The Activity of Cellulase Enzymes of Ganoderma applanatum and Ganoderma tropicum

Article history: Received on: 27/09/2016 Accepted on: 20/12/2016 Available online: 30/04/2017 Ganoderma can live as saprobes on rotting stumps and roots by decomposing lignin as well as cellulose and related polysaccharides. It is because of mushrooms cellulolytic activities. Objective: The aim of this study was to compare the cellulolytic activity of the crude extract of Ganoderma applanatum and Ganoderma tropicum from Garut (Indonesia). Method: Colorimetric method using dinitrosalicylic acid (DNS). Results: The cellulolytic activity of G. applanatum and G. tropicum in the pH range of 3.5-10.0 was 0.0510.109 and 0.063-0.131 UI/mL, respectively. The cellulolytic activity of G. applanatum and G. tropicum in the temperature range of 35-65 °C was 0.100-0.184 and 0.077-0.110 UI/mL, respectively. Conclusion: The activity of cellulase enzymes were affected by temperature and pH. The cellulolytic activity of G. applanatum was the same as G. tropicum.


INTRODUCTION
The genus Ganoderma (Ganodermataceae) found in subtropical and tropical regions, because they can survive under hot and humid conditions.Ganoderma species contain chemical constituents such as polysaccharides, proteins, amino acids, fatty acids, terpenoids, steroids, alkaloids, and phenolic compounds, with potential nutritional and therapeutic values (Mizuno, 1995, Paterson, 2006, Singh et al., 2013).Ganoderma species grow as a facultative parasite that can live as saprobes on rotting stumps and roots (Pilotti et al., 2004) by decomposing lignin as well as cellulose and related polysaccharides (Hepting, 1971).We believe these mushrooms have cellulolytic activity which controlled and processed by cellulase system.Currently, cellulase enzyme was used in various industrial processes, such as biofuels (Vaithanomsat et al., 2009), triphase biomethanation (Chakraborty et al., 2000), plants and agriculture waste processing (Mswaka andMagan, 1998, Lu et al., 2004).We see the opportunities of usage of G. applanatum and G. tropicum in agriculture waste processing.This study aimed to compare the cellulytic activity of the cellulase enzyme of G. applanatum and G. tropicum which grow on a dead stump of bitter bean (Parkia speciosa) in various pH and temperature.

Materials
Ganoderma applanatum and Ganoderma tropicum were collected from a dead stump of bitter bean (Parkia speciosa) in the Cipangramatan village, Garut sub-district, Indonesia, in July 2015.Mushrooms were identified by School of Biological Sciences and Technology, Bandung Institute of Technology, Indonesia with No. 1996/I1.CO2.2/PL/2015.All chemical reagents are analytical grade (Merck), including glucose and dinitrosalicylic acid (Sigma Aldrich).

Preparation of Dinitrosalicylic Acid (DNS) Reagent
A total of 5 g of sodium hydroxide, 91 g of potassium sodium tartrate, 5 g of sodium sulfite, 1% phenol, and 5 g of DNS were dissolved in 500 mL distilled water.The mixture was homogenized using a magnetic stirrer.The solution was stored in amber bottles and low temperature (Benfeld, 1955).

Determination of Standard Curve
A total of 1 mL of the glucose solution (0.25, 0.50, 0.75, 1.00, and 1.25 g/mL) in a different tube was added by 1 mL of DNS reagent.The color was then developed by boiling the mixture for 5 min.Optical densities were measured at 575 nm against a blank containing all the reagents minus glucose (Miller, 1959).

Extraction of Crude Enzyme
Mushrooms were washed and dried to a constant weight.A total of 5 g of mushrooms and 25 mL of phosphate buffer pH 7.0 were mashed for 5 min.The extract was filtered, then centrifuged for 15 min at 4000 rpm to obtain supernatant which contain the cellulase enzyme.

Determination of the Cellulolytic Activity
A total of 1 mL of the supernatant, 0.5 mL of phosphate buffer, and 1 mL of 1% CMC were mixed and incubated for 30 min at 25 C.The reaction was terminated by adding 1 mL of DNS reagent.The color was then developed by boiling the mixture for 5 min.Optical densities were measured at 575 nm against a blank containing all the reagents minus the crude enzyme (Miller, 1959).The cellulolytic activity was determined at various pH (3.5, 5.8, 7.0, 8.0, and 10.0), and various temperature (25, 35, 45, 55, and 65 C).

Statistical Analysis
Results are presented as the mean + standard error of the mean (SEM).Data comparisons between groups were done by two way ANOVA followed by Tukey-Kramer post hoc test.Values were considered statistically significant at P < 0.05.

RESULTS AND DISCUSSION
Both of ganoderma in this study were grown on a dead stump of bitter bean.The size of G. applanatum was 27 ± 2 cm in length, 20 ± 3 cm in width, 5 ± 1 cm in height, and brown colored.The size of G. tropicum was 25 ± 3 cm in length, 15 ± 2 cm in width, 5 ± 2 cm in height, and white brownish colored because of spores (Fig. 1).Both mushrooms have morphology according the literature.The growth of Ganoderma is influenced by the environmental conditions, specific host species, and the habitat nature (Turner, 1981).Different species of Ganoderma produced different feature and pathogenecity (Wong et al., 2012).

Determination of Standard Curve
The reaction between glucose and DNS involves the oxidation of the aldehyde functional group in the glucose and 3,5dinitrosalicylic acid (DNS) is reduced to 3-amino,5-nitrosalicylic acid under alkaline conditions, simultaneously (Miloski et al., 2008).The correlation coefficient value was close to 1.00 (R 2 = 0.9961, Fig. 2) met the criteria by ICH (ICH, 2005) and showed that the instrument response was proportional to the glucose concentration.

Extraction of Crude Enzyme
Cellulase enzymes were extracted using phosphate buffer pH 7.0 to maintain the stability and function of cellulase enzymes y = 0.0207x + 0.1722 and R² = 0.9961 0.17 in the crude extract.The extract were cloudy solution, so conducted the centrifugation to separate the supernatant from the residues.The color of clear supernatant was yellowish-white for G. applanatum, and brown for G. tropicum.

Determination of the Cellulolytic Activity
The cellulase system consists of three classes of soluble extracellular enzymes, i.e 1,4--endoglucanases, 1,4-exoglucanases, and -glucosidases (-Dglucoside glucohydrolases or cellobiases) (Shewale, 1982).These enzymes hydrolyze cellulose to glucose (Ryu and Mandels, 1980).Substrates for the cellulolytic activity assay was carboxy methyl celulose (CMC) which dispersed in water, the hydrophilic moieties of CMC absorb the water absorbing, then swelling, so the viscosity increased.The efficient enzymatic degradation of insoluble polysaccharides, such as cellulose, requires a tight interaction between the enzymes and substrates.In the cellulose degradation, many cellulases are known to bind to crystalline and/ or amorphous cellulose via cellulose binding domains (CBDs) which are distinct from the catalytic domains (Aderemi et al., 2008).
The cellulase enzyme of G. tropicum is more active than G.applanatum at pH 3.5 -10.0, but at pH 7.0, both enzymes have equal activity (Fig. 3).We suggested that the cellulase enzyme of G. tropicum is more stable to pH alteration compared to G. applanatum.The unfavorable pH may result in reduced enzyme activities by reducing accessibility of the substrate (Bakri et al., 2008).The cellulase enzyme of G. applanatum is more active than G. tropicum at temperature of 25-65 °C, but at 35 °C, both enzyme have equal activity (Fig. 4).We suggested that the cellulase enzymes of G. applanatum more stable to temperature alteration compared to G. tropicum.The cellulases catalytic activity is inhibited at higher temperatures.Increasing temperature was supposed to denature and unfold the proteins, thus may cause two different effects, i.e. more ionic groups of the proteins become exposed to the surface and are available to dye fixation dan more tryptophan residues formerly hidden inside the 3D-structure are revealed and thus may lead to an increase in hydrophobic interactions between protein and cellulose.Higher temperatures (above 30 C) alter the cell membrane composition and stimulate protein catabolism, causing cell death (da Silva et al., 2005).

Statistical Analysis
There was statistically significant on temperature (P = 0.0064) and pH (P = 0.0087), but there was no statistically significant on Ganoderma species for various temperature (P = 0.1576) and various pH (P = 0.1081).

CONCLUSION
The activity of cellulase enzymes were affected by temperature and pH.The cellulolytic activity of G. applanatum was the same as G. tropicum.