Cloning and expression of beta-glucuronidase from Lactobacillus brevis in E. coli and application in the bioconversion of baicalin and wogonoside.
The beta-glucuronidase (GUS) gene from Lactobacillus brevis RO1 was cloned and expressed in Escherichia coli GMS407. The GUS gene was composed of 1812 bp, encoding a 603-amino-acid protein belonging to the glycosyl hydrolase family 2 with three conserved domains. The amino acid similarity was higher than 70% with the beta-glucuronidases of various microorganisms, yet less than 58% with the beta-glucuronidase of L. gasseri ADH. Overexpression and purification of the GUS was performed in beta-glucuronidase-deficient E. coli GMS407. The purified GUS protein was 71 kDa and showed 1284 U/mg of specific activity at optimum condition of pH 5.0 and 37 degrees C. At 37 degrees C, the GUS remained stable for 80 min at pH values ranging from 5.0 to 8.0. The purified enzyme exhibited a half-life of 1 h at 60 degrees C and more than 2 h at 50 degrees C. When the purified GUS was applied to transform baicalin and wogonoside into their corresponding aglycones, 150 microM of baicalin and 125 microM of wogonoside were completely transformed into baicalein and wogonin, respectively, within 3 h.
Kim
Hyun Sung
HS
Interdisciplinary Program in Genetic Engineering, Seoul National University, Seoul 151-742, Korea.
Kim
Jin Yong
JY
Park
Myeong Soo
MS
Zheng
Hua
H
Ji
Geun Eog
GE
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism*; D044950:Flavanones / Q000096:biosynthesis / Q000378:metabolism*; D005419:Flavonoids / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism*; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D052196:Levilactobacillus brevis / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D044950:Flavanones; D005419:Flavonoids; D005960:Glucosides; C038044:baicalin; C006680:baicalein; D005966:Glucuronidase; C473995:wogonoside; C085514:wogonin