title: Controlled Aggregation and Increased Stability of β-Glucuronidase by Cellulose Binding Domain Fusion.
abstract: Cellulose-binding domains (CBDs) are protein domains with cellulose-binding activity, and some act as leaders in the localization of cellulosomal scaffoldin proteins to the hydrophobic surface of crystalline cellulose. In this study, we found that a CBD fusion enhanced and improved soluble β-glucuronidase (GusA) enzyme properties through the formation of an artificially oligomeric state. First, a soluble CBD fused to the C-terminus of GusA (GusA-CBD) was obtained and characterized. Interestingly, the soluble GusA-CBD showed maximum activity at higher temperatures (65°C) and more acidic pH values (pH 6.0) than free GusA did (60°C and pH 7.5). Moreover, the GusA-CBD enzyme showed higher thermal and pH stabilities than the free GusA enzyme did. Additionally, GusA-CBD showed higher enzymatic activity in the presence of methanol than free GusA did. Evaluation of the protease accessibility of both enzymes revealed that GusA-CBD retained 100% of its activity after 1 h incubation in 0.5 mg/ml protease K, while free GusA completely lost its activity. Simple fusion of CBD as a single domain may be useful for tunable enzyme states to improve enzyme stability in industrial applications.
authors.affiliation: Synthetic Biology & Bioengineering Research Center, KRIBB, Yuseong-gu, Daejeon, Korea.
authors.forename: Soo-Jin
authors.initials: SJ
authors.lastname: Yeom
authors.affiliation: Synthetic Biology & Bioengineering Research Center, KRIBB, Yuseong-gu, Daejeon, Korea.
authors.forename: Gui Hwan
authors.initials: GH
authors.lastname: Han
authors.affiliation: Synthetic Biology & Bioengineering Research Center, KRIBB, Yuseong-gu, Daejeon, Korea.|Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, Korea.
authors.forename: Moonjung
authors.initials: M
authors.lastname: Kim
authors.affiliation: Synthetic Biology & Bioengineering Research Center, KRIBB, Yuseong-gu, Daejeon, Korea.
authors.forename: Kil Koang
authors.initials: KK
authors.lastname: Kwon
authors.affiliation: Synthetic Biology & Bioengineering Research Center, KRIBB, Yuseong-gu, Daejeon, Korea.
authors.forename: Yaoyao
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Synthetic Biology & Bioengineering Research Center, KRIBB, Yuseong-gu, Daejeon, Korea.
authors.forename: Haseong
authors.initials: H
authors.lastname: Kim
authors.affiliation: Synthetic Biology & Bioengineering Research Center, KRIBB, Yuseong-gu, Daejeon, Korea.
authors.forename: Hyewon
authors.initials: H
authors.lastname: Lee
authors.affiliation: Synthetic Biology & Bioengineering Research Center, KRIBB, Yuseong-gu, Daejeon, Korea.|Biosystems & Bioengineering, University of Science & Technology, Yuseong-gu, Daejeon, Korea.
authors.forename: Dae-Hee
authors.initials: DH
authors.lastname: Lee
authors.affiliation: Synthetic Biology & Bioengineering Research Center, KRIBB, Yuseong-gu, Daejeon, Korea.
authors.forename: Heungchae
authors.initials: H
authors.lastname: Jung
authors.affiliation: Synthetic Biology & Bioengineering Research Center, KRIBB, Yuseong-gu, Daejeon, Korea.|Biosystems & Bioengineering, University of Science & Technology, Yuseong-gu, Daejeon, Korea.
authors.forename: Seung-Goo
authors.initials: SG
authors.lastname: Lee
mesh_terms: D001665:Binding Sites; D002482:Cellulose / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D000432:Methanol / Q000737:chemistry; D011485:Protein Binding / Q000502:physiology; D000072417:Protein Domains / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D002482:Cellulose; D005966:Glucuronidase; D019286:Endopeptidase K; D000432:Methanol