49NISR Research GRANT
2010 Young Investigator Research Grant

Development of Biomass Conversion System with Hydrophilic
Ionic Liquid and Enzyme from Extremophile
Atsushi KURATA
Department of Applied Biological Chemistry, Graduate School of Agriculture, Kinki University

Research aims
Ionic liquids (ILs) are composed of a bulky asymmetricalcation and a small anion, both of which are easily replaced;
therefore, numerous compositions of ILs are possible. Unlike conventional organic solvents, ILs are able to dissolve
many compounds. Thus, ILs are useful as reaction media1).
Using a hydrophobic IL as reaction solvent, we developed a convenient procedure for enzymatic synthesis of 3cyclohexylpropyl caffeate, which exhibits a strong antiproliferative activity on human tumor cells2). However, wefound difficulties in using hydrophilic ILs as reaction solvents in the enzyme reaction3). It is suggested that hydrophilic ILs can cause changes in enzyme conformationand thereby enzyme denaturation, probably through theireffect of reducing water activity4). Thus, it would be desirable to develop enzymes that are stable and functional inhydrophilic ILs.
The purpose of this research was to elucidate the mechanism of resistance to hydrophilic ILs in hydrophilic IL-tolerant microorganisms, and to identify enzymes that are stable in hydrophilic ILs.

([BMIM][CF 3SO 3]), and 1-butyl-3-methylimidazolium
chloride ([BMIM][Cl]), and the hydrophobic ILs were 1butyl-3-methylimidazolium hexafluorophosphate([BMIM][PF6]) and 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([BMIM][NTf2]). The bacterial isolates were identified by 16S rRNA gene sequence
analysis. Using LC-MS (Waters, MA), the [BMIM] cation(m/z 139.1 as molecular ion, m/z 83.1 as fragment ion) wasquantified. Enzyme activities in the culture supernatantcontaining the hydrophilic IL were detected with theAPIZYM system (bioMérieux, France).
Results

Methods(1) Isolation of bacteria that could grow in the presence ofhydrophilic ILs
Mixed bacterial floras from kusaya gravy and Korean
pickled cabbage were inoculated into a broth medium containing [BMIM][PF6] or [EMIM][CF3SO3]. From the bacterial communities grown in the media, we obtained bacterial
isolates that could grow in broth media containing hydrophilic or hydrophobic IL. The 16S rRNA gene sequences of all isolates had the highest similarity to those ofbacteria belonging to the genus Staphylococcus.

Using various food samples as bacterial sources, we obtained a number of bacterial isolates that could grow inbroth media containing a hydrophilic or hydrophobic IL.
The hydrophilic ILs used were 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([EMIM][CF3SO3]), 1butyl-3-methylimidazolium trifluoromethanesulfonate(2) Enzyme activities detected in culture containing hydrophilic IL
A mixed bacterial flora from kusaya gravy was inoculated into a broth medium containing hydrophilic IL (10%[EMIM][CF3SO3]). Good growth was observed with theculture. In the supernatant of the bacterial culture thus ob-Fig. 1. Enzyme activities detected in the supernatant of culture broth containing hydrophilic IL ([EMIM][CF3SO3]), in which a mixed bacterial flora fromkusaya gravy was grown (A) and growth of Staphylococcus sp. SM-1-W in the presence of the hydrophilic IL (B)50tained, alkaline phosphatase activity and esterase activity
were detected with the APIZYM system (Fig. 1A).
(3) Characterization of an IL-tolerant Staphylococcus sp.
strainThe bacterial isolate Staphylococcus sp. SW-1-M wasfound to be capable of growing in broth containing 10%[EMIM][CF3SO3], 10% [BMIM][Cl], 10% [BMIM][PF6],
or 10% NaCl, but not in broth containing 10%[BMIM][NTf2] or 10% [BMIM][CF3SO3]. LC-MS analysisof the culture broth samples suggested that [BMIM] cation
was not degraded during cultivation. Thus, it was indicatedthat Staphylococcus sp. SW-1-M is a hydrophilic IL([BMIM][Cl])-tolerantbacterium(doublingtime102.2 min) (Fig. 1B).
Conclusion
Bacterial isolates obtained from food samples were examined for growth in broth containing hydrophilic or hydrophobic ILs. One isolate, Staphylococcus sp. SW-1-M,
grew adequately in the broth containing 10% [BMIM][Cl].
When a mixed bacteria population from kusaya gravy
was grown in culture medium containing 10%[EMIM][CF3SO3], alkaline phosphatase activity and esterase activity were detected in the culture supernatant. It
was reported that cellulose is soluble in certain hydrophilicILs, namely, [BMIM][Cl] and N-ethyl-N-methylimidazolium methylphosphonate5,6). We have already obtained
bacterial communities capable of growing adequately in thepresence of carboxymethyl cellulose or [BMIM][Cl]. Thus,
using those IL-tolerant bacteria and hydrophilic ILs, a novel
system for efficient conversion of cellulose could be constructed.
References
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