Chapter 6 Figure 6.3.1
2.2 Cellulose hydrolysing enzymes (cellulases)
2.2.4 Purification, characterization and application of cellulase .1 Methods of purification
Shaking speed or agitation is other important culture parameter which is related to the maintenance of homogeneity in fermentation broth, dispersion of dissolved oxygen into smaller bubbles thereby increasing the interfacial area and oxygen mass transfer rate resulting to the enhancement of both substrate utilization and microbial activity (Singh et al. 2000). However, greater agitation speed may attribute to increased shear stress on the cells leading to reduced enzyme production (Purkarthofer et al. 1993). Bacillus amyloliquefaciens UMAS 1002 showed a 2 fold enhancement in cellulase activity under shaking at a rate of 100 rpm (Khan and Hussaini 2006). Trivedi et al. (2011) reported agitation speed of 120 rpm as optimum for cellulase production by Bacillus flexus isolated from green sea weed Ulva lactuca. Optimum shaking speed for B. subtilis AS3 was found to be 121 rpm in shake flask level cellulase production (Deka et al. 2013b). In the present study shaking speed optimization for cellulase production from B. amyloliquefaciens SS35 also resulted in similar observation with a value of 120 rpm (Singh et al. 2014a).
2.2.4 Purification, characterization and application of cellulase
protein mixture is further processed for purification of desired enzyme either by ion- exchange or gel filtration or by both employed stepwise. For purification of cellulase generally DEAE-cellulose (Bajaj et al. 2009, Vijayaraghavan and Vincent 2012), DEAE-sepharose (Trivedi et al. 2011, Sadhu et al. 2013, Deka et al. 2013a, Singh et al. 2014c) have been used as matrix. However, for cellulase purification by hydrophobic interaction chromatography, HiTrapTM Q column has been preferred (Lee et al. 2008, Kim et al. 2009). Gel filtration using sephadex (Vijayaraghavan and Vincent 2012), sephacryl (Sadhu et al. 2013), DEAE-sephadex (Singh et al. 2001, Trivedi et al. 2011) have also been used. Singh et al. (2001) reported purification of cellulase from Bacillus sp. VG1 by ammonium sulphate fractionation (40-80%
saturation) followed by two-step gel filtration chromatography using DEAE Sephadex A-50 and Sephadex G-75. Lee et al. (2008) and Kim et al. (2009) carried out a three step purification employing ammonium sulphate precipitation, HiTrap Q and two cycles of ion exchange chromatography with Mono Q HR column. Deka et al. (2013a) in their study used ammonium sulphate precipitation (0-80% saturation) of cell-free supernatant of B. subtilis AS3, followed by ion-exchange chromatography using DEAE-sepharose.
2.2.4.2 Characterization of cellulase
2.2.4.2.1 Effect of temperature and pH on cellulase activity and stability
Literature available on cellulase from naturally isolated Bacillus spp. indicated that cellulase activity and stability varied in a wide range of temperature (40-75oC) and pH (4.0-10.0), this is considered to be a common characteristic of cellulase from
Bacillus spp. Bajaj et al. (2009) reported that cellulase produced by Bacillus sp. M-9 showed maximal CMCase activity at 60oC and pH 5.0. However, enzyme was stable at 50-70oC for 1 h and retained more than 60% residual CMCase activity at pH 4.0- 6.0 for 1 h. Cellulase from Bacillus sp. DUSELR13 exhibited maximum activity at 75oC and pH 5.0 and it was a thermostable enzyme with 78% residual activity at 70oC for 1 day (Rastogi et al. 2010). On the other hand, cellulase from B. flexus NT, isolated from a sea weed showed maximal activity at 45oC and pH 10.0 and stability studies revealed that the enzyme was stable at 15-20oC and pH 9.0-11.0 with more than 60% residual activity for 30 min (Trivedi et al. 2011). Another report by Lin et al. (2012) has revealed that cellulase from B. thuringiensis had an optimum activity at 40oC and pH 4.0 and at 40-60ºC enzyme retained 50% of its initial activity for 1 h.
CMCase from B. subtilis AS3 showed maximal activity at 45oC and pH 9.2, however, enzyme was stable in a range of temperature 30-50oC and pH 12.0 for 1 h with more than 60% residual activity (Deka et al. 2013a). Cellulase from another strain of B. subtilis SU40 isolated from soil exhibited maximum activity at pH 8.0 and 45oC (Asha and Sakthivel 2014).
2.2.4.2.2 Effect of metal ions on cellulase activity
The effect of most commonly used metal ions Co2+, Ca2+,K+, Na+ and Mn2+
Zn2+, Fe3+ and Hg2+, have been investigated on cellulases of different origins.
Generally, Co2+, Ca2+,K+, Na+ and Mn2+ ions enhance the cellulase activity and Zn2+, Fe3+ and Hg2+ inhibit the activity. However, these trends may vary for the cellulase from different source. For example, CMCase from Bacillus sp. VG1 (Singh et al.
2001) and B. flexus NT(Trivedi et al. 2011) showed the above mentioned trends of effect of metal ions and cellulase from B. subtilis GN156 on the other hand showed the inhibition effect on enzyme activity by Co2+ and Mn2+ ions (Apiraksakorn et al.
2008). In another report by Asha and Sakthivel (2014) cellulase produced by B.
subtilis SU40 exhibited enhancement in activity in presence of Ca2+ and Na+ and the cellulase activity was inhibited by Mn2+, Zn2+ and Hg2+ ions.
2.2.4.2.3 Substrate specificity of cellulase
Many cellulases of bacterial origin are known to have multi-substrate specificity, for example cellulase from B. amyloliquefaciens DL-3 exhibited activity against avicel, CMC, cellobiose, β-glucan and xylan (Lee et al. 2008). However, the enzyme was not able to hydrolyze 4-nitrophenyl β-D-glucopyranoside (pNPG) which is a substrate for detecting β-glucosidase activity. In another study by Kim et al.
(2009) cellulase produced by B. subtilis subsp. subtilis A-53 showed detectable activity against CMC, cellobiose, filter paper and xylan. Cellulase from B. subtilis AS3 showed significant activity towards CMC, lichenan and barley β-D-glucan (Deka et al. 2013a). Cellulase from B. subtilis SU40 interestingly, showed the presence of all the three cellulase activities viz. exoglucanase, endoglucanase and β- glucosidase, indicated by hydrolysis of avicel, CMC and pNPG, respectively (Asha and Saktivel 2014). It also hydrolyzed glucan and xylan significantly.
2.2.4.3 Applications of cellulases
Cellulases are regarded as third most important enzymes for applications in various industries viz. pulp and paper, textile, food and beverages, animal feed, detergent and currently in bioethanol industry (Nigam and Pandey 2009, Kuhad et al.
2011). However, few applications in waste management (Kuhad et al. 2010a), extraction of olive oil and carotenoids (Cinar 2005) have also been reported.
2.2.4.3.1 Paper and pulp industry
Mechanical pulping process in presence of cellulases (bio-mechanical pulping) has been preferred over mechanical pulping alone, which lead to decreased viscosity of processed material (Bhat 2000). Interestingly, several mills used cellulases for removing fine fibrils in dissolved substances for improved drainage (Kuhad et al.
2011).
2.2.4.3.2 Food processing and beverages
Cellulases have been used for extraction and clarification of fruit and vegetable juices as a part of macerating enzyme complex which involves cellulases, xylanases and pectinases (Youn et al. 2004, de Carvalho et al. 2008). This enzyme complex has been used to increase cloud stability and texture of purees prepared from fruits and to decrease its viscosity (Baker and Wicker 1996). Cellulases also play an important role in producing alcoholic beverages viz. beer and wine. Cellulase enzymes are added either during mashing or primary fermentation which results in increased dissolution of glucan, reduced viscosity of wort and thus improved filterability
(Bamforth 2009). The enzymes are also employed to obtain improved skin degradation, color extraction, clarification and improved quality and stability of the wine (Caldini et al. 1994).
2.2.4.3.3 Animal feed
Cellulases are useful in improving nutritive quality of forage diet of ruminants as well as cereal based diet of poultry and pigs and thus health and performance of animals (Cowan 1996, Fontes et al. 2004). Since the forage diet of ruminants is cellulose rich, the cellulases have been used as supplement to improve feed conversion rate when the animal suffers inadequate digestion, for example in the post-weaning period. Cellulases have been used for de-hulling and elimination of anti-nutritional factors present in cereals and grains such as starch rich grain with non-starch polysaccharide, cellulose. Reduced viscosity and thus improved emulsification of high fibre based feeds in poultry and pig has been achieved by using cellulases (Karmakar and Ray 2011, Kuhad et al. 2011).
2.2.4.3.4 Textile and detergent
Cellulases in textile industry have been used in the process of bio-polishing, which involves de-piling, ageing, scouring and bleaching of the fabric (Sreenath et al. 1996). Cellulases are also being used for bio-stoning of denim as an alternative to conventional pumice stone treatment (abrasion). In this process, cellulases dissolve the small fiber ends on the fabric surface, thereby loosening the dye with less damage of the fibers (Galante et al. 1998). Cellulase preparations are added to laundry
detergents to modify the structure of fibrils which in turn results to color brightness, hand feel and dirt removal from cotton garments (Karmakar and Ray 2011).
2.2.4.3.5 Lignocellulosic ethanol
Currently, the use of cellulases in bioethanol production process is the most popular application being investigated. Bioethanol production from lignocellulosic substances involves three major steps, viz. pretreatment of raw biomass to yield cellulose, enzymatic hydrolysis of cellulose to simple sugars and fermentation of sugars to ethanol. Lignocellulosic biomass in the form of agricultural or forest residues such as rice straw (Binod et al. 2010), wheat straw (Talebnia et al. 2010), corn cob (Itelima et al. 2013), sugarcane bagasse (Canilha et al. 2013), hard wood (Lim et al. 2013), saw dust (Shea tree sawdust) (Ayeni et al. 2013) and grasses/weeds, for example reed canary grass (Dien et al. 2006), switch grass (Bals et al. 2010), Kans grass (Kataria and Ghosh 2011), water hyacinth (Satyanagalakshmi et al. 2011), red sage (Lantana camara) (Kuhad et al. 2010b) and Prosopis juliflora (Gupta et al. 2009) are being utilized as potent feedstock for ethanol production.
Since enzymatic hydrolysis of cellulosic substrate is an important step of bioethanol production, the efficiency of the process should be enhanced in terms of sugar yield and saccharification rate. The performance of cellulases can also be improved to reduce the cost of enzyme which will make cellulosic ethanol commercially viable.
The efforts are being carried out by enzyme engineering methods (Kazlauskas and Bornschcucr 2009), high expression (Handelsman 2005), immobilization, coenzyme regeneration, multienzyme system, enzyme coupling with fermentation, asymmetric
biosynthesis and nonaqueous biocatalysis, etc. (Li et al. 2012).