title: Characterization of a novel thermostable chitin-binding domain and its application in immobilization of a multifunctional hemicellulase.
abstract: A novel thermostable chitin-binding domain (Tt-ChBD) of chitinase A1 from Thermoanaerobacterium thermosaccharolyticum DSM571 was cloned, characterized, and compared for its binding activity with another mesophilic chitin-binding domain (Bc-ChBD). Recombinant protein with Tt-ChBD exhibits stronger affinity to chitin than those with Bc-ChBD at temperatures from 65 °C to at least 75 °C, but not to other polysaccharides including xylan, chitosan, cellulose, and agarose. For repeated production of xylose from arabinoxylan-containing feedstocks, a best-characterized trifunctional chimeric enzyme Xar-L1-Xyn (XX) constructed in our previous work was attempted to be immobilized on chitin efficiently by genetically fusing Tt-ChBD to the N-terminal region of XX (named CXX) and the C-terminal region of XX (named XXC), respectively. The fusing position of Tt-CBD affected the affinity-binding activity to chitin. Recombinant XX, XXC, and CXX were purified to homogeneity and characterized. According to the xylosidase activities, the optimum temperature and pH profiles of the CXX and XXC both in free and immobilized form were the same as those of XX. However, the thermal and pH stabilities of the immobilized XXC and CXX were both greatly improved in the range from 70 to 90 °C and pH 4.2-8.2. The immobilized multifunctional hemicellulase exhibited high stability to producing xylose for at least 19 or 30 times in continuous operation with the achievement of 60% or 80% conversion yield at temperatures up to 65 °C. These results indicate the usefulness of Tt-ChBD as an affinity tag for the simultaneous purification and immobilization of the enzyme on chitin and the great potential applications for thermophilic enzyme immobilization at higher temperatures.
authors.affiliation: Department of Food Science and Nutrition, GinLing College, Nanjing Normal University, Nanjing, People's Republic of China 210097.
authors.forename: Qilei
authors.initials: Q
authors.lastname: Wang
authors.forename: Yemin
authors.initials: Y
authors.lastname: Xue
authors.forename: Xixi
authors.initials: X
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002686:Chitin / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D064251:Multifunctional Enzymes / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046950:Thermoanaerobacterium / Q000201:enzymology; D014994:Xylose / Q000096:biosynthesis
chemical_list: D004800:Enzymes, Immobilized; D064251:Multifunctional Enzymes; D011993:Recombinant Fusion Proteins; D002686:Chitin; D014994:Xylose; D006026:Glycoside Hydrolases; C023305:hemicellulase