Research Projects

I. Enzyme Activation and Stabilization in Ionic Liquids and Other Solvent Media

Ionic liquids (ILs), usually consisting of organic cations (Fig 1) and inorganic anions, are liquids at low temperature (< 100 ºC). An important feature of ILs is their extremely low vapor pressure. For this reason, they are called ‘green’ solvents, in contrast to traditional volatile organic compounds (VOCs). ILs have many attractive properties, such as chemical and thermal stability, nonflammability, high ionic conductivity, and a wide electrochemical potential window. Therefore, they have shown strong potentials as 'green' media or co-catalysts in various chemical reactions, including enzymatic reactions. They have also been investigated as novel engineering fluids in various applications, such as liquid-liquid extractions, heat-transfer processes, etc. Our study is to evaluate the enzyme activity in different types of ionic liquids, and to conduct biocatalysis using these new organic salts.

Our research focuses on the effect of ionic liquid properties on the enzyme stabilization and activation. In aqueous solutions, ionic liquid dissociate into individual ions. Therefore, the enzyme activity is affected the ion's kosmotropicity (known as the Hofmeister's series): kosmotropic anion and chaotropic cation stabilize the enzyme, while chaotropic anion and kosmotropic cation destabilize it (Fig 2). On the other hand, in nearly dried ionic liquid, the enzyme activity could be influenced by several factors, including hydrophobicity, hydrogen-bond basicity of anions, ionic association of anions, and substrate ground-state stabilizations.

Ionic liquids (ILs) are increasingly being used as neoteric solvents in a variety of enzymatic reactions. However, it is not well understood what properties of ILs govern the enzyme stabilization, and whether the microwave irradiation could activate enzymes in ILs. To tackle these two important issues, the synthetic activities of immobilized Candida antarctica lipase B (CaLB, known as Novozyme 435) were examined in more than twenty ILs under microwave irradiation. Under microwave irradiation, enhanced enzyme activities were observed in the presence of a small amount of water. However, such enhancement diminished when the reaction system was dried. To understand the effect of ionic liquid properties, the enzyme activities under microwave irradiation were correlated with the viscosity, polarity and hydrophobicity (log P) of ILs. The initial reaction rates bear no simple relationship with the viscosity and polarity (in terms of dielectric constant and ) of ILs, but have a loose correlation (a parabolic shape) with the log P values (Fig 3). The enzyme stabilization by ILs was explained from aspects of anion nucleophilicity, hydrogen-bonding basicity of anions, ionic association strength of anions, and substrate ground-state stabilization by ILs.

Fig. 3 Correlation of enzyme activities with solvent log P values (40 °C, Novozyme 435).

Recently, there is a rising interest in dissolving a variety of carbohydrates (such as sugars, starch and cellulose) in ionic liquids (ILs). The solutions of carbohydrates are then conveniently subject to chemical or physical modifications. However, one serious disadvantage of these ILs is their strong tendency in denaturing enzymes. This drawback prohibits the dissolved carbohydrates from being transformed by enzymatic reactions. In the present study, we designed a series of ILs that are able to dissolve carbohydrates but do not considerably inactivate the immobilized lipase B from Candida antarctica. These ILs consist of glycol-substituted cations and acetate anions. They could dissolve more than 10% (wt) cellulose and up to 80% (wt) D-glucose. The transesterification activities of the lipase in these ILs are very comparable with those in hydrophobic ILs. The hydrogen-bond forming anions, oxygen-containing cations, and low cation bulkiness promote the carbohydrate dissolution, while the low anion concentration seems essential for the enzyme stabilization. Therefore, an optimization could be achieved through a fine design of IL structures. To demonstrate the potential applications of these ILs, we performed the enzymatic transesterifications of methyl methacrylate with D-glucose and cellulose (Fig 4) respectively, both fully dissolved in ionic media. In the case of D-glucose, conversions up to  80% were obtained; and in the case of cellulose, conversions up to 89% and isolated yields up to 66% were achieved.

 Fig. 4 The enzymatic transesterification of cellulose and methyl methacrylate (both dissolved in an ionic liquid) was catalyzed by the immobilized lipase B from Candida antarctica. The FTIR-KBr spectrum of the cellulose product (b) suggested the formation of ester-bond (1745 cm^-1 band) as compared to that of cellulose (a). No ester linkage (c) was found between 6-O-trityl-cellulose and methyl methacrylate, indicating the transesterification did not occur on the two secondary hydroxyls (2,3-OH) of cellulose, but regioselectively on the primary hydroxyls (C-6 position).

Publications:

Green Chemistry, 10 (6), 696-705 (2008).

II. Dissolution and Enzymatic Hydrolysis of Cellulose Using Ionic Liquids

The world is heavily depending on the crude oil to meet the increasing need of energy. As a major source of energy, the crude oil is non-renewable and its production is expected to decline dramatically in the next 50 years. Campbell and Laherrere predicted the global oil production would decrease from the current 25 billion barrels to 5 billion barrels in 2050. Therefore, exploration of alternative energy sources has been a research focus in recent years. Ethanol is a renewable energy source because it can be produced from sugar fermentation. Ethanol has been added into gasoline fuels (up to 10% v/v) to reduce the use of fossil fuels and eliminate the toxic additive – methyl tertiary butyl ether (MTBE). However, fuel ethanol is more expensive than gasoline fuels because ethanol is mainly produced through a cornstarch-based technology. Therefore, alternate strategies of ethanol production are much needed, one of which is to use inexpensive lignocellulosic materials (Fig 5) such as wood chips, crop residues, grasses, and solid animal waste.

Fig. 5 A typical structure of cellulose as polymer of β-D-glucose.

According to the U.S. Energy Department (http://genomicsgtl.energy.gov/biofuels/), combustion of cellulosic ethanol produces less green house gases compared to both sugar-based ethanol and gasoline. Combustion of ethanol made from sugar reduces green house gas emissions by 18 percent to 29 percent compared to gasoline but cellulose ethanol reduces greenhouse gases emissions by up to 85 percent. Another advantage of cellulosic ethanol is the plentiful supply of the raw material. Agricultural waste like straw and grass can be used as sources of cellulose. Because the plants are not grown as food supplies, plants such as switch grass can be grown on land unsuitable for food production.

As early as in 1934, a molten salt, N-ethylpyridinium chloride, was found to dissolve cellulose under basic conditions.33 However, this salt has a high melting point (118 ºC) and is not considered useful in practice. A mixture of this salt (> 50%) with pyridine and dimethylformamide exhibits a lower melting point (about 75 ºC) and can dissolve cellulose.34 Only recently, pure ILs, without the addition of organic solvents, were found capable of dissolving carbohydrates (including cellulose) in high concentrations. Chloride and dicyanamide based ILs are able to break more hydrogen-bonds of cellulose than many other ILs do. Therefore, these two types ILs have been investigated in a number of studies for dissolving carbohydrates. A few examples are listed in Table 1.

Table 1 Solubility of carbohydrates in ILs

                                                              
Note: BMIM = 1-butyl-3-methylimidazolium, AMIM = 1-allyl-3-methylimidazolium, BMPy= 3-methyl-N-butylpyridinium, MoeMIM = 1-methoxyethyl-3-methylimidazolium, MomMIM = 1-methoxymethyl-3-methylimidazolium, dca = dicyanamide.

One of the major obstacles in producing cellulosic ethanol (Fig 6) is the low enzymatic hydrolysis rate and high cost of the enzyme (cellulase). The low hydrolysis rate is mainly due to the cellulose being an insoluble porous substrate. Therefore, in such a heterogeneous system, the reaction rate is limited by external/internal surfaces and crystallinity of cellulose. The crucial stages of enzymatic hydrolysis are adsorption of enzymes on cellulosic particles and the formation of enzyme substrate complexes (ES).
 

Fig. 6 Traditional Cellulosic Biomass Conversion to Ethanol Based on Concentrated Acid Pretreatment Followed by Hydrolysis and Fermentation (U.S. DOE. 2006. Breaking the Biological Barriers to Cellulosic Ethanol: A Joint Research Agenda, DOE/SC/EE-0095, U.S. Department of Energy Office of Science and Office of Energy Efficiency and Renewable Energy, http://genomicsgtl.energy.gov/biofuels/).

Our approach is to dissolve cellulose in ionic liquids, followed by an enzymatic hydrolysis of cellulose. In our study, several chloride and acetate-based ILs were investigated in dissolving cellulose. The cellulose was then regenerated from ILs by the addition of water. The regenerated cellulose exhibited much lower crystallinity than the untreated one as confirmed by infrared spectra, and higher accessible surfaces as suggested by the cellulase adsorption isotherm. As a result, the enzymatic hydrolysis of regenerated Avicel, filter paper and cotton proceeded much faster than those of untreated samples. A complete hydrolysis of Avicel could be achieved in 6 hrs given the Trichoderma reesei cellulase/substrate ratio (wt/wt) of 3:20 at 50 ˚C. The regenerated cellulose was also able to improve the thermal stability of cellulase.

References

[1] Swatloski, R. P.; Spear, S. K.; Holbrey, J. D.; Rogers, R. D. J. Am. Chem. Soc. 2002, 124, 4974.
[2] Heinze, T.; Schwikal, K.; Barthel, S. Macromol. Biosci. 2005, 5, 520.
[3] Barthel, S.; Heinze, T. Green Chemistry 2006, 8, 301.
[4] Xie, H.; Li, S.; Zhang, S. Green Chem. 2005, 7, 606
[5] Zhang, H.; Wu, J.; Zhang, J.; He, J. Macromolecules 2005, 38, 8272.
[6] Wu, J.; Zhang, J.; Zhang, H.; He, J.; Ren, Q.; Guo, M. Biomacromolecules 2004, 5, 266
[7] Liu, Q.; Janssen, M. H. A.; van Rantwijk, F.; Sheldon, R. A. Green Chem. 2005, 7, 39.
[8] Fukaya, Y.; Sugimoto, A.; Ohno, H. Biomacromolecules 2006, 7, 3295.
[9] Park, S.; Kazlauskas, R. J. J. Org. Chem. 2001, 66, 8395.

III. Study of Liquid Microstructures by Fractal and Equation of State (EOS)

A liquid is known to have a characteristic of short-range orderliness and long-range non-orderliness. The fractal geometry, a new non-linear science, has been attempted to describe the complicated non-linear behaviors in the molecular distribution. A simple coordination number model was proposed for regular liquids based on a self-similar structure. Furthermore, an EOS was proposed using the coordination number model and applied in the calculations of  thermodynamic properties. However, this is a very simple model system. The real fluid has a more complicated structure than a simple self-similar fractal distribution of molecules. The fluid at critical conditions is known have some kind of fractal behavior although it is not fully understood. A real fluid under normal conditions may contain certain fractal information to be explored by advanced tools such as multifractal.

Publications:

Chemical Engineering Communications, 192(2), 145-154 (2005).
Chemical Engineering Communications, 189(9), 1155-1195 (2002).

IV. Optimization of a Process for Carboxymethyl Cellulose (CMC) Preparation by a Mixing Solvent

A benzene-ethanol-water mixture was investigated as the solvent medium in preparing carboxymethyl cellulose (CMC). The process conditions of carboxymethylation were optimized to produce CMC with DS> 1.0, solution viscosity about 1500 mpa×s, and with uniform distribution of substituents. It seems promising to scale up this process using industrial kneading machine.

Publications:

International Journal of Polymeric Materials, 52(9), 749-759 (2003).
Acta Polymerica Sinica, (5), 524-529 (1997).

____________________________________________________________________________________________

Terra E. Person (Fall 2004 - Spring 2005)
Rasheed A. Lawal (Fall 2004 - Spring 2005)
Artez L. Sims (Fall 2004 to Spring 2005)
LaDena A. Holley (Fall 2004)
Sophia M. Campbell (Spring 2005 - Summer 2005 - Fall 2005 - Fall 2006)
Jonathan Solomon (Spring 2005)
Shannon Watts (Fall 2005)
Lee Jackson (Fall 2005 - Spring 2006)
Janet Cowins (Summer 2006)
Darkeysha Peters (Spring 2007 - Summer 2007)
Tanisha Crittle (Spring 2007 - Summer 2007)
Elliott Goins (Spring 2007 - Summer 2007)
Lavezza Zanders (Summer 2007)
Vernecia Person (Spring 2008)
Janet Cowins (Spring 2008, Summer 2008, Fall 2008)