Comparative analysis of two catalytically distinct endoglucanases from Aspergillus nidulans

Abstract

This study reports purification and characterization of two catalytically distinct endoglucanases (EGI and EGII) from a thermotolerant fungus Aspergillus nidulans. The endoglucanases (EGI and EGII) exhibited molecular masses of 56 and 31 kDa and pIs of 3.6 and 3.8, respectively. EGI was putatively classified as GH7 family member catalyzed carboxymethyl cellulose, xyloglucan, barley β-glucan as well as pNP-β-D-lactopyranoside and pNP-cellobioside, and was optimally active at 50°C and pH 4.0. Whereas, EGII lacking CBD preferentially recognized barley β-glucan when compared substrate CMC, xyloglucan and lichenan and was putatively classified as GH12 member. Interestingly, EGII was characterized to be thermoacidophilic exhibiting 96% its activity at pH 2.0 and at 60 °C. Hydrolysis of barley β-glucan and CMC by EGI and EGII liberated cellobiose as a major product. HPLC analysis showed that barley β-glucan hydrolysate obtained by action of EGI showed high levels of glucose in addition to cellobiose indicating towards an exo type action of this enzyme. Key words:Thermoacidophilic endoglucanases, GH7, GH12, barley β-glucan 1.INTRODUCTIONCellulose has a great potential as a renewable energy source and has gained interest of biotechnology community on their exploration for biofuel production and other value added products. Cellulose is a linear homo-polymer of D-glucose monomers linked together through β-1-4 glycosidic bonds and mainly forms the structural cell wall component in both the lower and higher plants [1]. The biodegradation of complex polymeric structure of cellulose is primarily attributed to multi-component enzyme system that works in a synergistic manner. These enzymes are comprises of endoglucanase (EC 3.2.1.74), which attack cellulose in amorphous zone and release oligomers, cellobiohydrolases (CBHI & CBHII) (EC 3.2.1.91), that liberate cellobiose from reducing and non-reducing ends, and β-glucosidase (EC 3.2.1.21), which hydrolyze cellobiose to glucose [2]. Endoglucanases are widely classified on the basis of different characteristics into families GH 5,6,7,8,9,12, 45 & 74 [3]. Due to intensive research on the structural, catalytic and functional roles, both bacterial and fungal cellulases have led their use in various fields [4]; [5]. In addition to their potential use in the biofuel industry, for degradation of agro-residual waste into simple sugars which can be further fermented, cellulases are widely employed in increasing the yield of fruit juices, beer filtration, paper and pulp industry as well as improving animal feed stock [6]; [7]. With increasing demand for endoglucanases, . * Corresponding Author E-mail: baljit_sohal@rediffmail.comidentification of new EGs, especially thermoacidophilic, with good properties for improved performance is highly desirable [8]. Different endoglucanases from the same family show difference in their substrate specificity as well as different optimum pH and temperature and therefore needs to be characterized [9]. In the present study, we have reported the comparative...

Hydrolysis Kinetics of Oil Palm Empty Fruit Bunch in Ionic Liquids and Cellulase Integrated System

“…The value of k cat was between 5.6 and 11.5 min -1, and k cat /K M was between 42 to 400. Reports have also revealed that endoglucanases vary in their affinity towards polysaccharides as supported by the observed values of K M (Kaur, Oberoi, & Chadha, 2015). Glucose and [EMIM]OAc acted as uncompetitive inhibitors for β-glucosidase (BG), which in this context indicated the probability of IL binding to the E-S complex.…”