Characterization of Cellulase Enzyme Produced by Two Selected Strains of Streptomyces Macrosporeus Isolated from Soil in Indonesia

This study was conducted to characterize the cellulase enzymes produced by Streptomyces macrosporeus BB 32 and S. microspores KRC 21. D, which were isolated from Tanjung Pendam, Belitung Island, and from Cibodas Botanical Garden, Indonesia, respectively. The optimal activity of the enzymes was analyzed using parameters such as incubation time, pH, temperature, carboxymethylcellulose (CMC) concentration. The effect of the addition of some metal ions as activators or inhibitors was also analyzed spectrophotometrically at  540 nm. Results demonstrated that the activity of the cellulase enzymes of S. macrosporeus BB 32 and S. macrosporeus KRC 21 D reached the optimum level after 2 and 5 days of incubation and at pH values of 8.0 and 6.0, temperatures of 35 °C and 50 °C, and CMC concentrations of 1.75% and 2%, respectively. S. macrosporeus BB 32 cellulase was activated by the cations CuCl2, MgCl2, and ZnCl2 but inhibited by NaCl and CoCl2, reducing its activity. The cellulase of S. macrosporeus KRC 21.D was activated by the cation NaCl and by the divalent cations CoCl2, CuCl2, MgCl2, and ZnCl2. S. macrosporeus BB 32 was deposited at the Indonesian Culture Collection with the collection number InaCC A144.

Enzymes can be sequestered from various sources such as plants, animal tissues, and microbes [5]. The majority of enzymes that have been used traditionally originate from plants and animals. However, issues such as low availability and high cost of production are limitations in the growth of enzyme industry.
Currently, microbial enzymes have been found to be economically beneficial as microbial culturing can be done in an easier and a more rapid manner than culturing plant or animal cells or tissues. Microbial enzymes produced industrially from actinomycetes bacteria, fungi, and protozoa with desired quantity or quality can be easily manipulated genetically [6,7]. Numerous cellulases are used in industries because of their role as biocatalysts, which efficiently enhances the development of the enzyme. This beneficial aspect can be identified by the continuously increasing commercial value of enzymes [8]. As cellulose has a high commercial demand, it is necessary to produce this enzyme in high quantities for the required consumers [9]. Cellulase has several commercial applications in terms of food processing such as in the coffee industry, wherein it is used for hydrolyzing cellulose during the drying process of coffee beans. In addition, cellulase is generally used in the textile industry and in laundry detergents. It is also used in the pulp and paper industry for various purposes. Cellulases are even used for pharmaceutical applications [10]. Actinomycetes is a soil bacterium with important functions and exhibits high diversity and abundance in the soil [11].
This study was conducted to characterize the cellulase enzyme from actinomycetes bacteria isolated from June 2019  Vol. 23  Cellulolytic actinomycetes were selected qualitatively using media containing 1% CMC with the following composition: 2.0 g KH 2 PO 4 , 1.4 g (NH 4 ) 2 SO 4 , 0.3 g MgSO 4 .7H 2 O, 0.1 g CaCl 2 , 1.0 g peptone, and 2.0 g agar dissolved in 100 mL aquadest [12]. Strains that showed a clear zone were grown in media with the following composition: 2.0 g KH 2 PO 4 , 1.4 g (NH 4 ) 2 SO 4 , 0.3 g MgSO 4 .7H 2 O, 0.1 g CaCl 2 , 1.0 g peptone, and 2.0 g agar. Then, the microbial cultures were incubated at 37°C for 6-7 days on slant agar. Cellulolytic enzyme was produced using medium comprising 1% CMC, 2.0 g KH 2 PO 4 , 1.4 g (NH 4 ) 2 SO 4 , 0.3 g MgSO 4 .7H 2 O, 0.1 g CaCl 2 , and 1.0 g peptone dissolved in 100 mL aquadest [12]. The enzyme sample was shaken in a shaker incubator at 120 rpm and 37°C for 5 days. The cellulase activity was analyzed daily using a spectrophotometer at λ 540 nm. Then, before analysis, the microbial culture was centrifuged at 13,000 g and 4°C for 10 min to separate the enzyme solution (supernatant) from the particles of the substrate or the cells. The obtained supernatant was stored in a freezer (−10°C) for further measurement of cellulase activity [13]. The activity of the cellulolytic enzyme was examined by the DNS method [14] based on the magnitude of sugar reduction achieved by enzyme activity during the hydrolysis of 1% CMC substrate. The activity of the cellulase enzyme was determined qualitatively, wherein each actinomycetes strain was inoculated onto 1% CMC agar media. Each strain was concentrated on the surface of media, incubated for 3-7 days, and observed daily. Growing colony was treated by flooding with the indicator 0.1% Congo red, allowed for 15-30 minutes, and then washed with 2% NaCl [15].
Formation of a clear zone around a colony indicated the presence of cellulolytic enzyme activity. Quantitative examination of the cellulolytic enzyme activity was performed by the following procedure: 0.125 mL of enzyme solution was added to 0.125 mL of the substrate (1% CMC in phosphate buffer solution, pH 7.0) and then incubated at 40°C for 30 min. Subsequently, 0.25 mL of 3 5-dinitrosalicylic acid (DNS) was added and boiled for 5 min. DNS was added to stop the reaction [16]. Immediately after cooling the sample under flowing water, 2.5 mL of aquadest was added. Measurement was performed using a spectrophotometer at λ 540 nm.
Cellulase activity was calculated using the following formula: One unit of enzyme activity was defined as the amount of enzyme required to form 1 mmol of product per unit time for each ml of enzyme. Dissolved protein content was determined to evaluate the specific cellulolytic activity. Protein content was examined using Coomassie Brilliant Blue (CBB) G250 stain in an acidic solution, which produced a bluish color [17]. The procedure was as follows: 0.01 mL of enzyme extract was added to 0.5 mL of CBB reactant, which was then vortexed and incubated for 10 min at room temperature; then, the light intensity was measured using a spectrophotometer at λ 595 nm. For enzyme extract blank, 0.15 N NaCl solution was used. The standard was prepared using 0.2 mL of bovine serum albumin solution at different concentrations ranging between 0 and 800 μg/mL.
Because characterization of the cellulase enzyme involves determination of the incubation time, pH, temperature, optimum concentration of CMC substrate, and addition of metal ions [18], this study included all these parameters. The effect of incubation time on enzyme activity was analyzed using an enzymatic reaction solution after 1-5 days of incubation duration. The effect of pH on enzyme activity was assessed based on the reaction with the enzyme solution at a substrate concentration of 1% CMC incubated in 0.05 N acetic buffer (pH 5.0), 0.05 N phosphate buffer at pH values of 6.0, 7.0, and 8.0-9.0, for which Glycine-NaOH Buffer as used. The effect of temperature on enzyme activity was analyzed using the enzymatic reaction solution with CMC at various pH values and temperatures of 35°C, 40°C, 45°C, 50°C, 60°C, and 70°C.
The effect of CMC on enzyme activity was determined using CMC at various concentrations of 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, and 1.75% at pH and optimum temperature which obtained from previous analysis. Furthermore, the effect of metal ions on the cellulase activity was analyzed using the ions Na + , Co 2+ , Cu 2+ , Mg 2+ , and Zn 2+ obtained from NaCl, CoCl 2 , CuCl 2 , MgCl 2 , and ZnCl 2 , respectively, wherein the concentration of each salt was 1 mM for each CMC solution. CMC June 2019 Vol. 23  No. 2 solution without the addition of metal ions was used as control treatment.

Results and Discussion
The success of bioconversion of cellulose material depends particularly on the characteristic of the source of cellulose, the source of cellulolytic enzymes, and the optimum condition for catalytic activity and enzyme production [19]. Cellulose is a renewable biological resource found abundantly in agricultural wastes. Cellulose material waste can be hydrolyzed to produce glucose and other soluble sugars using the cellulase enzyme isolated from bacteria and fungi [20]. Qualitative examination using carboxymethyl cellulose based on clear zone formation can be performed to determine whether an isolated microbe can produce cellulase [21,22].
According to an earlier research [15], cellulolytic microbes could be rapidly filtered out by measuring the clear zone (qualitative). Quantitative filtering can be done for confirmation, although the results are not consistent, exact, and similar to those obtained by filtering the clear zone (qualitative). This indicates that if there is a clear zone formation in the bacterial colony, then the microbes can decompose cellulose to yield glucose with the help of cellulase [21]. The potential index value of bacteria is obtained using a ratio obtained between the diameter of a clear zone and the diameter of the colony [23].
Congo red used in the cellulolytic assay binds with 1,4β glycoside bonds in cellulose, resulting in a red color, whereas a clear color around the colony of microbes indicates that cellulose has been decomposed to produce a monosaccharide. Since 1,4-β glycoside bonds are released through the action of cellulase produced by microbes, Congo red cannot bind with glucose, resulting in the formation of a clear zone [24]. The pH of Congo red has an influence on the media color. A clear zone is formed around the colony due to the interaction between (1,4)-β-d-glucan and (1,3)-β-d-glucan in the presence of the reaction of cellulose hydrolysis by the fungal strain to produce the enzyme cellulase [25].
The results of this study demonstrated that only two actinomycetes strains, S. macrosporeus BB 32 and S. macrosporeus KRC 21.D, possessed the ability to make a clear zone around the colony after incubation for 48 h (Figure 1). Furthermore, both strains had the ability to produce cellulolytic enzymes on 1% CMC agar media. The potential of cellulolytic actinomycetes to produce cellulase has been explored earlier [26]. The distribution and abundance of microbial populations such as the actinomycetes Micromonospora, Nocardia, and Streptomyces can support nutrient availability, temperature, and ecology of the habitat, and these strains can also degrade several complex substances such as cellulose and chitin [27] and photosynthetically they can convert sunlight into energy releasing oxygen as a by-product that can used in aerobic processes [28]. Streptomyces has the potential to produce antibiotics, vitamins, antimicrobial compounds, and enzymes that are important in both industries and agricultural sector [29,30]. Streptomycetes is one of the genera that is extremely important and represents almost 70% of the soil population among all actinomycetes [31]. It has been reported that S. macrosporeus BB 32 produces the enzyme chitinase [32] and S. macrosporeus KRC 21.D also possesses a similar property [33]. In addition, an earlier study [34] has demonstrated the antimicrobial activity S. macrosporus.
Previous studies [35,11] have also reported that actinomycetes, especially Streptomyces, can produce other cellulase enzymes as cellulose decomposer, which depends on the pH, aeration, temperature, texture, humidity, and also the minimal nutrition content of soil. The results of the present study revealed that the amount of the soluble protein of S. macrosporeus BB 32 was 0.0168 μg/mL, whereas that of S. macrosporeus KRC 21.D was not detected.
Actinomycetes is a soil microorganism commonly found in several types of soil [36]. Actinomycetes bacteria are saprophytes and actively decompose organic matter, which increases soil fertility. Actinomycetes bacteria are one of the microorganisms that can degrade cellulose other than bacteria, molds, and yeast [37]. Modification of the fermentation medium is an essential step in the design of successful laboratory experiments, pilot-scale development, and manufacturing processes [38].
In the process of enzyme production by using other raw material, microbial strains used and environmental factor are essential. The important parameters those will be considered are incubation time, substrate concentration, temperature, pH, and metal ions addition as activator or inhibitor [12,39,40,41].
In this study, S. macrosporeus KRC 21.D exhibited a higher cellulase activity of 4.9 U/mL on the 5 th day than S. macrosporeus BB 32, whose activity was 4.0 U/mL ( Figure 2) on day 2. This result was according to an earlier research that the cellulase enzyme of different isolates by using the same medium would show different optimum activity of the enzyme. This result was according to an earlier research [42] that the cellulase enzyme of different isolates by using the same medium would show different optimum activity of the enzyme.
Regarding the effect of pH on the cellulolytic enzyme activity, the optimum cellulolytic enzyme activities of S. macrosporeus BB 32 and S. macrosporeus KRC 21.D were 5.9 and 6.3 U/mL at pH values of 8.0 and 6.0, respectively ( Figure 3). An earlier study [43] had reported that the pH of the fermentation medium affected microbial growth and enzyme production. Several reports have also demonstrated that an optimum pH, especially about 5.0-11, of the fermentation medium used for the production of cellulase by Streptomyces sp. could increase the enzyme activity [44,45,46].
The optimum cellulolytic enzyme activities of S. macrosporeus BB 32 and S. macrosporeus KRC 21.D were 7.6 and 7.3 U/mL, which were obtained at 35°C and 50°C, respectively ( Figure 4). The activity of carboxymethyl cellulase enzyme at low or high temperatures can be considered as a major physiological adaptation on an enzymatic level, such as to compensate the reduction of the chemical reaction rate that was caused by low or high temperatures [47,48]. It has been reported that S. griseorubens AB184139 exhibited the optimum activity of cellulose enzyme at pH 7.0, temperature 45°C, and 6 days of incubation duration [49]. It is known that Streptomyces can use several carbon sources for deriving energy for its growth. A previous study [35] showed that Streptomyces has an optimum temperature ranged of 25°C-35°C. Several bacterial species that can grow between psychrophilic and thermophilic temperatures are abundant in the soil habitat and compost at an optimum pH of 6.5-8.0, similar to S. microspores BB 32 or S. microspores KRC 21. D whose optimum temperatures are 35°C (at pH 6.0) and 50°C (at pH 8.0), respectively. The acidity level of the environment can also be highly affected. S. griseorubens have been reported to have a wide range of temperatures and pH values for growth and cellulolytic activity [49]. This strongly indicates that actinomycetes can grow in different habitats with differences in environmental conditions, pH, and temperature. This important capability will be useful not only for achieving better production but also for achieving the end products, including bioethanol and other compounds.
Regarding the effect of the concentration of CMC as a substrate on the enzyme activity, the optimum enzyme activities of S. macrosporeus BB 32 and S. macrosporeus KRC 21.D were 5.9 and 6.8 U/mL, which were obtained at the CMC concentrations of 1.75% and 2%, respectively ( Figure 5). According to an earlier study [50], the reaction of enzymes is basically dependent on the enzyme concentration, the activity of existing enzymes, and the substrate availability. The ability of each microorganism to use carboxymethyl cellulose substrate to produce cellulase enzyme varies according to its environment [49].
There are particular chemical substances that can increase the enzyme activity (activators) or inhibit the activity (inhibitors). This study demonstrated the cellulolytic enzyme activity of S. macrosporeus BB 32 was induced by the divalent cations CuCl 2 , MgCl 2 , and ZnCl 2 , whereas the monovalent cation NaCl and the divalent cation CoCl 2 inhibited the activity. The cellulolytic enzyme activity of S. macrosporeus KRC 21.D was induced by the monovalent cation NaCl and the divalent cations CoCl 2 , CuCl 2 , MgCl 2 , and ZnCl 2 ( Figure 6). It has been reported that several metals ions such as monovalent, divalent, and trivalent such as Na + , K + , Ca 2+ , Mg 2+ , Mn 2+ , Fe 2+ , Co 2+ , Cu 2+ , Ni 2+ , Zn 2+ , Hg 2+ , and Fe 3+ could act as inhibitors and activators of enzyme activity [51].

Conclusions
S. macrosporeus BB 32 and S. macrosporeus KRC 21.D possessed the capability of degrading carboxy methyl cellulose (CMC) indicated by the formation of a clear zone. The optimum activities of BB 32 cellulase were as follows: 4 U/mL at an incubation duration of 2 days, 5.9 U/mL at pH 8.0, 7.6 U/mL at a temperature of 35°C, and 5.9 U/mL at a CMC concentration of 1.75%.
The divalent metal cations CuCl 2 , MgCl 2 , and ZnCl 2 induced the enzyme activity, whereas NaCl and the divalent cation CoCl 2 acted as inhibitors decreasing the enzyme activity. The optimum activities of cellulase isolated from the KRC 21.D strain were as follows: 4.9 U/mL at an incubation duration of 5 days, 6.3 U/mL at pH 6.0, 7.3 U/mL at a temperature of 50°C, and 6.8 U/mL at a CMC concentration of 2%. In addition, the monovalent cation NaCl and the divalent cations CoCl 2 , CuCl 2 , MgCl 2 , and ZnCl 2 induced the enzyme activity.