time of filtration and enhance the production at the end of the process (da Silva et al., 2005;Serrat et al., 2011;Mehraj Pasha et al., 2013).
Pectinase production used in industry has been reported from microorganisms including bacteria (Swain and Ray, 2010;Roosdiana et al., 2013), actinomycetes (Kuhad et al., 2004;Jacob and Prema, 2006), filamentous fungi in particular of the Aspergillus niger (Solis-Pereira et al., 1993), Penicillium pinophlilum (Ahmed and Mostafa, 2013), Penicillium viridicatum (Silva et al., 2002), Mucor circinelloides (Thakur et al., 2010) and some yeast (da Silva et al., 2005;Oliveira et al., 2006;Serrat et al., 2011;Taskin, 2013). Aspergillus niger is known worldwide for production of secondary metabolites and extracellular enzymes of commercial value, including industrial production of pectinases Yeasts have advantages compared to filamentous fungi with regard to the production of pectinases, because they are unicellular, the growth is relatively simple and the growth medium does not require an inducer. Yeasts have a great potential for the production of microbial enzymes for the food industry and they offer an alternative source of these enzymes (da Silva et al., 2005;. Although some yeasts produced one or two types of pectinolytic enzymes, the production of pectinase is not so widespread in them. Very few yeasts show this ability namely those belonging to the genera Saccharomyces, Kluyveromyces, Cryptococcus, Rhodotorola and Candida (Schwan et al., 1997;Miura et al., 2001;Moyo et al., 2003).
At present filamentous fungi, produce almost all the pectinolytic enzymes used for industrial applications.
There are a few reports of pectinase production by yeast strains. Therefore, aim of the present study, was to select yeast strains, which are able to secrete PGase and characterize some of the properties of the enzymes produced by submerged fermentation (SmF) processes. In addition, enzyme productions on various agroindustrial residues, nutritional and environmental conditions required for the production of extracellular pectinase activity have been described.

Selection for Pectinolytic Activity (Plate Assays)
For plate assay, yeasts were spot inoculated in the solid medium including citrus pectin (1%) and incubated for 72 h at 28ºC. After, iodine-potassium iodide solution or 6 M HCI was added to detect clearance halos around the colonies. Strains presenting large clearing zones were used for enzyme production assays on liquid medium. The strain, Kluyveromyces marxianus NRRL-Y-1109 with the best PGase production in the qualitative assay, was used for further experiments.

Selection of Suitable Culture Conditions for the Optimum Production of PGase
Various different cultural parameters such as pH, temperature, carbon and nitrogen sources and time incubation in hours were studied to monitor their effect on PGase production of the K. marxianus was optimized.
To determine, influence of initial pH value of culture medium on growth and enzyme production; the strain was cultivated in the medium with different initial pH values (3.5-7). The pH was adjusted using 0.1 N hydrochloric acid or 0.1 N sodium hydroxide. The initial pH of culture medium achieved maximum enzyme production was used for subsequent study.
The optimum temperature for cell growth and enzyme yield was assayed by incubating the production medium at different temperatures varying from 20-45°C, maintaining all other conditions at optimum levels at original concentration.
For determinations of the influence of different carbon (glucose, glycerol, starch, fructose, galactose, xylan, carboxymethyl cellulose and sucrose) and nitrogen sources (yeast extract, peptone, tryptone, asparagine, alanine, potassium nitrate, soybean meal and urea) were added to the pre-optimized medium before sterilization at a concentration of 1% (wt./vol.) and 0.1% (wt./vol.), respectively. The carbon and nitrogen source supporting the maximum production of enzyme were selected for the further studies. The temperature and pH were set to 30°C and at 6, respectively.
During fermentation, up to 72 h at a regular interval of 12 h, cell free supernatant of fermentation broth was harvested by centrifugation at 11000 rpm for 10 min at 4°C and assayed for enzyme activity.

Determination of the Influence of Agro-Industrial Residues for PGase Activity
Wheat straw, beet molasses, wheat bran, grape waste, brewer's malt and corncob were ground to particles of 0.2-0.5 cm. For delignification, 100 g of substrate in 500 mL Erlenmeyer flasks were soaked in 1% NaOH and autoclaved at 121°C for 20 min. After the alkali treatment, the materials were washed with tap water until neutral and oven-dried. Treated substrates were passed through 0.5 mm screens (Pham et al., 1998).
The pectinase activity of the strains (mm): ≥15-Very good producers; 10-15-good producers; 5-10-weak producers; +, 4 > poor producers. a Data presented as the mean value of three determinations ± standard deviation, 0-negative, clear zones not in Agro-industrial wastes as a carbon sources replaced with citrus pectin, by adding them separately at a final concentration of 1.5% (wt./vol.) in the optimized medium.

PGase Production and Fermentation
For PGase production, initially in a 100 mL Erlenmeyer flask containing 25 mL of propagation medium the strain was inoculated with a single loopful of a 48 h yeast culture from YPG plates for development of inoculums. The culture was then incubated at 30ºC in an orbital shaker at 150 rpm for 12 h, after which the growing culture (1%, v/v) was transferred into a 250 mL Erlenmeyer flask containing 50 mL of propagation medium and incubated for an additional 10 h under the same conditions. This culture was used as the inoculum in subsequent experiments. SmF was carried using 250 mL Erlenmeyer flasks with 40 mL of optimized production medium in a rotary shaker (120 rpm) at 30°C. After 48 h the biomass was separated by centrifugation at 11000 rpm for 10 min at 4°C and the supernatant was used to evaluate PGase activity.

Assay of PGase Activity in Supernatants
Assay of PGase activity was determined by measuring the release of reducing groups using the modified dinitrosalicylic acid (DNS) method described by Miller (1959). Briefly, the reaction mixture containing 250 µL of 1% citric pectin in 250 µL citrate-phosphate, pH 6.0 buffer and 100 µL of cell free culture supernatant, was incubated at 30 ºC for 5 min under static conditions. The reaction was stopped using the 3, 5-dinitrosalicyclic acid reagent followed by keeping at 100°C for 5 min for development of color. After heating for 5 min in boiling water, the reaction mixture was centrifuged (8000 rpm for 5 min) to separate out the insoluble pectinolytic materials formed during reaction. A control mixture deprived of pectin and another mixture deprived of cell free supernatant were assayed in parallel tests. The absorbance read at 540 nm using Varian Carry50 UV-visible Spectrophotometer. One Unit (U) of PGase activity was defined as the amount of enzyme required to liberate one µmol of galacturonic acid per minute under the assay conditions.

Effect of Temperature, pH and Salt Concentration on PGase Activity
The optimum temperature of PGase activity was determined by incubating the enzyme at different temperatures ranging from 30 to 70°C at pH 5.5 and the pH profile (3.5-8) at 45°C was evaluated in the presence of citrus pectin solubilized in appropriate buffers. Residual enzyme activity was measured by the standard assay. The effect of the presence of different concentrations of NaCI (1, 2, 5 and 10%) on PGase activity was also measured under optimized conditions. Thermal stability of the enzyme extract was determined by first incubating the enzyme at 50°C for intervals of 5, 25, 45, 50 and 60 min at pH 5.5 and the residual enzyme activity measured.

Pectinolytic Activity of the Yeast Strains
Twenty yeast strains were evaluated for their potential to pectinase activity in solid medium containing citrus pectin. Based on the size of the clear zones, among the strains, only nine were positive for pectinase activity (45%). Yeast strains were classified as very good producers of pectin depolymerizing enzymes when presented clear halos around colonies of at least 15 mm (Kluyveromyces marxianus NRRL-Y-1109), good producers when the halos were of at least 10 mm (Pichia pastoris, Candida rugosa, Saccharomyces cerevisiae NRRL-Y-12632 and Kluyveromyces marxianus TEM-4), weak producers when halos were at least 5 mm (4 strains), poor producers when no pectinolytic activity and no clear lysis zones were observed (11 strains) ( Table 1). The yeast K. marxianus NRRL-Y-1109 was found excrete high level of extracellular pectinolytic activity on pectin agar and was selected for further studies.

Optimization of the Suitable Culture Conditions for the PGase Production
Preliminary fermentation studies were conducted in medium containing pectin as the primary carbon source to production of pectinase of yeast strain at pH 6 and temperature at 28°C under shaking conditions (150 rpm). Maximum pectinase values of K. marxianus were ranging from 0.4 to 3.6 U/mL, obtained at 72 h incubation (Data not shown). Various different cultural parameters were tested such as pH (3.5-7), temperature (20-45°C), commercial carbon and nitrogen sources and time incubation in hours (12-72) were studied to monitor their effect on optimum PGase production of K. marxianus. The optimum pectinase activity was achieved at pH 6, 30°C and shaking conditions at 120 rpm after 48 h of incubation (Table 2). PGase production of K. marxianus in liquid medium has been shown to be influenced by the type and source of carbon and nitrogen. The highest (4.2 U/mL) of PGase was produced on citrus pectin (1% wt./vol.) as a pectinase inducer, was the best source followed by glucose (2 U/mL), galactose (1.8 U/mL) and starch (1.6 U/mL), (Table 3). On the other hand, amongst various nitrogen sources tested for PGase activity, best nitrogen sources were found yeast extract and peptone (0.1%, 1:1) (4.8 U/mL) supplemented to production medium under optimized physiological conditions (Table 3). Yeast extract (3.8 U/mL) and soybean meal (3.6 U/mL) alone gave comparable activity. When the K. marxianus was grown on urea, as a sole nitrogen source, pectinase activity was detected in the supernatant very low (0.4 U/mL). As seen from Table 3 nitrogen free production medium decreased the PGase activity (1.6 U/mL).

Influence of the Agro-Industrial Residues for PGase Activity
The influence of different agro-industrial residues on extracellular PGase production was investigated. In these experiments, substrat dosage has been chosen according to the literature reported value and further optimization will have to be carried out. Enzyme activities were measured after 48 h of incubation at 30°C at pH 6. The highest of PGase was produced on citrus pectin (4.8 U/mL) following treated wheat bran (2.2 U/mL) and treated grape waste (1.8 U/mL) whereas much lower levels of activities were produced with wheat straw and corncob (0.6 U/mL) ( Table 4).

Effect of Temperature, pH and Salt Concentration on PGase Activity
The optimum reaction pH, temperature and NaCI concentrations were investigated with crude culture supernatant. The optimum pH and temperature for the enzymatic activity were 5.5 and 40-45°C, respectively. When the effect of NaCI concentrations was studied, optimum PGase activity observed at 1% NaCI and was not stable up to 5 and 10% NaCI. The extracellular pectinase of K. marxianus was relatively thermostable and the long-term temperature stability of crude PGase at pH 5.5 was stable at temperatures up to 45°C even after 50 min as it retained over 100% for activity (Table 5).   a In production medium, pH and temperature were 6 and 30°C, respectively. Mean values, n = 3, enzyme activity was expressed as the U/mL ± 0.1 Table 4. Effect of different agro-industrial residues on production of PGase by K. marxianus

Discussion
It has been reported that strains belonging to the yeast species, K. marxianus have been isolated from a great variety of habitats, which results in a high metabolic diversity and a substantial degree of intraspecific polymorphism. This yeast is most important in different biotechnological applications: Production of enzymes (βgalactosidase, β-glucosidase, inulinase and polygalacturonases, among others), of single-cell protein, of aroma compounds and of ethanol (including hightemperature and simultaneous saccharification-fermentation processes); reduction of lactose content in food products; production of bioingredients from cheese-whey; bioremediation; as an anticholesterolemic agent; and as a host for heterologous protein production have been investigated (Serrat et al., 2004;Fonseca et al., 2008).
Twenty yeast strains subjected to the preliminary screening in solid medium, only nine of them were positive for pectinase activity. K. marxianus selected further screening to verify its consistent enzymatic activity. Schwan et al. (1997) isolated 12 yeast strains from cocoa fermentations, among them only four showed extracellular pectinase activity. They also reported K. marxianus was the most pectinolytic activity.
Results of the primary fermentation studies suggested that the maximum pectinase values of K. marxianus were ranging from 0.4 to 3.6 U/mL in medium containing pectin as the sole carbon source at pH 6 and temperature at 28°C under shaking conditions (150 rpm) obtained at 72 h incubation. On the other hand, in the optimized culture conditions: Optimum pectinase activity was achieved at pH 6, 30°C and shaking conditions at 120 rpm after 48 h of incubation ( Table 2). The pectinase secreted by K. wickerhamii has optimum conditions were as follows: pH 5.0, Temperature, 32°C and an incubation period of 91 h (Moyo et al., 2003). Earlier, the optimal conditions for PGase activity (9.8 U/mL) reported from K. marxianus were pH, 4.6 and temperature, 31°C when beet juice was the used as a substrate (Serrat et al., 2011). In our study, the highest (4.2 U/mL) of PGase was produced on citrus pectin, was the best carbon source followed by glucose (2 U/mL), galactose (1.8 U/mL) and starch (1.6 U/mL). The wildtype yeast Wickerhamomyces anomalus (Pichia anomala) was able to produce PGase in liquid medium containing glucose and citrus pectin (51 U/mL) . PGase from Aspergillus niger in SmF, was strongly decreased when glucose or sucrose (3%) was added to culture medium containing pectin (Solis-Pereira et al., 1993). PGase activity from culture broths which including the different nitrogen sources in a basal medium were determined for testing maximum enzyme production, best nitrogen sources were found yeast extract and peptone (0.1%, 1:1) (4.8 U/mL) under optimized physiological conditions (Table 3). Swain and Ray (2010) indicated the optimum temperature, pH and incubation period for exo-PGase production (82-83.2 U/mL) from Bacillus subtilis were 50°C, 7 and 36 h, respectively. However, in their study, culture medium with peptone (1%) was found best nitrogen source for enzyme production. It has been reported that maximum an extracellular PGase activity of Mucor circinelloides, was obtained in 48 h at 30°C and pH 4 with pectin methyl ester (1% wt./vol.) as carbon source and a combination of casein hydrolysate (0.1% wt./vol.) and yeast extract (0.1% wt./vol.) as nitrogen source (Thakur et al., 2010).
It has been reported that wheat bran was proved the best substrate for Streptomyces lydicus (Jacob and Prema, 2006), Penicillium viridicatum (Silva et al., 2002), Moniliella sp. and Penicillium sp. (Martin et al., 2004) for the maximal activity value of PGase in submerged fermentation. Similarly, in this study the highest of PGase was achieved with treated wheat bran (2.2 U/mL) and treated grape waste (1.8 U/mL) as an agro-industrial substrate. It should be noted that the differences and lower PGase production by yeast strains grown on agro-industrial end products might be attributed to less accessible sites in the substrate for enzyme attack due to the presence of pectin.
Effect of pH, temperature and NaCI concentration on PGase activity of K. marxianus were 5.5, 40-45°C and 1%, respectively. The optimum pH and temperature are also consistent with values that have been found for K. marxianus pectinase production of 5 and 40°C respectively (Schwan et al., 1997). Our results are in agreement with those reported by other authors (Masoud and Jespersen, 2006; who indicated the most yeasts secreted typical of pectinolytic enzymes.
Based on the results of these experiments, the yeast strain K. marxianus produced PGase possessing different activity of pH, temperature, NaCI concentration and thermal stability. The enzyme solution of yeast in this study has specific properties that may be applied directly to fruit juice industries without the pH modification. Nevertheless, because of the temperature stability of the enzyme offer advantages at processing temperature of 40-50°C, this is sufficient for industrial process, although enzyme preparation could be improved.