title: Site-directed mutagenesis of α-L-rhamnosidase from Alternaria sp. L1 to enhance synthesis yield of reverse hydrolysis based on rational design.
abstract: The α-L-rhamnosidase catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides and is widely used due to its applications in a variety of industrial processes. Our previous work reported that a wild-type α-L-rhamnosidase (RhaL1) from Alternaria sp. L1 could synthesize rhamnose-containing chemicals (RCCs) though reverse hydrolysis reaction with inexpensive rhamnose as glycosyl donor. To enhance the yield of reverse hydrolysis reaction and to determine the amino acid residues essential for the catalytic activity of RhaL1, site-directed mutagenesis of 11 residues was performed in this study. Through rationally designed mutations, the critical amino acid residues which may form direct or solvent-mediated hydrogen bonds with donor rhamnose (Asp252, Asp257, Asp264, Glu530, Arg548, His553, and Trp555) and may form the hydrophobic pocket in stabilizing donor (Trp261, Tyr302, Tyr316, and Trp369) in active-site of RhaL1 were analyzed, and three positive mutants (W261Y, Y302F, and Y316F) with improved product yield stood out. From the three positive variants, mutant W261Y accelerated the reverse hydrolysis with a prominent increase (43.7 %) in relative yield compared to the wild-type enzyme. Based on the 3D structural modeling, we supposed that the improved yield of mutant W261Y is due to the adjustment of the spatial position of the putative catalytic acid residue Asp257. Mutant W261Y also exhibited a shift in the pH-activity profile in hydrolysis reaction, indicating that introducing of a polar residue in the active site cavity may affect the catalysis behavior of the enzyme.
authors.affiliation: State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan, 250100, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.affiliation: State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan, 250100, People's Republic of China.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Liu
authors.affiliation: Qilu Pharmaceutical Co., Ltd., Jinan, 250100, People's Republic of China.
authors.forename: Zhenhao
authors.initials: Z
authors.lastname: Yin
authors.affiliation: Academy of State Administration of Grain, Beijing, 100037, People's Republic of China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Liu
authors.affiliation: State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan, 250100, People's Republic of China.
authors.forename: Lili
authors.initials: L
authors.lastname: Lu
authors.affiliation: State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan, 250100, People's Republic of China. minxiao@sdu.edu.cn.
authors.forename: Min
authors.initials: M
authors.lastname: Xiao
mesh_terms: D000528:Alternaria / Q000201:enzymology*; D020134:Catalytic Domain; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D012210:Rhamnose / Q000378:metabolism
chemical_list: D006027:Glycosides; D050505:Mutant Proteins; D011134:Polysaccharides; D006026:Glycoside Hydrolases; C022525:alpha-L-rhamnosidase; D012210:Rhamnose
keywords: 3D structural modeling; Reverse hydrolysis reaction; Rhamnose-containing chemicals; Rhamnosidase; Site-directed mutagenesis