chitinolytic and chitosanolytic activities from crude cellulase
TRANSCRIPT
J. Microbiol. Biotechnol. (2011), 21(12), 1312–1321http://dx.doi.org/10.4014/jmb.1106.06036First published online 19 August 2011
Chitinolytic and Chitosanolytic Activities from Crude Cellulase ExtractProduced by A. niger Grown on Apple Pomace Through Koji Fermentation
Dhillon, Gurpreet Singh1, Satinder Kaur Brar
1*, Surinder Kaur
1,2, Jose R. Valero
1, and Mausam Verma
3
1INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec, G1K 9A9, Canada2Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University (BHU), Varanasi-221005, India3Institut de recherche et de développement en agroenvironnement inc. (IRDA), 2700 rue Einstein, Québec (Québec), G1P 3W8,Canada
Received: June 16, 2011 / Revised: July 16, 2011 / Accepted: August 15, 2011
Enzyme extracts of cellulase [filter paper cellulase (FPase)
and carboxymethyl cellulase (CMCase)], chitinase, and
chitosanase produced by Aspergillus niger NRRL-567
were evaluated. The interactive effects of initial moisture
and different inducers for FP cellulase and CMCase
production were optimized using response surface
methodology. Higher enzyme activities [FPase 79.24±
4.22 IU/gram fermented substrate (gfs) and CMCase
124.04±7.78 IU/gfs] were achieved after 48 h fermentation
in solid-state medium containing apple pomace supplemented
with rice husk [1% (w/w)] under optimized conditions [pH
4.5, moisture 55% (v/w), and inducers veratryl alcohol
(2 mM/kg), copper sulfate (1.5 mM/kg), and lactose 2%
(w/w)] (p<0.05). Koji fermentation in trays was carried
out and higher enzyme activities (FPase 96.67±4.18 IU/gfs
and CMCase 146.50±11.92 IU/gfs) were achieved. The
nonspecific chitinase and chitosanase activities of cellulase
enzyme extract were analyzed using chitin and chitosan
substrates with different physicochemical characteristics,
such as degree of deacetylation, molecular weight, and
viscosity. Higher chitinase and chitosanase activities
of 70.28±3.34 IU/gfs and 60.18±3.82 to 64.20±4.12 IU/gfs,
respectively, were achieved. Moreover, the enzyme was
stable and retained 92-94% activity even after one month.
Cellulase enzyme extract obtained from A. niger with
chitinolytic and chitosanolytic activities could be potentially
used for making low-molecular-weight chitin and chitosan
oligomers, having promising applications in biomedicine,
pharmaceuticals, food, and agricultural industries, and in
biocontrol formulations.
Keywords: Cellulase, chitooligomers, chitinase, chitosanase,
response surface methodology
Cellulases are increasingly gaining importance owing to theirwidespread applications in biofuels, food, textile, and paperindustries, formulation of animal feed supplements, extractionof fruit and vegetable juices, among others [25, 32]. Recently,cellulases have been finding potential applications owingto their multifunctional activities, such as nonspecifichydrolysis of chitin and chitosan to chitooligomers andlow-molecular-weight chitosans (LMWCs). These productsfind innumerable applications in various fields, such asbiomedical, pharmaceutical, biotechnological, food, andagricultural sectors [36]. The cellulases are preferred overspecific chitinases and chitosanases because of their lowproduction costs and are even comparable, albeit withsuperior chitosan hydrolysis rates [17, 20, 38].
Generally, chitinolytic and chitosanolytic enzymes areused for depolymerization of chitin and chitosan to makeLMWCs and chitooligomers [6]. However, the applicationsof chitosanases are hindered by their high cost and loweravailability. Various other enzymes, such as cellulases,lipases, lysozyme, papain, and pectin lyase, have beenutilized for hydrolysis of chitosan to chitosan preparationswith different molecular masses [19, 20, 31, 36]. Cellulase-chitosanase dual activity has been reported for Streptomyces
griseus MUT6037, Myxobacter A-L1, and Bacillus megaterium
P1 [15, 26, 27].Conventionally, the production of enzymes is very
costly, and raw materials mainly contribute higher share inthe economics of the process. Utilization of various agro-industrial wastes provides a promising way to reduce the
*Corresponding authorPhone: +1-418-654-3116; Fax: +1-418-654-2600;E-mail: [email protected]
1313 Dhillon et al.
cost of the enzymes. The higher enzyme production can beachieved by optimizing the fermentation medium and processparameters. Filamentous fungi, such as Trichoderma andAspergillus niger strains, are the most widely exploitedmicroorganisms owing to their ability to grow on complexsolid substrates and produce an array of extracellularenzymes [5, 7].
The successful scale-up of the process demands optimizationof critical parameters that influences microbial growth andproduct formation. In solid-state fermentation (SSF) orkoji fermentation, moisture content is a critical parameterfor cell growth and enzyme production, thus affecting theoutcome of the fermentation processes [8]. The enzymesecretion by the fungus can be further increased by the use ofvarious inducers, such as lactose (LAC), veratryl alcohol(VA), and copper sulfate (CS). Studies have demonstratedthe positive effect of copper ions and veratryl alcohol onligninolytic enzyme production by some basidiomycetes,such as Ceriporiopsis subvermispora and Phanerochaete
chrysosporium [1, 12]. Similarly, the potential of lactose asan inducer for enzyme production by the filamentousfungus T. reesei has also been demonstrated [24].
Our objectives were to optimize cellulases productionvia koji fermentation by A. niger NRRL-567 using applepomace (AP) supplemented with 1% (w/w) rice husk. A.
niger NRRL-567 is well known for its hyper citric acidbioproduction [8] ability that can be inevitably attributedto efficient secretion of hydrolytic enzymes, but has notbeen investigated for production of hydrolytic enzymes sofar. The initial moisture and inducer concentrations wereoptimized using response surface methodology (RSM),which otherwise is an often arduous and time-consumingtask. RSM is the statistical technique used to evaluate thesignificance of various factors, especially when interactionsexist among factors. pH was optimized using the ‘‘one-factor-at-a-time’’ approach. Taking into account the optimumparameters, solid-state tray fermentation was also conducted.The nonspecific dual chitinase and chitosanase activities ofcrude cellulase extract were also determined. To the best ofour knowledge, the nonspecific chitinase and chitosanaseactivities of cellulases from A. niger have not been evaluatedso far.
MATERIALS AND METHODS
Microorganism, Inoculum, and Substrate Preparation
Aspergillus niger NRRL-567 (Agricultural Research Services (ARS)
Culture Collection, IL, USA) was used. The culture conditions,
maintenance, and inoculum preparation have already been described
in Dhillon et al. [8]. The spore suspension having 1 × 107 spores/ml
was adjusted using a hemocytometer.
The solid substrate comprised apple pomace (Lassonde Inc.,
Rougemont, Montreal, Canada). The AP used was already
supplemented with 1% (w/w) rice husk as a general practice during
processing of apples in the apple industry. The moisture of the AP
was analyzed using a moisture analyzer (HR-83 Halogen, Mettler
Toledo, Switzerland).
Solid-State Cellulolytic Enzyme Production in Flasks and in a
Tray Bioreactor
Fungal growth and the production of enzymes were carried out at
30±1oC for 7 days using 500 ml Erlenmeyer flasks containing 40 g
of AP having particle sizes of 1.7-2.0 mm. The required initial
moisture was adjusted with distilled water. The inducers were added
in required concentrations (Table 1) and the medium was sterilized
and inoculated with a spore suspension having 1 × 107
spores/g
of substrate, and the cultures were incubated at 30±1oC in an
environmental chamber under controlled relative humidity (RH) for
7 days. All experiments were conducted in triplicates.
Using the optimization parameters, static solid-state tray fermentation
was conducted in plastic trays having the approximate dimensions of
40 cm (length), 25 cm (breadth), and, 12 cm (height). About 500 g
of AP was supplemented at optimized inducer concentration and
desired moisture, followed by sterilization at 121±1oC for 30 min.
After cooling down the contents, the AP was transferred to trays
and inoculated with spore suspension having 1 × 107 spores/g
substrate and incubated at 30±1oC for 24 h in an environmental
chamber at 55% RH. Samples were analyzed for cellulase activity
and nonspecific chitinase and chitosanase activities.
Enzyme Extraction
Enzyme activities were analyzed at every 24 h intervals from extracted
samples. A wet fermented sample (1 g) was harvested from each
flask after every 24 h in aseptic conditions and dispensed in
100 mM citrate buffer at pH 4.8 [15:1 (v/w)]. The enzyme extraction
was carried out by incubating the samples in a wrist action shaker
(Burrell Scientific, PA, USA) for 30 min. After incubation, the
sample was centrifuged at 9,000 ×g and 4±1oC for 15 min, and the
Table 1. Experimental range of the four variables studied using CCD in terms of actual and coded factors.
Independent variablesSymbol
Coded range and levels
-α* Low (1) Mid (0) High (+1) +α
Moisture % (v/w) X1 50 60 70 80 90
VA (mM/kg) X2 0 1 2 3 4
CS (mM/kg) X3 0.5 1.0 1.5 2.0 2.5
LAC % (w/w) X4 0 1 2 3 4
*α= 2.0.
Abbreviations: VA, veratryl alcohol; CS, copper sulfate; LAC, lactose.
FUNGAL CELLULASES AND HEMICELLULASES 1314
Table 2. Results of experimental plan by central composite design for apple pomace (shaded cells represent higher enzyme activity,CFUs, and total extracellular protein at optimum process conditions).
Trial X1 X2 X3 X4 FPase (IU/gfs) CMCase (IU/gfs) Viability of fungus (CFUs/gfs) Total protein (mg/gfs)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
60.0
60.0
60.0
60.0
60.0
60.0
60.0
60.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
50.0
90.0
70.0
70.0
70.0.
70.0.
70.0
70.0
70.0
70.0
70.0
70.0
70.0
70.0
70.0
1.0
1.0
1.0
1.0
3.0
3.0
3.0
3.0
1.0
1.0
1.0
1.0
3.0
3.0
3.0
3.0
2.0
2.0
0.0
4.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
1.0
1.0
2.0
2.0
1.0
1.0
2.0
2.0
1.0
1.0
2.0
2.0
1.0
1.0
2.0
2.0
1.5
1.5
1.5
1.5
0.5
2.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.0
3.0
1.0
3.0
1.0
3.0
1.0
3.0
1.0
3.0
1.0
3.0
1.0
3.0
1.0
3.0
2.0
2.0
2.0
2.0
2.0
2.0
0.0
4.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
39.54
41.71
36.64
37.42
41.52
41.96
39.11
39.13
23.72
18.70
17.33
28.78
14.56
30.35
29.43
32.58
70.78
17.72
38.81
39.41
39.60
44.75
19.91
38.69
38.00
35.96
36.41
37.83
37.20
37.32
37.89
52.20
57.80
47.50
52.10
50.20
51.44
51.50
53.60
26.60
26.60
14.90
32.20
17.44
32.90
34.10
34.20
92.50
19.44
45.02
44.70
43.80
46.40
29.60
44.90
36.20
35.41
36.40
35.10
35.90
36.40
36.10
3.89E+08
3.92E+08
3.65E+08
3.46E+08
3.98E+08
4.0E+08
3.8E+08
4.1E+08
1.4E+08
1.0E+08
9.78E+07
1.41E+08
8.96E+07
1.49E+08
1.42E+08
1.73E+08
8.17E+09
9.98E+07
3.79E+08
3.65E+08
3.92E+08
5.01E+08
2.48E+08
3.43E+08
3.9E+08
3.54E+08
3.92E+08
3.78E+08
3.9E+08
3.31E+08
3.43E+08
9.28
9.95
9.28
9.03
9.13
8.75
10.97
8.82
6.60
5.51
7.0
6.15
5.67
6.54
7.55
5.93
11.67
3.88
7.69
8.13
8.17
9.34
7.02
8.98
8.23
8.52
8.44
8.90
8.70
8.51
8.95
supernatant was analyzed for enzyme activities and total protein
content.
Enzyme Activity and Total Extracellular Protein Assays
The total extracellular protein content in crude enzyme extract was
analyzed by the method of Lowry et al. [21]. Assays of the FP
cellulase (FPase) and carboxymethyl cellulase (CMCase; endoglucanase)
activities in the crude enzyme extract were done following the
methods of Ghose [14] and Wood and Bhat [35], respectively.
Chitinase and chitosanase activities were determined by the
modified methods of Ueda and Arai [34] and Liu and Xia [20]. The
reaction mixture contained 0.5 ml of crude enzyme and 1.5 ml of
substrate [1% (w/v) chitin/chitosan in 50 mM sodium acetate buffer,
pH 4.8]. Three chitosan substrates having different physicochemical
properties were used (Substrate 1, Fischer, MW 600-800 kDa,
degree of deacetylation (DD) >90%, and viscosity 200-500 mPa·s;
substrate 2, Sigma, medium MW 190-310 kDa, DD 82%, and
viscosity 522 cps); and substrate 3, Sigma, high MW 310-375 kDa,
DD 77%, and viscosity 1,120 cps). The reactions were carried out
by incubating at 50±1oC for 30 min. The reaction was stopped by
boiling the reaction contents for 5 min. The liberated reducing
sugars were measured by the 3,5-dinitrosalicylic acid (DNS) method
[23]. One international unit was defined as the amount of enzyme
that liberates 1 µmol of reducing sugar per ml/min. All the
experiments were conducted in triplicates and the enzyme activities
described are the mean of three replicates and expressed as IU/gfs
(units per gram fermented substrate).
Viability Assay
Viability assay was used as an indicator/measure of the amount of
living fungal biomass in solid-state fermentation. The viability of
broken fungal mycelium was assayed using the most probable number
(MPN) method. The details of the viability method are described in
Dhillon et al. [9]. The results are given in colony forming units
(CFUs) as calculated according to the following formula:
MPN = P ÷ (Nn × Nk)0.5
where P= the number of positives in all the accounted series; Nn=
amount of sample in the negative parallels (g); Nk= amount of
sample in all the accounted series (g).
1315 Dhillon et al.
Experimental Design and Optimization
In order to identify the significant factors that affect the responses,
an attempt was made to improve the composition of the medium by
comparing different levels of several factors on the production of
cellulase enzymes by A. niger NRRL-567. The impacts of four
independent quantitative variables, including moisture (X1), veratryl
alcohol (X2), copper sulfate (X3), and lactose (X4), were evaluated
by a factorial central composite design (CCD) to find the optimal
concentrations of these four factors. In this regard, a set of 31
experiments including 7 center points (0, 0, 0) and 8 axial points (α
= 2.0) and 16 points corresponding to a matrix of 24, which
incorporates 16 experiments including 3 variables (+1, -1, 0), were
carried out. Each variable was studied at two different levels (-1, +1)
and center point (0), which is the midpoint of each factor range. The
minimum and maximum range of variables investigated and the full
experimental plan with respect to their actual and coded values are
listed in Tables 1 and 2, respectively. A multiple regression analysis
of the data was carried out by STATISTICA 6 of STATSOFT Inc.
(Thulsa, USA) by surface response methodology and the second-
order polynomial equation that defines predicted responses (Yi) in
terms of the independent variables (X1, X2, X3, and X4):
Yi = b0i + b1iX1 + b2iX2 + b3iX3 + b4iX4 + b11iX21+ b22iX
22 + b33iX
23
+ b44iX24 + b12iX1X2 + b13iX1X3 + b14iX1X4 + b23iX2X3 + b24iX2X4
+ b34iX3X4 (1)
where Yi = predicted response, boi is the intercept term, b1i, b2i, b3i,
b4i are linear coefficients, b11i, b22i, b33i, b44i squared coefficients, and
b12i, b13i, b14i, b23i, b24i, b34i interaction coefficients, and i refers to the
response. A combination of factors (such as X1X2) represents an
interaction between the individual factors in the respective term.
There are 2 different responses, FPase and CMCase production.
These responses are a function of the level of factors. The response
surface graphs indicate the effect of variables as individual response
and in combination, and determine their optimum levels for maximal
FPase and CMCase production.
The data were fitted into a second-order polynomial function [Eq.
(1)] and a correlation was drawn between experimental data and the
predicted values by the model (Fig. 1A and 1B). Linear, quadratic,
and interaction coefficients of variables under study that were found
to be significant at p<0.05 were retained in reduced models. The
quality of the model fit was evaluated by the coefficient R2, and its
statistical significance was determined by an F-test. R2 represents
the proportion of variation in the response data that can be
explained by the fitted model. High R2 indicates the applicability of
the model in the range of variables included. It should be noted that
a R2 value greater than 0.75 indicates the suitability of the model.
The coefficients of determination (R2) are presented in Table 3.
Statistical Analysis
Data means and standard deviation were analyzed with individual
Student’s t-tests to distinguish differences among treatments. The
test was performed at the level of P <0.05 to determine the
significance of the difference between treatments to produce enzymes.
RESULTS AND DISCUSSION
A. niger NRRL-567 was selected as a suitable microorganismfor bioprocessing of AP through SSF for enzymeproduction. AP was used as the substrate as it containsabout 62% carbohydrates and 5.7% (v/w) protein and othervital micro and macronutrients [7]. Initial moisture leveland inducer concentrations were varied to determine theoptimum combination of factors to produce the maximumyield of cellulase enzymes.
Effects of Variables on Cellulase Production
The central composite design was applied to find theoptimized values of the variables on cellulase productionby using AP as a solid substrate by A. niger NRRL-567.Tables 1 and 2 represent the results of CCD experimentsthat consist of experimental data for studying the effect offour independent variables (moisture, VA, CS, and LAC)on cellulases (FPase and CMCase) response production byA. niger. The responses at various coded levels of moistureand inducer concentrations are shown in Fig. 2A, 2B, 2C;
Fig. 1. Parity plots showing the distribution of experimentalversus predicted values of (A) FPase activity and (B) CMCaseactivity using apple pomace by A. niger NRRL-567.
FUNGAL CELLULASES AND HEMICELLULASES 1316
3A, 3B, 3C; 4A, 4B, 4C; and 5A, 5B, 5C, respectively,for cellulase production. The effect of the pairwiseinteractions is clearly depicted in the 3-D graphs keepingthe other two parameters constant. It represents an infinitenumber of combinations of two test variables.
Statistical analysis was performed with the data havinghigher cellulase production at 48 h of incubation period, as
no significant increase (p<0.05) in cellulase productionwas observed after 48 h of incubation time. Cellulaseproduction exhibited different responses with variedconcentrations of three inducers (VA, CS, and LAC) anddifferent moisture levels. The FPase production variedfrom 14.56 IU/gfs (trial 13) to 70.78 IU/gfs (trial 17), andCMCase activity from 14.90 IU/gfs (trial 11) to 92.50 IU/
Table 3. Model coefficients estimated by central composite design and best selected prediction models.
CoefficientFPase (IU/gds)
Coefficient t-value p-valueCMCase (IU/gds)
Coefficient t-value p-value
Constant 37.2* 16.07* 0.000* 35.92* 16.37* 0.000*
Linear
M -19.0* -7.58* 0.000* -28.62* -12.08* 0.000*
VA 2.2 -0.87 0.399 1.24 0.52 0.608
CS 1.6 0.62 0.543 0.83 0.35 0.729
LAC 5.5* 2.21* 0.042* 6.41* 2.70* 0.0157*
Interactions
M × VA 1.5 0.49 0.632 2.65 0.91 0.375
M × CS 4.2 1.35 0.194 2.34 0.81 0.432
M × LAC 2.7 0.90 0.384 2.43 0.84 0.415
VA × CS 1.9 0.63 0.539 4.74 1.63 0.122
VA × LAC 1.3 0.41 0.688 -1.08 -0.37 0.713
LAC × CS 0.3 0.08 0.935 0.23 0.08 0.936
Quadratic
M 1.3 0.56 0.583 7.98* 3.68* 0.002*
VA -1.3 -0.56 0.583 2.43 1.12 0.279
CS 0.2 0.11 0.916 2.56 1.18 0.256
LAC -6.2* -2.70* 0.016* -1.38 -0.064 0.533
R2 value 0.825 0.916
*Significant (p<0.05). Abbreviations: M, moisture; VA, veratryl alcohol; CS, copper sulfate; LAC, lactose.
Reduced equations for cellulase production (best selected models):
FPase = 37.2 – 19.0 M + 2.2 VA +1.6 CS + 5.5 LAC + 1.3 M - 1.3 VA + 0.2 CS – 6.2 LAC + 1.5 M × VA + 4.2 M × CS + 2.7 M × LAC + 1.9 VA × CS + 1.3
VA × LAC – 0.3 CS × LAC
CMCase = 35.92 – 28.62 M + 1.24 VA + 0.83 CS + 6.41 LAC + 7.98 M +2.43 VA + 2.56 CS – 138 LAC + 2.65 M × VA + 2.34 M × CS + 2.43 M × LAC +
4.74 VA × CS - 1.08 VA × LAC +0.23 CS × LAC
Fig. 2. Response surface graphs illustrating the effects of (A) moisture and veratryl alcohol; (B) moisture and copper sulfate; and (C)moisture and lactose on cellulase activity measured as FPase/g fermented substrate.
1317 Dhillon et al.
gfs (trial 17), as shown in Table 2. The higher FPase andCMCase activities of 70.78 IU/gfs and 92.50 IU/gfs,respectively, were obtained with 50% (w/w) initial moisturelevel and inducers [VA 2 mM/kg, CS 1.5 mM/kg, and LAC2% (w/w)], which declined by 56.22 IU/gfs [VA 3 mM/kg,CS 1 mM/kg, and LAC 1% (w/w)] and 77.60 IU/gfs [VA1 mM/kg, CS 2 mM/kg, and LAC 1% (w/w)], respectively,on increase in the initial moisture content to 80% (w/w).The reduction in both activities was probably due to thevariation in moisture content and interactive effect ofdifferent inducers with varied concentrations. Moisturecontent is important for fungal growth and enzymeproduction. A lower moisture level results in a lowerdegree of substrate swelling and higher water tension, andreduces the solubility of nutrients. A higher moisture leveldecreases porosity, changes particle structure, promotesdevelopment of aggregates due to the sticky nature of some
substrates (e.g., AP), decreases diffusion, lowers oxygentransfer, and increases formation of aerial hyphae [8].
The results of the second-order response surface modelfitting in the form of ANOVA are given in Table 3. Datawere best fitted by a second-order polynomial equation[Eq. (1)], as it can be inferred from the good agreement ofexperimental data with those predicted by the model (Fig. 1and 2). In all models, a higher coefficient of determination(i.e., R2 value; 0.825 for FPase and 0.916 for CMCaseproduction) indicated that the models fitted well in theexperimental results, representing about 82.5-91.6%variability in the responses.
The significance of each coefficient was evaluated withthe help of p-values [13]. The analysis of variance and thecorresponding p-values (Table 3) indicated that the linearmodel terms of X1 (moisture) have a significant negativeeffect on cellulases production (FPase and CMCase)
Fig. 3. Response surface graphs illustrating the effects of (A) veratryl alcohol and copper sulfate; (B) veratryl alcohol and lactose; and(C) lactose and copper sulfate on cellulase activity measured as FPase/g fermented substrate.
Fig. 4. Response surface graphs illustrating the effects of (A) moisture and veratryl alcohol; (B) moisture and copper sulfate; and (C)moisture and lactose on cellulase activity measured as CMCase (carboxymethyl cellulase activity)/g fermented substrate.
FUNGAL CELLULASES AND HEMICELLULASES 1318
(p<0.05). This could be explained by the fact that furtherincrease in the initial moisture level showed negative interactiveeffects on cellulase production. The initial moisture plays avital role in the growth and metabolism of microorganisms,which finally affect the yield of any metabolite. Althoughthe solid-state fermentation is carried out in the absence offree water, the substrate must still possess enough moistureto sustain the growth of microorganisms [7, 8]. On the otherhand, X4 (LAC) was found to have significant positiveinteraction on FPase and CMCase production (p<0.05) byA. niger using AP. However, X2 (VA) and X3 (CS) were notfound to have any significant effect (p<0.05) on FPase andCMCase production by A. niger. The initial moisture levelexerted significant negative effects on both FPase and CMCaseproduction. The increase in VA and LAC concentration ledto an increase in FPase and CMCase production with lowerlevels of moisture. However, with increase in moisturecontent, the inducers did not show any increase in cellulaseactivity. It is evident from Table 3 that the interactiveeffects of variables are not significant on cellulaseproduction. All the variables showed positive insignificanteffects (p<0.05) on FPase and CMCase production. It canbe inferred that the variables alone may have positivesignificant effect on the response. The quadratic term of X4
(LAC) showed a negative significant effect on FPaseproduction, and all the other terms showed appositiveinsignificant effects on FPase and CMCase production byA. niger. The interactive effects of moisture, VA, CS, andLAC on cellulase production have not been reportedearlier in the literature.
There are few reports about the successful utilization oflactose as an inducer for cellulases production by filamentousfungi [11, 24, 33]. Taking into consideration the enhancing
effect of lactose on cellulases production, it has been usedas an inducer to evaluate their effect on the cellulasesproduction. The copper ions exert a positive effect onligninolytic enzyme production, particularly laccase bysome basidiomycetes, such as Ceriporiopsis subvermispora
and Phanerochaete chrysosporium [1, 12]. Alvarez et al.[1] isolated and characterized a transcription factor similarto ACE-1 from C. subvermispora (Cs-ACE 1) found to beessential for laccase induction by copper ions. There is apossibility that copper ions can also induce highertranscriptional factors related to other hydrolytic enzymes,such as cellulases produced by other related filamentousfungi [2].
Determination of Total Extracellular Protein
Table 2 represents the total extracellular protein productiontrend during cellulose production optimization throughcentral composite design after a 48 h incubation period. Tocompare cellulase synthesis in SSF through differenttreatments, various enzyme activities and the proteinconcentration of crude enzymes were measured. The totalextracellular protein production correlated well with thecellulase production trend in different treatments duringoptimization. As evident from Table 2, the protein contentpeaked in treatment 17, with initial moisture content 50%(v/w) also having the higher cellulose activity. However, intreatment 18 having initial moisture 90% (v/w), the proteincontent was lower than all other treatments. The lowerprotein concentration might be due to the inability of thefungus to grow in a high moisture content. The total proteincontent observed in different treatments mostly correspondsto the potential of A. niger for higher cellulase enzymeproduction.
Fig. 5. Response surface graphs illustrating the effects of (A) veratryl alcohol and copper sulfate; (B) veratryl alcohol and lactose; and(C) lactose and copper sulfate on cellulase activity measured as CMCase (carboxymethyl cellulase activity)/g fermented substrate.
1319 Dhillon et al.
Fungal Viability
The growth of A. niger (CFUs/gfs) during cellulaseproduction through RSM is given in Table 2. The growthof the fungus is directly related to the formation of anymetabolite. As evident from the Table 2, the fungus grewefficiently in the treatments having initial moisture level of50% (v/w). However, the treatments having higher moisturelevel resulted in poor growth, and higher sporulation wasobserved, which resulted as an adaptation to the environmentalconditions.
Optimum Moisture and pH Studies
Taking into account the parameters optimized by CCD, thecellulose production was examined at upper and lowerlevels of optimized moisture [50% (v/w)] to find the optimummoisture level for higher cellulase production. The lower[45% (v/w)] and upper levels [55% (v/w)] were tested inindependent experiments, taking into account all optimizedparameters. The results demonstrated that maximum FPase(75.15±5.21 IU/gfs) and CMCase (98.60±5.44 IU/gfs) activitiescan be achieved by cultivating A. niger under optimumconditions with 55% (v/w) initial level of moisture andwith inducers VA (2 mM/kg), CS (1.5 mM/kg), and LAC[2% (w/w)], respectively.
Initial pH produced direct effect on the uptake ofmineral nutrients that are present in the fermentationmedium. An independent experiment was conducted toevaluate the effect of varied pH conditions (pH 3.5, 4.0,4.5, 5.0, 5.5, and 6.0) on cellulase production. Slightlyacidic pH value of 4.5 proved to be optimum for obtaininghigher FPase and CMCase production by A. niger. It mightbe due to the fact that fungal cultures require slightlyacidic pH for their growth and enzyme biosynthesis. Themaximum levels of cellulase production by A. niger (FPase79.24±4.22 IU/gfs and CMCase 124.04±7.78 IU/gfs) wereobtained with pH 4.5, initial moisture level of 55% (v/w),along with inducers VA (2 mM/kg), CS (1.5 mM/kg), andLAC [2% (w/w)], respectively. Increasing the pH above 5.0significantly decreased the cellulase production by A. niger.
Cellulolytic, Chitinolytic, and Chitosanolytic Activities
of Crude Enzyme Extract Produced Through Koji
Fermentation in Trays
The method of experimental factorial design and responsesurface analysis was used to determine the optimalconditions to achieve high enzyme activities. The validityof the model was proved by fitting different values of thevariables into the model equation. The experiments atoptimum model values of the variables were scaled-up inthe laboratory using simple and cheap solid-state trayfermentation technology (koji fermentation). As comparedwith the flasks, koji fermentation in trays resulted in highercellulase activities (IU/gfs) as follows: FPase 96.67±4.18(214.82 IU/gds basis) and CMCase 146.50±11.92 (325.44 IU/gds
basis). Similarly, nonspecific enzyme activities were obtained(IU/gfs) as follows: chitinase 70.28±3.34, chitosanase (1)64.20±4.12, chitosanase (2) 62.58±4.42, and chitosanase(3) 60.18±3.82. The best results were obtained with optimumconditions having pH 4.5, initial moisture level of 55% (v/w), along with inducers VA (2 mM/kg), CS (1.5 mM/kg),and LAC [2% (w/w)], respectively. Chitosanase activity ofthe cellulase enzyme extract was 0.66 times the FPaseactivity and 0.44 times CMCase the activity. There was nosignificant difference (p<0.05) found between the treatmentshaving chitosan substrates with different DD’s and MW’s.
Ike et al. [17] obtained dual activity from T. reesei
cellulase enzyme with CMCase activity (34.9 mU/mg) andchitosanase activity (350 mU/mg). Liu and Xia [20] purifiedand characterized the bifunctional enzyme with chitosanaseand CMCase activities from commercial cellulase (SinopharmMedicine Holding Co. Ltd.) from Trichoderma viride. Theauthors observed CMCase activity of 0.36 U/mg andchitosanase activity of 0.40 U/mg, respectively. Similarly,Zhou et al. [38] obtained a cellulase activity of 15 U/mg andchitosanase activity of 0.92 U/mg. da Silva et al. [22] studiedthe optimization of chitosanase production by Trichoderma
koningii sp. grown on wheat bran in solid-state fermentationusing CCD. There was lower chitosanase production of4.84 IU/gds. Owing to the higher production using low-cost substrates, cellulases are widely employed for thehydrolysis of chitosan in order to make low-molecular-weight chitosan and chitooligomers having more specificapplications in biomedicine, food, and agriculture sectors.
Qin et al. [28] studied the antitumor activity of cellulase-treated chitosan. The results indicated that the hydrolysisproducts of cellulase-treated chitosan inhibited the growthof sarcoma 180 tumor cells in mice with maximum inhibitoryrates of up to 50% by different routes of administration.Seo et al. [29] demonstrated that chitooligosaccharidescould activate murine peritoneal macrophages for tumorcell killing in the presence of IFN-γ. The cellulasesproduced by A. niger can be employed for possiblehydrolysis of chitosan and chitin to high-value products.
Recently, studies demonstrated the potential of koji
fermentation for obtaining higher cellulase activities byusing various agro-residues [3, 7]. In the present case, wedemonstrated the utilization of apple pomace supplementedwith rice husk and without any pretreatment for cellulasesproduction. The rice bran contains appreciable amount ofhemicellulose fraction, which is known to induce cellulaseproduction [3, 7]. No expensive media are required, andfurthermore, the use of inexpensive agro-industrial wasteswill have important economical and environmental advantages.
Stability of Chitinase and Chitosanase Activities
Displayed by Cellulases Enzyme Extract
It is evident from the literature that chitinases play asynergistic role in enhancing entomotoxicity of biocontrol
FUNGAL CELLULASES AND HEMICELLULASES 1320
agents, such as bacteria, fungi, and actinomycetes [4, 10,16, 30]. Brar et al. [4] demonstrated that chitinases play asynergistic role in Bacillus thuringiensis spp. kurstaki HD-1 (Bt)-based formulations and enhance its entomotoxicitylevel up to 1.2-fold. However, the biocontrol-agent-basedformulations have a certain shelf-life before their applicabilityin the fields and there is a possibility of inactivation ofenzymes during this shelf-life. The antifungal biocontrolagent Streptomyces lydicus WYEC108 produces highlevels of chitinase enzyme and was capable of damagingcell walls of fungal hyphae and germinating oospores ofPhythium ultimum [37]. The authors also demonstratedthat the S. lydicus produced high levels of chitinases wheninduced with fungal cell wall chitins in the growth medium.Similarly, S. griseus, S. albocinaceus, and S. violaceusniger
XL-2 secrete high levels of chitinases over a range of pHand inhibit the growth of various phytopathogenic fungi[10, 16, 30].
In view of the applicability of cellulases in biocontrolformulations, a stability profile of nonspecific chitinase andchitosanase activities displayed by cellulase enzyme wasdetermined using the 48 h crude enzyme extract (Fig 6).The enzyme extract was amended with glycerol 1% (v/v)and stored at 4±1oC. Samples were drawn at the interval of2 days for a month to determine the chitinase and chitosanaseactivities. The enzyme stability can be further enhanced byamendment with various adjuvants, such as glycerol,sorbitol, and sodium metabisulfite and their combinations.
The potential of A. niger NRRL-567 for rapid andhigher production of cellulases was demonstrated for thefirst time. The cellulases so produced possess multifunctionalchitinase and chitosanase activities. During laboratory
level scale-up, cellulase production in solid-state trayfermentation with optimized parameters showed maximumactivity after a 48 h incubation time. The cellulases showedcomparable chitinase and chitosanase activities to FPcellulase and carboxymethyl cellulase activities and werestable for over one month. Thus, A. niger showed thepotential of cellulases for chitin/chitosan hydrolysis inorder to obtain their higher value depolymerized productsfor wide-range applications, and in the preparation ofvarious biocontrol formulations increasing their efficiency.
Acknowledgments
The authors are sincerely thankful to the Natural Sciencesand Engineering Research Council of Canada (DiscoveryGrant 355254, Canada Research Chair), FQRNT (ENC125216) MAPAQ (No. 809051) and Inde Initiative 2010(Ministère de l'Éducation, du Loisir et du Sport) forfinancial support. The views or opinions expressed in thisarticle are those of the authors.
REFERENCES
1. Alvarez, J. M., P. Canessa, R. A. Mancilla, R. Polanco, P. A.
Santibanez, and R. Vicuna. 2009. Expression of genes encoding
laccase and manganese-dependent peroxidase in the fungus
Ceriporiopsis subvermispora is mediated by an ACE1-like
copper-fist transcription factor. Fungal Genet. Biol. 46: 104-
111.
2. Aro, N., A. Saloheimo, M. Ilmen, and M. Penttila. 2001.
ACEII, a novel transcriptional activator involved in regulation
of cellulase and xylanase genes of Trichoderma reesei. J. Biol.
Chem. 276: 24309-24314.
3. Brijwani, K., H. S. Oberoi, and P. V. Vadlani. 2010. Production
of a cellulolytic enzyme system in mixed-culture solid-state
fermentation of soybean hulls supplemented with wheat bran.
Proc. Biochem. 45: 120-128.
4. Brar, S. K., M. Verma, R. D. Tyagi, J. R. Valero, and R. Y.
Surampalli. 2007. Bacillus thuringiensis fermentation of
wastewater and wastewater sludge:Presence and characterization
of chitinases. Environ. Technol. 29: 161-170.
5. Cen, P. and L. Xia. 1999. Production of cellulase in solid state
fermentation. In T. Scheper (ed.). Recent Progress in
Bioconversion of Lignocellulosics. Advances in Biochemical
Engineering/Biotechnology, 6.5, Springer; Berlin.
6. Dahia, N., R. Tewari, and G. S. Hoondal. 2006. Biotechnological
aspects of chitinolytic enzymes: A review. Appl. Microbiol. 71:
773-782.
7. Dhillon, G. S., H. S. Oberoi, S. Kaur, S. Bansal, and S. K. Brar.
2011. Value-addition of agricultural wastes for augmented
cellulase and xylanase production through solid state tray
fermentation employing mixed-culture of fungi. Indus. Crops
Prod. 34: 1160-1167.
8. Dhillon, G. S., S. K. Brar, M. Verma, and R. D. Tyagi. 2011.
Enhanced solid-state citric acid bioproduction using apple
Fig. 6. Stability profile of enzyme produced by A. niger NRRL-567 using AP in solid-state tray fermentation with optimizedparameters [moisture 55% (v/w), VA 2 mM/kg; CS 1.5 mM/kg;LAC 2% (w/w), pH 4.5, and temperature 30
oC].
Three chitosan substrates having different physicochemical properties were
used: (Substrate 1, Fischer, MW 600-800 kDa, degree of deacetylation
(DD) > 90% and viscosity 200-500 mPa·s; substrate 2, Sigma, medium
MW 190-310 kDa, DD 82% and viscosity 522 cps; and substrate 3,
Sigma, high MW 310-375 kDa, DD 77%, and viscosity 1120 cps).
1321 Dhillon et al.
pomace waste through response surface methodology. J. Appl.
Microbiol. 110: 1045-1055.
9. Dhillon, G. S., S. K. Brar, and J. R. Valero. 2011. Bioproduction
of hydrolytic enzymes using apple pomace waste by Aspergillus
niger: Applications in biocontrol formulations and for
hydrolysis of chitin/chitosan. Bioproc. Biosyst. Eng. DOI:
10.1007/s00449-011-0552-9.
10. El-Sayed, E. S. A., S. M. Ezzat, M. F. Ghaly, M. Mansour, and
M. A. El-Bohey. 2000. Purification and characterization of two
chitinases from Streptomyces albovinaceus S-22. World J.
Microbiol. Biotechnol. 16: 87-89.
11. Fang, X., S. Yano, H. Inoue, and S. Sawayama. 2008. Lactose
enhances cellulase production by filamentous fungus Acremonium
cellulolyticus. J. Biosci. Bioeng. 106: 115-120.
12. Gassara, F., S. K. Brar, R. D. Tyagi, M. Verma, and R. Y.
Surampalli. 2010. Screening of agro-industrial wastes to
produce ligninolytic enzymes by Phanerochaete chrysosporium.
Biochem. Eng. J. 49: 388-394.
13. Ghodkey, S. K., L. Ananthenarayan, and L. Rodrigues. 2009.
Use of response surface methodology to investigate the effects
of milling conditions on damaged starch, dough thickness and
chapatti quality. Food Chem. 112: 1010-1015.
14. Ghose, T. K. 1987. Measurement of cellulase activities. Pure
Appl. Chem. 59: 257-268.
15. Hedges, A. and R. S. Wolfe. 1997. Extracellular enzyme from
Myxobacter AL-1 that exhibits both β-1,4-glucanase and
chitosonase activities. J. Bacteriol. 120: 844-853.
16. Hoster, F., J. E. Schmitz, and R. Danial. 2005. Enrichment of
chitinolytic microorganisms: Isolation and characterization of a
chitinase exhibiting antifungal activity against phytopathogenic
fungi from a novel Streptomyces strain. Appl. Microbiol.
Biotechnol. 66: 434-442.
17. Ike, M., Y. Ko, K. Yokoyama, J. I. Sumitani, T. Kawaguchi, W.
Ogasawara, H. Okada, and Y. Morikawa. 2007. Cellobiohydrolase
I (Cel7A) from Trichoderma reesei has chitosonase activity. J.
Mol. Cat. B Enz. c47: 159-163.
18. Ilmen, M., A. Saloheimo, M. L. Onnela, and M. E. Penttila.
1997. Regulation of cellulase gene expression in the filamentous
fungus Trichoderma reesei. Appl. Environ. Microbiol. 63:
1298-1306.
19. Lin, H., H. Y. Wang, C. H. Xue, and M. Ye. 2002. Preparation
of chitosan oligomers by immobilized papain. Enz. Microbial
Technol. 31: 588-592.
20. Liu, J. and W. Xia. 2006. Purification and characterization of a
bifunctional enzyme with chitosanase and cellulase activity
from commercial cellulase. Biochem. Eng. J. 30: 82-87.
21. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.
1951. Protein measurement with the Folin phenol reagent. J.
Biol. Chem. 193: 265.
22. Luis da Silva, C. A., T. L. Honorato, T. T. Franco, and S.
Rodrigues. 2010. Optimization of chitosanase production by
Trichoderma koningii sp. under solid-state fermentation. Food
Bioproc. Technol. DOI: 10.1007/s11947-010-0479-1.
23. Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for
determination of reducing sugars. Anal. Chem. 31: 426-428.
24. Morikawa, Y., T. Ohashi, O. Mantani, and H. Okada. 1995.
Cellulase induction by lactose in Trichoderma reesei PC-3-7.
Appl. Microbiol. Biotechnol. 44: 106-111.
25. Oberoi, H. S., N. Babbar, S. S. Dhaliwal, S. Kaur, P. V. Vadlani,
V. K. Bhargav, and R. T. Patil. 2010. Enhanced oil recovery by
pre-treatment of mustard seeds using crude enzyme extract
obtained from mixed-culture solid-state fermentation of kinnow
(Citrus reticulata) waste and wheat bran. Food Bioproc.
Technol. DOI 10.1007/s11947-010-0380-y.
26. Ohtakara, A. 1988. Chitosanase from Streptomyces griseus.
Methods Enzymol. 161: 505-510.
27. Pelletier, A. and J. Sygush. 1990. Purification and characterization
of the chitosanase activity from Bacillus megaterium P1. Appl.
Environ. Microbiol. 56: 644-648.
28. Qin, C., B. Zhou, L. Zeng, Z. Zhang, Y. Liu, Y. Du, and L.
Xiao. 2004. The physicochemical properties and antitumor
activity of cellulase-treated chitosan. Food Chem. 84: 107-115.
29. Seo, W. G., H. O. Pae, and H. T. Chung. 2000. Synergistic
cooperation between water soluble chitosan oligomers and
interferon-γ for induction of nitric oxide synthesis and
tumoricidal activity in murine peritoneal macrophages. Cancer
Lett. 159: 189-195.
30. Shekhar, N., D. Bhattacharaya, K. Dishant, and R. K. Gupta.
2006. Biocontrol of wood-rotting fungi using Streptomyces
violaceusniger XL-2. Can. J. Microbiol. 52: 805-808.
31. Shin-Ya, Y., M. Y. Lee, H. Hinode, and T. Kajiuchi. 2001.
Effect of N-acetylation degree on N-acetylated chitosan
hydrolysis with commercially available and modified pectinases.
Carbohydr. Res. 7: 85-88.
32. Sukumaran, R. K., R. R. Singhania, G. M. Mathew, and A.
Pandey. 2009. Cellulase production using biomass feed stock
and its application in lignocellulose saccharification for bio-
ethanol production. Renew. Energy 34: 421-424.
33. Sun, H., X. Ge, Z. Hao, and M. Peng. 2010. Cellulase
production by Trichoderma sp. on apple pomace under solid-
state fermentation. Afr. J. Biotechnol. 9: 163-166.
34. Ueda, M. and M. Arai. 1992. Purification and some properties
of chitinase from Aeromonas sp. No. 10S-24. Biosci. Biotechnol.
Biochem. 56460-56464.
35. Wood, T. M. and K. M. Bhat. 1988. Methods for measuring
cellulase activities. Methods Enzymol. 160: 87-112.
36. Xia, W., P. Liu, and J. Liu. 2008. Advances in chitosan
hydrolysis by non-specific cellulases. Bioresour Technol. 99:
6751-6762.
37. Yuan, W. M. and D. L. Crawford. 1995. Characterization of
Streptomyces lydicus WYEC108 as a potential biocontrol agent
against fungal root and seed rots. Appl. Environ. Microbiol. 61:
3119-3128.
38. Zhou, G., Z. P. He, G. H. Deng, Z. Y. Huang, and X. C. Tan.
2003. Enzyme kinetics of amylase and cellulase on hydrolyzing
chitosan. China Marine Sci. 27: 59-63.