Improvement of cyclodextrin glucanotransferase as an antistaling enzyme by error-prone PCR.
In an effort to improve the properties of cyclodextrin glucanotransferase (CGTase) as an antistaling enzyme, error-prone PCR was used to introduce random mutations into a CGTase cloned from alkalophilic Bacillus sp. I-5 (CGTase I-5). A mutant CGTase[3-18] with the three mutations M234T, F259I and V591A was selected by agar plate assay. Sequence alignment of various CGTases indicated that M234 and F259 are located in the vicinity of the catalytic sites of the enzyme and V591 in the starch binding domain E. The cyclization activity of CGTase[3-18] was dramatically decreased by 10-fold, while the hydrolyzing activity was increased by up to 15-fold. These mutations near subsite +1 (M234T) and at subsite +2 (F259I) are likely to alter the enzyme activity in a concerted manner, promoting hydrolysis of substrate while retarding cyclization. The addition of CGTase[3-18] reduced the retrogradation rate of bread by as much as did the commercial antistaling enzyme Novamyl during 7-day storage at 4 degrees C. No cyclodextrin (CD) was detected in bread treated with CGTase[3-18], whereas 21 mg of CD per 10 g of bread was produced in bread treated with wild-type CGTase.
Shim
Jae-Hoon
JH
National Laboratory for Functional Food Carbohydrate, Center for Agricultural Bio-Materials and School of Agricultural Biotechnology, Seoul National University, Korea.
Kim
Young-Wan
YW
Kim
Tae-Jip
TJ
Chae
Hye-Young
HY
Park
Jin-Hee
JH
Cha
Hyunju
H
Kim
Jung-Wan
JW
Kim
Yong-Ro
YR
Schaefer
Thomas
T
Spendler
Tina
T
Moon
Tae-Wha
TW
Park
Kwan-Hwa
KH
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D001939:Bread; D002152:Calorimetry, Differential Scanning; D003500:Cyclization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D006026:Glycoside Hydrolases; C120909:glucan 1,4-alpha-maltohydrolase