Gopal Das, current Head of the Center, for his support during the completion phase of my dissertation. I would also like to thank my sisters-in-law and brothers-in-law for their best wishes at all stages of my life.
General Introduction
Cellulose degrading enzymes and mechanism of action
- Cellulases
- Mechanism of action of cellulase: Induction and regulation
Other substrates such as cellobiose, lactose and sophorose are also known inducers of cellulase activity (Mandels et al. The generally accepted theory of induction of cellulase gene expression by insoluble and polymeric substrate is that low levels of cellulase activity are constitutively produced by microorganisms that initiate cellulose hydrolysis into soluble sugars (Ilmen et al. 1997; Bhat and Bhat, 1997).
Screening of microorganisms producing cellulase
Cellulase production
Acharya fi Chaudhury (2011) fi Hirasawa fi kkf. 2006) pH 9.0 ol ta’e oomisha seeloleesiif akka mijatu gabaasan. Peeptooniin Baasilas amiilooliikeefaasiins DL-3 keessatti sochii seeloleesiidhaaf yuuriyaa fi amooniyeemii naayitreetii caalaa bu’a qabeessa ture (Jo et al. 2008).
Optimization strategies for cellulase production
Of these, a central composite design was used for cellulase production (Techapun et al. 2002; Singh et al. 2009). A central composite design with 22 complete factors was used to determine the relationship between enzyme production and three medium factors, namely CMC, yeast extract and peptone.
Purification of cellulase
- Precipitation techniques
- Chromatography
At a certain concentration of ammonium sulfate (NH4)2SO4, an insufficient amount of unbound water will be available to hold a given protein species in solution, resulting in precipitation of that protein. Many proteins can also be precipitated by the addition of water-miscible organic solvents, such as ethanol and acetone, which displace water from the protein surfaces, resulting in a decrease in the solvation layer around the protein.
Characterization of cellulase
- Optimum pH and stability of cellulase
- Optimum temperature and thermal stability of cellulase
- Substrate specificity
But the cellulases from Bacillus spp. 2010) reported maximum cellulase activity against CMC of Bacillus sp. Some alkaline cellulases from Bacillus spp. 2008) reported significant activity with avicel and filter paper of Bacillus sp.
Applications of cellulases
- Cellulases in pulp and paper industry
- Cellulases in textile and detergent industries
- Cellulases in beer and wine industry
- Cellulases in animal feed
- Cellulases in the lignocellulose to bioethanol process
Their role is to (i) eliminate anti-nutritional factors present in grains and vegetables, (ii) break down certain grain compounds to improve the nutritional value of feed, (iii) supplement digestive enzymes in animals when these are insufficient, especially in the post-weaning period, (iv) improve feed turnover rate and (v) enable the utilization of cheaper feed components (Fontes et al. 2004). To improve hydrolytic efficiency, it is necessary to optimize cellulase production and develop an efficient cellulase-based catalysis system (Sukumaran et al. 2005).
Bioethanol production by Simultaneous saccharification and fermentation…. 22
- Physical pretreatment
- Physico-chemical pretreatment
- Chemical pretreatment
Orgaanizimoonni sukkaara peentoosii (C5) dammaqsuuf yeroo baay’ee itti fayyadaman Pichia stipitis, Pachysolen tannophilus fi Candida shehatae dha (Olofsson et al. 2008). Haa ta’u malee, malli kun sababa itti fayyadamaa fi deebisanii argachuu ammooniyaatiin baasii guddaa kan gaafatudha (Mosier et al. 2005).
Objectives of the present study
2006) Production of cellulase by Trichoderma reesei WX pretreatments to improve the digestibility of lignocellulosic biomass. Pilot-scale production of cellulase using Trichoderma reesei RUT-C-30 in fed-batch mode. 2008) Purification and characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 using rice husks.
Introduction
The major barrier to wider application of fungal enzymes is their slow growth and sensitivity to high temperatures and pH (Chowdary et al. 2001). These properties led to the search for fast-growing bacteria capable of synthesizing active cellulases under harsh conditions (Maki et al. 2009). The existence of enormous spontaneous diversity often leads to ambiguity at the phylum and even species level (Rondon et al. 2000).
Materials and methods
- Chemicals, Reagents and substrates
- Screening and isolation of cellulolytic microorganisms
- Cellulase activity determination
- Calculation of enzyme activity
- Estimation of protein concentration
- Morphological and biochemical characterization of the selected isolate AS3 63
- Optimization of the inoculum concentration
- Effect of different cellulosic substrates on cellulase production…
- Optimization of the CMC concentration
- Effect of different nitrogen sources on cellulase production
Protein concentration was determined by the Lowry method using bovine serum albumin (BSA) as a standard (Lowry et al. 1951) as described in section 2.2.3.2. The cell-free supernatant was analyzed for enzyme activity (U/mL) and protein concentration (mg/mL) as described in section 2.2.3 from which the specific activity (U/mg) was calculated. The cell-free supernatant was analyzed for enzyme activity (U/mL) and protein concentration (mg/mL) as described in section 2.2.3 from which the specific activity (U/mg) was calculated.
Results and Discussion
- Screening and isolation of cellulolytic microorganisms
- Selection of isolate based on cellulase activity
- Morphological and biochemical characterization of isolate AS3
- Optimization of incubation time for cellulase production
- Optimization of the inoculum concentration
- Effect of different cellulosic substrates on cellulase production…
- Optimization of CMC concentration for growth of Bacillus subtilis AS3 74
Cellulase production was significantly affected when cellulosic substrates with different linkages were used at 1% as a substitute for CMC. Similar reports are available where 2% CMC was optimal for maximum cellulase production (Kim et al. Similar results were reported earlier where organic nitrogen served better than inorganic nitrogen for cellulase production (Singh et al.
Conclusions
The influence of culture conditions on mycelium structure and cellulase production by Trichoderma reesei RUT C-30. Alkaline cellulases for detergents: production by alkalophilic strains of Bacillus and some properties of the crude enzymes. A new reagent for the determination of sugars. 2009) Degradation of wood and enzyme production by Ceriporiopsis subvermispora.
Optimization of medium composition for enhanced cellulase
Introduction
Response surface methodology (RSM) is a collection of mathematical and statistical techniques based on fitting a polynomial equation to the experimental data, which describes the behavior of a data set with the aim of making statistical predictions (Box and Hunter, 1975). It can be used effectively to study the effects of factors and to determine optimum levels of parameters for desired responses. The effects of different carbon sources such as lichen and barley β-glucan were also tested as alternatives to carboxymethyl cellulose (CMC) in optimized medium.
Materials and Methods
- Microorganism and reagents
- Sterilization and aseptic techniques
- Maintenance and sub-culturing
- Inoculum preparation and production of cellulose
- Assay of enzyme activity
- Optimization procedure and experimental design
- Screening of the most significant medium components by Plackett-
- Central composite design (CCD) and statistical analysis
- Experimental validation of the optimized conditions
- Effect of different cellulosic substrates on cellulase production
A total of 20 experiments were performed in duplicate and the average of the cellulase activity was taken as the response (Table 3.3.1). Where xi is the dimensionless value of an independent variable, Xi is the real value of an independent variable, X0 is the value of Xi at the midpoint, and ∆Xi is the step change. Where Y is the predicted response, k is the number of factor variables, Xi and Xj are independent variables, β0 is the model constant, βi is the linear coefficient, βii is the quadratic coefficient, and βij is the interaction coefficient.
Results and Discussion
- Screening of the significantly influencing medium components by Plackett-
- Optimization of medium components by CCD
- Experimental validation of the optimized medium composition in flask 103
From Table 3.3.5 it is observed that the regression coefficients of linear and quadratic terms for all the factors in the model were found to be highly significant (P < 0.06), except that the quadratic coefficient of CMC indicated insignificance on the responses (P > 0.9). The model predicted a maximum cellulase activity of 0.49 U/mL appearing at: CMC (18 g/L), peptone (8 g/L) and yeast extract (4.8 g/L) by setting the other components at their median levels keep. Three-dimensional response surface plot for alkaline cellulase production showing the interactive effects of medium components in g/L: (A) Yeast extract and CMC median level: peptone; 5g/L (B) Yeast extract and peptone median level: CMC, 10 g/L;.
Conclusions
A photometric adaptation of the Somogyi method for the determination of glucose. 1998) Optimization of a culture medium for the production of xylanase by Bacillus sp. 1946). 2009) Nutrient optimization for cellulase production using basal medium palm oil mill effluent. 2010) Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains. 2004) Isolation and characterization of a cellulolytic Geobacillus thermoleovorans T4 strain from sugar refinery wastewater. 2005) Optimization of α-amylase production by Bacillus sp. 2009) Enhanced production of endoglucanase enzyme by Aspergillus terreus; application of Plackett-Burman for optimization of process parameters.
Enhanced production of alkaline cellulase from Bacillus subtilis by
Introduction
Statistical design techniques have been successfully used in many studies, for example cellulase production by Trichoderma reesei (Alam et al. 2008) and Bacillus subtilis AS3 (Deka et al. 2011), xylanase production by Bacillus pumilus (Nagar et al. 2010) . In this study, physical process parameters such as initial pH, temperature, and culture stirring speed were optimized by a central composite design technique using multiple response analysis to enhance alkaline cellulase activity from Bacillus subtilis AS3. The optimal levels of the physical process parameters predicted by the model were verified both in the flask and in the laboratory bioreactor.
Materials and Methods
- Microorganism and reagents
- Inoculum preparation and production of cellulase
- Assay of enzyme activity
- Cell growth measurement
- Optimization procedure and experimental design
- Optimization of culture conditions using Response surface method
- Multiple response optimizations
- Validation of the experimental model
Cell growth was determined by measuring absorbance at an optical density of 600 nm using a UV-visible spectrophotometer (Perkin Elmer, model Lambda-45) and absorbance values were expressed as cell dry weight using an optical density calibration curve ( OD600) versus dry cell weight. cell weight (g/l) of the sample. Xi and These values are combined to determine the composite or overall desirability of the multi-response system.
Results and Discussion
- Optimization of culture conditions using RSM
- Experimental validation of the model in flask and bioreactor
And to illustrate the accuracy of the models in predicting the responses, the normal probability plot of the residuals for cellulase activity and cell growth is also shown in Fig. And in the accuracy of the models in predicting the reactions is the normal probability plot of the residues for cellulase activity and cell growth as well. Temperature is also one of the most important parameters affecting enzyme activity and is essential for a fermentation process (Rastogi et al. 2010).
Conclusions
2008) Statistical optimization of process conditions for cellulase production by liquid bioconversion of domestic sewage sludge. 2008) Production of bacterial endoglucanase from pretreated oil palm fruit bunch by Bacillus pumilus EB3. 2006) Effect of sulfur-containing compounds on Bacillus cellulosome-associated CMCase and Avicelase activities. 2008) Effect of culture pH and agitation rate on growth and xylanase production of Aspergillus oryzae in spent sulfite liquor. Cellulase production by Aspergillus niger MS82: effect of temperature and pH. 2005) Optimization of α-amylase production by Bacillus sp.
Purification and characterization of an alkaline cellulase from
Introduction
After the purification process, the homogeneity of the enzyme was confirmed by either polyacrylamide gel electrophoresis or iso-electric focusing (IEF) before further characterization of the enzyme. Commercialization of cellulases depends on their stability during isolation, purification and storage as well as their robustness to solvents and surfactants (Annamalai et al. 2011; Trivedi et al. 2011). Therefore, the study of kinetics and catalytic behavior of enzyme purified from any new strain is crucial (Wang et al. 2009).
Materials and Methods
- Microorganism and reagents
- Assay of enzyme activity
- Estimation of protein concentration
- Inoculum preparation and production of cellulase
- Purification
- Ammonium sulfate precipitation
- Ion exchange chromatography
- Analysis of purification by SDS-PAGE
- Effect of temperature on enzyme activity and stability
- Effect of pH on enzyme activity and stability
- Substrate specificity of the enzyme
- Kinetic parameters
The total protein content of the cell-free extract was estimated by the method of Lowry et al. The optimal pH of purified cellulase was determined by incubating 100 µL of a reaction mixture containing 20 µL of purified enzyme (1.16 U/mg, 0.5 mg/mL) and 80 µL of 2% CMC in the presence of appropriate buffers at 45 °C. C 10 min. The substrate specificity of the enzyme was determined by incubating 20 µL of the purified enzyme (1.16 U/mg, 0.5 mg/mL) with 80 µL of 2% (w/v) cellulose substrates: CMC, hydroxyethyl cellulose, lichenan, laminarin, avicel, steam-exploded bagasse and barley β-glucan in 50 mM glycine-NaOH buffer (pH 9.2) at 45 °C for 10 min.
Results and Discussion
- Purification of cellulase
- SDS-PAGE analysis of fractions of ion exchange chromatography
- Molecular size characterization of cellulase by zymogram analysis
- Effect of temperature on activity and stability of the purified cellulase… 153
- Substrate specificity of the enzyme
- Kinetic characterization of cellulase
SDS-PAGE analysis of the column purified fractions showed the presence of multiple protein bands (Fig. Plot showing Rf versus log Mw to determine molecular weight of the purified cellulase from Bacillus subtilis (AS3). Relative activity is expressed as a percentage of the maximum enzyme activity under standard assay conditions.
Conclusions
Size and charge isomer separation and estimation of protein molecular weights by disk gel electrophoresis. 2004) Purification and characterization of a cellulase (CMCase) from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. Alkaline cellulases for laundry detergents: production by alkalophilic strains of Bacillus and some properties of crude enzymes. 2004) Purification and characterization of alkaline cellulase produced by a new isolate of Bacillus sphaericus JSI. 2009) Purification and characterization of a novel halostable cellulase from Salinivibrio sp. 2010) Purification and characterization of a cellulase from Bacillus subtilis YJ1.
Introduction
Clostridium thermocellum contains genes encoding exocellular multienzyme complexes called cellulosomes that exhibit endoglucanase and exoglucanase activity (Taylor et al. 2005). Glycoside hydrolase (GH5) family 5 gene from Clostridum thermocellum belongs to a set of enzymes with varying substrate specificity with high cellulase activity (Bharali et al. 2005). Bacillus subtilis (AS3) produces a thermostable cellulase system which remains active under varying pH conditions and broad substrate specificity (Deka et al. 2011).
Materials and Methods
- Reagents and substrates
- Microorganisms and culturing conditions
- Production of Recombinant cellulase (GH5)
- Pretreatment of substrate
- Steam explosion
- Ammonia fibre explosion (AFEX)
- Phosphoric acid-acetone
- Simultaneous saccharification and fermentation using 1% (w/v) thatch grass
- Simultaneous saccharification and fermentation using AFEX pretreated 5%
- Simultaneous saccharification and fermentation using AFEX pretreated 5%
- Analytical methods
- Assay of enzyme activity
- Estimation of protein concentration
- Ethanol estimation
- Structural carbohydrates estimation
During the third wash, pH was adjusted to 5-6 using NaOH (Li et al. 2009). The pretreated substrate was then used for SSF studies. Similar enzymatic and fermentative combination was applied to 1% (w/v) acid-acetone and AFEX pretreated thatch grass. The same enzyme and fermentative microbe were included in SSF using 1% (w/v) acid-acetone and AFEX pretreated thatch.
Results and Discussion
- Structural carbohydrates determination
- Pretreatment of substrate (Thatch grass)
- SSF involving B. subtilis cellulase along with S. cerevisiae and C. shehatae
At the same time, phosphoric acid-acetone pretreatment gave an ethanol titer of 1.56 g/l from reducing sugar concentration of 1.6 g/l. An ethanol titer of 1.4 g/l from reducing sugar concentration of 1.43 g/l was obtained from phosphoric acid-acetone pretreated straw. The phosphoric acid-acetone pretreatment gave an ethanol titer of 1.66 g/l from reducing sugar concentration of 1.68 g/l (Table 6.3.2).
Conclusions
