TY - JOUR
AU1 - Jabbar, Abdul
AU2 - Rashid, Muhammad Hamid
AU3 - Javed, Muhammad Rizwan
AU4 - Perveen, Raheela
AU5 - Malana, Muhammad Aslam
AB - Abstract Gymnoascella citrina produced two isoforms of endoglucanases (CMCase-I and -ІІ) under solid-state condition. Purified CMCase-I was novel because it was apparently holoenzyme in nature. The enzyme was monomeric as its native and subunit mass were almost the same, i.e., 43 and 42 kDa, respectively. E a for carboxymethylcellulose (CMC) hydrolysis was 36.2 kJ mol−1. The enzyme was stable over a pH range of 3.5–6.5, while temperature optimum was 55 °C. V max, K m and k cat for CMC hydrolysis were 39 U mg−1 protein, 6.25 mg CMC mL−1 and 27.5 s−1, respectively. The pKa1 and pKa2 of ionizable groups of active site were 2.8 and 7.4, respectively. Thermodynamic parameters for CMC hydrolysis were as follows: ΔH* = 33.5 kJ mol−1, ΔG* = 70.42 kJ mol−1 and ΔS* = −114.37 J mol−1 K−1. The removal of metals resulted into complete loss of enzymatic activity and was completely recovered in the presence of 1 mM Mn2+, whereas inhibition initiated at 5 mM. The other metals like Ca2+, Zn2+ and K1+ showed no inhibition up to 7 mM, Co2+ completely inhibited the activity, while Mg2+ could not recover the initial activity up to 7 mM. So we are reporting for the first time, kinetics and thermodynamics of CMCase-Ι from G. citrina. Introduction Carboxymethylcellulases (β-1,4-d-glucan-4-glucanohydrolase, EC 3.2.1.4) are members of cellulase system, which is a consortium of enzymes mainly comprised of endoglucanases (EC 3.2.1.4), exoglucanases (EC. 3.2.1.91) and cellobiases (EC. 3.2.1.21). These enzymes act in synergy, though each has different profile [11, 45]. Cellulases have application in paper and pulp industry [17] as well as in alcohol and beverage industry [8]. Furthermore, cellulases have been widely used in detergents and in textile industry for desizing, stain removing, fabric softening, depilling, pilling prevention as anti-redepositors, colour care agents, stone washing, biopolishing, biofinishing and smooth surfacing of cotton fabric [15, 37]. Other uses of cellulases, which are of great ecological and commercial importance are: amelioration of municipal, forestry, agricultural and industrial wastes to control environmental pollution; biocomposting to produce natural organic fertilizers; production of food and feed supplements for cattle and poultry feed stocks; production of plant protoplast for genetic manipulation; preparations of pharmaceuticals; baking; malting and brewing; extraction of fruit juices and processing of vegetables; botanical extraction for maximum oil yield; processing of starch and fermentating tea and coffee [6, 11, 29]. Although there are many reports on isolation and characterization of cellulases but on an average less than 1% of the potential microbes have been identified [13, 29]. So the need to isolate and identify organisms, which are either hyper-producers and/or sufficiently robust to withstand conditions of the intended application and/or are producers of novel enzymes is highly significant. In terms of enzyme novelty from an applications perspective, interest is focused on not only finding enzymes, which could break down lignocellulose much more rapidly but also enzymes, which could withstand pH, temperature and inhibitory agents more resiliently depending on the intended application [16]. Enzymes may require metal ions for their maximal catalytic activity and the enzymes requiring cofactors in the form of tightly bound metals for their activity are termed as holoenzymes. Therefore, chelation of metals results into complete loss of their activity, however, addition of metals reactivate them. The ions most commonly found in metallo-enzymes are the transition metals such as iron, zinc, copper, manganese and cobalt, etc. [43]. Metal ions may be essential for the enzyme-catalyzed reactions to proceed at a measurable rate (essential activators). Alternatively, activators may act to promote a reaction, which is capable of proceeding at an appreciable rate in its absence (non-essential activators). The tightness of binding depends on conditions of pH and temperature used, and in addition activator may combine with other components of the system [7]. Gymnoascella citrina (synonym Arachniotus citrinus) is a filamentous mesophilic ascomycetous fungus [1] isolated from sheep dung and dog dung in Pakistan. It is a novel strain as there are few reports on enzymes from fungi belonging to the genus Gymnoascella. Previously, we successfully immobilized the glucoamylase and endoglucanase of G. citrina using gel entrapment technique [28, 35]. Solid state fermentation is gaining great interest from researchers as it offers several economical and practical advantages, e.g., higher product concentration, improved product recovery, very simple cultivation equipment, reduced waste water output, lower capital investment and plant operation costs [24, 36, 37]. Hence, we are reporting for the first time purification, kinetic and thermodynamic characterization of a novel apparently metal requiring CMCase (endoglucanase) produced under solid-state growth condition by G. citrina. Materials and methods All chemicals used were of analytical grade and mostly purchased from Sigma Chemical Company, Missouri, USA. Microbial strain Gymnoascella citrina was obtained from Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan. The culture was maintained on Malt extract peptone agar slants, which were prepared according to the method 90 of DSM-catalogue of strains [9]. Inoculum preparation Vogel’s medium (100 mL) was added in 500 mL conical flask having about 20 chromic acid washed marble gravels to break the fungal mycelia and autoclaved for 20 min at 121 °C (1.1 kg cm−2). Glucose stock (50% w/v) was autoclaved at 121 °C (1.1 kg cm−2) for 5 min and was aseptically transferred to each flask to get a final concentration of 2% (v/v). Platinum wire loop full of spores of G. citrina was transferred aseptically to each flask and the flasks were kept on orbital shaker at 110 rpm at 30 °C for 1 day [31]. Alkali treatment of corn cobs Corn cobs obtained from CPC Rafhan, Faisalabad, Pakistan, were grinded to 40 mesh size and soaked for 24 h in aqueous solution of NaOH (2% w/v) at a ratio of 1:5 (w/v) and filtered through muslin cloth. The treated corn cobs were washed thoroughly with tap water and rinsed with distilled water till neutrality (pH: 7) and dried in oven at 70 °C for 48 h. Production of CMCases Gymnoascella citrina was grown under solid-state growth conditions as reported [26]. Briefly 50 conical flasks of 500 mL capacity, containing 30 g of wheat bran (40 mesh size) and alkali treated corn cob at a ratio 1:1 (w/w) were used for the production of CMCases. The substrates were soaked with 60 mL of distilled water and the flasks were plugged with cotton, covered with aluminum foil and autoclaved at 121 °C (1.1 kg cm−2) for 20 min. Afterwards, 10 mL of inoculum was sprinkled aseptically on the surface of carbon source and incubated at 30 °C. Thick fungal mat covered whole carbon source on fourth day. Isolation of crude enzyme The crude CMCases were harvested after 1 month by adding 30 mL of distilled H2O and shaken vigorously on orbital shaker at 100 rpm for 1 h. The crude enzyme was filtered through muslin cloth, centrifuged at 15,600×g at 4 °C for 30 min and further centrifuged at 39,200×g at 4 °C for 10 min to increase the clarity and then concentrated by freeze drying. The concentrate was dialyzed using 15 kDa cut-off cellulosic dialysis tubing. Total proteins and CMCase activity was determined before and after dialysis. Protein estimation Total proteins were estimated by Bradford assay [2] and bovine serum albumin (BSA) was used as standard. CMCase assay Appropriate amount of CMCase (100 μl) was incubated for 15 min at 40 °C in the presence of 1 mL Carboxymethyl cellulose sodium salt solution (CMC) [1.5% CMC (w/v) in 50 mM sodium acetate buffer: pH 5]. The reducing sugars were determined by adding 3 mL of DNS reagent and the reaction mixture was boiled in a water bath for 10 min, cooled on ice and absorbance was measured at 550 nm. Glucose (50–400 μg) was used to prepare standard curve [41]. CMCase units were calculated as follows: $$ {\text{U ml}}^{ - 1} {\min} ^{- 1} = \frac{\Updelta {\text{A}}_{550}\;{\text{of sample}} \times {\text{Glucose}}\;{\text{standard}}\;{\text{factor}}\,(500) \times {\text{Total vol}}{\text{. of reaction mixture}}\,(2.1\,{\text{ml}})} {{\text{Enzyme vol }}(0.1\,{\text{ml}}) \times {\text{Incubation time}}\;(15\,\min) \times {\text{Reaction mix vol for color development}}(2.1\,{\text{ml}})} $$ Glass cuvettes with different volume capacity and brands showed variations in results. Therefore, we recommend that whole study may be carried out with the cuvette of same specification. One unit of CMCase activity was defined as “μmol of glucose equivalent liberated min−1 under defined conditions”. Purification of CMCase The harvested crude enzyme was subjected to five-step purification procedure comprising of fractional precipitation by ammonium sulphate, Hiload anion exchange, Hydrophobic interaction, Mono-Q anion-exchange and Gel filtration chromatography on fast protein liquid chromatography (FPLC) system [26, 30, 41]. Ammonium sulfate precipitation Solid ammonium sulfate was added to 1 mL of crude, dialyzed, concentrated CMCase in eppendorf tubes to get 10–90% saturation at 0 °C and vertimixed. The tubes were left at 4 °C for about 5 h and centrifuged at 12,000 rpm for 15 min. The supernatant was assayed for residual endoglucanase (CMCase) activity. After optimization, the crude enzyme concentrate was placed on ice and solid ammonium sulfate was dissolved bit by bit to attain initially 35% saturation at 0 °C and left overnight at 4 °C. Later on, it was centrifuged at 18,000 rpm (39,200×g) for 30 min at 4 °C and the pellet was discarded, and more solid ammonium sulfate was added in supernatant to attain 65% saturation at 0 °C. Again kept for a night at 4 °C, centrifuged as mentioned before and supernatant was discarded. The pellet was dissolved in water and dialyzed extensively against distilled water for 24 h, with four changes of equal intervals. Total proteins and CMCase activity was determined before and after dialysis. The volume of enzyme solution was reduced by freeze drying [26]. Hiload anion-exchange chromatography After ammonium sulfate precipitation the CMCases were loaded on FPLC Hiload-Q Sepharose column, using superloop of 50 mL at a rate of 2 mL min−1. The linear gradient of NaCl (0–1 M) in 20 mM Tris/HCl pH 7.5 was used as elution buffer. The fractions (4 mL fraction−1) containing endoglucanase activity were pooled and dialyzed against distilled water. The pools were assayed for enzyme activity and total proteins. Hydrophobic-interaction chromatography (HIC) Active fractions of CMCase-І from Hiload column were mixed with ammonium sulfate to get final concentration of 2 M and then filled in superloop by peristaltic pump and loaded on Phenyl Superose column at a rate of 0.5 mL min−1. The elution was carried out with a linear gradient of ammonium sulfate (2–0 M) in 50 mM phosphate buffer pH 7. Active fractions (2.5 mL fraction−1) were pooled, dialyzed and assayed for enzyme activity and total proteins. Mono-Q anion-exchange chromatography The dialyzed, purified CMCase from HIC was loaded on Mono-Q column at a flow rate of 1 mL min−1 and a linear gradient of NaCl (0–1 M) in 20 mM Tris/HCl pH 7.5 was used as elution buffer. Active fractions (2 mL fraction−1) were pooled, dialyzed and assayed for total enzyme activity and proteins. Gel filtration chromatography The purified CMCase-І after Mono-Q was loaded on Superose column to get purification to homogeneity level and to determine the native molecular weight. The sample (200 μl run−1) was loaded using “loop TMS program” of FPLC and 100 mM Tris/HCl, pH 7 having 0.15 M NaCl was used as elution buffer at a flow rate of 0.5 mL min−1. A measure of 1 mL size fractions were collected. Native molecular mass The purified CMCase and different marker proteins were applied separately on FPLC gel filtration chromatography and native molecular mass was determined as described [36]. Sub-unit molecular mass Sub-unit molecular mass of CMCase was determined by using sodium dodecyl-sulphate polyacrylamide gel electrophoresis (10% SDS-PAGE) [20], which was performed using BRL apparatus and gels were stained by Coomassie blue R-250. Standard curve, which was drawn between R f values of standard protein bands versus their log molecular weight was used to determine the exact molecular mass. Temperature and pH optimum CMCase-І was assayed at different temperatures ranging from 10 to 80 °C and energy of activation (E a) was determined by applying Arrhenius plot. The effect of temperature on the rate of reaction was expressed in terms of temperature quotient (Q 10), which is the factor by which the rate increases due to a raise in the temperature by 10 °C. Q 10 was calculated by rearranging the equation given by Dixon and Webb [7]. $$ Q_{{10}} \, = \,{\text{antilog }}_{\varepsilon } {\text{ }}(E\, \times \,10/RT^{2} ) $$1 where E = E a = activation energy. Activity of the enzyme was also determined at 40 °C against different pH, ranging from 2 to 9.6 and Dixon plot was applied to determine the pKa of ionizable groups of active site residues [41]. Effect of metals The holoenzyme nature of CMCases of G. citrina was determined by chelating the bound metals. Apo-CMCase was made by dialyzing the enzyme against 5 mM EDTA dissolved in 50 mM “MOPS”/KOH pH 7.0 for 20 h with three changes. The EDTA was removed by dialyzing apoenzyme intensively against 30 L of distilled deionised water in 24 h (four changes) [43]. Activity of apo-CMCase was determined in the presence of different metals: Ca2+, Zn2+, Mg2+, Mn2+, Co2+ and K1+. Effect of substrate Michaelis–Menten kinetic constants (V max, K m) were determined by assaying the enzyme at different CMC concentrations ranging from 0.5 to 2.5% (w/v) and Lineweaver–Burk plot was applied [41]. Similarly, effect of 1.5 mM Mn2+ on kinetic constants for CMC hydrolysis by CMCase-І was also determined. Thermodynamics of CMC hydrolysis The thermodynamic parameters (ΔG*, ΔH* and ΔS*) for CMC hydrolysis were calculated using the Michaelis constants determined under effect of substrate by rearranging the Eyring’s absolute rate equation derived from the transition state theory [41]. $$ K_{{{\text{cat}}}} \, = \,(K_{{\text{b}}} T/h){\text{ e}}^{{( - \Updelta H^*P/RT)}} {\text{ e}}^{{(\Updelta S^*/R)}} $$2 where h Planck’s constant = 6.63 × 10−34 J s K b Boltzman’s constant (R/N) = 1.38 × 10−23 J K−1 R gas constant = 8.314 J K−1 mol−1 N Avogadro’s No = 6.02 × 1023 T absolute temperature. $$ E_{{\text{a}}} \, = \, - {\text{Slope}}\, \times \,R $$3 $$ \Updelta H^*\, = \,E_{{\text{a}}} -RT $$4 where ΔH* is the enthalpy of activation of CMC hydrolysis $$ \Updelta G^*\, = \, - RT\,\ln(K_{{{\text{cat}}}} h/K_{{\text{b}}} T) $$5 where ΔG* is the free energy of activation of CMC hydrolysis. $$ \Updelta S^*\, = \,(\Updelta H^* - \Updelta G^*)/T $$6 where ΔS* is the entropy of activation of CMC hydrolysis. $$ \Updelta G^*_{{{\text{E}} - {\text{S}}}} \, = \, - RT\,\ln\,K_{{\text{a}}}({\text{free energy of CMC binding}}) $$7 where K a = 1/K m $$ \Updelta G^*{\text{ }}_{{{\text{E}} - {\text{T}}}} \, = \, - RT\,\ln\,K_{{{\text{cat}}}} /K_{{\text{m}}} ({\text{free energy of transition state formation}}) $$8 Results and discussion Solid-state fermentation offers several economical and practical advantages such as: higher product concentration, improved product recovery, etc. Therefore, G. citrina, a mesophillic fungus was grown under solid-state growth conditions. The production of CMCases after 30 days at 30 °C was 4 units mg−1 protein. Purification of CMCases Crude enzyme was purified to homogeneity level after subjecting to ammonium sulfate precipitation, Hiload anion exchange, hydrophobic interaction, Mono-Q anion exchange and gel filtration chromatography on Pharmacia FPLC unit. The five-step purification procedure resulted into 27.3-fold purification and final recovery of CMCase-І was 25.5% (Table 1). The purity of CMCase-І was apparently to homogeneity level, which was confirmed on 10% SDS-PAGE. Three step purification procedure for CMCase of Cellulomonas biazotea and four step procedure for that of A. niger resulted into an increase in specific activity of 9- and 12-fold, respectively [40]. Purification of CMCases from Gymnoascella citrina Treatment . Total units . Total protein (mg) . Specific activity (U mg−1) . Purification factor . % Recovery . Crude 3,418 810.0 4.2 1.0 100 (NH4)2 SO4 precipitation 2,941 403.0 7.3 1.7 86 FPLC Hiload® chromatography 1,343 43.4 30.9 7.3 39 Hydrophobic interaction chromatography 1,211 22.8 53.2 12.6 35 FPLC Mono-Q® chromatography 1,071 14.2 75.4 17.9 31 Gel filtration chromatography 874 7.6 115 27.3 25.5 Treatment . Total units . Total protein (mg) . Specific activity (U mg−1) . Purification factor . % Recovery . Crude 3,418 810.0 4.2 1.0 100 (NH4)2 SO4 precipitation 2,941 403.0 7.3 1.7 86 FPLC Hiload® chromatography 1,343 43.4 30.9 7.3 39 Hydrophobic interaction chromatography 1,211 22.8 53.2 12.6 35 FPLC Mono-Q® chromatography 1,071 14.2 75.4 17.9 31 Gel filtration chromatography 874 7.6 115 27.3 25.5 Where, all quoted values were taken after dialysis against distilled water Open in new tab Purification of CMCases from Gymnoascella citrina Treatment . Total units . Total protein (mg) . Specific activity (U mg−1) . Purification factor . % Recovery . Crude 3,418 810.0 4.2 1.0 100 (NH4)2 SO4 precipitation 2,941 403.0 7.3 1.7 86 FPLC Hiload® chromatography 1,343 43.4 30.9 7.3 39 Hydrophobic interaction chromatography 1,211 22.8 53.2 12.6 35 FPLC Mono-Q® chromatography 1,071 14.2 75.4 17.9 31 Gel filtration chromatography 874 7.6 115 27.3 25.5 Treatment . Total units . Total protein (mg) . Specific activity (U mg−1) . Purification factor . % Recovery . Crude 3,418 810.0 4.2 1.0 100 (NH4)2 SO4 precipitation 2,941 403.0 7.3 1.7 86 FPLC Hiload® chromatography 1,343 43.4 30.9 7.3 39 Hydrophobic interaction chromatography 1,211 22.8 53.2 12.6 35 FPLC Mono-Q® chromatography 1,071 14.2 75.4 17.9 31 Gel filtration chromatography 874 7.6 115 27.3 25.5 Where, all quoted values were taken after dialysis against distilled water Open in new tab Ammonium sulfate precipitation The onset of CMCases precipitation occurred at 35% saturation of ammonium sulfate at 0 °C, while complete precipitation was at 65% saturation. The cellulases were 1.73-fold purified after precipitation. The CMCases from A. niger precipitated between 45 and 65% ammonium sulfate saturation at 0 °C [41]. Hiload anion exchange chromatography Partially purified CMCases, after ammonium sulfate precipitation were further purified on FPLC Hiload anion exchange chromatography and two isoforms (CMCase-I and -ІІ) were recovered. Majority of CMCases belonged to CMCase-I, which eluted just at the onset of sodium chloride gradient, while CMCase-II present in minute quantity eluted at about 500 mM NaCl. The elution pattern indicated that CMCase-I was slightly acidic in nature (Fig. 1). Endoglucanases from A. niger elute at 550 mM NaCl on Hiload column [41]. The production of CMCase-II was very low with respect to total CMCases, so CMCase-I was further purified for characterization. Fig. 1 Open in new tabDownload slide FPLC Hiload anion exchange chromatography of CMCases on Q-Sepharose column using 0–1 M NaCl gradient for elution Hydrophobic interaction chromatography Partially purified CMCase-I from Hiload column was further purified by applying on phenyl superose column and was eluted at 286 mM ammonium sulphate (Fig. 2). The elution of CMCase-I from HIC column showed that it was tightly adsorbed on the column and hence had highly hydrophobic surface. On the other hand, the CMCases from A. niger elute at about 95 mM (NH4)2SO4 on HIC [41]. Ammonium sulfate inhibited CMCase activity, which was recovered by dialysis against distilled water. Fig. 2 Open in new tabDownload slide FPLC Hydrophobic interaction chromatography of CMCase-I on FPLC Phenyl Superose column using 2–0 M (NH4)2 SO4 gradient Mono-Q anion exchange chromatography Purified CMCase-I from HIC was further purified on FPLC Mono-Q anion exchange column and was recovered before the start of NaCl gradient (Fig. 3). The A. niger endoglucanases were eluted at about 640 mM NaCl on Mono-Q column [41]. Fig. 3 Open in new tabDownload slide FPLC Mono-Q anion exchange chromatography of CMCase-I using 0–1 M NaCl gradient Gel filtration chromatography The purified CMCase-I was finally applied on gel filtration column (Fig. 4) and purity was at homogeneity level on SDS-PAGE (Fig. 5). The CMCase-I was about 27-fold purified (Table 1). Fig. 4 Open in new tabDownload slide FPLC Gel filtration chromatography of CMCase-I on Superose column Fig. 5 Open in new tabDownload slide 10% SDS-PAGE of CMCase-I from Gymnoascella citrina: Fermentas protein marker #SM0661 10–200 kDa (lane-1) and CMCase-I (lane-2) Molecular mass The CMCase-I was monomeric in nature because its native (43 kDa) and sub-unit (42 kDa) molecular masses were almost the same. Molecular weights of CMCases produced by variety of microbes have been reported, e.g. native and SDS-PAGE molecular weight of Bacillus sp. was 33 kDa [38], Neurospora crassa have CMCases with 70 kDa molecular mass [46] and the endoglucanases obtained from Trichoderma viride had multiple forms with molecular weights 38, 42, 52 and 60 kDa [18]. Effect of temperature Temperature optimum of CMCase-I was 55 °C. Arrhenius plot for energy of activation (E a) showed a biphasic trend (Fig. 6) and ES-complex formation at optimum temperature (55 °C) required E a of 36.2 kJ mol−1. The activation energies for CMCases from A. niger showed a triphasic trend and E a up to temperature optima were 53 and 18 kJ mol−1 [41] while CMCase from C. biazotea have E a 35 kJ mol−1 [40]. It was found that removal of non-covalently bound polysaccharides from CMCases of A. niger have changed the activation energy profile and E a of polysaccharides free and complexed were 17 and 55, and 19 and 21 kJ mol−1, respectively [39]. Endoglucanase isolated from recombinant strain of E. coli has 38.9 kJ mol−1 E a between temperatures 23 and 53 °C [23]. We have also calculated the temperature quotient of the enzyme, which was 1.01. Fig. 6 Open in new tabDownload slide Arrhenius plot for effect of temperature on activity and determination of activation energy for CMcellulose hydrolysis by CMCase-I of Gymnoascella citrina. Data presented are average values ± SD of n = 3 experiments Effect of pH CMCase-I had pH optima in the range of 3.5–6.5 and Dixon plot was applied to determine pKa of ionizable groups of the active site residues. It was found that CMCase-I involved two polar ionizable residues in catalysis (Fig. 7). The pKa1 for proton donating ionizable group was 2.8, which showed that carboxyl groups may be acting as proton donor whereas, pKa2 for proton receiving residue was 7.4, which on comparison with pKa values for amino acids present in proteins indicated that proton receiving group may be imidazole [14]. Hakamada et al. [12] deduced active site residues of a thermostable alkaline endoglucanases from an alkaliphilic Bacillus sp. by site directed mutagenesis and declared that histidine, glutamic acid, arginine and tyrosine are playing important role in catalysis. The active site residues for A. niger CMCases have been determined and found that both proton donating and receiving residues contain carboxyls as ionizable group with pKa values of 3.5 and 5.5, respectively [41]. Endoglucanases from Schizophyllum commune involve the amino acid residues with pKa values of 3.7 and 6.1 for CMC catalysis [29]. Fig. 7 Open in new tabDownload slide Dixon plot for the effect of pH on activity and determination of pKa of ionize able groups of active site residues of CMCase-I from Gymnoascella citrina. Data presented are average values ± SD of n = 3 experiments Effect of metals The removal of metals from CMCase-I resulted into almost complete loss of enzymatic activity and apoenzyme of CMCase-I displayed only about 3.4% residual activity. The activity of apo-CMCase-I was completely recovered in the presence of 1 mM Mn2+. We consider that the apo-CMCase-I was not absolutely free from all metals due to which it gave minute activity. Due to limited lab facilities we could not evaluate the quality of apoenzyme. Therefore, based on % residual activity, we believe that CMCase-I was apparently metallo in nature. The enzymes requiring cofactors in the form of tightly bound metals for their activity are termed as holoenzymes and chelating metals results into complete loss of their activity, however, addition of metals reactivate them. Hence, holoenzymes contain firmly bound metal ions at their active sites. Therefore, we conclude that CMCase-I is apparently holoenzyme and this property made it novel in nature. The effect of different metal ions like Ca2+, Zn2+, Mg2+, Mn2+, Co2+and K1+ on the reactivation of apo-CMCase-I was also determined. No inhibition up to 7 mM of Ca2+, Zn2+ and K1+ was observed while Mn2+ showed onset of inhibition at 5 mM. On the other hand Mg2+ could not recover the initial activity up to 7 mM while Co2+ completely inhibited the activity (Fig. 8). Endoglucanases from Cellulomonas uda activated by 1 mM Mn2+ [37], while, that of C. biazotea and A. niger showed activation at 1.5 mM Mn2+ [40]. Furthermore, 1.25 mM Hg2+ completely inhibited and Pb2+ partially inhibited the endoglucanases of Arthrobotrys oligospora [19]. Endoglucanase from Chalara paradoxa were inhibited by Hg2+, Ag1+ while, partial inhibited by 10 mM Zn2+, Fe2+, Mg2+ but stimulated by Mn2+ [22]. Fig. 8 Open in new tabDownload slide Effect of metals on the reactivation of apo-CMCase-I from Gymnoascella citrina. Error bars represent the standard deviation Effect of substrate CMCase-I was assayed in the presence of different CMC concentrations at 50 °C, pH 5.0. The V max was 39 U min−1 mg−1 protein and K cat was 27.5 catalytic events s−1, while K m was 6.25 mg CMC mL−1. The Specificity constant (V max/K m) was 6.24, while the enzyme concentration used was 2.143 × 10−4 μmol or 9 μg (Fig. 9). Fig. 9 Open in new tabDownload slide Lineweaver–Burk plot for the determination of Michaelis kinetic constants (V max, K m) for CMcellulose hydrolysis at 50 °C, pH 5 by CMCase-I of Gymnoascella citrina. Turn over (K cat) = s−1 = V max/[e], where [e] is the enzyme concentration = 2.143 × 10−4 μmol, K m = mg CMC mL−1 and K cat/K m = s−1 mg−1CMC mL−1. Data presented are average values ± SD of n = 3 experiments The native CMCase-I was twelve fold activated by 1.5 mM Mn2+. Kinetic rate constants in the presence of Mn2+ were: V max = 454 U min−1 mg−1 protein, K cat = 318 s−1, K m = 40 mg CMC mL−1 and V max/K m = 11.35. The Michaelis constant (K m) value in the presence of Mn2+ become very high which confirmed that the affinity of CMC in the presense of Mn2+ was seriously affected and the enzyme required high amount of CMC to saturate it. The K cat values confirmed that the Mn2+ binding has activated the conversion of transition complex into products. Theberge et al. [42] found that endoglucanase from Streptomyces lividans IAF 74 have V max of 24.9 U mg−1 and K m of 4.2 mg mL−1. CMCases of Thermomonospora curcata, T. reesei and Alternaria alternata hydrolyze CMC substrate with V max of 833 μmol glucose min−1, 405.5 μmol glucose h−1 and 18 μmol glucose min−1 mg−1 protein, respectively whereas, their K m for CMC were 7.33 mg mL−1, 1.32% (w/v) and 0.43 mg mL−1, respectively [10, 21, 45]. The endoglucanase from E. coli harboring the genes of endoglucanase of Fibrobacter succinogenes S85 displayed V max of 152 IU mg−1 and K m of 0.49% w/v [4]. CMCases isolated by Wittmann et al. [44], Rashid and Siddiqui [32], Siddiqui et al. [41] and Lucas et al. [22] from S. lividans IAF9, A. niger and C. paradoxa had V max of 110 IU mg−1, K cat of 1,000 min−1 and V max of 1.1 μmol min−1, respectively whereas, their K m was 1.3, 70 and 8.3 mg mL−1, respectively. Thermodynamics of CMC hydrolysis Thermodynamic parameters for CMC hydrolysis by CMCase-I of G. citrina were determined as described [41]. The enthalpy (ΔH*) of activation of CMC hydrolysis was 33.5 kJ mol−1. Gibbs free energy of substrate binding (ΔG*E-S) was 4.92 kJ mol−1, while change in free energy (ΔG*E-T) for the formation of activated complex (ES*) was −3.98 kJ mol−1. The conversion of transition state to products exhibited ΔG* of 70.42 kJ mol−1 and the entropy of activation of CMC hydrolysis (ΔS*) was −114.37 J mol−1 K−1. Previously, we report about thermodynamics of CMcellulose hydrolysis by native CMCase of A. niger, which hydrolyzed CMC with the following thermodynamic parameters: ΔG* (69 kJ mol−1), ΔG*E-T (−13 kJ mol−1), ΔG*E-S (5.1 kJ mol−1), ΔH* (50 kJ mol−1) and ΔS* (−61 J mol−1 K−1) [41]. The lower enthalpy value of CMCase from G. citrina as compared to CMCase of A. niger showed that the formation of transition state or activated complex between enzyme-substrate was very efficient. Conclusion In the light of our results, we concluded that endoglucanase of G. citrina was apparently metallo in nature. The capability of CMCase-I to resist against high concentration of metals (7 mM) signifies their importance for possible applications in industries like textile, detergents, and paper and pulp, etc. Acknowledgments The project was funded by Pakistan Atomic Energy Commission (PAEC), Islamabad. Technical assistance of G. A. Waseer is appreciated. We are thankful to Dr. M. Sajjad Mirza (Plant Micro-biotechnlogy Division, NIBGE) for proof reading of the manuscript. Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan, is highly acknowledged for providing the culture of Gymnoascella citrina. References 1. 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TI - Kinetics and thermodynamics of a novel endoglucanase (CMCase) from Gymnoascella  citrina produced under solid-state condition
JF - Journal of Industrial Microbiology and Biotechnology
DO - 10.1007/s10295-008-0310-4
DA - 2008-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/kinetics-and-thermodynamics-of-a-novel-endoglucanase-cmcase-from-NDrMii4ZoA
SP - 515
EP - 524
VL - 35
IS - 6
DP - DeepDyve
ER -