Indian Journal of Biotechnology Vol 7, July 2008, pp 333-340
Extracellular collagenase from Rhizoctonia solani: Production, purification and characterization Hossam S Hamdy* Biology Department, Faculty of Education, Ain Shams University, Roxy 11757, Cairo, Egypt Received 2 March 2007; revised 5 September 2007; accepted 10 December 2007 Potentiality of R. solani grown on Sabouraud-glucose-collagen medium to produce glycosylated metallo-proteinase with collagenolytic activity was optimized and maximum production (212.33 U/mL) was recorded after 108 h of submerged incubation (175 rpm) at pH 5.5 and 30°C of temperature. Two-step column chromatography technique on DEAE-cellulose and Sephadex G150 was adopted to purify the partially purified enzyme produced by ammonium sulfate (40%, w/v) precipitation. Yield of purification was 60.49% of the original activity with specific activity of 18064.7 × 103 U/mg protein and 18.72-folds of purification. The purified enzyme showed maximum activity at 40°C and pH 5 ,which was stimulated by ions of Ca, Co, Cu, K, Mg, Na or Zn and inhibited by ions of Fe and Hg. Metal composition of the purified enzyme revealed that it contains Ca2+ and Zn2+. Tm (midpoint of thermal inactivation) was recorded at 65°C and 55°C after 1 and 6 h of exposure, respectively and T1/2 was found to be 7 and 5 weeks at 15°C and 4°C, respectively. Molecular mass, Kmg/mL, Vmax of the enzyme were found to be 66 ± 4 kDa, 0.033 kDa and 0.28 U/mg min-1, respectively. Activities toward gelatin and casein were also detected. Keywords: Collagenase, production, purification, characterization, Rhizoctonia solani.
Introduction Collagenases [E.C. 126.96.36.199] are the proteolytic enzymes responsible for degradation of helical regions of native collagen to small fragments1. In contrast to mammalian collagenases, which cleave the collagen helix at a single site, microbial collagenases attack multiple sites along the helix2. Importance of collagenase is extended and has found widespread applications, such as use of the purified collagenase in estimation of collagen levels, isolation of specific cell types from attendant connective tissue2 and pulmonary mast cells from bovine lungs3. Moreover, there is much interest in the application of collagenase in the experimental transplantation of pancreatic islet cells to alleviate diabetic symptoms4. The role of collagenase in controlling some pathogens of plants has been ascertained5. Its role in dental caries6 and bacterial vaginosis7 was also recorded. Collagenolytic activity also contributes to tenderizing dry-cured meat products and in generating taste and flavour in meat industry8. Recently, collagenases were used to anchor signaling molecules to collagencontaining tissues, presenting a great potential for targeted drug delivery of anti-arthritic and anticancer reagents9. __________ *E-mail: [email protected]
Production of collagenase is associated with a variety of microorganisms including fungi: Aspergillus flavus10, A. fumigatus11, A. repens, Penicillium purpurogenum and P. frequentan12, Cunninghamella elegans13, P. chrysogenum8, some pathogenic species of Aspergillus and Candida causing aspergillosis and candidiosis14, some dermatophyte fungi15, Histoplasma capsulatum var. capsulatum and var. dubosii16, as well as some bacterial species17,18 and Streptomycetes19. However, little was found in the literature about purification and characterization of collagenolytic enzymes produced by fungi. Moreover, most of such work discussed collagenolytic activity as an activity included within other proteolytic activities and there were no separate reports to optimize its production as a target. Thus, the present work is an attempt to bridge this gap of knowledge about fungal collagenase by studying production, purification and characterization of collagenase produced by Rhizoctonia solani. Materials and Methods Microorganism
Rhizoctonia solani (J. G. Kuhn CBSnr 126.08) and the other fungal strains used throughout the screening were collections of our laboratory previously isolated, and identified (Centraalbureau Voor Schimmelcultures, Netherlands) and routinely subcultured on malt extract agar medium.
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Sabouraud-glucose-collagen (SGC) medium containing 0.2% insoluble collagen type I (from bovine Achilles tendon, Sigma Chemicals, Germany) as a soul nitrogen source was used as a fermentation medium11. Preparation of Spore Suspension
A heavy spore suspension (27 × 105 spore mL-1) was used to inoculate the medium, and the growing mycelial spheres were aseptically transferred to slants of SGC medium. These slants were incubated for 7 d at 30°C, subcultured and used in the subsequent work to prepare the spore suspension. Enzyme Production
Triplicate sets of 250 mL flasks were inoculated with 1 mL of the spore suspension (27 × 104 spore mL-1) and incubated at 30°C for 4 d in a GFL shaking incubator at 150 rpm and initial pH value of 4.5. At the end of the growth period, the biomass was removed by filtration and the supernatant (cell-free filtrate, CFF) served as crude enzyme preparation. Enzyme and Protein Assays
Collagenolytic activity (CA) was assayed according to the method of Mandle et al20, in which 0.1 mL of the enzyme solution was incubated for 5 h at 37°C with 25 mg native collage (type I, from bovine Achilles tendon, in 5 mL of 0.01 M acetate buffer (pH 4.5) containing 10 mM calcium acetate, and the extent of collagen breakdown was determined using the colorimetric Ninhydrin method21. One unit of collagenolytic activity equals one micromole of L-leucine equivalents released from collagen under the specified conditions. Caseinolytic activity (PA) was assayed as follow22, 1 mL of 2% casein solution (dissolved in 0.01 M acetate buffer, pH 4.5) and 1 mL of the enzyme solution were incubated at 40°C for 10 min. Two mL of 0.44 M trichloroacetic acid (TCA) were added, and mixed well to terminate the reaction and the amount of TCA soluble hydrolysate in 1 mL of the reaction mixture was estimated23. Gelatin hydrolyzing activity was determined viscometrically by measuring its liquefaction where 1 unit of enzyme was considered as that amount of enzyme required to reduce the viscosity of 1 mL of 5% gelatin to half a minute at 30°C24. Hydrolyzing activities toward ovalbumin and hemoglobin were performed as in casein.
Protein content was determined using bovine serum albumin as standard25. Enzyme Purification
Crude enzyme preparation was dialyzed for 2 d against several changes of 0.01 M acetate buffer, pH 4.5. Protein content of cell-free dialysate (CFD) was precipitated with 40% w/v, (NH4)2SO4, collected by centrifugation (12 × 103 g for 15 min), dissolved in the previous buffer containing 10 mM calcium acetate24 and salted out by passing through Sephadex G25. This enzyme preparation was then purified by 2 successive steps of column chromatography, i.e., ion exchange and gel exclusion using DEAE-cellulose (Dimethylaminoethyl cellulose, Sigma Chemicals, Germany) or Sephadex G150 (Pharmacia, Uppsala, Sweden, equilibrated with 0.01 M acetate buffer containing 10 mM calcium acetate), respectively26. Two mL of the enzyme preparation was cautiously applied onto the column (2.5 × 82 cm2) and 5 mL fractions were eluted with a linear gradient of calcium acetate (0.0 to 0.5 M prepared in acetate buffer) in case of ion exchange chromatography or with acetate buffer containing 10 mM calcium acetate in case of gel exclusion. Collagenase activity and protein content of the different fractions were determined and used to calculate the specific activity where the fractions of highest specific activities were pooled, salted out, lyophilized and used in the following experiments. Characterization of Collagenase
Dependence of collagenase activity on pH was determined by measuring the enzyme activity at different pH values using different buffers (0.05 M citrate for pH 3.0-3.5, 0.01 M acetate for pH 4-5.5, 0.1 M phosphate for pH 6.0-7.0 and Tris-HCl for pH 7.09.0) containing 10 mM calcium acetate. To test the pH-dependence of collagenase stability, aliquots of enzyme solution were dialyzed for 18 h against the same buffers used for estimating the pH activity at different pH values (3 to 9). After this time, samples achieved a pH equals to that of the dialysis buffer and kept at this value for 1 or 6 h. The residual activity in each treatment was assayed under the standard assay conditions. For determination of the optimum temperature for collagenase activity, reactions carried out at different temperatures (25 to 80°C) and enzyme activity was measured in each case. However, the enzyme was
HAMDY: RHIZOCTONIA SOLANI EXTRACELLULAR COLLAGENASE
incubated at pH 5.0 for 1 or 6 h at a fixed temperature for determination of thermal stability. The molecular mass (Mr) of the enzyme preparation was estimated by gel filtration on Sephadex G75 using standard proteins (100 mg for each) with sample volume of 5 mL26. Elution of standard proteins was monitored at 280 nm while elution of Blue Dextrane, used to determine the void volume, was monitored at 540 nm. Statistical Analysis
The data (correspond to the mean of triplicates) was analyzed through T-test and considered as highly significant, significant or non-significant where P < 0.01, > 0.01 and < 0.05, > 0.05, respectively27. Results and Discussion Optimization of Collagenase Production
R. solani grown on SGC medium was selected in this work as a source of collagenolytic enzyme for its relative highest productivity (147.77 U/mL) in comparison to other 25 fungal species screened (data
not shown). Production of collagenase by R. solani was also recorded on peptone-containing medium at a level of 83.45 U/mL and its ratio to proteolytic activity (0.989 U/mL) was only 84.4. In this respect, production of collagenase by T. schoeleinii either in Sabouraud's medium or collagen-containing medium (0.2% w/v) has been reported15. Since use of different lots or types of collagen as substrate will produce varying amounts of enzyme activity20 and collagenases of various microbial origins are specific to different collagen types, therefore, comparison of production levels by different microorganisms on different media should be undertaken with full care. Optimization of enzyme production in response to incubation period, initial pH value of the fermentation medium, incubation temperature and velocity of shaking was performed and the optimum conditions which enabled the maximum enzyme production for each experiment were settled for the next one. Data revealed that maximum productivity (212.33 U/mL) was achieved after 108 h of incubation (Fig. 1-a),
Fig. 1⎯Optimization of extracellular collagenase production (CA, ―♦―) by R. Solani and the related activities, i.e., proteolytic activity (PA, ――) and the ratio of collagenolytic to proteolytic activity (CA/PA ―S―). a: Time course of CA & PA production, CA/PA and dry biomass (―×―) grown at 30°C and pH was initially adjusted to 4.5. b: pH-dependence of CA, PA & CA/PA. c: Temperature dependent variation of CA, PA & CA/PA d: Influence of shaking velocity on CA, PA and CA/PA. * Buffer solutions used for controlling pH's were: 0.05 M citrate for pH 3.0-3.5; 0.01 M acetate for pH 4-5.5; 0.1 M phosphate for pH 6.0-7.0; Tris-HCl buffer for 7.0-9.0.
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pH 5.5 (Fig. 1-b), temperature 30°C (Fig. 1-c), and shaking velocity 175 rpm (Fig. 1-d) which represents 1.44-folds increase in productivity. Under these conditions CA/PA was 433.33. Most of collagenases, however, also exhibited proteolytic activity and only those isolated from Clostridium are considered real specific collagenases28.
obtained confirming that the enzyme was purified to electrophoretic homogeneity (Fig. 4). Effect of pH value on Activity and Stability of Purified Collagenase
Although Sela et al18 stated that the maximum activity of most collagenases has been found within a pH range of 7-7.5, in this work pH 5 was found
Purification of Collagenase
Table 1 represents summary of the purification steps. After dialysis, the total activity of collagenase decreased to 95.57% of the original activity most probably due to the loss of certain activating ions during dialysis. Enzyme activity of the protein precipitate produced by ammonium sulfate was 178.64 × 103 U, representing a yield of 84.13% with specific activity of 1768.71 × 103 U/mg protein. This protein fraction was subjected to ion exchange chromatography through DEAE-cellulose and fraction Nos 16 to 20 were found to contain the maximum amount of collagenase (133.25 × 103 U) with specific activity of 8830.35 × 103 U/mg protein (Fig. 2). These fractions were pooled and subjected to further fractionation on a column of Sephadex G150. Maximum collagenolytic activity was recorded in a single sharp peak representing fractions 18 to 23 that contained 128.44 × 103 U with specific activity of 18064.7 × 103 U/mg protein (Fig. 3). Yield of purification was 60.49% with 18.72 purification folds, while for collagenase from T. schoenleinii, the yield was only 12% with 19.3 purification folds15. This enzyme preparation was desalted, the concentration adjusted to 100 U/mL and kept at 4°C for use in the subsequent work. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed29 to judge purity of the enzyme, and a single band was
Fig. 2⎯Fractionation of collagenase of R. solani on DEAEcellulose. Collagenolytic activity [CA, (U × 103), ―O―]; total protein, (mg × 10-1), ―S― and molarity (M) of calcium acetate prepared in acetate buffer.
Fig. 3⎯Further fractionation of collagenase of R. solani on Sephadex G150. Collagenolytic activity [CA, (U × 103), ―O―] and total protein (mg) [―S―].
Table 1⎯ Purification of extracellular collagenase of R. oryzae Step
Cell-free filtrate (CFF) Cell-free dialysate (CFD) Protein precipitate of 40% w/v, (NH4)2SO4 Ion exchange chromatography Gel filtration
Total activity CA PA (U × 103) (U × 103)
Total protein (mg)
Specific activity (U/mg protein × 103)
Total volume used was 1000 ml of CFF Data were approximated to 1 decimal
HAMDY: RHIZOCTONIA SOLANI EXTRACELLULAR COLLAGENASE
Fig. 4⎯SDS-PAGE of collagenase of R. solani. 15 µg protein was applied onto 12.5% polyacrylamide gel and stained with Coomassie Brilliant Blue R-25.
optimum for the purified collagenase from R. solani (Fig. 5). Moreover, collagenases from Sporothrix schenckii17, H. capsulatum16 and T. schoenleinii30 were optimally active at pH values ranged from 4 to 6.5. Collagenase from R. solani showed complete stability in the pH range of 4-5 and reasonable stability in the pH range of 3-6.5 after 1 h of exposure. At pH 3, the enzyme restored 90% and 70% of the original activity after 1 h and 6 h of exposure, respectively (Fig. 5). Microbial enzymes possessing high activity and stability at relatively low pH values are industrially important because such conditions help in avoidance of microbial contamination. Therefore, this may provide collagenase from R. solani as enzyme of industrial importance. Effect of Temperature on Activity and Stability of Purified Collagenase
Optimum temperature for the activity of the purified collagenase was recorded at 40°C (Fig. 6), which is in complete accordance with the optimum temperature reported for other collagenases from H. capsulatum16, Streptomyces candidus and S. cremeus19. The purified collagenase showed a complete stability up to 40°C after 1 h of exposure, then a gradual decline in enzyme activity was observed (Fig. 6) and the midpoint of thermal inactivation (Tm) was found at 65°C and 55°C after 1 and 6 h of exposure, respectively. In this regard,
Fig. 5⎯Effect of pH on the activity and stability of the purified collagenase. The enzyme preparation was held at the indicated pH under the standard assay conditions for measuring the activity (___♦__) and for 1 h (------) or 6 h (----▲---) at the indicated pH, after which the residual activity was assayed at pH 5, 37°C and other standard assay conditions in case of stability.
Fig. 6⎯Effect of temperature on the activity (__♦___) and stability (1 h, ------ & 6 h, ---▲---) of the purified collagenase. Reaction carried out at pH 5 and other conditions are those described in legend to Fig. 4.
collagenase from Bacillus cereus is thermally stable from 4-40°C18, while those from S. candidus and S. cremeus are thermally stable between 40-80°C19. T1/2 of the purified enzyme preparation (dissolved in acetate buffer, pH 5.0) was estimated in presence or absence (control) of Ca2+, Mg2+ or Zn2+ (added as chloride at a final concentration of 10 mM) at 15°C or 4°C. At 15°C, T1/2 was found to be 7 wk in control or any of the other treatments. At 4°C, T1/2 was found to be 5 wk. For control, 6 wk in presence of Mg2+ or Zn2+, and 8 wk in presence of Ca2+.
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Effect of Metal Ions and Inhibitors on Eenzyme Activity
Activity of R. solani collagenase as affected by the presence of some metal ions presented in Table 2 shows that Ca2+, Co2+, Cu2+, K+, Mg2+, Na+ or Zn2+ had a stimulatory effect upon the enzyme activity; Fe3+ and Hg2+ had an inhibitory effect, while Ba2+ or Mn+ showed a non-significant effect. It is observed that Ca2+ or Mg2+ activated the enzyme in a progressive way up to 10 mM, which may suggest an important role for these cations. Collagenase of A. flavus was also found to be Zn stimulated and had a Zn cofactor10. Collagenases were previously reported as a group of Ca2+- and Zn2+-dependent enzymes18. The present results are contrary to the results reported for collagenase of Pseudomonas marinoglutinosa, where it was inhibited by Zn ions and increasing the concentration of Co2+ reversed its activating action24. With regard to the effect of some enzyme inhibitors on the purified collagenase activity, Table 2 shows Table 2⎯Effect of metal ions and enzyme inhibitors on relative activity of the purified collagenase Metal ions Control
Relative activity as affected by the concentration of 1 mM 5 mM 10 mM 100
Ba2+ Ca2+ Co2+ Cu2+ Fe3+ Hg2+ K+ Mg2+ Mn+ Na+ Zn2+
102 + 4.3*** 147 + 4.5* 123 + 5.2* 106 + 4.2* 75 + 2.3* 43 + 1.2* 126 + 6.2* 152 + 5* 104 + 4.5*** 112 + 2.0* 142 + 2.3**
100 + 3.6*** 181 + 4.4* 131 + 6.5* 110 + 2.1* 66 + 2.5* 31 + 1.3* 132 + 3.2* 173 + 7.6* 106 + 3.1** 111 + 5.6** 141 + 5.8*
99 + 2.9*** 198 + 2.9* 132 + 4.3* 108 + 2.0* 40 + 1.9* 12 + 0.4* 146 + 2.8* 212 + 8.1* 106+4.9*** 110 + 3.5* 140 + 4.1*
Enzyme inhibitor EDTA Iodoacetate Sodium arsenate Sodium arsenite 2,4-dinitrophenol Sodium azide Cysteine
62 + 1.6* 44 + 1.7* 76 + 3.8* 79 + 3.6* 64 + 2.4* 78 + 2.4* 101 + 3.3***
50 + 1.9* 34 + 1.0* 50 + 2.1* 60 + 1.9* 60 + 2.5* 63 + 2.7* 98 + 2.9***
31 + 0.6* 18 + 0.7* 38 + 1.2* 13 + 0.3* 54 + 0.7* 41 + 1.5* 79 + 3.1*
[Data represent the mean of 3 readings approximated to the nearest integer number] The investigated metal ion, as chloride at the indicated concentration, was incubated with the enzyme for 30 min. at 30°C before adding substrate. Activity of the enzyme in complete absence of such compounds served as control (100 % activity) to which other data were statistically compared as described in Materials and Methods.* = highly significant, ** = significant & *** = ns
that the enzyme activity was inhibited by Hg2+, SH group specific inhibitors (i.e. iodoacetate, arsenate and arsenite) as well as cystein at 10 mM, which specifically acts on disulfide bonds24. Such findings suggest the possible participation of SH group in the enzyme structure. Enzyme was inhibited by EDTA, which also showed an inhibitory effect upon other collagenases, i.e., T. schoenleini30, A. fumigatus31 and P. marinoglutinosa24. To attain better understanding of the effect of metal ions and EDTA upon the enzyme activity, aliquots of EDTA-inhibited enzyme (10 mM) were separately incubated with different metal ions at a concentration of 10 mM for 1 h at 30°C. Enzyme activity was then assayed under standard assay conditions and the relative activity of the enzyme was calculated as % to the EDTA-treated enzyme. Surprisingly, with the exception of Hg that synergistically with EDTA increased the severity of inhibition, all the added metal ions reactivated the EDTA-inhibited enzyme to various extents (Table 3). Ions of Ca, Zn and Mg recorded the highest capabilities of reactivation at levels of 199, 188 and 170%, respectively. It seems that reactivation is non-specific to the metals with activating potentiality most probably because EDTA binds to free metal easier than binding to a metal contained in an enzyme structure. Restoring the activity of EDTA-inhibited enzyme by inhibitory metal ions has been reported for another enzyme, i.e., glucoamylase of A. niger32. Meanwhile, collagenase of T. schoenleinii was irreversibly inhibited by Table 3⎯Effect of metal ions on EDTA-inactivated collagenase Metal ion (10 mM)
Relative activity (% to EDTAtreated enzyme)
Native enzyme Native enzyme + EDTA (10 mM)• Ba2+ Ca2+ Co2+ Cu2+ Fe3+ Hg2+ K+ Mg2+ Mn+ Na+ Zn2+
100 31 + 0.6 119 + 5.7* 199 + 8.4* 152 + 5.5* 112 + 3.1* 107 + 5.4 88 + 2.5* 140 + 5.2* 170 + 6.8* 100 + 4.2*** 118 + 3.4* 188 + 7.5*
• = control
HAMDY: RHIZOCTONIA SOLANI EXTRACELLULAR COLLAGENASE
Table 4―Km, Vmax, Kcat and their ratios for the purified collagenase against collagen, gelatin and casein Substrate Collagen Gelatin Casein
Vmax (U/mg min-1)
Kcat (mg U-1min-1)
0.033 1.25 0.2
0.28 1.25 1.175
2.8 1.25 1.175
8.48 1.00 5.87
0.280 0.0125 0.0117
*Due to the indefinite molecular weight of collagen, casein or gelatin, units of weigh (mg) instead of molarity were used in determination of Km, Vmax, Kcat and their ratios.
EDTA35, while that of P. marinoglutinosa restored its activity by Ca2+. Metal composition of the purified collagenase was performed in the Central Laboratory, Faculty of Science, Ain Shams University, using Perkin-Elmer3100 Atomic Absorption, where Ca2+, 0.45% and Zn2+, 0.09% were detected in an appreciable amount. From the activation effect shown by Ca2+ and Zn2+, their ability to restore the EDTA-inactivated enzyme and as evidenced from the metal composition of the enzyme, it is clear that collagenase of R. solani is a typical metalloproteinase enzyme. Collagenases from A. fumigatus, S. cerevisiae31 and T. schoenleinii15 are also metalloproteinases. Other Enzymatic Properties
Molecular mass of the purified enzyme determined by gel filtration and was found to be 66 ± 4 kDa. Collagenases from other microbial sources were found to have different molecular masses 20 and 82 kDa for collagenases produced by T. schoenleinii30 and A. fumigatus31, respectively. The use of phenol/sulfuric acid method―with glucose as standard33―revealed that 4.9% of the composition of the secreted collagenase is carbohydrate suggesting that the enzyme is a glycoprotein. In this respect, Tomee et al11 found that collagenase from A. fumigatus is highly glycosylated with mannose-containing structures. Protein hydrolysate of the purified enzyme preparation (the purified enzyme, equals to 10 mg protein, was boiled in 10 mL of 6 N HCl at 110°C for 24 h in vacuum-sealed ampoules) by paper chromatography34,35 revealed the presence of amino acids, alanine, arginine, asparagines, glycine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine and valine. Although other microbial collagenases from H. capsulatum var. dubosii and var. capsulatum, B. cereus, P. marinoglutinosa and C. histolyticum2,16,18,24
have been purified to electrophoretic homogeneity, they show other proteolytic activities towards casein, gelatin, egg- or bovine serum-albumin and other proteins. In this regard, Sela et al 18 declared that gelatin is a denatured form of collagen and can be degraded by collagenase and/or any non-specific protease. In the present work, the purified collagenase showed hydrolyzing activities toward casein (0.23 U/mg enzyme) and gelatin (33.23 U/mg enzyme) in addition to collagen (120 U/mg enzyme), while it was inactive against hemoglobin. Activity of the enzyme against different concentrations of such proteins was estimated and the data were used to construct the Lineweaver-Burk plots of the reciprocals of initial velocities and collagen, gelatin or casein concentrations. Also Km, Vmax, Kcat and ratios of both Vmax/Km and Kcat/Km for the 3 substrates were calculated (Table 4). From the enzyme kinetics point of view, the relative Kcat/Km values for the hydrolysis of a series of substrates are the correct kinetic quantities to evaluate the substrate specificity of the enzyme36. The best substrate for the enzyme as determined by the ratios Vmax/Km (8.485) and Kcat/Km (0.028) was collagen, indicating that the enzyme preparation is a collagenolytic enzyme. References 1 2
3 4 5
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