PEDS Advance Access originally published online on April 19, 2004
Protein Engineering Design and Selection 2004 17(3):205-211; doi:10.1093/protein/gzh035
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2004 Oxford University Press
Improvement of cyclodextrin glucanotransferase as an antistaling enzyme by error-prone PCR
1National Laboratory for Functional Food Carbohydrate, Center for Agricultural Bio-Materials and School of Agricultural Biotechnology and 5Department of Biological Resources and Materials Engineering, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-742, 3Department of Food Science and Technology, Chungbuk National University, Cheongju 361-763, 4Department of Biology, University of Incheon, Incheon 402-749, Korea and 6Novo Nordisk A/S, Krogshojvej 36, 2880 Bagsvaerd, Denmark 2Present address: Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada
7 To whom correspondence should be addressed. e-mail: parkkh{at}plaza.snu.ac.kr
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°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.
Received November 19, 2003; revised April 1, 2004; accepted April 5, 2004 Edited by Stephen Withers