Production and assay of cellulolytic enzyme activity of Enterobacter cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya abbatoir, Indonesia

Aim: This study aims to produce and assay cellulolytic enzyme activity (endo-(1,4)-β-D-glucanase, exo-(1,4)-β-D-glucanase, and β-glucosidase, at optimum temperature and optimum pH) of Enterobacter cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya Abbatoir, Indonesia. Materials and Methods: To produce enzyme from a single colony of E. cloacae WPL 214, 98 × 1010 CFU/ml of isolates was put into 20 ml of liquid medium and incubated in a shaker incubator for 16 h at 35°C in accordance with growth time and optimum temperature of E. cloacae WPL 214. Further on, culture was centrifuged at 6000 rpm at 4°C for 15 min. Pellet was discarded while supernatant containing cellulose enzyme activity was withdrawn to assay endo-(1,4)-β-D-glucanase, exo-(1,4)-β-D-glucanase, and β-glucosidase. Results: Cellulase enzyme of E. cloacae WPL 214 isolates had endoglucanase activity of 0.09 U/ml, exoglucanase of 0.13 U/ml, and cellobiase of 0.10 U/ml at optimum temperature 35°C and optimum pH 5. Conclusion: E. cloacae WPL 214 isolated from bovine rumen fluid waste produced cellulose enzyme with activity as cellulolytic enzyme of endo-(1,4)-β-D-glucanase, exo-(1,4)-β-D-glucanase and β-glucosidase.


Introduction
Rumen is an excellent environment for microbial growth consisting of bacteria, fungi and protozoa which are widely known to play important role in the fermentation process of ruminant cattle feed [1]. Cellulase is an enzyme produced by cellulolytic microbes capable of hydrolizing β-1,4 glycoside bond in cellulose, a polysaccharide structure often found in plants [2].
Cellulose degradation by cellulolytic bacteria is a product of synergy in a group of cellulase enzymes. Cellulase enzyme system consists of three groups of hydrolytic enzymes, i.e. (1) endo- (1,4 [3].
The cost of using commercial cellulose enzymes is still expensive, making it less economical when applied in livestock industry in relation to provision and improvement of feed quality. Thus, other cellulolytic microbes capable of degrading fibrous feed stuffs need developing. Biodegradation by Enterobacter cloacae WPL 214 rumen cellulolytic bacteria is expected to be able to be used as degrading source material for fibrous feed stuffs at a cheaper price compared to commercial cellulose enzymes.

Measurement of E. cloacae WPL 214 growth curve
10 ml of cultured E. cloacae WPL 214 isolates was taken and transferred to100 ml growth medium in Erlenmeyer flask. Culture suspensions were incubated in a shaker incubator (37°C, 120 rpm). Optical density (OD) was measured at λ 600 nm by taking as much as 1 ml sampling with interval of 2 h for 30 h (h 0; 2; 4; 6; 10; 12; 14; 16; 18; 20; 22; 24; 26; 28; 30). The first sampling was done at 0th h and continued until OD values showed a clear decline. OD was measured with UV-vis spectrophotometer at λ 600 nm. Growth curve was obtained from the result of absorbance measurement on the time.

Measurement of E. cloacae WPL 214 optimum temperature and pH
1 ml of cultured cellulolytic bacteria isolates of E. cloacae WPL 214 was taken and transferred into 10 ml Luria Bertani growth medium in erlenmeyer flask. Culture suspensions were incubated for 24 h in a shaker incubator at 30°C, 35°C, 40°C and pH 4, 5, 6, 7, 8 with shaking at 120 rpm. OD measurement was conductedby taking 1 ml sampling. OD was measured using UV-Vis spectrophotometer at λ 600 nm.

Cellulase enzyme production of E. cloacae WPL 214
Enzyme activity assay was conducted on enzyme produced from optimum growth time and temperature in the following way: Single colony of E. cloacae of 98 × 10 10 CFU/mlwas put into 20 ml of liquid medium and incubated in a shaker incubator for 16 h at 35°C. Afterwards, culturewas centrifuged at 6000 rpm at 4°C for 15 min. Pellet was discarded while supernatant containing cellulase enzyme was taken to assay endo-(1,4)-β-D-glucanase, exo-(1,4)-β-D-glucanase, and β-glucosidase enzyme activity.

Assay of endo-(1,4)-β -D-glucanase E. cloacae WPL 214 activity
Assay of endo-(1,4)-β-D-glucanase activity was conducted using 3.5-dinitrosalisilat acid (DNS) method by using CMC as a specific substrate. Endo-(1,4)-β-D-glucanase activity was assayed by mixing 100 ml enzyme with 100 ml substrate(1% CMC in 0.1M citratephosphate bufferat pH7) and incubating it in a water bath at 50°C for 30 min. Enzyme activity of an amount of formed reducing sugars was measured using DNS method by adding 600 μl DNS into tube and placing it in a boiling water bath for 15 min together with control (containing 100 μl enzyme mixed with 600 μl and 100 μl substrate, without incubation) and finally cooling it in ice water for 20 min. Total volume of enzyme activity assay in this study was 800 μl. Absorbance measurement was afterward conducted using cuvettes. Absorbance was read using spectrophotometer at λ 550 nm. 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 [7,8].

Assay of exo-(1,4)-β-D-glucanase E. cloacae WPL 214 activity
To measure exo-(1,4)-β-D-glucanase activity, 100 μl enzyme mixed with 900 μl pNPC (1 mMol pNPC in 10 ml of citrate phosphate buffer at pH5) was incubated for 30 min at optimum temperature 35°C. Reaction was stopped by adding 100 μl 1M Na 2 CO 3 . Liberation of p-nitrophenol was read using spectrophotometer at λ 405 nm. A sa blank, 100 μl aquadest and 900 μl substrate that were treated the same as sample condition were used and reaction was stopped by adding 100 μl of 1M Na 2 CO 3 . One unit of enzyme activity is equivalent to the amount of enzyme required to produce 1 μmol p-nitrophenol/min [9].
p-nitrophenol standard was made in the range of 0.1-0.5 mM p-nitrophenol from 10 mM p-nitrophenol stock in phosphate citrate buffer solvent pH 5. 100 μl of each standard p-nitrophenol solution was mixed with 900 μl of phosphate citrate buffer pH 6 and incubated at 35°C for 30 min. Reaction was stopped by adding 100 μl of 1 M Na 2 CO 3 and absorbance was read using UV-Vis spectrophotometer at λ 405 nm.

Assay of β-glucocidase E. cloacae WPL 214 activity
Measurement of β-glucocidase activity was assayed using pNPG method. β-glucocidase activity was measured by mixing 100 μl enzyme with 900 μl substrate (1 mM pNPG in 10 ml of phosphate citrate buffer pH5) and incubating it at 35°C for 30 min. Reaction was stopped by adding 100μl of 1M Na 2 CO 3 . As blank, 100 μl aquadest and 900 μl substrate that were treated the same as sample condition were used. Liberation of p-nitrophenol was read with spectrophotometer at λ 405 nm and then compared top-nitrophenol standard curve. One unit of enzyme activity was equivalent to the amount of enzyme required to produce 1 μmol p-nitrophenol/min [9].

Statistical analysis
This research was descriptive research so that the duplo data collected and analyzed in descriptive and expressed in the form of narrative and figures.

E. cloacae WPL 214 growth curve
Growth curve of E. cloacae WPL 214 cellulolytic bacterial inoculants is shown in Figure-1. The highest growth logarithmic phase occurred at the 16 th h with absorbance (λ 600 nm) of 3.122.

Optimum temperature and pH of cellulase enzyme E. cloacae WPL 214
Incubation temperature was specified at 30°C, 35°C, 40°C and 45°C and phosphate citrate at pH 4, 5, 6, 7, 8. The highest cellulose enzyme activity was obtained at 35°C as much as 0.154 U/ml (Table-1) and at pH5 as much as 0.606 U/ml (Table-2).

Cellulase enzyme production of E. cloacae WPL 214
In addition to growth curve observation, measurement of cellulase enzyme production was also conducted. Optimum production of cellulase enzyme occurred at the end of stationary phase, at the same time as the peak of microbial growth which occured at the 22 nd h (Figure-2).

E. cloacae WPL 214 growth curve
Growth curve describes gradual growth process of a microorganism, from the beginning until the end of activity. This consists of four main phases: Lag, exponential, stationary, and death [10]. During this phase mass or cell accretion has not happened yet. Therefore, phase curve is generally flat. Lag phase interval depends on the compatibility between activity and environment setting. In this research, this phase occurs before in the first 2 h of E. cloacae WPL 214 isolate growth and followed by exponential phase.
Exponential or logarithmic phase is a phase when transformation activity increases and the accretion of microorganism growth reaches maximum speed so that the curve is in exponential form. This increasing activity should be offset by many factors among others: Biological factors, such as shape and nature of the microorganism to its environment, life association between related organism, and non-biological factors, such as temperature, pH, and nutrient content in the growth medium. Exponential phase of E. cloacae WPL 214 isolates occurred during the first 2 h of incubation up to the 8 th h of incubation.
Stationary phase is a phase when increased and decreased activities come to a balance. In colony growth, it means the rate of individual death equals the rate of individual birth. Therefore, this phase forms a flat curve. This phase also occurs due to diminishing nutrient sources, inhibitory compounds formation, and unfavorable environmental factors. Stationary phase of E. cloacae WPL 214 isolates occurred from the 8 th up to the 28 th h of incubation with optimum growth at 16th h of incubation.
Death phase is the phase when cessation of activity starts, or in colony growth, the event of death begins to exceed individual birth. Death phase of E. cloacae WPL 214 isolate soccured after 30 h of incubation.

Cellulase enzyme production of E. cloacae WPL 214
In addition to growth curve observation, measurement of cellulase enzyme production was also conducted. Optimum production of cellulase enzyme occurred at the end of stationary phase, at the same time as the peak of microbial growth which occurred at the 22 nd h (Figure-2). Cellulolytic activity of cellulose enzyme was assayed by performing reaction between cellulase enzyme and p-nitrophenol substrate derivatives. Exoglucanase was assayed using pNPC specific substrate while cellobiase was assayed using p-nitrophenyl-β-D-glucopyranocide (pNPG) specific substrate. Enzyme activity was determined by measuring the amount of p-nitrophenol released [11]. Observations on the amount of p-nitrophenol released was observed by using spectrophotometry at λ 405 nm. One unit of cellulase enzyme activity is defined as the amount of enzyme producing 1 μmol of p-nitrophenol in 1 min (U/ml) in experimental conditions. Cellulase enzyme activity curve was used to determine the optimum time required to obtain maximum cellulase enzyme activity from enzyme production process.   Measurement of exoglucanase and β-glucocidase enzyme activity was conducted using p-nitrophenol derivative substrates. Exoglucanase was assayed using pNPC specific substrate while cellobiase was assayed using pNPG specific substrate. Assays were carried out using the enzyme produced when enzyme activity reached optimum time, at the 22 nd h. Assay was also carried out at optimum temperature of 35°C and optimum pH of 5. Data of exoglucanase and cellobiase enzyme activity are presented in Table-3. A unit of enzyme activity is defined as the amount of enzyme required to form 1 μmol product per minute.
Endo -(1,4)-β-D-glucanase enzyme activity was measured simultaneously with the making of the growth curve. Sample was taken every 2 h and the enzyme extracted. Cellulase enzyme activity curve was used to determine the optimum time required to obtain maximum cellulase enzyme activity from enzyme production process. Figure-2 shows data of optimum time needed to obtain optimum growth curve and optimum enzyme activity, which occurred at the 22nd h with enzyme activity of 0.09 U/ml.
Research data of E. cloacae WPL 214 isolates showed that the optimum time to produce cellulose enzyme was at the 22 nd h with endoglucanase enzyme activity of 0.09 units/ml (U/ml). Results of enzyme activity measurement using specific substrates of p-nitrophenol derivatives on E. cloacae WPL 214 isolates showed exoglucanase activity of 0.13 U/ml while cellobiose enzyme activity was at 0.10 U/ml. This result indicated that these isolates had activity on endoglucanase, exoglucanase and cellobiose enzymes. Cellulose hydrolysis requires synergistic activity of various cellulase enzymes with different specifications to produce multi enzyme system. It also requires coordination in cellulose molecule breaking, product resulted, and catalytic movement of cellulose chain [12,13]. Multi enzyme system of cellulose is a strategy of microorganisms to improve the effectiveness of cellulase hydrolysis, in which each enzyme has a specific function [14].
Not all Enterobacter bacteria produce cellulase enzyme. Isolation and identification have also been made from several plants, i.e. 53 endophyticentero bacteria from some plants including citrus, coconut, eucalyptus, sugarcane, and soybean. These studies identified E. cloacae, Pantoea agglomerans, Hafniaalvei, and Pantoea ananatis. The lowest cellulase enzyme production is obtained from coconut plants as much as 20% (2 of 10), equivalent to that produced by citrus crop as much as 20% (4 of 20). The level of cellulase production obtained, from the highest to the lowest, is as follows: Sugarcane, 100% (4 of 4), eucalyptus, 84.6% (11 of 13), and soybean, 83% (5 of 6) [15]. Isolation and identification of cellulolytic bacteria from insects show the existence of cellulolytic bacteria: Enterobacter chrysanthemi, E. cloacae, and Proteus mirabilis, Erwinia chrysanthemi, among others. These microorganisms are already known to have cellulolytic activity. Cellulolytic bacteria found in the digestive tract of insects have higher cellulolytic activity ability as they are naturally involved in the digestion of lignocellulosic substrates found in insect feed. Genetic engineering (recombinant) is performed on those cellulolytic bacteria and is further used in its role of converting cellulosic biomass (CMC substrate and sugarcane bagasse) into bioethanol product more quickly and efficiently through process of fermentation at 37°C and pH 7. Cellulosic ethanol products from cellulolytic bacteria aim to reduce dependence on petroleum [16].

Conclusion
Based on the research results, it can be concluded that cellulolytic enzyme having activity ofendo-(1,4)β-D-glucanase, exo-(1,4)-β-D-glucanase and β-glucosidase can be produced from cellulolytic isolates of E. cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya Abbatoir, Indonesia. This indicates that E. cloacae WPL 214 can be used to hydrolyze fibrous feeding material containing lignocellulose.

Authors' Contributions
WPL designed the research, collected and processed samples. DSN helped in designing the research. ABY carried out the data collection and gathering assay samples. TN and KS assisted in manuscript preparation; WPL, DSN, TN, and KS collected materials for manuscript. All authors have read and approved the final manuscript.