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PEDS Advance Access published online on April 19, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh035
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Received December 18, 2003
Revised March 26, 2004
Accepted April 1, 2004

Article

Improvement of Cyclodextrin Glucanotransferase as an Antistaling Enzyme by Error-Prone PCR

Jae-Hoon Shim 1, Young-Wan Kim 2, Tae-Jip Kim 3, Hye-Young Chae 1, Jin-Hee Park 1, Hyunju Cha 1, Jung-Wan Kim 4, Yong-Ro Kim 5, Thomas Schaefer 6, Tina Spendler 6, Tae-Wha Moon 1, Kwan-Hwa Park 1*

1 National Laboratory for Functional Food Carbohydrate, Center for Agricultural Bio-Materials and School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, S. Korea
2 National Laboratory for Functional Food Carbohydrate, Center for Agricultural Bio-Materials and School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, S. Korea; Current address: Department of Chemistry, University of British Columbia, Vancouver, BC, V6T 1Z1, Canada
3 Department of Food Science and Technology, Chungbuk National University, Cheongju 361-763, S. Korea
4 Department of Biology, University of Incheon, Incheon 402-749, S. Korea
5 Department of Biological Resources and Materials Engineering College of Agriculture and Life Sciences, Seoul National University, Seoul 151-742, S. Korea
6 Novo Nordisk A/S, Krogshojvej 36, 2880 Bagsvaerd, Denmark

* To whom correspondence should be addressed. E-mail: parkkh{at}plaza.snu.ac.kr.


  Abstract

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 bread was produced in bread treated with wild-type CGTase.

Keywords: antistaling enzyme/CGTase/error-prone PCR/random mutagenesis/ retrogradation


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