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KMID : 0380020060210040279
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Korean Journal of Biotechnology and Bioengineering
2006 Volume.21 No. 4 p.279 ~ p.285
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Comparison of Enzymatic Activity and Cleavage Characteristics of Trypsin Immobilized by Covalent Conjugation and Affinity Interaction
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Jang Dae-Ho
Seong Gi-Hun
Lee Eun-Kyu
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Abstract
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We investigated the effects of immobilization chemistry on the yield of immobilization and the bioactivity of the immobilized enzymes. Trypsin as a model protein and macroporous polymer beads (Toyopearl AF 650M, Tosho Co., Japan) was used as a model matrix. Four methods were used to immobilize trypsin; covalent conjugation by reductive amination (at pH 10.0 and pH 4.0) and affinity interaction via streptavidin-biotin, and double-affinity interaction via biotin-streptavidin-biotin system. The covalent conjugation immobilized 3~4 mg/ml-gel, ca. 3-fold higher than the affinity method. However, the specific activity of the covalently (pH 10.0) and affinity-immobilized trypsin (via streptavidin-biotin) are ca. 37% and 50%, respectively, of that of the soluble enzyme (on the low-molecular-weight BAPNA substrate). When the molecular size of a substrate increased, the affinity-immobilized trypsin showed higher clavage activity on insulin and BSA. This result seemed to indicate the streptavidin-biotin system allowed more steric flexibility of the immobilized trypsin in its interaction with a substrate molecule. To confirm this, we studied the molecular flexibility of immobilized trypsin using quartz crystal microbalance-dissipation. Self-assembled monolayers were formed on the Q-sensor surface by aminoalkanethiols, and gultaraldehyde was attached to the SAMs. Trypsin was immobilized in two ways: reductive amination (at pH 10.0) and the streptavidin-biotin system. The dissipation shift of the affinity-immobilized trypsin was 0.8 × 10-6, whereas that of the covalently attached enzyme was almost zero. This result confirmed that the streptavidin-biotin system allowed higher molecular flexibility. These results suggested that the bioactivity of the immobilized enzyme be strongly dependent on its molecular flexibility.
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KEYWORD
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Immobilized trypsin, covalent immobilization, streptavidin-biotin system, quartz crystal microbalance, molecular flexibility
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