earticle

영어

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.

한국어

본 연구에서는 trypsin을 모델 단백질로 하여 단백질 본연의 활성을 유지할 수 있는 고정화 방법을 찾기 위하여 공유결합방법과 친화력 결합방법을 이용하여 trypsin을 고정화하였다. 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.