title: Effects of a non-conservative sequence on the properties of β-glucuronidase from Aspergillus terreus Li-20.
abstract: We cloned the β-glucuronidase gene (AtGUS) from Aspergillus terreus Li-20 encoding 657 amino acids (aa), which can transform glycyrrhizin into glycyrrhetinic acid monoglucuronide (GAMG) and glycyrrhetinic acid (GA). Based on sequence alignment, the C-terminal non-conservative sequence showed low identity with those of other species; thus, the partial sequence AtGUS(-3t) (1-592 aa) was amplified to determine the effects of the non-conservative sequence on the enzymatic properties. AtGUS and AtGUS(-3t) were expressed in E. coli BL21, producing AtGUS-E and AtGUS(-3t)-E, respectively. At the similar optimum temperature (55°C) and pH (AtGUS-E, 6.6; AtGUS(-3t)-E, 7.0) conditions, the thermal stability of AtGUS(-3t)-E was enhanced at 65°C, and the metal ions Co(2+), Ca(2+) and Ni(2+) showed opposite effects on AtGUS-E and AtGUS(-3t)-E, respectively. Furthermore, Km of AtGUS(-3t)-E (1.95 mM) was just nearly one-seventh that of AtGUS-E (12.9 mM), whereas the catalytic efficiency of AtGUS(-3t)-E was 3.2 fold higher than that of AtGUS-E (7.16 vs. 2.24 mM s(-1)), revealing that the truncation of non-conservative sequence can significantly improve the catalytic efficiency of AtGUS. Conformational analysis illustrated significant difference in the secondary structure between AtGUS-E and AtGUS(-3t)-E by circular dichroism (CD). The results showed that the truncation of the non-conservative sequence could preferably alter and influence the stability and catalytic efficiency of enzyme.
authors.affiliation: School of Chemical Engineering and Technology, Tianjin University, Nankai District, Tianjin, People's Republic of China.
authors.forename: Yanli
authors.initials: Y
authors.lastname: Liu
authors.forename: Jie
authors.initials: J
authors.lastname: Huangfu
authors.forename: Feng
authors.initials: F
authors.lastname: Qi
authors.forename: Imdad
authors.initials: I
authors.lastname: Kaleem
authors.forename: Wenwen
authors.initials: W
authors.lastname: E
authors.forename: Chun
authors.initials: C
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D055162:Biocatalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability / Q000235:genetics; D005966:Glucuronidase / Q000737:chemistry* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D017384:Sequence Deletion; D013696:Temperature
chemical_list: D005966:Glucuronidase