Dhillon, Gurpreet Singh
(INRS-ETE, Universite du Quebec)
,
Brar, Satinder Kaur
(INRS-ETE, Universite du Quebec)
,
Kaur, Surinder
(INRS-ETE, Universite du Quebec)
,
Valero, Jose R.
(INRS-ETE, Universite du Quebec)
,
Verma, Mausam
(Institut de recherche et de developpement en agroenvironnement inc. (IRDA))
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 optimiz...
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 $79.24{\pm}4.22$ IU/gram fermented substrate (gfs) and CMCase $124.04{\pm}7.78$ 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 $96.67{\pm}4.18$ IU/gfs and CMCase $146.50{\pm}11.92$ 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 $70.28{\pm}3.34$ IU/gfs and $60.18{\pm}3.82$ to $64.20{\pm}4.12$ 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.
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 $79.24{\pm}4.22$ IU/gram fermented substrate (gfs) and CMCase $124.04{\pm}7.78$ 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 $96.67{\pm}4.18$ IU/gfs and CMCase $146.50{\pm}11.92$ 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 $70.28{\pm}3.34$ IU/gfs and $60.18{\pm}3.82$ to $64.20{\pm}4.12$ 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.
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제안 방법
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.
The enzyme extract was amended with glycerol 1% (v/v) and stored at 4±1℃. Samples were drawn at the interval of 2 days for a month to determine the chitinase and chitosanase activities. The enzyme stability can be further enhanced by amendment with various adjuvants, such as glycerol, sorbitol, and sodium metabisulfite and their combinations.
The validity of the model was proved by fitting different values of the variables into the model equation. The experiments at optimum model values of the variables were scaled-up in the laboratory using simple and cheap solid-state tray fermentation technology (koji fermentation). As compared with the flasks, koji fermentation in trays resulted in higher cellulase activities (IU/gfs) as follows: FPase 96.
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.
대상 데이터
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.
데이터처리
Data means and standard deviation were analyzed with individual Student’s t-tests to distinguish differences among treatments.
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.
이론/모형
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.
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.
성능/효과
1 and 2). In all models, a higher coefficient of determination (i.e., R2 value; 0.825 for FPase and 0.916 for CMCase production) indicated that the models fitted well in the experimental results, representing about 82.5-91.6% variability in the responses.
Statistical analysis was performed with the data having higher cellulase production at 48 h of incubation period, as no significant increase (p<0.05) in cellulase production was observed after 48 h of incubation time.
82. The best results were obtained with optimum conditions 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 of the cellulase enzyme extract was 0.
The maximum levels of cellulase production by A. niger (FPase 79.24±4.22 IU/gfs and CMCase 124.04±7.78 IU/gfs) were obtained with 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.
The results demonstrated that maximum FPase (75.15±5.21 IU/gfs) and CMCase (98.60±5.44 IU/gfs) activities can be achieved by cultivating A. niger under optimum conditions with 55% (v/w) initial level of moisture and with inducers VA (2 mM/kg), CS (1.5 mM/kg), and LAC [2% (w/w)], respectively.
[28] studied the antitumor activity of cellulasetreated chitosan. The results indicated that the hydrolysis products of cellulase-treated chitosan inhibited the growth of sarcoma 180 tumor cells in mice with maximum inhibitory rates of up to 50% by different routes of administration. Seo et al.
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