Characterization of a Cellobiohydrolase (MoCel6A) Produced by Magnaporthe oryzae

Three GH-6 family cellobiohydrolases are expected in the genome of Magnaporthe grisea based on the complete genome sequence. Here, we demonstrate the properties, kinetics, and substrate specificities of a Magnaporthe oryzae GH-6 family cellobiohydrolase (MoCel6A). In addition, the effect of cellobiose on MoCel6A activity was also investigated. MoCel6A contiguously fused to a histidine tag was overexpressed in M. oryzae and purified by affinity chromatography. MoCel6A showed higher hydrolytic activities on phosphoric acid-swollen cellulose (PSC), β-glucan, and cellooligosaccharide derivatives than on cellulose, of which the best substrates were cellooligosaccharides. A tandemly aligned cellulose binding domain (CBD) at the N terminus caused increased activity on cellulose and PSC, whereas deletion of the CBD (catalytic domain only) showed decreased activity on cellulose. MoCel6A hydrolysis of cellooligosaccharides and sulforhodamine-conjugated cellooligosaccharides was not inhibited by exogenously adding cellobiose up to 438 mM, which, rather, enhanced activity, whereas a GH-7 family cellobiohydrolase from M. oryzae (MoCel7A) was severely inhibited by more than 29 mM cellobiose. Furthermore, we assessed the effects of cellobiose on hydrolytic activities using MoCel6A and Trichoderma reesei cellobiohydrolase (TrCel6A), which were prepared in Aspergillus oryzae. MoCel6A showed increased hydrolysis of cellopentaose used as a substrate in the presence of 292 mM cellobiose at pH 4.5 and pH 6.0, and enhanced activity disappeared at pH 9.0. In contrast, TrCel6A exhibited slightly increased hydrolysis at pH 4.5, and hydrolysis was severely inhibited at pH 9.0. These results suggest that enhancement or inhibition of hydrolytic activities by cellobiose is dependent on the reaction mixture pH.Cellulose, composed of β-1,4-linked glucosyl units, is the most abundant naturally produced biopolymer on earth and can be utilized as a sustainable and renewable energy resource in place of fossil fuel. Establishing conditions for the efficient degradation of cellulose will contribute to the enhanced use of bioethanol, a biobased alternative to gasoline, which will increase biomass recycling and reduce carbon dioxide emissions (21, 30). Hence, efficient degradation of cellulose is an issue of great importance today.Bacteria and fungi produce cellulases that catalyze the hydrolysis of β-1,4-glycosidic bonds and are involved in the degradation of cellulose. Cellulases are divided into three major types according to their substrate specificities and the mode of hydrolysis: endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21). The most efficient hydrolysis of cellulose is thought to result from the combined synergistic actions of cellulases, whereby the enzymatic activity of an enzyme mixture is substantially higher than the sum of the activities of the individual enzymes. Several types of synergy have been described as the cooperative actions of endo- and exo-acting enzymes (20, 25, 32, 33, 45). Such cellulose-degrading enzymes are routinely used in the manufacture of beverages and industrial products, e.g., beer and wine, animal feed, paper, textiles, laundry detergents, and food ingredients (5). Hence, reducing cellulase manufacturing costs by increasing the productivity of cellulases with high specific activities through biotechnological modification is a desired research goal.Fungal cellobiohydrolases belong to glycosyl hydrolase families 6 and 7 (GH-6 and -7) and act most efficiently on highly ordered crystalline cellulose, hydrolyzing from either the reducing or the nonreducing terminus to liberate predominantly cellobiose (C2) with a minor amount of cellotriose (C3) (6, 39, 40). Also, Trichoderma reesei Cel6A can hydrolyze 1,3-1,4-β-glucan (1, 18), but it is unclear whether in vivo it is hydrolysis of 1,3-1,4-β-glucan that occurs mainly or hydrolysis of cellulose derivatives. The resulting accumulation of cellobiose inhibits the activity of cellobiohydrolase (13, 15, 28, 31, 36, 37, 44). Some microorganisms possess cellulosomes, multienzyme complexes that contribute to the efficient degradation of cellulose. Cellobiohydrolase is a documented component of cellulosomes in Clostridium thermocellum (2, 31).The three-dimensional (3D) structures of two GH-6 family members have been elucidated, including the cellobiohydrolase of T. reesei and that of Humicola insolens in complex with glucose, cellooligosaccharide, and a nonhydrolyzable substrate analogue (35, 41-43). The proposed structures have identified the significant amino acids associated with the catalytic core domain, where the catalytic site is buried inside a tunnel-shaped cavity and an enzyme-cellooligosaccharide hydrogen bond network. The structure suggests that the mode of action proceeds in a processive manner as cellobiohydrolase progresses along the cellulose chain (7, 19, 34, 40).The ascomycete fungus Magnaporthe grisea is the pathogen that causes rice blast, the most devastating fungal disease of rice. Since the complete genome sequence of M. grisea has been published (10), mining the database for candidate genes involved in pathogen-plant interactions, cell wall degradation, etc., is quite feasible. The cell wall-degrading enzymes of the genus Magnaporthe that are involved in the infection process have been of particular interest (22-24). Based on the complete genome sequence, M. grisea has three putative GH-6 family cellobiohydrolases and four GH-7 family cellobiohydrolases. Using primers designed from the database of M. grisea cellobiohydrolases, we cloned putative GH-6 and GH-7 family cellobiohydrolases, designated MoCel6A and MoCel7A, respectively, from Magnaporthe oryzae by PCR. The cloned MoCel6A and MoCel7A from M. oryzae were completely identical to those of M. grisea. In this paper, we demonstrate the properties of MoCel6A prepared by homologous overexpression in M. oryzae and examine the effects of cellobiose on the hydrolytic activity of MoCel6A. Furthermore, the effects of cellobiose on the activities of both MoCel6A and a T. reesei GH family 6 cellobiohydrolase (TrCel6A, formerly referred to as CBH II), which were overexpressed in Aspergillus oryzae, were also examined.