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 Co2+, Ca2+ and Ni2+ 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.
Highlights
Glycyrrhizin (GL), the main constituent of licorice extract (Glycyrrhiza glabra), is a natural edulcorant as well as an important ingredient of traditional Chinese medicine [1,2,3]
It showed that the gene cloned was a b-glucuronidase gene named after catalysis by b-glucuronidase (AtGUS) (Genbank accession No JF894133), which was found very similar to PGUS(Genbank accession No EU095019) from P. purpurogenum Li-3 and AuGUS (Genbank accession No JN247805) from A. ustus Li-62, especially in the sugar-binding, immunoglobulin-like beta-sandwich, and TIM barrel domain (Table 1)
The b-glucuronidase (GUS) gene was first cloned in 1987 [19], and in subsequent years, many GUS genes were cloned and registered in the GenBank. This gene has never been cloned for the hydrolysis research of GL into glycyrrhetinic acid monoglucuronide (GAMG) or/and glycyrrhetinic acid (GA), with more valuable merits
Summary
Glycyrrhizin (GL), the main constituent of licorice extract (Glycyrrhiza glabra), is a natural edulcorant as well as an important ingredient of traditional Chinese medicine [1,2,3]. As an important derivative of GL, GAMG displayed stronger physiological functions than GL such as antiviral, anti-inflammatory, anti-tumor functions, and so on; and it is 1000-fold sweeter than saccharose [4]. A fungal strain, Aspergillus terreus Li-20 was screened, which can use GL as a carbon source and produce GAMG and GA after catalysis by b-glucuronidase (AtGUS). The main disadvantages of AtGUS were low enzyme productivity, low catalytic efficiency, and pathogenicity, which rendered it unsafe for use in the food and medical industries
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