Structure-based protein engineering of bacterial ß-xylosidase to increase the production yield of xylobiose from xylose.

Xylobiose consists of two molecules of xylose and has been highly recognized as a food supplement because it possesses high prebiotic functions. ß-xylosidase exhibits enzymatic activity to hydrolyze xylobiose, and the enzyme can also catalyze the reverse reaction in the presence of high concentrations of xylose. Previously, ß-xylosidase from Bacillus pumilus IPO (BpXynB), belonging to GH family 43, was employed to produce xylobiose from xylose. To improve the enzymatic efficiency, this study determined the high-resolution structure of BpXynB in a complex with xylobiose and engineered BpXynB based on the structures. The structure of BpXynB deciphered the residues involved in the recognition of the xylobiose. A site-directed mutation at the residue for xylobiose recognition increased the yield of xylobiose by 20% compared to a similar activity of the wild type enzyme. The complex structure of the mutant enzyme and xylobiose provided the structural basis for a higher yield of the engineered protein. This engineered enzyme would enable a higher economic production of xylobiose, and a similar engineering strategy could be applied within the same family of enzymes.

Identification of a naturally-occurring 8-[alpha-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl]daidzein from cultivated kudzu root.

INTRODUCTION: Kudzu root (Radix puerariae) is a rich source of isoflavones that are effective in preventing osteoporosis, heart disease and symptoms associated with menopause. The major isoflavonoids in kudzu root extracts were reported as puerarin, daidzin and daidzein. Recently, an unknown isoflavonoid (compound 1) was detected from one-year-old kudzu root cultivated in Vietnam. OBJECTIVE: To identify a novel compound 1 in kudzu root extract and determine the structure of the compound by ESI(+) TOF MS-MS, (1)H-, (13)C-NMR and enzymatic hydrolysis. METHODOLOGY: Samples were prepared by extraction of one-year-old kudzu root with 50% ethanol and the isoflavonoids were purified using recycling preparative HPLC. Unknown compound 1 was detected using UV-light at 254 nm in TLC and HPLC analyses. The molecular weight of 1 was determined using a TOF mass spectrometer equipped with an electrospray ion source. The structure of 1 was determined from the (13)C and (1)H NMR spectra recorded at 100.40 and 400.0 MHz, respectively. RESULTS: ESI(+) TOF MS-MS analysis shows that 1 is a puerarin diglycoside. The interglycosidic linkage of diglycoside determined by (1)H-, (13)C-NMR, and enzymatic hydrolysis suggests that 1 has a glucosyl residue linked to puerarin by an alpha-1,6-glycosidic bond. This compound is the first naturally-occurring 8-[alpha-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl]daidzein in kudzu root. The concentration of glucosyl-alpha-1,6-puerarin in kudzu root was 2.3 mg/g as determined by HPLC. CONCLUSION: The results indicate that puerarin diglycoside is one of the major isoflavonoids in kudzu root and has a significant impact on the preparation of highly water-soluble glycosylated puerarin.