Designing Ionic Liquids As Novel Solvents For Enzymatic Synthesis Of Starch Esters

Enzymatic starch esterification is usually carried out in organic solvents,such as dimethyl sulfoxide?DMSO?and N,N-dimethylacetamide/lithium chloride?DMAC/LiCl?.However,these volatile organic solvents are usually unfriendly to the environment and even unrecyclable.To solve these problems,two novel imidazolium ionic liquids?[Me?OCH₂CH₂?₂-Al-Im]Ac and [Me?OCH₂CH₂?₃-Al-Im]Ac?were synthesized and applied for biosynthesis of two kinds of starch esters.Also,the effects of novel ionic liquids?ILs?on starch dissolution and lipase activity were tested.Based on starch solubility,lipase activity and physicochemical properties of ILs,the anions and cations of 8 kinds of commercial ILs were screened.To verify the screening results,the substitution degrees of starch esters enzymatically synthesized in ILs were studied.Results showed that among all the tested anions?Cl-,BF4-,PF6-,Ac-and NO3-?,Ac-worked best;introducing long alkyl chain to imidazolium cations?such as [C₆MIm]NO₃ and [C₈MIm]NO₃?could improve the lipase activity,but the melting point and viscosity of ILs were also increased,which limited the mass transfer and reduced the solubility of starch.Long alkyloxy and allyl substituents,which could reduce melting point and viscosity of ILs,were simultaneously grafted onto the imidazolium ring and two novel ILs?[Me?OCH₂CH₂?₂-Al-Im]Ac and [Me?OCH₂CH₂?₃-Al-Im]Ac?were synthesized.Technologies of LC-MS and NMR were used to identify the structure of ILs,and physicochemical properties?such as melting point,viscosity and thermal stability?were also determined.Results showed that this study successfully synthesized two novel ILs?[Me?OCH₂CH₂?₂-Al-Im]Ac and [Me?OCH₂CH₂?₃-Al-Im]Ac?and they were both room temperature ILs?melting point <-50??with good thermal stability?decomposition temperature > 200??and low viscosity?109.49 and 93.32 mPa·S,25??.Solubility of starch in novel ILs was measured,and the dissolution process was observed by polarizing microscope.Besides,technologies of SEM,XRD,DSC and HPSEC-RID-MALLS were used to study apparent morphology,crystal structure,thermal stability and molecular weight of dissolved starch.Results showed that dissolving 10.5 wt.%?100??starch in these novel ILs could still form a stable and transparent solution.The particle structure of corn starch was destroyed with the agglomeration occurred,the polarization cross disappeared gradually and the crystalline structure was destroyed?from type A crystal changed into amorphous structure?during dissolution.In addition,the degradation of corn starch dissolved in novel ILs occurred at 100 ?,but the degree of degradation was relatively low,which could meet the requirements of being solvents for dissolving starch.To investigate the effects of novel ILs on lipase activity,fluorescence spectroscopy was used to inspect the changes of lipase structure.Results showed that the lipase in novel ILs was more stable than that in [C₄MIm]NO₃,but less stable than that in [C₄MIm]PF₆.In addition,lipase in [Me?OCH₂CH₂?₃-Al-Im]Ac had higher structure stability than that in [Me?OCH₂CH₂?₂-Al-Im]Ac.Compared with DMSO,two kinds of starch esters were enzymatically synthesized in novel ILs.Besides,technologies of FT-IR,SEM,XRD and TGA were used to study the structure and thermal stability of starch esters.Results showed that DS values of starch esters synthesized in novel ILs were higher than those in DMSO.DS values of starch palmitate increased from 0.078?in DMSO?to 0.089 and DS values of starch stearate increased from 0.067?in DMSO?to 0.074.Then the novel ILs were chosen as solvents for synthesizing starch esters.Firstly,starch palmitate was synthesized in [Me?OCH₂CH₂?₂-Al-Im]Ac with the addition of Novozym 435.The maximum DS valus of starch palmitate?0.153?was reached under the following conditions: molar ratio of methyl palmitate and AGU was 3:1,concentration of Novozym 435 was 6 wt.%,reaction temperature was 50 ? and reaction time was 4 h.Secondly,starch stearate was enzymatically synthesized in [Me?OCH₂CH₂?₃-Al-Im]Ac.The maximum DS value of starch stearate?0.138?was reached under the conditions as follows: molar ratio of methyl stearate and AGU was 4:1,concentration of Novozyme 435 was 4 wt.%,reaction temperature was 50 ? and reaction time was 3 h.FT-IR spectrum confirmed that palmitate and stearate groups were successfully introduced into starch hydroxyl.SEM and XRD data showed that the morphology and crystal structures of starch esters were completely destroyed during esterification;TGA data showed that compared with native starch,the thermal stability of starch palmitate was lower but for starch stearate was higher.With the increase of DS values,the thermal stability of starch esters was improved.The novel ILs were recovered and reused for enzymatic synthesis of starch esters.The results showed that both two novel ILs had high recovery rates.After being recycled for 4 times,the DS values of starch esters synthesized in novel ILs were still more than 0.1.