Production of cellulase by Mucor ramanniacus using submerged fermentation and its applications in biodegradation of agro-industrial waste

This study was undertaken to isolate and identify a novel cellulase-producing strain from a waste site (7°28’11’’N 4°31’24’’E), optimise the growth conditions, partially purify and biochemically characterise the enzyme. The potentials of the puried cellulase to hydrolyse the lignocellulosic component of some agroindustrial wastes (e.g. orange peels etc.) was also investigated. The best cellulase-producing fungus was identied as Mucor ramanniacus and the optimum conditions for cellulase production were pH (4.5), inoculum size (12 mm), carbon and nitrogen sources were carboxymethyl cellulose and sodium nitrate respectively resulting in a specic activity of 1423 Units/mg protein. A purication fold of 1.56 and 45.37 % yield were obtained after purication. The optimum pH and temperature were at 9.0 and 40°C respectively. The kinetic parameters were 0.63 ± 0.495 mg/ml, 20.21 ± 11.28 U/ml, 1001.4s − 1 for K m and V max and k cat respectively. Na + , K + , Ca + , Cysteine, β-mercaptoethanol and SDS were activators while Tween 80, Triton X-100 EDTA, Hg 2+ and Ba 2+ inhibited the enzyme. M. ramanniacus cellulase hydrolysed all agro-industrial wastes used. The partially puried M. ramanniacus cellulase showed great potential in biodegradation of various lignocellulosic substrates and the biochemical characteristics exhibited makes it suitable in industrial applications.

Cellulase has attracted much scienti c and industrial attention. Cellulases are useful in cotton processing, paper recycling, laundry, ethanol production, juice extraction, and as animal feed additives. The world attention in the eld of scienti c development has shifted to environmental biotechnology due to the great challenge posed by accumulation of these wastes, cellulosic materials have become di cult to handle. These solid waste materials can be hydrolyzed e ciently by some classes of fungi known as lamentous fungi (Wood 1992;Subramaniyan and Prema 2004). Since microbial sources of enzyme is less capital intensive, the time required for growth is very short compared to plants and animals, the mode and amount to be produced at a time can as well be manipulated by the researchers. The majority of plant biomass, including stems and leaves are composed of lignocellulose which is the most abundant renewable biomass resource on earth. Lignocellulose is a complex and tightly organized matrix of three main polymers, hemicellulose, lignin and cellulose (the major component of agricultural wastes and municipal residues) (Gautam et al. 2010). The large volumes of cellulosic waste (35-45%) generated annually from forestry commercial, agricultural and industrial activities are less di cult to degrade and cause imbalances in the ecosystem (James and Natalie 2001).
In this study, a cellulase producing organism, Mucor ramannianus was isolated from sites of cellulosic waste deterioration. The cellulase was partially puri ed and its biochemical characteristics determined.
The potentials of the puri ed cellulase in the biodegradation of agro-industrial wastes were investigated. This is the rst report of cellulase production by M. ramannianus. The biotechnological and industrial potentials of the cellulase from this fungus were established.  Isolation was carried out by serial dilution and pure fungal isolates were obtained.
The pure fungal isolates were identi ed by observing macro and micro morphological characteristics using cotton blue in lactophenol stain technique (Alexopoulos et al. 1996). The morphological characteristics were noted and used in the preliminary identi cation (Kasana et al. 2008;Ademakinwa et al. 2016). Fungal strains isolated from the soil sample were screened for cellulase activity by spotting on cellulose-agar plates containing: NaNO 3 -2.0 g/L, MgSO 4 . 7H 2 O -0.5 g/L, KCl -0.5 g/L, K 2 HPO 4 -1.0 g/L, FeSO 4. 7H 2 O -0.01 g/L and high viscosity carboxymethyl cellulose (0.5% w/v). Plates were incubated at room temperature for 72 h. Thereafter, plates were ooded with iodine solution (1 % iodine crystals and 2 % potassium iodide) and incubated for 15 min at room temperature (Kasana et al. 2008). Excess stain was washed off for visual observation for zone of clearance around mycelia, indicating cellulolytic Page 4/28 activity. The medium composition for the submerged fermentation production of cellulase was NaNO 3 -2.0 g/L, MgSO 4 . 7H 2 O -0.5 g/L, KCl -0.5 g/L, K 2 HPO 4 -1.0 g/L, FeSO 4. 7H 2 O -0.01 g/L and high viscosity carboxymethyl cellulose (0.5% w/v). The medium was sterilized, cooled and inoculated with the fungal agar plugs.

Cellulase Assay
Cellulase activity was determined by estimating the amount of reducing sugars released in the reaction mixture containing carboxymethyl cellulose and the enzyme using the modi ed dinitrosalicyclic acid reagent method of Miller (1959). The reaction mixture consisted 0.5 ml of 0.1 % w/v CMC in citrate buffer (pH 4.8) and 0.5 ml of enzyme . One unit of cellulase activity was described as the amount of enzyme that liberated reducing sugar equivalent to 1 µg of glucose per minute under the speci ed assay conditions. The speci c enzyme activity was expressed as the unit of enzyme activity per mg of protein. The protein concentration was determined by the method of Bradford (1976) using bovine serum albumin (BSA) as standard.

Optimization of Cellulase Production
Optimization was studied for cellulase production were studied by maintaining all factors constant except the one being studied.

Effect of Incubation Period
The effect of incubation period on cellulase production was determined by varying the incubation time.
Fifty millilitres (50 ml) of sterilized culture medium was inoculated with 12 mm diameter disc of M. ramannianus and incubated at room temperature in a shaker for 8 days. Ten milliliters samples were withdrawn at 2 days intervals and centrifuged at 4000 x g for 15 min.
The supernatant served as the crude enzyme and was analysed for cellulase activity.

Effect of Inoculum Size on Cellulase Production
Inoculum sizes varying from 4-16 mm were used to determine its effect on cellulase production by the M. ramannianus. The agar plugs of the fungus were inoculated separately into 50 ml of the production medium and incubated for 4 days at room temperature. The extracellular enzyme was harvested from the culture medium by ltering using muslin cheesecloth and the ltrate (clear supernatant) was used as crude enzyme which was assayed for enzyme activity and inoculum size of 4 mm served as the control.

Optimum pH for Cellulase Production
The optimum pH for the production of cellulase was determined by varying the pH values of the basal salt medium ranging from 4 to 7. The clear supernatant was assayed for cellulase activity.

Determination of Optimum Temperature for Cellulase Production
The temperature optimal for cellulase production was determined by incubating the isolate in the basal medium at 30 to 60 o C with constant agitation. The supernatants obtained were assayed for cellulase activity. The incubation temperature at 30°C served as the control.

Effect of Nitrogen Sources on Cellulase Production
Different nitrogen sources were investigated for their effect on cellulase production by replacing the 2.0 g/L NaNO 3 in the basal salt medium with organic (gelatin, casein, yeast extract, beef extract and peptone) and inorganic (KNO 3 , NH 4 NO 3 , NH 4 H 2 PO 4 , (NH 4 ) 2 SO 4 and NH 4 Cl) nitrogen sources and sterilized at 121 o C for 15 min. The sterile modi ed media of appropriate nitrogen source was inoculated and incubated at the optimum temperature for 4 days. The content of the asks was ltered and the supernatants obtained were assayed for cellulase activity. The medium with NaNO 3 as nitrogen source served as the control.

Effect of Carbon Sources on Cellulase Production
The effect of carbon sources was studied by replacing the carboxymethylcellulose (CMC) at 1% concentration in the Basal Salt Medium (BSM) with agro-industrial wastes (orange peel, orange bagasse, cassava peel, potato peel, pineapple peel, banana peel and plantain peel ours) as crude carbon sources and re ned carbon sources (cellobiose and lter paper our) while other media components were kept constant. The medium was adjusted to pH 5.5 and sterilized at 121 o C for 15 min. This was followed by inoculation and incubation for 4 days at optimum temperature. The supernatants obtained after ltration were assayed for cellulase activity and the medium containing CMC as the carbon source served as the control.

Production of Cellulase under Optimized Condition and Determination of Fungal Growth
The enzyme production was carried out in two different 250 ml Erlenmeyer asks containing 50 ml medium each. The medium was then sterilized at 121 ºC for 15 min. The medium was cooled and then inoculated with one and half agar plugs of 12 mm of three days old fungus culture. The asks were incubated at optimum temperature in a shaker for 4 days. The extracellular enzyme was harvested from the culture medium by ltering using muslin cheesecloth and the ltrate (clear supernatant) was used as crude enzyme.

2.6
Puri cation of Cellulase The crude enzyme ltrate (70 ml) was lyophilized by freezing under atmospheric pressure (-25 °C). The powdered sample obtained after lyophilisation was re-suspended in 6 ml of 0.1 M citrate buffer. Preswollen Sephacryl S-200 was packed into a 1.5 x 65 cm column as described by (Amiola et al., 2018 andAgboola, 2020). Five millilitres of the re-suspended lyophilized enzyme was layered on the column. The column was eluted with 200 ml 0.1 M citrate buffer, pH 4.8. Fractions of 5 ml were collected from the column at a ow rate of 20 ml/h. Protein content of the fractions was monitored spectrophotometrically at 280 nm and the fractions were also assayed for cellulase activity. The active fractions were pooled, assayed and protein concentration was determined by methods described by Bradford (1976). The pooled enzyme was stored on ice.
The percentage yield was calculated thus: The puri cation fold was calculated thus: 2.7 Characterization of M. ramannianus Cellulase (MrC)

Determination of Subunit Molecular Weight
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Weber and Osborn (1975) using Tris-glycine buffer system to determine subunit molecular weight. The sample was prepared by mixing aliquots of the enzyme with electrophoresis sample buffer (60 mM Tris-HCl buffer, pH 6.8, containing 2% SDS, 0.5 M 2-mercaptoethanol and 0.05% bromophenol blue (a tracking dye) in ratio 2:1. SDS-PAGE was performed on 12% resolving or separating gel and 4% stacking gel.

Determination of Kinetic Parameters
The apparent kinetic parameters (K M , V max , k cat and k cat/ K M ) of cellulase were determined by varying concentrations of carboxymethyl cellulose (CMC) between 0.1 mg/ml and 0.5 mg/ml in 0.1 M citrate buffer, pH 4.8 and measuring the initial velocities (U/ml/min) at a xed volume of the puri ed enzyme. The assay mixture contained 0.5 ml of puri ed enzyme. The data were plotted according to Lineweaver-Burk (1934).

Effect of Temperature on Enzyme Activity and Stability
The effect of temperature on the activity of the puri ed cellulase was studied over a temperature range of 20 -90°C to determine the optimum temperature. The thermal stability of the enzyme was studied by pre-incubating the puri ed enzyme at the optimum temperature for 1 h. From the incubated solution, 0.2 ml of the enzyme was withdrawn at 10 min interval and added to the substrate containing 0.1% CMC in 0.1 M citrate buffer to initiate the reaction and then activity was determined under standard assay conditions.

Effect of pH and pH Stability
The activity of cellulase was monitored at different pH values 3.0 -11.0 by incubating the mixture of the enzyme and the substrate in the presence of the appropriate buffers. The buffer systems used were 50 mM acetate buffer (pH 3.0-5.0), 50 mM citrate buffer (pH 4.0-6.0), 50 mM Tris buffer (pH 7.0-9.0) and 50 mM borate buffer (pH 9.0-11.0). The stability was determined by incubating aliquots of the puri ed enzyme in 50 mM Tris buffer pH 9.0 for 1 h at room temperature (24±2 ˚C) which was withdrawn at 10 min intervals for assay.

Effect of Salts
The behaviour of the puri ed enzyme in the presence of metal ions in salts of chloride ions were studied in nal concentrations of 1 mM, 5 mM and 10 mM. The enzyme was incubated for 30 min with the chloride salts and the activity was checked under optimal assay conditions. The activity was considered 100% in the absence of any salt.

Effect of Inhibitors
The effect of some representative inhibitory compounds such as EDTA, SDS, L-cysteine, Tween 80, Triton X-100 and β-mercaptoethanol were examined in nal concentrations of 1.0 mM, 2.5 mM, 5.0 mM and 7.5 mM. Aliquots of the inhibitor was incorporated into a 1 ml reaction mixture containing 0.2 ml substrate solution (0.1% CMC in 0.1 M citrate buffer, pH 4.8), 0.2 ml buffer and 0.4 ml water. The reaction was initiated by the addition of 0.2 ml enzyme and incubated for 30 min at room temperature. The residual enzyme activity was assayed by measuring the amount of reducing sugars released and reaction mixtures without inhibitor were taken as control with 100% activity.

Effect of Organic Solvents
The puri ed enzyme was incubated in different organic solvent (diethyl ether, hexane, toluene, ethanol, methanol and acetone) in nal concentrations of 1.0%, 5.0%, 10% and 20% for 30 min. The relative cellulase activity was estimated against the control in which solvent was not present.

Activities of MrC with various Natural Substrates
The hydrolytic potential of puri ed MrC was investigated against a variety of substrates such as agroindustrial wastes (orange peel, orange bagasse, cassava peel, potato peel, pineapple peel, banana peel and plantain peel ours) as crude carbon sources, cellobiose and CMC as re ned carbon sources. Each substrate (0.1% w/v) was suspended in of 0.1 M citrate buffer, pH 4.8. To 0.2 ml of the substrate, 0.2 ml of the enzyme was added to initiate the reaction which was then incubated for 30 min at room temperature. The relative enzyme activity was assayed by measuring the amount of reducing sugars released and expressed as percentage relative activity.

Statistical Analysis
Data were expressed as mean ± standard error of mean (SEM). The signi cance of the results was evaluated using analysis of variance (ANOVA). Values of p<0.05 were regarded as statistically signi cant.

Discussion
Degradation of cellulosic materials is a complex process requiring participation by a number of microbial enzymes. This study showed that, of the eleven fungal strains were isolated from the dumpsite soil, eight had potential for cellulase production from which the best cellulase-producing strain was selected for further study. Based on morphological characterization, the organism was identi ed as Mucor ramannianus. Cellulases have been identi ed in a variety of fungal species such as Trichoderma sp., Penicillium sp. and Aspergillus sp. (Lakshmikant and Mathur 1990;Mandels and Reese 1985).
Eleven (11) fungal strains were isolated from the University dumpsite soil. Eight (8) out of the eleven (11) fungal strains isolated showed a zone of clearance with varying magnitude indicating the production of the enzyme and Mucor ramannianus which had the highest activity was selected for further studies. Aside from M. ramannianus, another notable fungus that elaborated cellulase was Aureobasidium pullulans (Table 1 a).The production of cellulase by A. pullulans has been reported by . From our previous studies, this fungus can also elaborate rhodanese , laccase (Ademakinwa et al., 2016) and fructosyltransferase (Ademakinwa et al. 2018). The isolates were further tested for cellulolytic activity in the culture broth using CMC as substrate (Table 1 a & b). The production of enzyme gradually increased with the fermentation (incubation) time and reached the peak point on the 4 th day of incubation. The inoculum size of 12 mm resulted in a relatively higher cellulase activity (61.38 U/mg. Maximum cellulase production was obtained at the temperature of 40°C with 124.53 U/mg in the temperature range tested (graphs not shown).
The optimum temperature for growth of fungus may not necessarily be the optimum temperature for production of enzymes, although incubation temperature is a critical factor in enzyme production (Kheng and Omar 2005). This study recorded an optimum temperature for maximum enzyme production at 40°C with speci c activity of 134.16 U/mg protein following a signi cant decline in cellulase production as temperature increased. Sabu et al. (2002) reported that as temperature increases, enzyme production decreases because enzyme is a secondary metabolite produced during exponential growth phase and incubation at high temperature could lead to poor growth and thus a reduction in enzyme yield.
The result for the cellulase production at 40˚C is in accordance with previous ndings for Aspergillus terreus QTC 828 (Ali et al. 1991), Aspergillus avus (Lin et al. 2010) and Aspergillus fumigatus (Sherief et al. 2010). In contrast, Asquieri and Park (1992) reported that the optimum temperature for maximum cellulase production from thermostable Aspergillus sp. was 37 ºC. Padmavathi et al. (2012) also observed a good enzyme yield with Mucor plumbeus at 37ºC.
In this study, the optimum pH of the medium was 4.5. Among the physical parameters, utilization of pH in growth medium is considered an important factor during enzyme production by inducing morphological changes in microbes and in enzyme secretion. According to Silva et al. (2009), most lamentous fungi are pH dependent. The pH change observed during the growth of microbes also affects product stability in the medium (Gupta et al. 2003). Padmavathi et al. (2012) reported a pH optimum of 3.0 for Mucor plumbeus, while Bhaskar et al. (2014) reported an optimum pH of 5.0 for maximum cellulase production by Penicillium variabile. Beldman et al. (1985) also reported that Aspergillus species grow and metabolize well in acidic pH medium between pH 3.0 -5.0. Most fungal cultures prefer a slightly acidic pH medium for growth and enzyme biosynthesis (Haltrich et al. 1996) which agrees with the result obtained in this study. It is noteworthy to highlight that the speci c activity was albeit lower during the optimization of the effect of pH compared with the values obtained during temperature optimization. This might be attributed to the physiological change that the fungus, M. ramannianus exhibits at altered pH values Research has shown that enzyme synthesis is correlated with the quality and concentration of nitrogen and carbon sources. These requirements differ from one organism to another (Pandey et al. 2001;Schnurer and Jarvis 2010). Maximum cellulase production (31.99 U/mg protein) was observed with sodium nitrate (NaNO 3 ) followed by potassium nitrate (KNO 3 ) with activity of 19.32 U/mg protein although an enzyme production of about 25 U/ml was obtained in Aspergillus terreus KJ829487 (Olanbiwonninu and Odunfa, 2016). Inorganic nitrogen source in the form of diammonium hydrogen phosphate (NH 4 H 2 PO 4 ), failed to support cellulase production as it inhibited the growth of Mucor ramannianus unlike Aureobasidium pullulans isolates Ap20 and Ap33 which grew and produced enzyme with speci c activity of 9.24 and 12.81 U/mg protein/h respectively (Kudanga and Mwenje, 2005).
Although ammonium sulphate ((NH 4 ) 2 SO 4 ), ammonium nitrate (NH 4 NO 3 ) and ammonium chloride (NH 4 Cl) gave speci c activity of 3.29,1.21, 2.19 U/mg protein respectively. This is very low compared to NaNO 3 in this study. Many studies have reported that ammonium compounds are the most favourable nitrogen sources for protein and cellulase synthesis. Sethi and Gupta (2014) reported ammonium sulphate as the best nitrogen source for Aspergillus niger.
This study found that gelatin, casein and peptone were poor sources of nitrogen for the production of cellulase by Mucor ramannianus while beef extract and yeast extract are best and second-best organic source of nitrogen. Mrudula and Murugammal, (2011) reported that peptone supported maximum enzyme production followed by beef extract, groundnut oilcake, yeast extract and casein in Aspergillus niger. Many researchers have reported good cellulase production with organic nitrogen sources but results from this study contradict that, clearly indicating that inorganic nitrogen sources are preferred by Mucor ramannianus to organic ones for cellulase production which may be due to the inability of the organism to hydrolyse the organic nitrogen sources during growth and enzyme production as opposed to other inorganic nitrogen sources. Thus, variability in the preference for nitrogen source could be explained by strain difference.
The study of the effect of carbon sources on the production of cellulase by Mucor ramannianus revealed that carboxymethylcellulose was the best with speci c activity of 592.06 Unit/mg, thus enhancing both nutrient uptake and the release of enzymes into culture medium. This result was also in agreement with Gomathi et al. (2012) and Sajith et al. (2014), where both authors reported carboxymethylcellulose as the best source of carbon for the strains of Aspergillus sp.
Endoglucanase, β-glucosidase and exoglucanase activities were tested using CMC, cellobiose and lter paper strips as substrates for assay and as the sole carbon source for the culture medium respectively. Endoglucanase activity (592.06 U/mg) was higher than exoglucanase activity (110.30 U/mg) possibly because it is believed to be the rate-limiting activity (Mandels 1982;Enari 1983). Speci c activity of 55.99 U/mg protein by Mucor ramannianus cellulase using cellobiose as carbon source showed β-glucosidase activity which means that the isolate is capable of carrying out complete cellulase hydrolysis. This shows appreciable levels of enzyme activity on both carboxymethylcellulose and lter paper when compared with the levels obtained for Aureobasidium pullulans Ap33 and Ap20 (Kudanga and Mwenje 2005) and cellulase from A. pullulans NAC8 (Ademakinwa and Agboola, 2019). Shankar and Isaiarasu (2011) showed that agro-industrial wastes are considered best for enzyme production. Some newly developed agro-industrial wastes such as orange peel, orange bagasse, cassava peel, potato peel, pineapple peel, banana peel and plantain peel ours which were used in this study as sole carbon source (added at 1% (w/v) to the growth medium) for cellulase production gave little or no activity when compared to CMC. Cassava and potato peel ours gave no enzyme activity while plantain peel, pineapple peel and orange peel ours gave activities of 91.17, 86.05 and 74.02 U/mg protein respectively which can still be used as low-cost materials for production of cellulase from Mucor ramannianus. Carbon sources therefore serve as important substrates for energy production (Shankar and Isaiarasu 2011). Thus, the type of strain, culture conditions, nature of the supplement and availability of nutrients are the important factors affecting enzyme production by microbes (Salihu and Aman 2012).
Production of the crude cellulase was subsequently carried out by growing the isolate in a basal salt medium containing CMC as the sole carbon source for 4 days after which the medium containing the crude cellulase was collected by ltering with cheese cloth.
Cellulase produced by Mucor ramannianus was puri ed and the physicochemical properties were investigated. The elution pro le on Sephacryl S-200 is shown in Fig. 1. A single peak of activity was obtained with a yield of 45.37% and a puri cation-fold of 1.56. Table 2.
The puri cation process of the crude cellulolytic enzyme gave 1.56-fold puri cation, 45.37% recovery and a speci c activity of 2214.29 U/mg protein. This result agreed with that reported by Shahid et al. (2016) who obtained a puri cation-fold of 1.90, 46.28% recovery and 3.51 U/mg protein. The puri cation fold is lower compared to cellulase puri ed from previous studies. For example, Ijaz et al. (2014) puri ed cellulase by gel ltration from Aspergillus niger up to 5.71-fold with a speci c activity of 232.5U/mg. Also, an extracellular endoglucanase gave a puri cation fold up to 408 from Mucor circinelloides using a combination of ethanol precipitation (75%, v/v), ion-exchange and gel ltration chromatography (Saha 2004) . Puri cation results differed with respect to the method applied.
The molecular weight of the native cellulase estimated by gel ltration on Sephacryl S-200 was 23.4 kDa.
The molecular weight is similar to that of an endoglucanase from Mucor circinelloides whose molecular weight was 25000 Da and 27000 Da as estimated by gel ltration and SDS-PAGE respectively (Saha 2004). The puri ed cellulase from Aspergillus niger resolved on SDS-PAGE was found to be a homogenous monomeric protein as evident by a single band corresponding to 43 kDa (Ijaz et al. 2014). Sajith et al. (2014) reported that the molecular weight of the cellulase produced by Aspergillus avus BS1 is about 23 kDa, which is similar to the result of this study although the partially puri ed enzyme from a strain of Aspergillus avus had a molecular weight of approximately 30.2 kDa (Ajayi et al. 2007). Devi and Kumar (2012) reported that the apparent molecular weights of the cellulase isoforms produced by Aspergillus niger were 33 and 24 kDa while Cole (1980) reported values within the range of 20 to 60 kDa for cellulase from different cellulolytic organisms. The molecular weight of the single cellulase from M. ramannianus described in this study is very much comparable to these cellulases.
The Michaelis-Menten constant (K M ) and maximum reaction velocities (V max ) values of MrC were 0.63 ± 0.15 mg/ml and 20.21 ± 1.28 U/ml respectively. The value of K M obtained in this study was low showing greater a nity for carboxymethyl cellulose as compared to some other studies with a bit higher K M values of 3.6 mg/ml, 1.90 mg/ml, 1.481 mg/mL and 1.167 mg/ml as reported by (Bakare et al. 2005;Tokuda and Watanabe 2007;Bai et al. 2017;Quadri et al. 2017) respectively. The K M of cellulase from Aspergillus niger and Rhizopus sp. was reported as 1.3 mg/ml and 1.0 mg/ml respectively (Ogwuche et al. 2012) which is a little close to the K M obtained in this study. The V max obtained in this study is greater than the values 0.0833 μg of glucose/mL/min and 5.37 μg/mL/min reported by (Quadri et al. 2017;Kumar et al. 2012) respectively but lower than the V max values of cellulase from Aspergillus niger given as 45.5 U/mL (Ijaz et al. 2014). K m is equivalent to the substrate concentration at which the reaction rate is half maximal and is often used as an indicator of the a nity of an enzyme for its substrate (Nelson and Coxx 2004), a high K M indicates weak binding, that is, low a nity of the enzyme for the substrate while a low K M indicates strong binding that is high a nity of the enzyme for the substrate (Berg et al. 2002). The maximal rate, V max , reveals the turnover number of an enzyme, which is the number of substrate molecules converted into product by an enzyme molecule in a unit time when the enzyme is fully saturated with substrate (Berg et al. 2002).
The turnover number, k cat , for M. ramannianus cellulase was 1001.04s -1 which is greater than earlier report of 1556.11 min -1 and 1475 min -1 (Salem et al. 2008; Shahid et al. 2016). The catalytic e ciency, k cat /K M is the rate constant for the interaction of substrate and enzyme and can be used as a measure of catalytic e ciency, to know how e cient an enzyme converts a substrate into product. The k cat /K M obtained for M. ramannianus cellulase was 1.59 x 10 3 M -1 s -1 which is lower than the value (1.3 x 10 6 M -1 s -1) earlier reported for Bacillus megaterium cellulase Shahid et al. 2016). Enzymes that have k cat /K M ratios at the upper limits (10 8 and 10 9 M -1 s -1 ) are said to have achieved catalytic perfection (Berg et al. 2002;Nelson and Coxx 2004) In order to convert substrate into product, temperature plays a vital role as enzyme has to collide and bind with the substrate at the active sites. A gradual increase was observed in the activity with increase in temperature up to 40°C (Fig. 2). An optimum temperature of 40°C was obtained for cellulase from M. ramannianus and at higher temperature above the optimum, enzyme activity decreased due to thermal denaturation (that is enzyme decomposition). This result is the same with the optimal temperature of 40°C that was reported for Aspergillus niger Z10 strain (Gokhan-Coral 2002). Similarly, Otajevwo and Aluyi (2010) reported peak cellulase activity at 40°C for Psuedomonas aeruginosa. Temperature optimum for puri ed cellulase was observed at 70°C for Mucor circinelloides (Saha 2004). Highest activity was recorded at 50°C, 30°C and 60°C for Cellulomonas sp., K. gibsonii CAC1 enzyme and Geobacillus sp. (Adu et al. 2015;Potprommanee et al. 2017;Bai et al. 2017) respectively. Cellulase activities from Trichoderma sp. and other mesophilic cellulolytic fungi are at their optimum when assayed at about 50°C (Mandels et al. 1974;Kawamori et al. 1987). The puri ed enzyme was quite stable at 40°C (optimum temperature) for 40 min by retaining 100% of its activity although it lost about 20% activity after 60 min of incubation time. Ogwuche et al. (2012) reported a loss of enzyme stability as temperature increased towards 70 °C for Rhizopus sp. and Aspergillus niger. Bai et al. (2017) also showed that the enzyme retains 50% activity at 60°C for 40 min of pre-incubation.
The in uence of pH on enzyme activity was found to be an important parameter and the pH optima for maximum cellulase activity in this study was found to be 9.0 (Fig. 3) an alkaline pH which is similar to what was obtained from Bacillus sp. cellulase (Fukumori et al. 1985). Mucor circinelloides displayed an optimum activity at pH 5.0 (Saha 2004). Potprommanee et al. (2017) reported the highest cellulase activity at pH 7.0 in phosphate buffer, and the enzyme was still active over a wide pH range while Ijaz et al. (2014) reported an optimum pH 7.0 (neutral pH) for Aspergillus niger. The pH stability of cellulase was determined at optimum pH and this shows that the enzyme was stable as it retained about 81% of its activity for 60 min of incubation time which showed that the pH did not really in uence enzyme stability after prolonged incubation. In previous reports, pH stability of cellulase was determined at different pH values and over these wide pH range (3-8) cellulase activity was retained after 5 h of incubation (Potprommanee et al. 2017). Cellulase from Mucor circinelloides is fairly stable and highly active over a broad pH range since it retained 80% activity at pH 3.0 and also at pH 8.0 upon 30 min incubation (Saha 2004) . Ijaz et al. (2014) also reported that cellulase was completely stable in a large pH range (5-9) for Aspergillus niger. Enzymatic activities are sensitive to changes in pH values and this may be due to ionic composition of the medium contributing to the stability of the enzyme.
Metal ions can be associated with proteins and can as well form complexes with other molecules linked to enzymes acting as electron donors or acceptors as Lewis´s acids, or as structural regulators (Riordan 1997). These ions can either activate or inhibit the enzymatic activity by interacting with amine or carboxylic acid group of the amino acids ( (Ishida et al. 1980) and several mono-, di-, and trivalent metal ions have reported the activation or inactivation of microbial cellulases (Mandels and Reese 1965). The carboxymethyl cellulase (CMCase) activity of cellulase in this study was greatly enhanced at various concentrations of 1 mM, 5 mM and 10 mM by Na + , K + , Ca 2+ but it was slightly inhibited by Hg 2+ with 9.96% loss of activity at 10 mM and completely inhibited by Ba 2+ with 100% loss of activity at 5 mM and 10 mM as compared to control (Fig. 4). Inhibition of cellulases by heavy metals Hg 2+ and Ba 2+ is related to the interaction with catalytic amino acids containing sulphur such as cysteine and histidine rich residues leading to oxidation and irregular formation of disulphide bonds (Tejirian and Xu 2010) , thus denature the enzyme and make them less soluble, there by precipitating them. Ba 2+ can complex with arginine, glutamine, proline, serine and valine (Bush et al. 2008) . This result is similar to that obtained by Potprommanee et al.(2017) which reported that CMCase activity of cellulase strongly increased in the presence of CaCl 2 (174.32%) followed by NaCl (158.11%) and KCl (117.57%) respectively from Geobacillus sp. HTA426. Gaur and Tiwari (2015) had also reported that CMCase activity was strongly stimulated by CaCl 2 , NaCl and KCl ions from Bacillus vallismortis, although Quadri et al. (2017) reported reduced enzyme activity by KCl (36.22%) and NaCl (61.45%) from Bacillus pantothenticus. Saha (2004) reported that cellulase activity was enhanced by 8% by Ca 2+ at 5 mM by Mucor circinelloides. Ijaz et al. (2014) reported that Hg 2+ showed inhibitory effect on cellulase at 5 mM for Aspergillus niger.
Activity of cellulase from M. ramannianus was greatly affected by a non-ionic surfactant, Tween 80, at the various concentrations examined which might be due to the fact that this surfactant does not have the capability to modify the surface property and help to minimize the irreversible inactivation of cellulase (Wu and Ju 1998) as compared to others. On the other hand, the enzyme was moderately active in the presence of Triton X-100 at concentrations of 1.0 mM and 2.5 mM but averagely active as concentration increases. Sodiumdodecyl sulphate (SDS) (Fig. 5) was also found as cellulase activator at 2.5 mM given a percentage relative activity of 142.87% when compared with control (100%) although at higher concentrations it showed an inhibitory effect. This result partially supports the results obtained from previous studies which showed that anionic and non-ionic surfactants were found as activators of cellulase. Potprommanee [65] reported that CMCase activity increased with SDS (129.73%), Triton X-100 (113.51%) and Tween-80 (105.41%). Increase in the cellulolytic activity with these surfactants has also been observed by Seki et al. (2015) and Ashaet al. (2012).
This study also reported EDTA, a metal chelating agent as inhibitory to the cellulase activity with a residual activity of 16.78% at 7.5 mM which corresponds to the reports of Bakare et al. (2005) and Ijaz et al. (2014) for cellulase from wild type of Pseudomonas uorescens and Aspergillus niger respectively. However, Saha (2004) reported that cellulase activity was not affected by EDTA at 10 mM for Mucor circinelloides. The observed inhibition by EDTA suggested that the cellulase has an inorganic group which forms inactive complexes with EDTA, thus, the enzyme is a metalloenzyme. CMCase activity was enhanced by L-cysteine (147.34%), a semi-essential proteinogenic amino acid because its thiol side chain participates in enzymatic reactions as a nucleophile and β-mercaptoethanol (115.78%) at a concentration of 7.5 mM. Gaur and Tiwari (2015) reported that β-mercaptoethanol retained full activity at 10 mM and explained that β-mercaptoethanol can reduce disulphide bonds and re-nature their activity, if the oxidation or aggregation of these enzyme proteins occurs during puri cation and storage, thus, suggesting that the active site of the enzyme contains -SH groups.
The results of different organic solvents tested showed that the activity of cellulase was not substantially inhibited. Water miscible (ethanol, methanol, acetone) organic solvents examined in nal concentrations of 1.0% to 20% (Fig. 6) greatly enhanced cellulase activity whereas water immiscible (hexane and toluene) organic solvent slightly inhibited the enzyme activity at 5% with residual activity of 54.89% and 90.17% respectively although diethyl ether at 5% and 10% nal concentration activated the enzyme.
These ndings were in accordance with Bai et al. (2017) where alcohols slightly reduced the activity at a concentration of 10% with residual activity above 90%. In another study, an endoglucanase residual activity of 68.6%, 67.4%, 63.6% and 78.1% were reported for methanol, ethanol, hexane and toluene respectively with obvious stimulation by acetone (127.3%) and dimethylformamide (118.4%) (Li and Yu 2012). Zaks and Klibanov (1988) suggested that stimulation of enzyme activity by organic solvents might be due to the residues of carried-over non-polar hydrophobic solvent providing an interphase, thereby keeping the enzyme in an open conformation resulting in stimulated activation. It is therefore evident from this study that MrC is stable with high activity in all the organic solvents examined at 5% v/v except for hexane and could be potentially useful for practical applications in biotechnological processes such as bioremediation of carbohydrate-polluted salt marshes and industrial wastewaters contaminated with organic solvents.
The activity of the puri ed enzyme on various natural substrates showed that cellulase was found to completely hydrolyse cellobiose, a microcrystalline cellulose and insoluble cellulosic substrates such as orange peel, orange bagasse, cassava peel, potato peel, pineapple peel, banana peel and plantain peel (each at 0.1%, w/v) where CMC served as the control ( Fig. 7 and Table 3). The enzyme has great potential to be used in enzymic sacchari cation of various lignocellulosic substrates. The higher activity of the enzyme for cellobiose (187.5%) than CMC (100%) infer the presence of exoglucanase and endoglucanase (Wood and Bhat 1988). Quadri et al. (2017) reported a higher value of microcrystalline cellulose than CMC, although the other substrates investigated gave low percentage relative activities.

Conclusion
The fungus M. ramannianus, produced cellulolytic enzyme under optimized conditions. One-factor-at-a time based optimization allowed for improved cellulase production by the fungus. The puri ed enzyme displayed a relative stability in organic solvent, heat etc. and was able to biodegrade the cellulose component of some locally sourced agro-industrial wastes. The hydrolytic properties as well as catalytic potentials of the puri ed MrC makes it of potential immense biotechnological or industrial importance such as in lignocellulosic waste management    Figure 1 Page 24/28 Elution Pro le of Cellulase from Mucor ramannianus on Sephacryl S-200. The column was equilibrated with 0.1 M Citrate buffer, pH 4.8. Fractions of 5 ml were collected from the column at a ow rate of 20ml/hour.

Figure 2
Effect of Temperature on Activity of Cellulase from Mucor ramannianus. The values shown represent the average from triplicate experiments. Error bars represent the standard deviation.

Figure 4
Page 26/28 Effect of Salts on Activity of Cellulase from Mucor ramannianus. The relative activity was determined by measuring cellulase activity in the control that contained no chloride salt and taken as 100%. The values shown represent the average from triplicate experiments.

Figure 5
Effect of Inhibitors on Activity of Cellulase from Mucor ramannianus. The relative activity was determined by measuring cellulase activity in the control that contained inhibitor and taken as 100%. The values shown represent the average from triplicate experiments.

Figure 6
Effect of Organic Solvents on Activity of Cellulase from Mucor ramannianus. The relative activity was determined by measuring cellulase activity in the control that contained no inhibitor and taken as 100%.
The values shown represent the average from triplicate experiments.