TY - JOUR
T1 - Spectrophotometric assay for protease activity in ionic liquids using chromogenic substrates
AU - Nakashima, Kazunori
AU - Maruyama, Tatsuo
AU - Kamiya, Noriho
AU - Goto, Masahiro
N1 - Funding Information:
This work was supported by a Grant-in-Aid for Scientific Research (no. 18045027) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. K.N. was supported by JSPS Research Fellowships for Young Scientists.
PY - 2008/3/15
Y1 - 2008/3/15
N2 - A new spectrophotometric assay has been developed to evaluate protease activity in ionic liquids (ILs). The assay consists of two strategies to enable real-time spectrometric analysis of enzymatic reaction in ILs. First, enzymes are modified with a comb-shaped poly(ethylene glycol), PM13, to obtain a transparent enzyme solution in IL. Second, a chromogenic substrate is used to follow the enzymatic reaction in IL. p-Nitroaniline-derivatized substrates are subjected to protease-catalyzed alcoholysis to release chromogenic p-nitroaniline that can be quantitatively detected by a UV-Vis spectrophotometer. By using this method, we can evaluate protease activity in ILs quite easily without separation of products from the reaction mixture. The availability of the novel assay system was demonstrated in a kinetic analysis of subtilisin-catalyzed reaction in the IL 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([Emim][Tf2N]) under different reaction conditions. Because two different serine proteases, subtilisin and α-chymotrypsin, substantially retained its original substrate specificity in the IL, the assay can be extended to other enzymes by using suitable chromogenic substrates.
AB - A new spectrophotometric assay has been developed to evaluate protease activity in ionic liquids (ILs). The assay consists of two strategies to enable real-time spectrometric analysis of enzymatic reaction in ILs. First, enzymes are modified with a comb-shaped poly(ethylene glycol), PM13, to obtain a transparent enzyme solution in IL. Second, a chromogenic substrate is used to follow the enzymatic reaction in IL. p-Nitroaniline-derivatized substrates are subjected to protease-catalyzed alcoholysis to release chromogenic p-nitroaniline that can be quantitatively detected by a UV-Vis spectrophotometer. By using this method, we can evaluate protease activity in ILs quite easily without separation of products from the reaction mixture. The availability of the novel assay system was demonstrated in a kinetic analysis of subtilisin-catalyzed reaction in the IL 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([Emim][Tf2N]) under different reaction conditions. Because two different serine proteases, subtilisin and α-chymotrypsin, substantially retained its original substrate specificity in the IL, the assay can be extended to other enzymes by using suitable chromogenic substrates.
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U2 - 10.1016/j.ab.2007.11.015
DO - 10.1016/j.ab.2007.11.015
M3 - Article
C2 - 18078800
AN - SCOPUS:39149112540
SN - 0003-2697
VL - 374
SP - 285
EP - 290
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -