Biosynthesis of 2-O-D-glucopyranosyl-l-ascorbic acid from maltose by an engineered cyclodextrin glycosyltransferase from Paenibacillus macerans.
In this work, the specificity of cyclodextrin glycosyltransferase (CGTase) of Paenibacillus macerans towards maltose was improved by the site-saturation engineering of lysine 47, and the enzymatic synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) with l-ascorbic acid and maltose as substrates was optimized. Compared to the AA-2G yield of the wild-type CGTase, that of the mutants K47F (lysine→phenylalanine), K47P (lysine→proline), and K47Y (lysine→tyrosine) was increased by 17.1%, 32.9%, and 21.1%, respectively. Under the optimal transformation conditions (pH 6.5, temperature 36°C, the mass ratio of l-ascorbic acid to maltose 1:1), the highest AA-2G titer by the K47P reached 1.12g/L, which was 1.32-fold of that (0.85g/L) obtained by the wild-type CGTase. The reaction kinetics analysis confirmed the enhanced maltose specificity of the mutants K47F, K47P, and K47Y. It was also found that compared to the wild-type CGTase, the three mutants had relatively lower cyclization activities and higher disproportionation activities, which was favorable for AA-2G synthesis. As revealed by the interaction structure model of CGTase with substrate, the enhancement of maltose specificity may be due to the removal of hydrogen bonding interactions between the side chain of residue 47 and the sugar at -3 subsite. The obtained mutant CGTases, especially the K47P, has a great potential in the large-scale production of AA-2G with maltose as a cheap and soluble substrate.
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
Long
L
Liu
Ruizhi
R
Han
Hyun-Dong
HD
Shin
Jianghua
J
Li
Guocheng
G
Du
Jian
J
Chen
D019943:Amino Acid Substitution; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D004795:Enzyme Stability; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D056507:Paenibacillus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011994:Recombinant Proteins; C065378:ascorbic acid 2-O-glucoside; D008320:Maltose; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D001205:Ascorbic Acid
2-O-Glucopyranosyl-l-ascorbic acid; Cyclodextrin glycosyltransferase (CGTase); Maltose; Site-saturation engineering; l-Ascorbic acid