Mutations of Lysine 47 in cyclodextrin glycosyltransferase from Paenibacillus macerans enhance beta-cyclodextrin specificity.
The nature of amino acid residue 47 shows a clear discrimination between the different groups of cyclodextrin glycosyltransferase (CGTase). The effects of amino acid side chain at position 47 on cyclodextrin product specificity were investigated by replacing Lys47 in the CGTase from Paenibacillus macerans strain JFB05-01 with arginine, histidine, threonine, serine, or leucine. All of the mutations reduced alpha-cyclodextrin-forming activity, whereas significant increases in beta-cyclodextrin-forming activity were achieved. Especially, the mutations of Lys47 into threonine, serine, or leucine converted P. macerans CGTase from alpha-type to beta/alpha-type. As a result, all of the mutants displayed a shift in product specificity toward the production of beta-cyclodextrin. Thus, they were more suitable for the industrial production of beta-cyclodextrin than the wild-type enzyme. The enhancement of beta-cyclodextrin specificity might be due to weakening or removal of hydrogen-bonding interactions between the side chain of residue 47 and the bent intermediate specific for alpha-cyclodextrin formation.
Li
Zhao-Feng
ZF
State Key Laboratory of Food Science and Technology, People's Republic of China.
Zhang
Jia-Yu
JY
Sun
Qi
Q
Wang
Miao
M
Gu
Zheng-Biao
ZB
Du
Guo-Cheng
GC
Wu
Jing
J
Chen
Jian
J
D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics; D007930:Leucine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D056507:Paenibacillus / Q000201:enzymology*; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry; D047391:alpha-Cyclodextrins / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000378:metabolism*
D047391:alpha-Cyclodextrins; D047392:beta-Cyclodextrins; D013912:Threonine; D012694:Serine; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D007930:Leucine; C031215:betadex; D008239:Lysine; C032613:alpha-cyclodextrin