Research

Publications:
ACS Omega, 4, 15234-15239 (2019)
Process Biochemistry, 98, 59-64 (2020)

II. Enzymatic synthesis of sugar-derived biosurfactants

Sugar fatty acid esters are non-toxic and biodegradable glycolipid-type biosurfactants that have wide pharmaceutical applications, including drug dissolution, absorption and permeation, and vesicles for controlled-release drug delivery. A more timely application of biosurfactants, based on their antiviral and amphiphilic properties, is the control of coronavirus-2 (SARS-CoV-2) spread by disrupting viral membrane, serving as handwashing and cleaning agents, and targeting and relieving the symptoms after infection. However, enzymatic synthesis of sugar fatty acid esters is challenged by the lack of a suitable reaction system that can dissolve both polar sugars and non-polar fatty acids, while maintaining high enzyme activity. Meanwhile, the fast-growing research on ionic liquids (ILs) has highlighted the potential of these ionic media for enzyme stabilization and activation. To bridge the gap, we will develop multifunctional ILs that can dissolve both substrates and that are also highly compatible with enzymes (lipases in most cases). The long-term goal is to mentor and guide a team of undergraduates to develop a general methodology for efficient synthesis of glycolipid-type biosurfactants. The main objective is for students to synthesize lipase-compatible functionalized ILs that can dissolve sugars and fatty acids/fatty acid esters, and conduct enzymatic preparation of sugar fatty acid esters in ionic media.

III. Carbon Dioxide Capture and Utilization

Ionic liquids (ILs) have been widely explored as alternative solvents for carbon dioxide (CO2) capture and utilization. However, most of these processes are under pressures significantly higher than atmospheric level, which not only levies additional equipment and operation costs, but also makes the large-scale CO2 capture and conversion less practical. In this study, we rationally designed glycol ether-functionalized imidazolium, phosphonium and ammonium ILs containing acetate (OAc–) or Tf2N– anions, and found these task-specific ILs could solubilize up to 0.55 mol CO2 per mole of IL (or 5.9 wt% CO2) at room temperature and atmospheric pressure. Although acetate anions enabled a better capture of CO2, Tf2N– anions are more compatible with alcohol dehydrogenase (ADH), which is a key enzyme involved in the cascade enzymatic conversion of CO2 to methanol. Our promising results indicate the possibility of CO2 capture under ambient pressure and its enzymatic conversion to valuable commodity.

Publications:
Green Chemistry Letters and Reviews, In press (2023). (doi: 10.1080/17518253.2022.2149280)

IV. Enzymatic synthesis of polylactide and other polyesters

Due to the increasing demand of polylactide and other polyesters in medical and food fields, there is a strong need to produce these polymers without the use of (toxic) metal catalysts. Conventional enzymatic polymerization in bulk (solvent-free) or organic solvents often leads to low molecular weight, poor yield and wide polydispersity. We propose the use of custom-designed ionic liquids for a better substrate dissolution, higher enzyme stability, and more efficient ring-opening polymerization of lactide and other cyclic lactones. We further propose to construct polyester-based cellulose nanocomposites via novel enzymatic copolymerization in functionalized ionic liquids (Scheme 3). This research is innovative and significant because custom designed solvents could provide a new direction for enzyme activation, and the synergistic combination of novel chiral solvents and enzyme mutants will lead to a general methodology for polyester synthesis. The fundamental understanding of the catalytic mechanism will offer a higher flexibility for manipulating the catalytic system.

Publications:
RSC Advances, 8, 36025-36033 (2018)
Journal of Chemical Technology and Biotechnology, 93, 9–19 (2018)
RSC Advances, 7, 48639–48648 (2017)

V. Cellulosic Ethanol: Enzymatic Hydrolysis of Cellulose Pretreated by 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 such as wood chips, crop residues, grasses, and solid animal waste.

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.

One of the major obstacles in producing cellulosic ethanol 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).

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.

Publications:
J. Biotechnol., 139, 47-54 (2009)
Biotechnol. Prog., 26, 127-133 (2010)

VI. Enzymatic liquefaction of coal using task-specific ionic liquids

Coal is a major feedstock of energy source in the US as an alternative to petroleum oil. Coal can be converted to liquid fuels from a synthesis gas intermediate by the thermochemical method but this process often requires high temperatures and pressures. While the enzymatic approach can be operated under much milder conditions to convert coal to fuel or aromatic compounds, this method has encountered numerous challenges primarily due to the poor solubility of coal in aqueous media resulting in insufficient enzyme-coal binding interactions. We propose to use less expensive, task-specific ionic liquids and deep eutectic solvents to improve the coal dissolution and thus, enhance the enzyme-coal interactions thereby leading to a much more efficient conversion of coal to liquid fuels. To achieve our objective, two Specific Goals are proposed: (1) Develop task-specific ionic solvent systems that can solubilize coal and are also compatible with enzymes (such as oxidases and reductases); (2) Provide the mechanistic understanding of enzymatic depolymerization of coal in non-aqueous ionic solvent systems.

Publications:
Journal of Chemical Technology and Biotechnology, 95, 2301-2310 (2020)

VII. Enzymatic Preparation of Biodiesel in (Eutectic) Ionic Liquids

There is an increasing demand for renewable energy sources, fearing the depletion of crude oil in the next 50 years. As one of the alternative biological sources, biodiesel (fatty acid monoester) is becoming an attractive, renewable and biodegradable fuel for diesel engines and heating systems as it can be produced from vegetable oils or animal fats. Other sources of lipids are also being considered, such as oleaginous microbial biomass, soybean oil deodorizer distillate (SODD), and pine trees. Biodiesel has comparable fuel economy as petroleum-based diesel, and can reduce the emissions of polluting substances (such as particulate matter, carbon monoxide and hydrocarbon).

The common synthetic route for biodiesel production is the transesterification of vegetable oils (or animal fats) with methanol (or ethanol). This reaction can be catalyzed by acids, alkaline metal hydroxides, alkoxides, and non-ionic bases (such as amines and amidines). However, these methods have several drawbacks: (1) acid/base processes are often related to corrosion and emulsification problems; (2) acid-catalyzed reactions are usually much slower than base-catalyzed processes; however, the base-catalysis technology may cause unnecessary saponification of fatty acids; (3) a large excess of alcohol is required to drive the equilibrium to the ester formation and to achieve the facile separation of biodiesel from the glycerol, however, it creates the recycling problems (such as cost and recovery of methanol); (3) practically, oils and fats are not soluble in alcohols, resulting in barriers for triglyceride conversions; (4) other issues such as being energy intensive, alkaline waste-water treatment, and interference of free fatty acids and water. Therefore, a number of new approaches have been vigorously pursued to circumvent these problems, such as the development of heterogeneous catalysts, alcoholysis in supercritical methanol, ionic liquids (ILs)-catalyzed transesterification, and the lipase-catalyzed transesterification.

In particular, the enzymatic transesterification method offers many advantages over chemical methods such as mild reaction conditions, low energy demand, low waste treatment, the reusability of enzymes, flexibility in choosing different enzymes for different substrates, also allowing small amount of water in substrates, etc. Unfortunately, the current lipase-catalyzed method exhibits several downsides that prevent this promising approach from being commercialized. These disadvantages include the high cost of enzymes, lipase inactivation by acyl acceptors such as methanol, lipase inactivation by impurities in crude and waste oils, etc. In addition, due to the poor miscibility between oils/fats and methanol, many enzymatic transesterification reactions are heterogeneous systems involving a complicated liquid-liquid interface.

Our approach is to take advantages of high lipase activity and stability in ionic liquids, and to perform the enzymatic transesterification of triglycerides in ionic liquids for biodiesel production. In particular, we prepare new, inexpensive and biodegradable eutectic ionic liquids that are also lipase-compatible for this purpose.

Publications:
Green Chemistry, 10, 696-705 (2008)
Appl. Biochem. Biotechnol., 162, 13-23 (2010)
Org. Biomol. Chem., 9, 1908-1916 (2011).

VIII. Synthesis of Betulinic Acid Derivatives with Anti-Cancer, Anti-HIV and Anti-Viral Activities

Betulinic acid (3β-hydroxy-lup-20(29)-en-28-oic acid) is a natural pentacyclic lupane-type triterpene that can be found in various plants including birch trees. This compound and its derivatives possess many favorable biological properties such as anticancer, anti-HIV-1 (human immunodeficiency virus type-1), antibacterial, anti-malarial, anti-inflammatory, and anthelmintic activities. We prepare a number of new ionic derivatives of betulinic acid, and evaluate their anti-cancer and anti-HIV activities.

Publications:
J. Enzyme Inhib. Med. Chem., 27, 715-721 (2012)
Bioorg. Med. Chem. Lett., 22, 1734–1738 (2012)
Bioorg. Med. Chem. Lett., 25, 3168–3171 (2015)

IX. DNA-Based Hybrid Catalysts for Asymmetric Catalysis

New hybrid catalysts can be prepared by binding metal complexes with DNA molecules as chiral scaffold. We customize the structures of double-stranded oligonucleotides so that the hybrid catalysts can be used with organic co-solvents (such as ionic liquids and deep eutectic solvents) with high catalytic efficiencies. These robust hybrid catalysts can be widely used in the asymmetric synthesis of Michael addition, Diels-Alder reaction and Friedel-Crafts alkylation.

Publications:
Journal of Chemical Technology and Biotechnology, 90, 19–25 (2015).
RSC Advances, 4, 54051-54059 (2014)
International Journal of Biological Macromolecules, 84, 367–371 (2016).

X. Deep Desulfurization of Liquid Fuel by Oxidative Extraction Using Ionic Liquids

With the growing concern over pollution caused by the burning of sulfur-containing petroleum-based liquid fuels, since 2005 more strict regulations have been imposed to reduce the sulfur content for gasoline and diesel oil to 10-30 ppm level. The conventional hydrodesulfurization (HDS) method has encountered a great challenge to meet this ultra-low sulfur limit, and is not always efficient in removing aromatic sulfur compounds such as benzothiophene, dibenzothiophene and their alkyl derivatives. Among various alternative deep desulfurization approaches, extraction and oxidative extraction using ionic liquids (ILs) have gained tremendous attention due to the favorable properties of ILs such as low volatility, high solubility of sulfur compounds, high thermal/chemical stability, and tunable physical properties. Unfortunately, most ILs explored for this application are based on imidazolium cations, which are costly and poorly biodegradable. Therefore, we propose to develop a new type of inexpensive ILs (glycol-functionalized ammoniums/piperidiniums, and choliniums), aiming to efficiently remove aromatic sulfur compounds from fossil fuels during the refining process. To achieve our objective, two Specific Goals are proposed: (1) Develop new IL systems for an efficient partition and extraction of aromatic sulfur compounds; (2) Identify strategies to improve the oxidation efficiency of aromatic sulfur compounds by H₂O₂ in the IL phase.

Publications:
Journal of Chemical Technology and Biotechnology, 91, 25–50 (2016)
ACS Sustainable Chemistry and Engineering, 4, 4771–4780 (2016)
Fuel, 189, 334–339 (2017)

XI. Enzyme Activation and Stabilization in Ionic Liquids and their Aqueous Solutions

Ionic liquids (ILs), usually consisting of organic cations 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.

• Enzymes in Aqueous Solutions of Ionic Liquids

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 1). 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.

Anions: (kosmotropic) PO₄^3- >SO₄^2- >CH₃COO^- >Cl^- >Br^- >I^- >BF₄^- >PF₆^- (chaotropic)

B-coefficients: 0.495→0.206→0.246→ -0.005→ -0.033→ -0.073→ -0.093→ -0.21

Cations: (chaotropic) (CH₃)₄N⁺ > K⁺ > Na⁺ > Li⁺ > Ca²⁺ > Mg²⁺> Al³⁺ (kosmotropic)

B-coefficients: 0.123→0.009→0.085→0.146→0.284→0.385→0.744

Fig. 1 The Hofmeister series of the ion effect on protein stability (The B-coefficients are from Marcus’ collection; (CH₃)₄N⁺ is a chaotrope, and its unusually large B-coefficient is due to the hydrophobic hydration).

Publications:
Journal of Molecular Catalysis B: Enzymatic, 37, 16-25 (2005)
Bioorganic Chemistry, 34(1), 15-25 (2006)
Tetrahedron: Asymmetry, 17(10), 1549-1553 (2006)
Journal of Chemical Technology and Biotechnology, 81(6), 877-891 (2006)
Chinese Journal of Chemistry, 24(4), 580-584 (April 2006)
Tetrahedron: Asymmetry, 17(3), 377-383 (2006)
Tetrahedron: Asymmetry, 17(17), 2491-2498 (2006)
Journal of Chemical Technology and Biotechnology, 82(3), 304 – 312 (2007).

• Enzymes in Neat Ionic Liquids

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. 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.

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 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.

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, 696-705 (2008)
J. Mol. Catal. B: Enzym., 57, 149-157 (2009)
Green Chemistry, 11, 1128-1138 (2009)
Biotechnol. Lett., 32(8), 1109-1116 (2010).

• Enzymes in Eutectic Ionic Liquids

Recently, it has been demonstrated that the mixture of a solid organic salt and a suitable complexing agent can sometimes liquify at temperatures below 100 °C, a so-called ‘deep eutectic IL’. The mechanism put forth is that the complexing agent (typically a hydrogen-bond donor) interacts with the anion, thereby increasing its effective size and shielding its interaction with the cation, in turn resulting in a depression in the melting point (T_m) of the mixture. A superb example is the mixture of choline chloride (T_m = 302 °C, 2-hydroxyethyl-trimethylammonium chloride, Scheme 1a) and urea (Tm = 133 °C) in a 1:2 molar ratio, resulting in a room-temperature deep eutectic IL (T_f = 12 °C). A major advantage of this approach is that inexpensive, bulk commodity, and non-toxic chemicals can frequently be employed, and the liquid properties can be fine-tuned by combining various organic salts and complexing agents in different ratios. Considering that many inexpensive quaternary ammonium salts are available and there exist a wide variety of amides, amines, carboxylic acids and alcohols to serve as complexing agents, the possibilities for forming new and inexpensive eutectic ILs are enormous. In particular, choline chloride, so-called vitamin B4, is produced on the Mtonne (million metric ton) scale annually as an additive for chicken feed, among other applications. In addition to its low cost, choline chloride is considered to be non-toxic and biodegradable. In fact, choline chloride is an essential micronutrient and human nutrient. Meanwhile, eutectic ILs can dissolve a variety of metal salts, acids (aromatic, amino, citric, benzoic), and polyols such as glucose or glycerol.

These eutectic solvents have favorable properties including low viscosity, high biodegradability, and excellent compatibility with Novozym® 435, a commercial immobilized Candida antarctica lipase B. Furthermore, in a model biodiesel synthesis system, we demonstrate high reaction rates for the enzymatic transesterification of Miglyol® oil 812 with methanol, catalyzed by Novozym 435 in choline acetate/glycerol (1:1.5 molar ratio). The high conversion (97%) of the triglyceride obtained within 3 hours, under optimal conditions, suggests that these novel eutectic solvents warrant further exploration as potential media in the enzymatic production of biodiesel.

In our work, the transesterification activities of cross-linked proteases (subtilisin and α-chymotrypsin), immobilized on chitosan, were individually examined in these novel deep eutectic solvents (DESs). In the 1:2 molar ratio mixture of choline chloride/glycerol containing 3% (v/v) water, cross-linked subtilisin exhibited an excellent activity (2.9 μmol min^–1 g^–1) in conjunction with a selectivity of 98% in the transesterification reaction of N-acetyl-l-phenylalanine ethyl ester with 1-propanol. These highly encouraging results advocate more extensive exploration of DESs in protease-mediated biotransformations of additional polar substrates and use of DESs in biocatalysis more generally.

Publications:
Org. Biomol. Chem., 9, 1908-1916 (2011)
J. Mol. Catal. B: Enzym., 72, 163-167 (2011).

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