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KMID : 0364820080440010074
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Korean Journal of Microbiology
2008 Volume.44 No. 1 p.74 ~ p.79
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Expression and Optimum Production of Cyclodextrin Glucanotransferase Gene of Paenibacillus sp. JB-13 in E. coli
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Kim Hae-Yun
Min Bok-Kee
Kim Hae-Nam
Jun Hong-Ki
Lee Sang-Hyun
Paik Hyung-Suk
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Abstract
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The purpose of this study is to clone cgt gene from Paenibacillus sp. JB-13 and to overexpress the protein in E.coli. For this purpose, the cgt gene was amplified from Paenibacillus sp. JB-13 genomic DNA by PCR using degenerate oligonucleotide primers. The sequence analysis results showed that the cgt gene from Paenibacillus sp. JB-13 has 98% homology with the cgt gene of Bacillus sp. To overexpress the protein, the cgt gene was cloned into pEXP7 expression vector and transformed into E. coli. The production of CGTase by recombinant E. coli was optimized under following conditions: 0.5% glucose, 3.0% polypeptone, 0.3% K2HPO4, 0.5% NaCl, and 7.0 of initial pH, 2.0% of inoculum, 37oC of culture temperature for 14 hr. And the optimal agitation was found at 0.1 vvm. The synthesis of 2-O-α-D-Glucopyranosyl L-Ascorbic acid (AA-2G) using the CGTase expressed in E. coli was identified as AA-2G by HPLC and HPLC confirmed that treating AA-2G made by cloned CGTase with α-glucosidase substantially produced AA and glucose.
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KEYWORD
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AA-2G, CGTase, cyclodextrin glucanotransferase, Paenibacillus sp. JB-13, transglucosylation
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