Ann Microbiol (2012) 62:1547–1556 DOI 10.1007/s13213-011-0409-0

ORIGINAL ARTICLE

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

Heba I. Abo-Elmagd & Manal M. Housseiny

Received: 25 June 2011 /Accepted: 16 December 2011 /Published online: 11 February 2012 # Springer-Verlag and the University of Milan 2012

Abstract The potential of 12 fungal strains to produce of 50 and 83 kDa for CMCase, and 47 kDa for protease on carboxymethyl cellulase (CMCase) and protease on Eich- SDS-PAGE. hornia crassipes (water hyacinth) wastes was investigated under conditions of solid state fermentation. Ulocladium Keywords Carboxymethyl cellulase . Protease . botrytis (Preuss) was selected as the best for the Ulocladium . 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 puri- fied by isopropanol (1:1) precipitation and column chroma- Introduction tography on Sephadex G-100 and DEAE-cellulose. Purification fold of 47.34 and 51.78, with 852.11 and Most aquatic systems are infested with one kind of aquatic 1,469.38 U/mg specific activity was achieved with 40.3 weed or the other. The most common one is Eichhornia and 56.25% recovery after purification of CMCase and crassipes (water hyacinth). As it is non-endemic, there are protease, respectively. The purified CMCase expressed its no natural control mechanisms such as insects and fishes maximal activity at 60°C and pH 5.2, showed good stability that feed on it (Gopal 1987). The cell wall of Eichornia in the pH range of 5.2–5.4 and its midpoint of thermal crassipes consists of cellulose, hemicellulose, pectin and inactivation (Tm) was 60°C after 75 min exposure. The lignin (Abd-El-Naby 1988). Moreover, it contains a high purified protease expressed its maximal activity at 35°C amount of protein (Louboudy et al. 2001; Gunnarsson and and pH 5.2, showed good stability in the pH range of 5.6– Petersen 2007). Several attempts have been made to 6.0 and its midpoint of thermal inactivation (Tm) was 35°C utilize this water plant in the production of some fungal after 75 min exposure. The best substrate concentration for cellulases (Louboudy et al. 2001; Ali and Saad El-Dein CMCase was 1.2% (w/v) Na-CMC and for protease, it was 2008;Deshpandeletal.2008) or bacterial and fungal 0.8% (w/v) casein. The best enzyme concentration for the pectinases (Louboudy et al. 2001; Bayoumi et al. 2008). two tested enzymes was 0.4 U/ml. Ions of Ca2+,Na+ and K+ As weed infestation in aquatic systems causes serious showed a stimulatory effect but sodium arsenate and iodoa- environmental problems, they must be eradicated. But all cetate showed an inhibitory effect. Moreover, Ag2+ and Hg2+ efforts to control the growth and spread of these weeds, inhibited both enzyme activities completely. The purified including physical, chemical and biological methods have enzymes from Ulocladium botrytis had molecular weights 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 H. I. Abo-Elmagd M. M. Housseiny (*) natural resources (Nagendra 2001; Calvert 2002). Department of Biological and Geological Sciences, Cellulase is used extensively in the textile and food Faculty of Education, Ain Shams University, Roxy, Cairo, Egypt industries, bioconversion of lignocellulosic wastes to alco- e-mail: [email protected] hol, animal feed industry as additive, in the isolation of plant 1548 Ann Microbiol (2012) 62:1547–1556 protoplasts, and in plant virus studies, metabolic investiga- sterilized, inoculated with 2 ml of an evenly prepared spore tions and genetic modification experiments (Evans and Bravo suspension from each of the tested fungi (∼ 105 ml−1 spores) 1983;PotrykusandShillito1986; Bhat 2000). and incubated for 7 days at 30°C. At the end of fermenta- Proteases are among the most important industrial enzymes tion, the contents of each flask were gathered, mixed thor- (Joo et al. 2002) and account for about 65% of the worldwide oughly with cooled distilled water (15 ml) and filtered sale of industrial enzymes in the world market (Johnvesly and rapidly through a Buchner funnel. The filtrate was then Naik 2001). subjected to enzyme activity assays for the determination Microorganisms serve as an important source of pro- of CMCase and protease production of the tested fungi in teases, due mainly to their shorter generation time, the ease order to select the best fungus, which was then used in of bulk production and the ease of genetic and environmen- further studies. tal manipulation (Patel et al. 2005). Proteolytic enzymes are by far the most important group Protease activity assay of enzymes produced commercially and are used in many areas of application, such as in detergents, and in the brew- Protease activity was determined according to Kunitz (1947) ing, photographic, leather and dairy industries (Yang et al. using casein as a substrate. The reaction mixture containing 2000). For these reasons proteolytic enzymes are also the 1 ml enzyme solution and 1 ml 1% (w/v) casein in 50 mM object of this study. citrate phosphate buffer pH 6.0 was incubated at 30°C for In the present work, different fungal species were tested 20 min. The reaction was stopped with 3 ml 10% (w/v) TCA for their capability to grow on water hyacinth (E. crassipes) and the mixture was centrifuged at 5,000 g for 10 min. The wastes as a potential organic substrate offering a cheap optical density of the supernatant was measured at 750 nm source of cellulase and protease by solid state fermentation after folin reaction according to the method of Lowry et al. (SSF) for the production of carboxymethyl cellulase (1951). Protease activity was defined as the amount of (CMCase) and protease. Ulocladium botrytis exhibited enzyme liberating 1 μg tyrosine/min under assay conditions. higher values of enzyme activities than the other tested Enzyme units were measured using tyrosine (30–300 μg) as fungi. a standard.

Carboxymethyl cellulase activity assay Materials and methods CMCase assay was investigated for CMC saccharifying Micro-organisms 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 The experimental isolates were obtained from NRRL (Ag- 30 min at 37°C. Sugars released were estimated by dinitro- ricultural Research Culture Collection) and ATCC (Ameri- salicylic acid (DNS) reagent (Miller 1959). One unit of can Type Culture Collection). These isolates are Aspergillus enzyme activity was defined as the amount of protein that candidus NRRL 4646, A. flavus NRRL 453, A. niger NRRL produced 1 mmol product per minute under standard assay 326, A. terreus NRRL 255, A. ustus NRRL 5856, Fusarium conditions. scirbi NRRL 26922, Penicillium chrysogenum NRRL 792, P. citrinum NRRL 1841, P. claviforme (P. vulpinum) NRRL 1001, P. velutinum NRRL 2069, Trichoderma viride ATCC Protein assay 8751 and Ulocladium botrytis ATCC 18042. The aforemen- tioned isolates were screened for CMCase and protease Protein content was measured at 750 nm according to the production. method of Lowry et al. (1951) using bovine serum albumin (30–300 μg) as a standard. Culture medium Effect of enriching the water hyacinth (E. crassipes) The cultures were kept on yeast extract agar at 4°C and medium cultured routinely. In order to study enzyme production, a triplicate set of 250 ml capacity Erlenmeyer conical flasks Medium enrichment with some natural additives, i.e., yeast, was used (for each treatment) containing 8 ml Waksman’s malt and beef extracts, soybean meal and corn-steep liquor medium [KH2PO4 1 g/l; CaC12 0.5 g/l, and MgSO4⋅7H2O 0.05% (w/v) was performed, and the levels of enzyme 0.5 g/l (without organic sources i.e., peptone or dextrose)] production in the enriched media were statistically com- and 20 g fresh ground whole Eichhornia crassipes plants. pared to that of the water hyacinth medium, which serves The pH was adjusted to 5.0. Each group of three flasks was as a control. Ann Microbiol (2012) 62:1547–1556 1549

Purification of CMCase and protease Effect of enzyme concentration

All purification steps were done at 4°C. The crude lyophi- Different purified CMCase and protease enzyme solutions lized culture supernatant (200 ml) was precipitated by iso- ranging from 0.2 to 1.4 (mg protein/ml) were prepared. The propanol (1:1, v/v) and dissolved in 50 mM citrate activity of each solution was then measured. phosphate buffer, pH 6.0, (5 ml) then dialyzed against the same buffer for 24 h at 4°C. The enzyme sample was gel- Effect of different metal ions and some enzyme inhibitors filtered through a Sephadex G-100 column (18×2 cm), pre- equilibrated with the same buffer. The elution was per- Various metal ions at final concentration of 1 and 10 mM formed by the same buffer at a flow rate of 20 ml/h. were added to the enzyme solution (KCl, NaCl, CaCl2, Fractions of 5 ml were collected and assayed for their MgSO4,FeSO4,CuSO4·5H2O, AgNO3,HgI2 and protein, CMCase and protease activities. The active frac- ZnSO4⋅H2O) then, each metal ion was incubated with the tions with the highest specific activity of each enzyme were two enzymes, before adding substrate. Activity of each pooled, mixed and dialyzed. The pooled fractions of each enzyme in the complete absence of such compounds enzyme were further fractionated separately through a served as control (100% activity). Also, the effect of var- – DEAE-cellulose column, and eluted with a 0 0.8 M-NaCl ious inhibitors such as EDTA, sodium arsenate, L-cysteine gradient in citrate-phosphate buffer (240 ml) at a flow rate of and Iodoacetate was investigated, then the activities of the 10 ml/h. For each enzyme, 5 ml fractions were collected and studied enzymes were measured. assayed for their protein and enzyme activities. The most active fractions were pooled, mixed and dialyzed once again Enzyme stability studies (Peterson and Sober 1962; Palmer 1991). These purified enzyme preparations were lyophilized and stored at 5°C pH stability for further investigations (Plummer 1978). To determine pH stability, each enzyme was incubated at pH Characterization of purified CMCase and protease 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 Effect of pH the residual activity for each enzyme was estimated under standard assay conditions. The results were expressed as The effect of pH on enzyme activities was assessed by relative activity (%) referred to the activity observed before adding 1 ml CMCase solution to 1 ml 1% (w/v) Na-CMC incubation. and 1 ml protease solution to 1 ml 1% (w/v) casein, respec- tively, at different pH values (from 3.6 to 5.2) obtained using 0.05 M citrate-phosphate buffer. After incubation at Thermal stability 30°C for 30 min for CMCase and at 30°C for 20 min for protease, each enzyme activity was measured under the For determination of enzyme thermal stability, the enzymes – standard assay conditions as described above. were incubated for varying times (0 60 min) at fixed temper- atures (60–80°C for CMCase; 35–55°C for protease). After Effect of temperature incubation, the residual activities of the enzymes were mea- sured in the usual manner. The effect of temperature on enzyme activities was assessed by incubating each enzyme with the corresponding substrate Sodium dodecyl sulfate-polyacrylamide gel electrophoresis as previously described at various temperatures ranging from 20 to 70°C. Each enzyme activity was measured as For determination of molecular weight, the purified enzyme mentioned before. preparations and known molecular weight markers were subjected to sodium dodecyl sulfate-polyacrylamide gel Effect of substrate concentration electrophoresis (SDS-PAGE) as described by Laemmli (1970) using a 12.5% (w/v) acrylamide slab gel with This experiment was carried out to study the effect of 25 mM Tris / 192 mM glycerin buffer (pH 8.3) that substrate concentrations on the activity of the purified contained 0.1% (w/v) SDS as the running buffer. After CMCase and protease, respectively. Different soluble con- electrophoresis, the gel was stained for 1 h with Coomassie centrations (0.4–1.6, % w/v) each of Na-CMC and casein Blue R250 dye in a methanol-acetic acid-water solution were used. Enzyme activity was measured to determine the (4:1:5, by volume) and then destained in the same solution optimum substrate concentration in each case. without the dye. 1550 Ann Microbiol (2012) 62:1547–1556

Table 1 Analysis of reason, CMCase and protease enzymes were chosen for Constituent % (dry weight basis) water hyacinth this study. composition Cellulose 21.5 Different fungal species were tested for their capability to Hemi-cellulose 33.90 grow on water hyacinth medium (WH) as a rich organic Fat 1.65 source under solid state fermentation (SSF). The superiority Crude protein 13.75 of Ulocladium botrytis in CMCase and protease activities Ash 12.10 (29.6 and 23U/g, respectively) compared to the other tested Lignin 7.01 fungi is clear from Fig. 1. Thus, U. botrytis was chosen for Total solids 5.01 further investigation. Carbon 45.5 Some natural additives, i.e., yeast, malt and beef extracts, Hydrogen 5.3–5.5 soybean meal and corn-steep liquor 0.05% (w/v) were used to Nitrogen 1.8–3.2 increase production of CMCase and protease enzymes by U. Sulfur 0.25–0.35 botrytis grown on WH medium. It was found that the maxi- mum 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 Statistical validation of treatment effects this regard, El-Gindy et al. (2008) stated that these additives promote the production of enzymes in solid state cultures. The mean, standard deviation, T-score and probability “P” A summary of purification steps of the CMCase and values of three replicates of the investigated factors and the protease produced by U. botrytis is recorded in Tables 2 control were computed according to the mathematical prin- and 3, respectively. The results indicate a 1.36-fold purifi- ciples described by Glantz (1992). Results were considered cation in the case of CMCase, and 1.27-fold in the case of highly significant, significant or non-significant where P< protease, with yields of 84.0% and 85.93%, respectively, 0.01, >0.01 and <0.05, >0.05, respectively. of the original activities obtained. Most of the CMCase and protease activities were applied onto Sephadex G-100 and recoveredin35–40 fractions representing 63.3% and Results and discussion 64.45% of the original activity, corresponding to specific activities of 106.74 and 299.46 U/mg protein (5.93- and The chemical composition of E. crassipes wastes is shown 10.55-fold purification, respectively). These fractions were in Table 1. The analytical data showed that leaves pooled, lyophilized and subjected to further fractionation contained 21.5% cellulose, 33.9% hemicellulose, 13.75% onto DEAE-cellulose using a linear sodium chloride gra- crude protein and other nutritional elements. For this dient as eluant. This resulted in 47.34- and 51.78-fold

Fig. 1 Screening of fungal 30 species for carboxymethylcellulase (CMCase; □) and protease (■) 25 production when grown on water hyacinth (Eichhornia 20 crassipes) wastes (WH) as a rich organic source under solid state fermentation (SSF) 15 conditions 10

5 Enzymatic activity (U/g)

0 A. candidusA. flavusA. nigerA. terreus A. A. ustus scirpiF. chrysogenum P. citrinum P. claviformP. velutinum P. verideT. botrytis U.

Fungus Ann Microbiol (2012) 62:1547–1556 1551

Fig. 2 Effect of enrichment 35 WH medium with some natural additives on the production of CMCase (□) and protease (■) 30

25

20

15

10

5 Enzymatic activity (U/g) 0 yeast Corn steep Beaf Malt Soyabean extract liquar extract extract Nitrogen source

purification, with yields of 40.3% and 56.25% of the initial the decreasing affinity of the enzyme to its substrate or due CMCase and protease activities, respectively. to an irreversible destruction of the enzyme protein. It may This purification procedure was also used by Po-Jui et al. also be due to the effect of pH on the stability of the enzyme (2004) for CMCase of Sinorhizobium fredii and resulted in (Dixon and Webb 1979). This optimum pH value (5.2) was 9.08-fold purification, the recovery yield was 26.4% and the identical to that found for CMCase from Macrophomina specific activity was 3.822 U/mg; and by Datta (1992) for phaseolina (Roy and Vora 1989). In contrast, these findings Phanerochaete chrysosporium protease, resulting in 37-fold are not in accordance with earlier reports showing pH optima purification. of 7–7.5 for protease from Aspergillus oryzae (Alagarsamy et The secreted enzyme level per total secretory protein al. 2005). level was calculated as 7.98% and 17.78% for CMCase The optimum incubation temperatures were 60°C and and protease, respectively. The accumulated extracellular 35°C for CMCase and protease, respectively (Table 4), enzyme activities were calculated by U/g dry cell weight while lower activities were seen at either side of these (dcw) during fermentation as 175 and 245 U/g dcw for values. Similarly, Roy and Vora (1989) stated that CMCase CMCase and protease, respectively. showed optimum activity at 65°C, while Usama and Saad El Testing the pH-dependence of the activity of CMCase Dein (2008) reported that the optimal temperatures of and protease revealed that pH 5.2 was the optimum for both CMCase produced from A. niger, A. nidulans and A. terreus CMCase and protease activities (Table 4); moreover, there were (30, 35 and 40°C), respectively. But for protease, was high inactivation of the two enzyme activities on both Abdul-Raouf (1990) stated that the optimum incubation the extreme sides of the pH curve. This can be attributed to temperature for purified protease activity was 35°C. In

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

Treatment Total protein (mg) Total activity Specific activity Recovery (%) Purification (fold) (μg/ml) (U/mg protein)

Cell free filtrate (200 ml) 83.4 1,500 18.0 100 1.0 Cell free precipitate (1:1 isopropanol: filtrate) 51.6 1,260 24.41 84.0 1.36 Gel filtration (Sephadex G-100) 8.9 950 106.74 63.3 5.93 Ion-exchange chromatography 0.71 605 852.11 40.3 47.34 (DEAE-Cellulose) 1552 Ann Microbiol (2012) 62:1547–1556

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

Treatment Total protein (mg) Total activity Specific activity Recovery (%) Purification (fold) (μg/ml) (U/mg protein)

Cell free filtrate (200 ml) 90.2 2560 28.38 100 1.0 Cell free precipitate (1:1 isopropanol: filtrate) 60.8 2200 36.18 85.93 1.27 Gel filtration (Sephadex G-100) 5.51 1650 299.46 64.45 10.55 Ion-exchange chromatography 0.98 1440 1469.38 56.25 51.78 (DEAE-Cellulose)

addition, Lee et al. (2002) reported that the optimum tem- were inhibited in the presence of Zn+ or Cu2+, and severely perature of purified protease ranged from 40°C to 50°C. inhibited in the presence of Ag2+ and Hg2+. Moreover, various Moreover, Ammar et al. (2003) reported that the optimum enzyme inhibitors were added to the reaction mixture to test temperature for thermostable purified protease was 55°C. their influence on both enzyme activities. The most injurious The effect of different substrate concentrations was also inhibitors were Ag2+,Hg2+ and sodium arsenate in addition to studied (Fig. 3). The protease reached its maximal activity iodoacetic acetate. These results were in accordance with with a casein concentration of 0.8% (w/v) but, for CMCase, those of Kim et al. (2009), who mentioned that CMCase the optimum substrate concentration was 1.2% (w/v) Na- activity was enhanced by some metal ions such as K+ but CMC. There is a decrease in both enzyme activities either inhibited by Hg2+. Similarly, Nongporn et al. (1999)stated side of these optimum values. This is complete agreement that Ca2+ was particularly effective in activating enzyme with Abdul-Raouf (1990), who also stated that an increase activity, causing 85% stimulation, while Mn2+ and Mg2+ at or decrease in substrate concentration led to a decrease in the same concentration showed a less positive effect. Similar enzyme activity. results were observed by Tsuchiya et al. (1987), who reported With regard to enzyme concentration, Fig. 4 shows that that protease isolated from Colosporium sp. was inhibited by the highest CMCase and protease activities were achieved at Hg2+; furthermore, Nehra et al. (2004)reportedthatMg2+ was an enzyme concentration of 0.4 U/ml. This behavior is in found to activate the alkaline protease enzyme produced by accordance with the observations of West et al. (1967), who Aspergillus sp. Metal ions may stimulate enzyme activity by stated that, within fairly wide limits, the speed of enzyme acting as a binding link between enzyme and substrate, com- activity is directly proportional to the enzyme concentration. bining with both and so holding the substrate in the active site The same finding also was reported by Abd El-Rahman of the enzyme (Mahmoud et al. 1968). The effect of metal ions (1990), El-Safey (1994), and El-Safey and Ammar (2003). on enzyme activity might also be due to changes in electro- The effect of some metal ions and enzyme inhibitors was static bonding that would change the tertiary structure of also studied. The results demonstrated (Table 5)thatCMCase enzymes (Roy et al. 1990). and protease activities increased significantly in the presence Incubation of each enzyme preparation at different of Ca2+,Mg2+,Na+ or K+. Activities of CMCase and protease pH values for either 20 or 60 min demonstrated that

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

Enzyme properties CMCase Protease

Optimum pH 5.2 5.2 pH stability 5.2–5.4 at 20 or 60 min 5.6–6.0 at 20 or 60 min Optimum temperature (°C) 60 35 Thermostability (min) Midpoint of thermal inactivation = Midpoint of thermal inactivation = 60°C for 75 min 35°C for 75 min

Vmax (U/mg protein) 100 40

Km (mM) 0.8 1.58 −1 Kcat (S ) 3.33 2 −1 −1 Kcat/Km (mlS mg ) 4.16 1.27 Molecular weight (kDa) 50 and 83 47 Ann Microbiol (2012) 62:1547–1556 1553

Table 5 Effect of different met- al ions and some enzymes Metal ions or inhibitors Relative activity inhibitors on the relative activi- ties of purified CMCase and CMCase Protease protease from Ulocladium botrytis 1 mM 10 mM 1 mM 10 mM

K+ 113±4.5* 123±3.24** 102±2.25ns 113±4.5* Na+ 110±5.3* 120±4.25** 108±4.76* 111±2.45* Ca2+ 105±4.6ns 112±5.01* 103±3.11ns 109±2.22* Hg2+ 0.0 0.0 0.0 0.0 Mg2+ 105±2.6ns 106±1.58ns 106±2.45ns 108±1.35* Fe3+ 111±3.5* 90±1.35* 107±1.67ns 98±2.85ns Ag2+ 0.0 0.0 0.0 0.0 Zn+ 60±4.2** 48±4.05** 70±2.53** 50±4.03** Cu2+ 90±3.11* 60±2.15** 89±1.78* 66±2.35** Data represent the mean of three ** ** ** ** readings approximated to the EDTA 68±2.13 50±5.02 58±3.12 56±5.11 nearest integer Sodium arsenate 50±3.14** 33±3.11** 44±2.26** 30±2.34** ns * ** ** ** Non significant, P>0.05; L-Cysteine 88±3.11 61±2.22 80±3.15 65±3.33 * significant, P≥0.01 and ≤ 0.05; Iodoacetate 45±1.16** 20±1.24** 50±4.36** 25±5.21** ** highly significant, P<0.01

CMCase was almost unaffected by incubation in the pH after heating to 35°C for 75 min, while no activity was range 5.2–5.4 for either 20 or 60 min (Table 4). These recorded after heating the enzyme at 50°C for 90 min and results were in accordance with the results of Coral et 55°C for 75 min (Table 4). The Tm was recorded at 35°C al. (2002), while protease was almost unaffected by after 75 min of exposure. The thermal inactivation of incubation in the pH range 5.6–6.0 for either 20 or enzymes is nearly always due to denaturation of the enzyme 60 min (Table 4). These results were in accordance with proteins (Dixon and Webb 1979). Temperature is a cardi- those of Datta (1992). nal factor affecting the amount and rate of growth of an Moreover, the midpoint of thermal inactivation CMCase organism (Garg et al. 1985) and increasing temperature (Tm) was determined and recorded at 60°C after 75 min of has the general effect of increasing enzyme activity exposure. The CMCase enzyme retained its original activity (Moor 1990), but the enzyme begins to suffer from after heating to 60°C for 75 min, while no activity was thermal inactivation at higher temperatures. recorded after heating the enzyme at 80°C for 75 min (Ta- The purified enzyme preparation was subjected to SDS- ble 4). The protease enzyme retained its original activity PAGE containing 0.2% (w/v) Na-CMC to determine the

Fig. 3 Effect of substrate 120 concentration on activity of purified CMCase (▲) and protease (■) from U. botrytis

80

40 Specific activitySpecific

0 0.4 0.6 0.8 1 1.2 1.4 1.6 Substrate concentration (%, w/v) 1554 Ann Microbiol (2012) 62:1547–1556

Fig. 4 Effect of enzyme 160 concentration on activity of purified CMCase (▲) and protease (■) from U. botrytis 120

80 Specific activitySpecific 40

0 0.2 0.4 0.6 0.8 1 1.2 1.4 Enzyme concentration (U/ml)

homogeneity and molecular weight of the enzyme. During 50 kDa for β-glucosidases (BGL) and cellobiohydrolases electrophoresis of the enzymes, two bands showing cellulytic (CBH), respectively (Mehdi et al. 2009), while the molecular activity were detected. As shown in Fig. 5, the molecular weight of the proteolytic enzyme was calculated to be about weights of these proteins were calculated to be about 83 and 47 kDa (Fig. 6).

Fig. 5 Separation of U. botrytis CMCase by SDS-PAGE. Electropho- Fig. 6 Separation of U. botrytis protease by SDS-PAGE. Electropho- resis was carried out in an SDS-polyacrylamide gel containing 0.2% resis was carried out in an SDS-polyacrylamide gel containing 0.1% CMC. Lanes: A CMCase from U. botrytis, M molecular weight casein. Lanes: A Protease from U. botrytis, M molecular weight markers markers Ann Microbiol (2012) 62:1547–1556 1555

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