Preparation of Trinitrophenyl Cellulose as Substrate for Cellulase Assay

Trinitrophenyl Cellulose의 조제

Two types of modified celluloses which contain trinitrophenyl groups as chromophore were synthesized from carboxymethyl cellulose Whatman CM 70 and CM 32. Diaminoethyl groups were added to the CM 70 and CM 32 to make DAE-CM celluloses and then the DAE-CM groups were substituted by 2,4,6-trinitrophenyl groups to produce TNP-celluloses. Average particle size of the TNP-cellulose from CM 32 was $44.6{\pm}9.6{\mu}m$ in diameter and $127.9{\pm}22.5{\mu}m$ in length, which was much smaller than those from CM 70, however its TNP-moiety per gram determined by using the molar extinction coefficient $1.33{\times}10^4$ of ${\varepsilon}$-TNP-lysine at 345 nm, was 0.68 millimoles, which was 5.6-fold greater than those from CM 70. The absorption spectrum of TNP-oligosaccharides which were the soluble products of TNP-celluloses by a cellulase preparation Onozuka R-10, showed a maximal peak at 344 nm. Increases in the absorbance during hydrolysis were linear with the enzyme concentration, and the differences of slope values between two types of TNP-celluloses that the more semsitive assay could be achieved by using those from CM 32 as substrate at the low range of the enzyme concentration.

Whatman carboxymethyl cellulose CM 32와 CM 70으로 부터 색소단으로서 trinitrophenyl를 함유하는 섬유소 유도체를 합성하였다. 먼저 diaminoethyl기를 부가하여 DAE-CM-celulose를 얻고 다시 DAE-CM기 부위를 2,4,6-trinitrophenyl기로 치환시켜 TNP-cellulose를 조제하였다. CM 32로 부터 얻은 TNP-cellulose는 입자의 평균 크기가 $44.6{\pm}9.6{\mu}mD{\times}127.9{\pm}22.5{\mu}mL$로서 CM 70의 것에 비하여 3분의 1 수준으로 길이가 짧은 반면 폭은 보다 팽윤된 상태이었으며, ${\varepsilon}$-TNP-lysine의 molar extinction coefficient $1.33{\times}10^4$을 적용하여 측정한 그램당 TNP의 몰분율은 0.68 millimole로서 CM 70의 것보다 5.6배 높았다. 섬유소 분해효소에 의한 TNP-cellulose의 분해산물인 TNP-oligosaccharide의 최대 흡수 파장 ${\lambda}_{max}$는 344 nm이었다. 이들 TNP-cellulose를 기질로 하여 Onozuka R-10 효소액을 반응시킨 결과 반응 생성물의 흡광도가 효소의 농도에 비례하는 관계를 나타내었으며 CM 32의 것을 사용한 경우에 그 기울기가 현저히 커서 낮은 효소 농도 범위에서도 감도가 높음을 확인하였다.