Waste Management 116 (2020) 58–65

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Waste Management
journal homepage: www.elsevier.com/locate/wasman

Cellulolytic bacteria isolation, screening and optimization of enzyme

production from vermicompost of paper cup waste
A. Karthika a,b, R. Seenivasagan c,e, R. Kasimani d, O.O. Babalola e, M. Vasanthy b,⇑
a
Department of Microbiology, Standard Fireworks Rajaratnam College for Women, Sivakasi 626123, Tamil Nadu, India
b
Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India
c
Department of Biotechnology, Kalasalingam Academy of Research and Education, Krishnankoil 626126, Tamil Nadu, India
d
Department of Microbiology, Nehru Arts and Science College, Coimbatore 641105, Tamil Nadu, India
e
Food Security and Safety Niche Area, Faculty of Natural and Agricultural Sciences, North-West University, Mmabatho 2735, South Africa

a r t i c l e i n f o a b s t r a c t

Article history: In the current scenario, used paper cups are disposed of without any proper treatment, thereby damaging
Received 18 April 2020 the environment. Hence, the vermicomposting technique is preferred for managing these wastes. The
Revised 14 June 2020 ability of bacterial strains on cellulase enzyme (Endoglucanase, exoglucanase and b-glucosidase) produc-
Accepted 25 June 2020
tion at altered pH and temperatures were focused in this study. Among nine bacterial strains
Acinetobacter baumannii was found to have high enzyme activity. HPLC analysis confirms that about
45% of cellulose degradation occurred due to the action of bacterial consortia at 37 °C with pH 7. The
Keywords:
overall period required for degradation takes only three months with the help of bacterial consortia while
Acinetobacter baumannii
Microbial consortia
comparing to our previous study, which takes six months. The insilico study on three cellulose-degrading
Cellulase enzyme enzymes sequence were retrieved from NCBI, and analysed for multiple sequence alignment and phylo-
Endoglucanase genetic tree construction. From the analysis, the endoglucanase SVK46152 (Acinetobacter baumannii)
Exoglucanase and b-glucosidase sequence got docked with cellopentaose with a high score value 11.07. Thereby we confirm that organ-
ism Acinetobacter baumannii was effective in paper cup degradation.
Ó 2020 Published by Elsevier Ltd.

1. Introduction Cellulase is the common enzyme degrading cellulose. Endo-b-

Currently, the amount of solid waste produced by Indian cities
is 600 M tons per day. By the year 2021, this amount is expected
to increase up to 1150 M ton solid waste (Vijay and Pandit,
2013). Another alarming fact is the amount of solid waste pro-
duced by the ubiquitous throwaway paper cups. Paper cups were
reported to generate about 253 million pounds of solid waste per
year (Whirley, 2008). Waste management by landfill and open
dumps would cause aesthetic pollution to the environment. In
the present study, vermicomposting of paper cup wastes was
examined. Vermicompost technology is one of the eco-friendly
methods for transforming waste into a nutrient-rich fertilizer,
through the joint action of earthworms and microorganisms. Paper
cups (Polypaper) are made up of 95% of high strength paper with
5% thin coating of Low-density polyethylene (LDPE). The polypaper
present in the paper cup contains about 90% of cellulosic fibres.
⇑ Corresponding author at: Department of Environmental Biotechnology,
Bharathidasan University, Tiruchirappalli, Tamil Nadu, India.
E-mail addresses: drmvasanthy02@gmail.com, drmvunit@gmail.com
(M. Vasanthy).
glucanase, Exo-b-glucanase and b-glucosidase are three main com-
ponents comprising in cellulase enzyme which have been shown to
act synergistically in the hydrolysis of cellulose to glucose (Emert
et al., 1974; Bajpai, 2018). This enzyme is produced by several
microorganisms, especially by bacteria and fungi (Immanuel
et al., 2006; Bansal et al., 2012; Meneses et al., 2016). Insilico tech-
nique is one of the simplest and time-saving techniques to screen
the bacterial enzyme based on the virtual screening approach. This
technique is helpful in understanding the cellulolytic activity and
the enzyme-substrate interaction based on the structural model.
The cellulolytic activity of the bacterial species is found to be
dependent on the source of occurrence (Saxena et al., 1993;
Thapa et al., 2020). However, the bioconversions of various com-
plex cellulosic waste materials such as bagasse (Kanosh et al.,
1999; Michelin et al., 2015), coir waste (Nagasathya, 2014) have
been already reported. No investigations are carried out both in
analytical & insilico study regarding the bacterial cellulolytic activ-
ity present in the vermicomposting of paper cup waste. In this
research, cellulolytic bacterial isolation, screening and optimiza-
tion of enzyme production was done. The insilico studies were per-
formed to test cellulose-degrading enzyme sequence, by multiple

https://doi.org/10.1016/j.wasman.2020.06.036
0956-053X/Ó 2020 Published by Elsevier Ltd.
 A. Karthika et al. / Waste Management 116 (2020) 58–65
sequence alignment and phylogenetic tree construction. The
selected endoglucanase enzyme SVK46152 (Acinetobacter bauman-
nii) was build using comparative modelling and docked with
cellopentaose.
2. Materials and methods
2.1. Sample collection
The paper cup waste was collected from Bharathidasan Univer-
sity canteen (latitude 10.6° North and longitude 78.74° East),
Tiruchirappalli, Tamil Nadu, India. The epigeic earthworm Eudrilus
eugeniae (Kinberg) was obtained from Periyar Maniyammai
University, Thanjavur, Tamil Nadu, India. The disposable paper
cup waste was vermicomposted by the bin method. The experi-
ment was carried out in a circular plastic container (diameter
40 cm and depth 9 cm) with a capacity of 2 kg and was labelled
as PCCD (1 kg of shredded paper cup waste + 1 kg of cow dung).
The vermibin was left for pre decomposition so that the wastes
become a suitable bed for the earthworms. After 15 days of pre
decomposition, (n = 15) earthworms were inoculated to the ver-
mibin. The moisture was maintained at about 60–80% during the
vermicompost process as the earthworms prefer the same for their
survival (Karthika et al., 2015).
2.2. Isolation of cellulolytic bacteria
The microorganisms obtained from the vermicompost were
tested for their cellulose degradation potential. Karthika et al.
(2015) reported that the microorganisms present in the vermicom-
post of paper cup waste were Bacillus anthracis (KM289159), B.
endophyticus (KM289167), B. funiculus (KM289165), B. thuringiensis
(KM289164), B. cereus (KM289160), B. toyonensis (KM289161), Vir-
gibacillus chiquenigi (KM289163), Acinetobacter baumannii
(KM289162) and Lactobacillus pantheries (KM289166). These bac-
terial strains were subjected to cellulose degradation by placing
them in the Czapek agar medium and were incubated for the per-
iod of 4–5 days at 30 °C. The growth of bacterial colonies was
observed after the incubation period. Colonies were isolated and
cultured individually on the Congo red plate. These cellulose
degrading bacteria were identified and labelled by observing the
zone of clearance in the Congo red medium plate.
2.3. In vitro study
The cellulase activity was determined by the amount of reduc-
ing sugars released per ml of sample per min under assay condi-
tion. Three sub enzymes were involved in the conversion of
cellulose to glucose. (i) Endoglucanase activity was determined
by 3,5-Dinitrosalicylic acid method (DNS) using CMC as a sub-
strate, (ii) Exoglucanase activity was determined by the DNS
method using filter paper as substrate and finally, (iii) b- glucosi-
dase activity was determined by pNPG assay (Miller 1959).
2.3.1. Endoglucanase activity
The culture filtrate was collected from the supplemented med-
ium (L-glutamic acid, 0.03; NH4NO3, 0.14; KH2PO4, 0.2; CaCl2, 0.03;
MgSO4, 0.03; Protease peptone, 0.75; FeSO4, 0.50; MnSO4,0.16;
ZnSO4, 0.14; Tween 80, 0.2%, Paper cup, 0.3 g/100 ml) containing
the consortia of nine isolates. About 0.5 ml of culture filtrate was
taken and equalized with 2 ml of DNS reagent and 2 ml of 1% of
CMC. The content was then heated in a boiling water bath for
5 min. After cooling to room temperature, the absorbance was read
at 540 nm using UV spectrophotometer (Systronics 2202 PC double
beam spectrophotometer). The amount of reducing sugars was
59
determined using the standard graph of glucose. One unit of activ-
ity was defined as the number of enzymes liberating 1 µm of
reducing sugars as glucose per minute under the assay conditions
(Miller, 1959; Ahmed et al., 2010; Lokapirnasari et al., 2015;
Rahman et al., 2018).
2.3.2. Exoglucanase activity
About 2 ml of crystalline cellulose solution (50 mg of filter
paper dissolved in 0.2 M sodium acetate buffer) was taken in the
test tubes. Accurately 0.5 ml of culture filtrate was added. The mix-
ture was then incubated at 35 °C for 1 h, and the reaction was ter-
minated by the addition of 2 ml of DNS reagent. Further, the
contents were heated in a boiling water bath for 5 min. The tubes
were allowed to cool, and the absorbance was read at 540 nm in
UV spectrophotometer (Barruetabena et al., 2019). One unit of
activity was defined as the amount of enzyme liberating 1 µm of
reducing sugars as glucose per minute under the assay conditions
(Lokapirnasariet al., 2015).
2.3.3. b- glucosidase activity
About 2 ml of P-nitrophenyl b-D-glucopyranoside (5 mM PNPG
in 0.02 M sodium acetate buffer (pH 4) was taken and 0.5 ml of cul-
ture filtrate was added. After boiling at 50 °C for 5 min, the reaction
was terminated by adding 2 ml of 0.2 M sodium borate buffer (pH
10). The release of p-nitrophenol was read at 400 nm using a UV
spectrophotometer. One unit is defined as the amount of enzyme
which liberates 1 µm of PNPG per minute under assay conditions
(Miller, 1959; Lokapirnasari et al., 2015).
2.4. Effect of pH and temperature on the production of cellulase
The supplemented media (SM) was prepared using a paper cup
substrate. Three different pH (3, 7 and 9); temperature (20, 37 and
60 °C) was set respectively in the medium. Later the consortia of
nine isolates were inoculated in the supplemented medium
(SMC) and was kept in a shaker (150 rpm) at 30 °C for 10 days. Cel-
lulase assay was carried out every day to optimize the pH and tem-
perature for the production of cellulase enzymes.
2.5. High-performance liquid chromatography analysis of degradation
product
HPLC analysis was carried out for Supplemented medium (SM)
and Supplemented medium with consortia of nine isolates (SMC).
Samples were taken for analysis after 20 days of the experimental
process. About 2 ml of culture filtrate was taken from both the
medium and was centrifuged at 12,000 rpm for 15 min. The super-
natant was collected and subjected to HPLC analysis. Supple-
mented medium without bacterial consortia served as control
(SM). The study was done in Shimadzu C18 column with a flow
rate of 1 ml/min and was detected in UV–Vis detector at 254 nm
(Kumar and Murthy, 2017).
2.6. Insilico study
2.6.1. Sequence and phylogenetic analysis of endoglucanase,
exoglucanase and b-glucosidase enzymes
Cellulose degrading bacterial enzymes such as endoglucanase
(1961 Sequence), exoglucanase (171 Sequence) and b-glucosidase
(151 Sequence) sequences were retrieved from NCBI database.
Individually the enzyme sequences were analyzed with multiple
sequence alignment, and a phylogenetic tree construction. From
the phylogenetic analysis, the origin/final enzyme sequence was
identified. The isolated effective cellulose degrading strain Acineto-
bacter baumannii having endoglucanase (SVK46152-Acinetobacter
baumannii) enzymes has already been reported in the NCBI data-
60 A. Karthika et al. / Waste Management 116 (2020) 58–65

base without structure. Hence the structure for identified enzyme 3.2. Quantification of cellulase activity
was modeled using automated comparative modeling online tool,
validated with PROCHECK and Verify with 3D (Laskowski et al., The assay for cellulase enzyme was performed by the ability of
1993; Luthy et al., 1992). Modelled structure was docked with cel- individual isolates to hydrolyze cellulose into reducing sugars. The
lopentaose using Auto Dock Vina and compared with the template conversion of crystalline cellulose into amorphous form is
structure and interactions. executed by the endoglucanase. The amorphous form is further
converted into cellobiose by exoglucanase. Further, which the
b-glucosidase converts cellobiose into glucose (Hanet al., 1995;
2.6.2. Docking experiment
George et al., 2001). The bacterial strains present in the vermicom-
Molecular docking investigations were carried out to study the
post were tested for their cellulase production DNS method. Table 2
molecular interactions between the enzyme and substrate. The
represents the amount of enzyme liberated by 1 µm of glucose per
structure of cellopentaose retrieved from Pubchem was chosen as
min under assay condition by the individual isolates. The results
the ligand for computational investigation. The structure of cellu-
confirm that the nine isolates were able to produce a sufficient
lose (cellopentaose) and modeled structures of endoglucanase
amount of cellulase to degrade the paper cup waste. The cellulase
was processed by adding hydrogen atom (http://mgltools.scripps.
activity of the bacterial strains was in the order of Lactobacillus
edu/). Docking studies were performed with the auto dock vina
pantheries (8.63 ± 0.02 unit/ml) > Bacillus funiculus (7.18 ± 0.02
algorithm with AMBER force field and Monte Carlo simulated
unit/ml) > B. endophyticus (6.67 ± 0.02 unit/ml) > B. anthracis
annealing (Trott and Olson, 2010). The structures of the three
(6.64 ± 0.02 unit/ml) > B. thuringiensis (6.36 ± 0.02 unit/ml) >
enzymes were kept as rigid, and cellulose (Cellopentaose) structure
B. toyonensis (6.14 ± 0.02 unit/ml) > B. cereus (5.85 ± 0.02 unit/ml)
was kept flexible throughout the docking process.
> Acinetobacter baumannii (5.54 ± 0.02 unit/ml) > Virgibacil-
luschiquensis (4.96 ± 0.02 unit/ml). The quantitative study of cellu-
3. Results and discussion lase enzyme was reported by several researchers. According to
Ekperigin et al. (2007) the enzyme production by A. anitratus and
Vermicompost is not only used in the agricultural field for their Branhamella sp. was 0.48 and 2.57 U/ml respectively. Liang et al.
beneficial effects on soil facilitating plant growth, in contrary, the (2014) studied the enzyme production in Paenibacillus terrae. Liu
process of vermicomposting is also widely used for waste manage- et al. (2014); Gupta et al. (2012) quantified the cellulose enzyme
ment. This study highlights the role of cellulose-degrading production by several bacterial strains involved in degrading the
microorganisms in the conversion of paper cup waste into organic cellulosic materials.
fertilizer.
3.3. Optimization of cellulase production
3.1. Isolation of cellulase producing bacteria
The optimum pH and temperature for cellulase production are
Cellulase production remains to offer an attractive prospect for considered the most important factor (Lehninger et al., 1993). pH
a longer time. Cellulose degrading bacteria are very much helpful is the very most important parameters required for the growth of
in the bioconversion of cellulose into value-added product. Many microbes. It directly influences the enzymatic production and
industries were involved in the production of glucose, alcohol, activities of the microbes while getting involved in denaturing
paper, nylon and plant protoplast by using cellulase (Kluepfel the subtracts (Subramaniyan and Prema, 2002). Consortia of nine
et al., 1986). Here the bacterial strains isolated from the vermicom- isolates were inoculated into the supplemented medium
post obtained from the paper cup waste were exploited for the cel- containing paper cup as substrate (SMC). At pH 4, the enzyme pro-
lulose degradation study (Karthika et al., 2015). The organisms duction was least on the 1st day (0.013 ± 0.02 U/ml), further
were identified based on their morphology and structure. While increased on the 2nd day of the process (0.082 ± 0.02 U/ml), then
screening the isolates for cellulase production, nine different it gradually decreased during the 4th day (-0.014 ± 0.02 U/ml). At
strains showed positive results on Congo red plate. The zone of pH 7, the enzyme production was the least on the 1st day
clearance produced by the individual microbes was measured (0.008 ± 0.02 U/ml), further increased on the 7th day of the
and presented in table 1. Of the nine different strains, Acinetobacter process (0.465 ± 0.02 U/ml), then gradually decreased during
baumannii shows the zone clearing value of 13 mm which indicates 10th day ( 0.08 ± 0.02 U/ml). At pH 9 the enzyme production
the highest cellulose degradation activity. This proves that the bac-
terial strains present in the vermicompost obtained from paper cup
Table 2
waste could be employed for cellulose degradation. Similar type of Microbial production of cellulase enzymes - exoglucanase, endoglucanase and b-
qualitative enzyme study was carried out by Lu et al. (2006) and glucosidase.
Gupta et al. (2012).
Bacteria Exoglucanase Endoglucanase b- glucosidase
(unit/ml) (unit/ml) (unit/ml)
Bacillus 2.51 ± 0.002 2.3 ± 0.002 1.56 ± 0.002
thuringiensis
Table 1
Bacillus anthracis 2.63 ± 0.002 1.92 ± 0.002 2.09 ± 0.002
Zone of inhibition formed by the cellulose degrading bacteria.
Bacillus 2.99 ± 0.002 1.70 ± 0.002 1.98 ± 0.002
Bacteria Cellulose activity (mm) endophyticus
Bacillus funiculus 3.03 ± 0.002 2.11 ± 0.002 2.04 ± 0.002
Bacillus thuringiensis 7 Bacillus cereus 3.52 ± 0.002 0.34 ± 0.002 1.99 ± 0.002
Bacillus anthracis 8 Bacillus 2.02 ± 0.002 2.05 ± 0.002 2.07 ± 0.002
Bacillus endophyticus 6 toyonensis
Bacillus funiculus 7 Acinetobacter 2.14 ± 0.002 1.68 ± 0.002 1.72 ± 0.002
Bacillus cereus 5 baumannii
Bacillus toyonensis 10 Lactobacillus 3.79 ± 0.002 2.65 ± 0.002 2.19 ± 0.002
Acinetobacter baumannii 13 pantheries
Lactobacillus pantheries 9 Virgibacillus 2.01 ± 0.002 1.52 ± 0.002 1.43 ± 0.002
Virgibacillus chiquenigi 4 chiquenigi
 A. Karthika et al. / Waste Management 116 (2020) 58–65
was the least on the 1st day (0.017 ± 0.02 U/ml), further it
increased on the 3rd day of the process (0.282 ± 0.02 U/ml), grad-
ually decreased during the 6th day (0.001 ± 0.02 U/ml). From this
experiment, it is clear that the consortia of the nine isolates exhib-
ited maximum enzyme production at pH 7 during the 7th day of
the experimental process compared to pH 4 and pH 9 (Fig. 1).
Importance of pH in enzyme production was studied by George
et al. (2001); Pathania et al. (2012).
The thermal stability of the enzyme is much more critical in
determining the significant application of enzymes in industries.
Therefore, every microorganism need an optimum temperature
to exhibit the stability of enzyme activities (Pathania et al.,
2012). The enzyme production by the consortia was stabilized at
a different set of temperatures (4, 35 and 60 °C). The experimental
results show that the output was least negative value (almost zero)
at 4 °C (-0.062 ± 0.02 U/ml) and 60 °C (-0.09 ± 0.02 U/ml) respec-
tively. At 37 °C the production was maximum during the 5th day
of the process (0.512 ± 0.02 U/ml) (Fig. 1). Hence from the study,
the production of cellulase enzyme was maximum at pH 7 and
37 °C during the 7th day of the experimental process. Similar
results were reported for cellulase production from Aspergillus
niger by Akiba et al. (1995); Bansal et al. (2012). According to
Banerjee et al. (2020), the maximum cellulase enzyme production
(0.69 ± 0.015 U/ml) obtained at 38 °C was achieved by a stain GAC
16.2. Therefore, in the present study, the optimum pH and temper-
ature for cellulase production was in good agreement with the
results of Jahangeer et al. (2005); Gilna and Khaleel, (2011).
3.4. Cellulose degradation
HPLC analysis was carried out to examine the hydrolysis of cel-
lulose present in the paper cup by the consortia of nine isolates.
Experiments were done in comparison with the supplemented
medium having consortia (SMC) and the supplemented medium
without consortia (SM) (Fig. 2).
According to Muramoto et al. (1987) and Han et al. (1995) the
cellulosic substance could be detected at 250–340 nm in a UV
detector. In Fig. 2a single peak was detected in SM at 254 nm with
the intensity of peak range at more than 5000 with the retention
time of 2.9. But in the case of SMC the intensity of the peak has
got decreased to 3000 with the same retention time and in the
same wavelength, after a period of 20 days (Fig. 2b). The area of
the peak was reduced from 380620 to 245,696 mAU. Similarly,
the height of the peak has also been reduced from 6061 to 3303
Fig. 1. Effect of cellulase production at different pH (4, 7 and 9) and temperature (4, 37 and 60 °C) by the inoculum during the incubation period of 10 days.
61
mAU. This variation may be attributed due to the activity of cellu-
lase producing bacteria. HPLC analysis makes us conclude that the
consortia of nine isolates have the ability to degrade cellulose and
also have the potential to achieve about 45% of degradation of the
paper cup within 20 days. Similar study detecting the presence of
monosaccharides was carried out by Khan et al. (2020); Yi et al.
(2020).
3.5. Enhancement of vermicomposting process
The vermicomposting is a process of joint action of earthworm
and microorganism. Hence by overloading the microbial concentra-
tion helps to fasten the vermicomposting process. Nine bacterial iso-
lates Bacillus anthracis (KM289159), B. endophyticus (KM289167),
B. funiculus (KM289165), B. thuringiensis (KM289164), B. cereus
(KM289160), B. toyonensis (KM289161), Virgibacillus chiquenigi
(KM289163), Acinetobacter baumannii (KM289162) and Lactobacil-
lus pantheries (KM289166) identified from the vermicompost of
paper cup waste were found to be effective in cellulose degradation.
Normally vermicompost of paper cup waste takes about 6 months
for the degradation process. Whereas the inoculation of the consor-
tia of nine isolates into the vermibin containing paper cup and cow
dung (PCCDB) has reduced the period required for the paper cup
degradation considerably. As Karthika et al. (2015) reported that
the earthworm separated the plastic lining coated inside the paper
cups and settled around the rim of the tub. Surprisingly, in PCCDB
vermibin the plastic coated in the paper cup waste started to sepa-
rate within the 2nd week of the process by the action of consortia,
but such separation was not noted in PCCB during the 2nd week of
the process (Fig. 3). This study substantiates the interrelationship
between the bacterial strains and the earthworm during the vermi-
composting process. Though earthworms play the role of degrada-
tion in the vermicomposting process, rapid degradation could be
achieved only with the help of additional microbes. The whole ver-
micomposting process has been completed within 3 months in
PCCDB, while PCCD took about 6 months.
3.6. Insilico studies
3.6.1. Phylogenetic tree analysis
Overall endoglucanase (1961), exoglucanase (1 7 1) and b-
glucosidase (1 5 1) sequence were retrieved from NCBI to deter-
mine multiple sequence alignment and phylogenetic tree using
the method of ClustalW on neighbor joints methods (Gap opening
62 A. Karthika et al. / Waste Management 116 (2020) 58–65

Fig. 2. Effect of bioinoculum in supplemented medium (SM and SMC) by cellulase enzyme detection through HPLC analysis. (a) SM represent the intensity of peak range at
more than 5000 with the retention time of 2.9, (b) SMC represent the intensity of the peak has got decreased to 3000 with the same retention time and in the same wave
length, after the period of 20 days.

Fig. 3. Cellulose degradation and plastic separation process in paper cup waste; (A) Bio inoculated vermin bin (PCCD), (B) plastic separated bin (2nd week) (PCCDB).

and extension penalty of 10.0, 0.10 and 10.0, 0.20) in MEGAX. From
this analysis, the origin / first sequence from three types of
enzymes (Supplementary Fig. 1) were retrieved. Among this, the
endoglucanase SVK46152 (Acinetobacter baumannii) reported
enzyme were matched with effectively cellulose-degrading strain
Acinetobacter baumannii (KM289162) of these studies.
Seenivasagan et al. (2016) reported a similar parameter and
method used for the construction of the phylogenic tree by MEGA
software.
3.6.2. Modelling and validation
SVK46152 (Acinetobacter baumannii) (Fig. 4a) enzyme structure
was theoretically modelled by online tools, using the template PDB
structure of 3QXQ. The modeled structure was validated and
energy minimisation was done by Procheck, WhatIF, Ramachan-
dran plot and Swiss pdb View with GROMOS96. From the valida-
tion, the Ramachandran plot results on SVK46152 enzyme
residue in most favoured regions (92.9%), allowed regions (7.1%),
disallowed regions (0.0%) and total sequence (3 6 7) (Fig. 4b). The
ProSA Z-scores for the model and template are much closer to
the middle region of scores observed for experimentally determin-
ing protein structures in the PDB, including the template structure
(Fig. 4c, d). Statistical based diagnostic tool ProSA, issued to ana-
lyze the protein structure (Wiederstein and Sippl, 2007).
3.6.3. Docking analysis
Modeled enzyme SVK46152was docked with cellopentaose. The
docking score was 11.07 kJ/mol. The cellopentaose interaction
with enzyme residues on seven conventional hydrogen bonding
with Asn110, Arg175, Tyr180 (H-donor), Gln55, Val 166 and
Gly167 (H-acceptor). Another typed three carbon hydrogen bond
with Gly167:HA1, Gly167:HA2 (H-donor) and Asp42 (H-acceptor)
and one pi-donor hydrogen bond with Trp94 (Pi-orbitals). These
enzymes and ligands interacting residues, atom position, hydrogen
bond, bond length, hydrogen donor and acceptor are shown in
Table 3 (Fig. 5).
Highly conserved Tyr180, Gly167, Asn110, and Gln55 residues
of endoglucanase (SVK46152) has good interaction between the
cellopentaose; these residues are conserved residues of template
of 3QXQ. Mazur and Zimmer (2011) reported that some of the sim-
ilar residues were interacting (H-bond) with cellopentaose (at
position 1), glucosyl residues reducing end H-bond with OH-1
 A. Karthika et al. / Waste Management 116 (2020) 58–65 63

Fig. 4. Comparative modelled structure and validation for Ramachandran plot and Z-core analysis of endoglucanase (SVK46152), (A) Predicted structure, (B) Ramachandran
analysis of predicted SVK46152 structure, (C) ProSA black dot analysis of predicted model fall in NMR based reliability with probable Z-score scale. (D) ProSA Z-scores for the
template structure (3QXQ) is similar like SVK46152 model and fall within NMR based reliability represented in black dot.

Table 3
Cellopentaose (CE52416) interacting residues of endoglucanase (SVK46152) and distance, types of hydrogen bond, donor and acceptor.

Name Distance Types of hydrogen bond H-Donor H-Acceptor

GLU53 3.17059 Conventional hydrogen bond CE52416 – O3A GLU53 – OE2
ASN110 2.65536 Conventional hydrogen bond ASN110 – HD21 CE52416 – O3C
ASN110 2.98371 Conventional hydrogen bond ASN110 – HD21 CE52416 – O6D
VAL166 3.33606 Conventional hydrogen bond CE52416 – O6D VAL166 – O
GLY167 3.05354 Conventional hydrogen bond CE52416 – O3D GLY167 – O
ARG175 2.41508 Conventional hydrogen bond ARG175 – HH22 CE52416 – O2D
TYR180 2.38069 Conventional hydrogen bond TYR180 – HH CE52416 – O6B
ASP42 3.74749 Carbon hydrogen bond CE52416 – C6A ASP42 – OD2
GLY167 2.63948 Carbon hydrogen bond GLY167 – HA1 CE52416 – O6D
GLY167 2.04386 Carbon hydrogen bond GLY167 – HA2 CE52416 – O5E
TRP94 3.4073 Pi-Donor hydrogen bond CE52416 – O2C TRP94

group (Gln55), OH-2 group (Asp243 and Tyr182) andOH-3
(Asp116). Trp96 indole ring stacking interaction with position-2
and OH-6H-bond with Ser113. In position-3, OH-3 group H-bond
with Asp110, Asn112 and stacking interaction with Phe170. Highly
conserved (Gly169) residue was H-bond with OH-2 group. This
study proves that there is a very good interaction occurred
between the cellulose substrate and the enzyme of Acinetobacter
baumannii. Thatoi et al. (2020) reported docking interaction of cel-
lulase and disaccharide by Aspergillus fumigates with significant
results.
64 A. Karthika et al. / Waste Management 116 (2020) 58–65
Fig. 5. Cellopentaose docked with endoglucanase (SVK46152) and control structure of 3QXQ (Structure of the bacterial cellulose synthase subunit Z in complex with
cellopentaose), (A) binding pocket of the endoglucanase (SVK46152) and residues interacting with cellopentaose, (B) cellopentaose hydrogen boing residues are labelled, (C)
2D diagram of endoglucanase (SVK46152) and cellopentaose, (D) 2D diagram of control structure 3QXQ.
4. Conclusion
The present study makes us understand the feasibility of sub-
jecting the paper cup waste for vermicomposting and substantiates
the importance of cellulose-degrading bacterial strains in paper
cup waste management. HPLC analysis proves that 45% of cellulose
degradation could be achieved by the consortia of the cellulase
producing bacterial isolates. In both insilico and in vivo method,
the enzymatic analysis proves that the organism Acinetobacter bau-
mannii was effective in paper cup degradation. Therefore, when
paper cup wastes and cow dung were blended in appropriate
quantities along with microbial consortia, it would be converted
into a good quality vermicompost in a comparatively short period
of time thereby reducing the waste to be taken to the landfills and
open dumps.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
TheMinistry of Environment and Forests (MOEF), New Delhi,
India supported the sanction of Major Research Project. The
authors acknowledge the DST- FIST, UGC NON – SAP and UGC –
SAP New Delhi for the support extended for the department.
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