Earticle

초록

영어

Oligosachharides in human milk have various useful biological functions for infant and applications in industry. Among them, sialyllactose(3’/6‘-SL) and fucosyllactose(2‘/3-FL) draw our atttention. To produce these oligosaccharides in large quantity, economic supply of CMP-Neu5Ac, highly active sialyltransferase(ST) and fucosyltransferase(FT) are keys to the success of the process development. Most sialylated oligosaccharides consist of Neu5Ac attached to galactose by an α2,3- or α2,6- linkage. In this research, α2,3-ST and α2,6-ST were engineered by hybrid approach to improve production of siayllactose. Hybrid approach is combined with rational design and directed evolution. This method can reduce library size by selecting target region such as substrate binding pocket and functional residues based on alanine scanning and computational mutation analysis. Saturation mutagenesis was done for selected residues to find best hits. First, multifunctional α2,3 ST from Pasteurella multocida was engineered by this approach. We selected non-conserved residues located in substrates binding site by alignment with STs in GT80 family. And we applied alanine scanning for the selected residues. Mutants which show neutral activity were selected for saturation mutagenesis, and a single mutant interacting with lactose showed 168% of increased specific activity. Also, specific activity of a combination mutant was increased 200% compared to wild-type. In addition, α2,6 side reaction was reduced significantly for R313 mutants. Second, α2,6 ST from Photobacterium damselae showed low activity and protein expression level. Therfore, it was engineered to increase catalytic activity. Substrate binding sites were predicted through homology modelling and functional residues were selected by the same method. Several mutants which show higher activity than wild type were screened by color assay method. Among them, single mutant interacting with CMP showed 4-5 times higher activity than wild type. Thus, α2,3- and α2,6-ST mutants obtained by hybrid approach will be an efficient tool for the improvement of production of sialyllactose. In addition, Fucosyltransferase is used for the synthesis of fucosyllactose(2‘/3-FL) and sialyl-LewisX derivatives, and the same hybrid approach was undertaken. The major problem of FTs was their inclusion body formation and low level of soluble expression. How we have overcome this problem will be discussed.

키워드

저자

• Byung-Gee KIM [ Interdisciplinary Program for Biochemical Engineering and Biotechnology, Institute of Molecular Biology and Genetics, Seoul National University,151-742, Seoul, South Korea. ]
• YUN-HEE CHOI [ Interdisciplinary Program for Biochemical Engineering and Biotechnology, Institute of Molecular Biology and Genetics, Interdisciplinary Program for Biochemical Engineering and Biotechnology, Institute of Molecular Biology and Genetics, Seoul National University,151-742, Seoul, South Korea. ]
• JONG-HOON KIM [ School of Chemical and Biological Engineering, Seoul National University, 151-742, Seoul, South Korea. ]
• HYUNG-WOO LEE [ School of Chemical and Biological Engineering, Seoul National University, 151-742, Seoul, South Korea. ]
• DAE-HEE KIM [ GeneChem Inc., Daejeon, 302-804, South Korea. ]
• JAE-KYUNG SONG [ SunMoon University, Dept. of Pharmaceutical Engineering. ]

참고문헌

발행기관

간행물

표지보기 관심저널 등록

• 간행물명

  한국생물공학회 학술대회

• 간기

  반년간

• 수록기간

  1985~2013

• 십진분류

  KDC 476 DDC 576