Abstract：As the major structural polysaccharide of plant cell walls, cellulose is the most abundant organic material on earth. Cellulose biodegradation offers the potential to produce fuels and other chemicals from renewable substrates. In cellulase biodegradation system, endogluconase attacks cellulose at random places, to break the long-chain polyer of glucose into short chains, exoglucanase attacks the ends of the cellulose, to release cellobiose, and β-glucosidase cuts cellobiose and cello-oligosaccharide into glucose. Endoglucanases are classified by the Carbohydrate-active Enzyme data into 13 glycosyl hydrolase families (5, 6, 7, 8, 9, 12, 44, 45, 48, 51, 74, 124 and 131) based on protein sequence similarity and catalytic domain structure. Glycosyl hydrolase family 5 (GH5) is one of the largest GH families, historically known as “cellulase family A” as it was the first cellulase family described. GH5 members are commonly found to be encoded as parts of multi-modular polypeptide chains mainly containing a catalytic domain and a cellulose-binding domain and play an important role in the cellulase biodegradation enzyme system. In this study, an endoglucanase (EGII) was isolated from thermophilic fungus Scytalidium thermophilum. Based on sequence alignment analysis, two catalytic residues of EGⅡ were identified by homology to the other GH5 structures as Glu 199 (acid-base) and Glu 306 (nucleophile), and 6 residues conserved among the glycoside hydrolase family 5 were also found. The 6 residues include Arg 115, His 159, Asn 198, His 264, Yyr 266, Trp 399. The His159 of the EGⅡ is highly conserved in the family 5 of glycoside hydrolases, but its function is unclear so far. To determine His159 function of catalysis, we cloned EGⅡ gene from S. thermophilum and constructed pPIC9K/egII recombinant plasmid and then mutated the His159 into Ala, Asp, Phe, Trp, Arg and Tyr by site-directed mutation, respectively. The mutant enzymes H159A, H159D, H159F, H159R, H159W, H159Y and wild-type enzyme WT (EGⅡ) were expressed in Pichia pastoris and purified using nickel affinity chromatography and characterized. Activity measurements indicated that the specific activity of all mutant enzymes was lower than that of WT. The specific activity of WT, H159A, H159D, H159F, H159R, H159W, H159Y is (11.02±0.41), (6.42±0.10), (2.09±0.07), (0.32±0.02), (0.66±0.01), (1.89±0.13), (0.19±0.02) U/mg, respectively. Further kinetic measurements indicated that Km values of all the mutant enzymes were higher than that of WT, indicating that the affinity of the mutant enzymes to the substrate is reduced. Compared with WT, kcat values of H159D, H159F, H159R, H159W and H159Y decreased but kcat value of H159A was significantly increased, indicating that His159 is involved in the binding of enzymes to substrates. The optimum reaction temperature was 70 ℃ for WT, 60 ℃ for H159A, H159D and H159F, and 55 ℃ for H159R, H159W and H159Y. After incubation at 60 ℃ for 10 minutes, H159A still retained more than 80% activity, H159D and H159W had a significant decrease, H159F, H159R and H159Y were substantially inactivated, indicating that His159 is also involved in EGⅡ thermostability. Our data provide insight into understanding of the function of His159 in the family 5 of glycoside hydrolases.
