Enzymatic synthesis of glycosylated puerarin using maltogenic amylase from Bacillus stearothermophilus expressed in Bacillus subtilis.
BACKGROUND
The maltogenic amylase from Bacillus stearothermophilus (BSMA) is a valuable biocatalyst that has been used to transglycosylate natural glycosides to improve solubility. To ensure safety, BSMA was produced in Bacillus subtilis, using new shuttle vector-based expression vectors. The transglycosylation of puerarin was also conducted with crude BSMA and analyzed.


RESULTS
Two expression systems, each containing one of the promoters from the genes encoding Bacillus licheniformis maltogenic amylase (BLMA) and an alpha-amylase from B. subtilis NA64 (amyR2), were constructed. The amyR2 promoter system was chosen as the best system; it yielded 107 mg of pure BSMA from a 2 L culture. In the transglycosylation reactions of puerarin using crude BSMA, relative amounts for maltosyl-alpha-(1 --> 6)-puerarin, glucosyl-alpha-(1 --> 6)-puerarin, glucosyl-alpha-(1 --> 3)-puerarin, and puerarin were determined as 26:18:7:49. A two-step purification process, including gel permeation chromatography, yielded 1.7 g of the transfer products from 3 g of puerarin.


CONCLUSIONS
The crude BSMA produced from a host generally recognized as safe (B. subtilis) can be used to transglycosylate various functional compounds. The expression system developed in this study will be helpful for the production of other food-grade enzymes by B. subtilis.
Choi
Chung-Hyo
CH
Center for Agricultural Biomaterials and Department of Food Science and Biotechnology, Seoul National University, Seoul 151-921, Korea.
Kim
Seung-Hee
SH
Jang
Jun-Hyuck
JH
Park
Jong-Tae
JT
Shim
Jae-Hoon
JH
Kim
Young-Wan
YW
Park
Kwan-Hwa
KH
D000681:Amylases / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D004274:DNA, Recombinant; D015870:Gene Expression; D005798:Genes, Bacterial; D005818:Genetic Engineering / Q000379:methods; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006031:Glycosylation; D007529:Isoflavones / Q000096:biosynthesis*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D029908:Pueraria / Q000737:chemistry; D012995:Solubility
D004274:DNA, Recombinant; D007529:Isoflavones; D000681:Amylases; C033607:puerarin