Purification and characterization of carboxymethyl cellulase and protease by Ulocladium botrytis Preuss ATCC 18042 using water hyacinth as a substrate under solid state fermentation

Abstract

The potential of 12 fungal strains to produce carboxymethyl cellulase (CMCase) and protease on Eichhornia crassipes (water hyacinth) wastes was investigated under conditions of solid state fermentation. Ulocladium botrytis (Preuss) was selected as the best fungus for the production of both enzymes. The best nitrogen sources for production of CMCase and protease were yeast extract and malt extract, respectively. CMCase and protease were purified by isopropanol (1:1) precipitation and column chromatography on Sephadex G-100 and DEAE-cellulose. Purification fold of 47.34 and 51.78, with 852.11 and 1,469.38 U/mg specific activity was achieved with 40.3 and 56.25% recovery after purification of CMCase and protease, respectively. The purified CMCase expressed its maximal activity at 60°C and pH 5.2, showed good stability in the pH range of 5.2–5.4 and its midpoint of thermal inactivation (Tm) was 60°C after 75 min exposure. The purified protease expressed its maximal activity at 35°C and pH 5.2, showed good stability in the pH range of 5.6–6.0 and its midpoint of thermal inactivation (Tm) was 35°C after 75 min exposure. The best substrate concentration for CMCase was 1.2% (w/v) Na-CMC and for protease, it was 0.8% (w/v) casein. The best enzyme concentration for the two tested enzymes was 0.4 U/ml. Ions of Ca²⁺, Na⁺ and K⁺ showed a stimulatory effect but sodium arsenate and iodoacetate showed an inhibitory effect. Moreover, Ag²⁺ and Hg²⁺ inhibited both enzyme activities completely. The purified enzymes from Ulocladium botrytis had molecular weights of 50 and 83 kDa for CMCase, and 47 kDa for protease on SDS-PAGE.

Introduction

Most aquatic systems are infested with one kind of aquatic weed or the other. The most common one is Eichhornia crassipes (water hyacinth). As it is non-endemic, there are no natural control mechanisms such as insects and fishes that feed on it (Gopal 1987). The cell wall of Eichornia crassipes consists of cellulose, hemicellulose, pectin and lignin (Abd-El-Naby 1988). Moreover, it contains a high amount of protein (Louboudy et al. 2001; Gunnarsson and Petersen 2007). Several attempts have been made to utilize this water plant in the production of some fungal cellulases (Louboudy et al. 2001; Ali and Saad El-Dein 2008; Deshpandel et al. 2008) or bacterial and fungal pectinases (Louboudy et al. 2001; Bayoumi et al. 2008).

As weed infestation in aquatic systems causes serious environmental problems, they must be eradicated. But all efforts to control the growth and spread of these weeds, including physical, chemical and biological methods have failed miserably or are too expensive to carry out on a regular basis. Hence the concept of eradication through utilization is being adopted by many countries, and researchers are focusing on new methods of utilizing these natural resources (Nagendra 2001; Calvert 2002).

Cellulase is used extensively in the textile and food industries, bioconversion of lignocellulosic wastes to alcohol, animal feed industry as additive, in the isolation of plant protoplasts, and in plant virus studies, metabolic investigations and genetic modification experiments (Evans and Bravo 1983; Potrykus and Shillito 1986; Bhat 2000).

Proteases are among the most important industrial enzymes (Joo et al. 2002) and account for about 65% of the worldwide sale of industrial enzymes in the world market (Johnvesly and Naik 2001).

Microorganisms serve as an important source of proteases, due mainly to their shorter generation time, the ease of bulk production and the ease of genetic and environmental manipulation (Patel et al. 2005).

Proteolytic enzymes are by far the most important group of enzymes produced commercially and are used in many areas of application, such as in detergents, and in the brewing, photographic, leather and dairy industries (Yang et al. 2000). For these reasons proteolytic enzymes are also the object of this study.

In the present work, different fungal species were tested for their capability to grow on water hyacinth (E. crassipes) wastes as a potential organic substrate offering a cheap source of cellulase and protease by solid state fermentation (SSF) for the production of carboxymethyl cellulase (CMCase) and protease. Ulocladium botrytis exhibited higher values of enzyme activities than the other tested fungi.

Materials and methods

Micro-organisms

The experimental isolates were obtained from NRRL (Agricultural Research Culture Collection) and ATCC (American Type Culture Collection). These isolates are Aspergillus candidus NRRL 4646, A. flavus NRRL 453, A. niger NRRL 326, A. terreus NRRL 255, A. ustus NRRL 5856, Fusarium scirbi NRRL 26922, Penicillium chrysogenum NRRL 792, P. citrinum NRRL 1841, P. claviforme (P. vulpinum) NRRL 1001, P. velutinum NRRL 2069, Trichoderma viride ATCC 8751 and Ulocladium botrytis ATCC 18042. The aforementioned isolates were screened for CMCase and protease production.

Culture medium

The cultures were kept on yeast extract agar at 4°C and cultured routinely. In order to study enzyme production, a triplicate set of 250 ml capacity Erlenmeyer conical flasks was used (for each treatment) containing 8 ml Waksman’s medium [KH₂PO₄ 1 g/l; CaC1₂ 0.5 g/l, and MgSO₄⋅7H₂O 0.5 g/l (without organic sources i.e., peptone or dextrose)] and 20 g fresh ground whole Eichhornia crassipes plants. The pH was adjusted to 5.0. Each group of three flasks was sterilized, inoculated with 2 ml of an evenly prepared spore suspension from each of the tested fungi (∼ 105 ml⁻¹ spores) and incubated for 7 days at 30°C. At the end of fermentation, the contents of each flask were gathered, mixed thoroughly with cooled distilled water (15 ml) and filtered rapidly through a Buchner funnel. The filtrate was then subjected to enzyme activity assays for the determination of CMCase and protease production of the tested fungi in order to select the best fungus, which was then used in further studies.

Protease activity assay

Protease activity was determined according to Kunitz (1947) using casein as a substrate. The reaction mixture containing 1 ml enzyme solution and 1 ml 1% (w/v) casein in 50 mM citrate phosphate buffer pH 6.0 was incubated at 30°C for 20 min. The reaction was stopped with 3 ml 10% (w/v) TCA and the mixture was centrifuged at 5,000 g for 10 min. The optical density of the supernatant was measured at 750 nm after folin reaction according to the method of Lowry et al. (1951). Protease activity was defined as the amount of enzyme liberating 1 μg tyrosine/min under assay conditions. Enzyme units were measured using tyrosine (30–300 μg) as a standard.

Carboxymethyl cellulase activity assay

CMCase assay was investigated for CMC saccharifying activity by incubating 0.5 ml enzyme solution with 0.5 ml Na-CMC 1% (w/v) in phosphate buffer (50 mM, pH 5.0) for 30 min at 37°C. Sugars released were estimated by dinitrosalicylic acid (DNS) reagent (Miller 1959). One unit of enzyme activity was defined as the amount of protein that produced 1 mmol product per minute under standard assay conditions.

Protein assay

Protein content was measured at 750 nm according to the method of Lowry et al. (1951) using bovine serum albumin (30–300 μg) as a standard.

Effect of enriching the water hyacinth (E. crassipes) medium

Medium enrichment with some natural additives, i.e., yeast, malt and beef extracts, soybean meal and corn-steep liquor 0.05% (w/v) was performed, and the levels of enzyme production in the enriched media were statistically compared to that of the water hyacinth medium, which serves as a control.

Purification of CMCase and protease

All purification steps were done at 4°C. The crude lyophilized culture supernatant (200 ml) was precipitated by isopropanol (1:1, v/v) and dissolved in 50 mM citrate phosphate buffer, pH 6.0, (5 ml) then dialyzed against the same buffer for 24 h at 4°C. The enzyme sample was gel-filtered through a Sephadex G-100 column (18 × 2 cm), pre-equilibrated with the same buffer. The elution was performed by the same buffer at a flow rate of 20 ml/h. Fractions of 5 ml were collected and assayed for their protein, CMCase and protease activities. The active fractions with the highest specific activity of each enzyme were pooled, mixed and dialyzed. The pooled fractions of each enzyme were further fractionated separately through a DEAE-cellulose column, and eluted with a 0–0.8 M-NaCl gradient in citrate-phosphate buffer (240 ml) at a flow rate of 10 ml/h. For each enzyme, 5 ml fractions were collected and assayed for their protein and enzyme activities. The most active fractions were pooled, mixed and dialyzed once again (Peterson and Sober 1962; Palmer 1991). These purified enzyme preparations were lyophilized and stored at 5°C for further investigations (Plummer 1978).

Characterization of purified CMCase and protease

Effect of pH

The effect of pH on enzyme activities was assessed by adding 1 ml CMCase solution to 1 ml 1% (w/v) Na-CMC and 1 ml protease solution to 1 ml 1% (w/v) casein, respectively, at different pH values (from 3.6 to 5.2) obtained using 0.05 M citrate-phosphate buffer. After incubation at 30°C for 30 min for CMCase and at 30°C for 20 min for protease, each enzyme activity was measured under the standard assay conditions as described above.

Effect of temperature

The effect of temperature on enzyme activities was assessed by incubating each enzyme with the corresponding substrate as previously described at various temperatures ranging from 20 to 70°C. Each enzyme activity was measured as mentioned before.

Effect of substrate concentration

This experiment was carried out to study the effect of substrate concentrations on the activity of the purified CMCase and protease, respectively. Different soluble concentrations (0.4–1.6, % w/v) each of Na-CMC and casein were used. Enzyme activity was measured to determine the optimum substrate concentration in each case.

Effect of enzyme concentration

Different purified CMCase and protease enzyme solutions ranging from 0.2 to 1.4 (mg protein/ml) were prepared. The activity of each solution was then measured.

Effect of different metal ions and some enzyme inhibitors

Various metal ions at final concentration of 1 and 10 mM were added to the enzyme solution (KCl, NaCl, CaCl₂, MgSO₄, FeSO₄, CuSO₄·5H₂O, AgNO₃, HgI₂ and ZnSO₄⋅H₂O) then, each metal ion was incubated with the two enzymes, before adding substrate. Activity of each enzyme in the complete absence of such compounds served as control (100% activity). Also, the effect of various inhibitors such as EDTA, sodium arsenate, l-cysteine and Iodoacetate was investigated, then the activities of the studied enzymes were measured.

Enzyme stability studies

pH stability

To determine pH stability, each enzyme was incubated at pH values ranging from 3.6 to 5.2 for two time intervals of 20 and 60 min. The original pH value was then restored and the residual activity for each enzyme was estimated under standard assay conditions. The results were expressed as relative activity (%) referred to the activity observed before incubation.

Thermal stability

For determination of enzyme thermal stability, the enzymes were incubated for varying times (0–60 min) at fixed temperatures (60–80°C for CMCase; 35–55°C for protease). After incubation, the residual activities of the enzymes were measured in the usual manner.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

For determination of molecular weight, the purified enzyme preparations and known molecular weight markers were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli (1970) using a 12.5% (w/v) acrylamide slab gel with 25 mM Tris / 192 mM glycerin buffer (pH 8.3) that contained 0.1% (w/v) SDS as the running buffer. After electrophoresis, the gel was stained for 1 h with Coomassie Blue R250 dye in a methanol-acetic acid-water solution (4:1:5, by volume) and then destained in the same solution without the dye.

Statistical validation of treatment effects

The mean, standard deviation, T-score and probability “P” values of three replicates of the investigated factors and the control were computed according to the mathematical principles described by Glantz (1992). Results were considered highly significant, significant or non-significant where P < 0.01, >0.01 and <0.05, >0.05, respectively.

Results and discussion

The chemical composition of E. crassipes wastes is shown in Table 1. The analytical data showed that leaves contained 21.5% cellulose, 33.9% hemicellulose, 13.75% crude protein and other nutritional elements. For this reason, CMCase and protease enzymes were chosen for this study.

Table 1 Analysis of water hyacinth composition

Different fungal species were tested for their capability to grow on water hyacinth medium (WH) as a rich organic source under solid state fermentation (SSF). The superiority of Ulocladium botrytis in CMCase and protease activities (29.6 and 23U/g, respectively) compared to the other tested fungi is clear from Fig. 1. Thus, U. botrytis was chosen for further investigation.

Fig. 1

Screening of fungal species for carboxymethylcellulase (CMCase; □) and protease (■) production when grown on water hyacinth (Eichhornia crassipes) wastes (WH) as a rich organic source under solid state fermentation (SSF) conditions

Some natural additives, i.e., yeast, malt and beef extracts, soybean meal and corn-steep liquor 0.05% (w/v) were used to increase production of CMCase and protease enzymes by U. botrytis grown on WH medium. It was found that the maximum productivity of CMCase (29.98 U/g) and protease enzymes (26.05 U/g) were recorded in the presence of yeast extract and malt extract 0.05% (w/v), respectively (Fig. 2). In this regard, El-Gindy et al. (2008) stated that these additives promote the production of enzymes in solid state cultures.

Fig. 2

Effect of enrichment WH medium with some natural additives on the production of CMCase (□) and protease (■)

A summary of purification steps of the CMCase and protease produced by U. botrytis is recorded in Tables 2 and 3, respectively. The results indicate a 1.36-fold purification in the case of CMCase, and 1.27-fold in the case of protease, with yields of 84.0% and 85.93%, respectively, of the original activities obtained. Most of the CMCase and protease activities were applied onto Sephadex G-100 and recovered in 35–40 fractions representing 63.3% and 64.45% of the original activity, corresponding to specific activities of 106.74 and 299.46 U/mg protein (5.93- and 10.55-fold purification, respectively). These fractions were pooled, lyophilized and subjected to further fractionation onto DEAE-cellulose using a linear sodium chloride gradient as eluant. This resulted in 47.34- and 51.78-fold purification, with yields of 40.3% and 56.25% of the initial CMCase and protease activities, respectively.

Table 2 Purification steps of carboxymethyl cellulase (CMCase) from Ulocladium botrytis grown on water hyacinth (WH) medium with yeast extract

Table 3 Purification steps of protease from U. botrytis grown on WH medium with malt extract

This purification procedure was also used by Po-Jui et al. (2004) for CMCase of Sinorhizobium fredii and resulted in 9.08-fold purification, the recovery yield was 26.4% and the specific activity was 3.822 U/mg; and by Datta (1992) for Phanerochaete chrysosporium protease, resulting in 37-fold purification.

The secreted enzyme level per total secretory protein level was calculated as 7.98% and 17.78% for CMCase and protease, respectively. The accumulated extracellular enzyme activities were calculated by U/g dry cell weight (dcw) during fermentation as 175 and 245 U/g dcw for CMCase and protease, respectively.

Testing the pH-dependence of the activity of CMCase and protease revealed that pH 5.2 was the optimum for both CMCase and protease activities (Table 4); moreover, there was high inactivation of the two enzyme activities on both the extreme sides of the pH curve. This can be attributed to the decreasing affinity of the enzyme to its substrate or due to an irreversible destruction of the enzyme protein. It may also be due to the effect of pH on the stability of the enzyme (Dixon and Webb 1979). This optimum pH value (5.2) was identical to that found for CMCase from Macrophomina phaseolina (Roy and Vora 1989). In contrast, these findings are not in accordance with earlier reports showing pH optima of 7–7.5 for protease from Aspergillus oryzae (Alagarsamy et al. 2005).

Table 4 General properties of carboxymethyl cellulase (CMCase) and protease enzymes

The optimum incubation temperatures were 60°C and 35°C for CMCase and protease, respectively (Table 4), while lower activities were seen at either side of these values. Similarly, Roy and Vora (1989) stated that CMCase showed optimum activity at 65°C, while Usama and Saad El Dein (2008) reported that the optimal temperatures of CMCase produced from A. niger, A. nidulans and A. terreus were (30, 35 and 40°C), respectively. But for protease, Abdul-Raouf (1990) stated that the optimum incubation temperature for purified protease activity was 35°C. In addition, Lee et al. (2002) reported that the optimum temperature of purified protease ranged from 40°C to 50°C. Moreover, Ammar et al. (2003) reported that the optimum temperature for thermostable purified protease was 55°C.

The effect of different substrate concentrations was also studied (Fig. 3). The protease reached its maximal activity with a casein concentration of 0.8% (w/v) but, for CMCase, the optimum substrate concentration was 1.2% (w/v) Na-CMC. There is a decrease in both enzyme activities either side of these optimum values. This is complete agreement with Abdul-Raouf (1990), who also stated that an increase or decrease in substrate concentration led to a decrease in enzyme activity.

Fig. 3

Effect of substrate concentration on activity of purified CMCase (▲) and protease (■) from U. botrytis

With regard to enzyme concentration, Fig. 4 shows that the highest CMCase and protease activities were achieved at an enzyme concentration of 0.4 U/ml. This behavior is in accordance with the observations of West et al. (1967), who stated that, within fairly wide limits, the speed of enzyme activity is directly proportional to the enzyme concentration. The same finding also was reported by Abd El-Rahman (1990), El-Safey (1994), and El-Safey and Ammar (2003).

Fig. 4

Effect of enzyme concentration on activity of purified CMCase (▲) and protease (■) from U. botrytis

The effect of some metal ions and enzyme inhibitors was also studied. The results demonstrated (Table 5) that CMCase and protease activities increased significantly in the presence of Ca²⁺, Mg²⁺, Na⁺ or K⁺. Activities of CMCase and protease were inhibited in the presence of Zn⁺ or Cu²⁺, and severely inhibited in the presence of Ag²⁺ and Hg²⁺. Moreover, various enzyme inhibitors were added to the reaction mixture to test their influence on both enzyme activities. The most injurious inhibitors were Ag²⁺, Hg²⁺ and sodium arsenate in addition to iodoacetic acetate. These results were in accordance with those of Kim et al. (2009), who mentioned that CMCase activity was enhanced by some metal ions such as K⁺ but inhibited by Hg²⁺. Similarly, Nongporn et al. (1999) stated that Ca²⁺ was particularly effective in activating enzyme activity, causing 85% stimulation, while Mn²⁺ and Mg²⁺ at the same concentration showed a less positive effect. Similar results were observed by Tsuchiya et al. (1987), who reported that protease isolated from Colosporium sp. was inhibited by Hg²⁺; furthermore, Nehra et al. (2004) reported that Mg²⁺ was found to activate the alkaline protease enzyme produced by Aspergillus sp. Metal ions may stimulate enzyme activity by acting as a binding link between enzyme and substrate, combining with both and so holding the substrate in the active site of the enzyme (Mahmoud et al. 1968). The effect of metal ions on enzyme activity might also be due to changes in electrostatic bonding that would change the tertiary structure of enzymes (Roy et al. 1990).

Table 5 Effect of different metal ions and some enzymes inhibitors on the relative activities of purified CMCase and protease from Ulocladium botrytis

Incubation of each enzyme preparation at different pH values for either 20 or 60 min demonstrated that CMCase was almost unaffected by incubation in the pH range 5.2–5.4 for either 20 or 60 min (Table 4). These results were in accordance with the results of Coral et al. (2002), while protease was almost unaffected by incubation in the pH range 5.6–6.0 for either 20 or 60 min (Table 4). These results were in accordance with those of Datta (1992).

Moreover, the midpoint of thermal inactivation CMCase (Tm) was determined and recorded at 60°C after 75 min of exposure. The CMCase enzyme retained its original activity after heating to 60°C for 75 min, while no activity was recorded after heating the enzyme at 80°C for 75 min (Table 4). The protease enzyme retained its original activity after heating to 35°C for 75 min, while no activity was recorded after heating the enzyme at 50°C for 90 min and 55°C for 75 min (Table 4). The Tm was recorded at 35°C after 75 min of exposure. The thermal inactivation of enzymes is nearly always due to denaturation of the enzyme proteins (Dixon and Webb 1979). Temperature is a cardinal factor affecting the amount and rate of growth of an organism (Garg et al. 1985) and increasing temperature has the general effect of increasing enzyme activity (Moor 1990), but the enzyme begins to suffer from thermal inactivation at higher temperatures.

The purified enzyme preparation was subjected to SDS-PAGE containing 0.2% (w/v) Na-CMC to determine the homogeneity and molecular weight of the enzyme. During electrophoresis of the enzymes, two bands showing cellulytic activity were detected. As shown in Fig. 5, the molecular weights of these proteins were calculated to be about 83 and 50 kDa for β-glucosidases (BGL) and cellobiohydrolases (CBH), respectively (Mehdi et al. 2009), while the molecular weight of the proteolytic enzyme was calculated to be about 47 kDa (Fig. 6).

Fig. 5

Separation of U. botrytis CMCase by SDS-PAGE. Electrophoresis was carried out in an SDS-polyacrylamide gel containing 0.2% CMC. Lanes: A CMCase from U. botrytis, M molecular weight markers

Fig. 6

Separation of U. botrytis protease by SDS-PAGE. Electrophoresis was carried out in an SDS-polyacrylamide gel containing 0.1% casein. Lanes: A Protease from U. botrytis, M molecular weight markers