한국산 파인애플에서 분리한 bromelain의 정제와 특성

Purification and characteristics of bromelain from Korean pineapple

한국산 파인애플로부터 bromelain을 추출하여 황산암모늄염석, DEAE-cellulose ion-exchange chromatography, gel filtration을 이용하여 약 21배 정제하였고, 정제효소는 전기영동상 단일밴드를 나타내었으며, 분자량은 약 22,000이었다. 정제된 bromelain은 glycine과 serine이 가장 많이 함유되어 있었으며 최적작용 pH와 온도는 6.0, $60^{\circ}C$였다. $pH\;5{\sim}7,\;50^{\circ}C$이하에서 안정성을 보였으며 $Mn^{2+}$에 의해 활성이 촉진되었고, $Mg^{2+},\;Fe^{2+}$ 등의 금속이온에 의해 급격한 활성저해가 관찰되었다. 정제효소는 p-chloromercuribenzoic acid에 의해 저해되어 SH효소로 추측되었으며, Km과 Vmax값는 각각 $5.747{\times}10^{-4}M,\;131.58\;{\mu}g/min$ 이었다. 돈육 조직에 효소를 처리하였을 때 효소 농도가 증가함에 따라 육단백질이 가수분해되어 유리되는 수용성 질소량과 아미노태 질소량이 증가되어 연육효과가 있음이 확인되었다.

Bromelain was purified from Korean pineapple, Ananas comosus, L. The enzyme was purified about 21 fold by DEAF-cellulose ion-exchange chromatography and gel filtration on Sephadex G-150. Purified enzyme was confirmed as active single band by polyacrylamide electrophoresis and the molecular weight was estimated to be about 22,000 by SDS-PAGE. The optimum pH and temperature were 6.0 and $60^{\circ}C$, respectively. The range of its stability to the pH and temperature were respectively 5.0 to 7.0 and below $50^{\circ}C$. It was found that $Mn^{2+}$ increased the enzyme activity, whereas $Mg^{2+}\;and\;Fe^{2+}$ decreased it abruptly. The purified enzyme was inhibited by p-chloromercuribenzoic acid, indicating that reactive SH groups are required for the enzyme activity. The reaction of the enzyme followed typical Michaelis-Menten kinetics with Km value of $5.747{\times}10^{-4}\;M\;and\;Vmax\;of\;131.58\;{\mu}g/min$ for casein. When meat was treated with the enzyme, free soluble nitrogen and amino acid nitrogen increased as enzyme concentration increased.