Comparison of Enzymatic Activity and Cleavage Characteristics of Trypsin Immobilized by Covalent Conjugation and Affinity Interaction

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 $3{\sim}4$ 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 $0.8{\times}10^{-6}$, 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.

공유결합과 친화력결합에 의한 고정화 Trypsin의 효소역가와 절단특성 비교

본 연구에서는 trypsin을 모델 단백질로 하여 단백질 본연의 환성을 유지할 수 있는 고정화 방법을 찾기 위하여 공유결합방법과 친화력 결합방법을 이용하여 trypsin을 고정화 하였다. Streptavidin-biotin system을 이용한 고정화 방법은 bioactivity 유지측면에서 공유결합 방법보다 우수함을 확인하였다. 하지만 streptavidin-biotin system을 이용하였을 때 고정화 수율이 낮은 것은 해결해야 할 과제이다. 분자량이 다른 기질들(BAPNA, insulin, BSA)을 대상으로 고정화 trypsin의 부위 특이적 절단 특성을 분석한 결과 streptavidin-biotin에 의해 고정화된 trypsin이 절단효율도 높고 sequence coverage도 높은 것으로 확인되었다. 또한 공유결합된 trypsin은 견고한 분자구조를 나타낸 반면 streptavidin-biotin system으로 고정화된 trypsin은 유연성이 높은 것을 QCM-D를 이용하여 관찰할 수 있었다. 따라서 streptavidin-biotin system에 의한 고정화 방법에서 streptavidin-biotin 결합이 일종의 spacer arm 역할을 하면서 고정화된 trypsin의 분자유연성을 향상시켜 절단반응의 부위특이성과 절단수율을 향상시키는 것으로 판단되었다.