Overview of biocatalysis and Candida antarctica lipase B catalysed kinetic
Enzymes as biocatalysts for organic synthesis
Biocatalysis refers to the chemical processes facilitated by biological catalysts, primarily enzymes, which promote reactions between organic components under various conditions, including extreme environments Enzymes, predominantly proteins made of folded chains of amino acids, are responsible for catalyzing these reactions, with their activity concentrated in a small region known as the active site Typically, only a few amino acid residues within this site are crucial for the catalytic mechanism For enzymes to function effectively, they must achieve a specific three-dimensional structure, influenced by post-translational modifications and interactions among amino acid side chains This inherent complexity in enzyme conformation presents both advantages and disadvantages in their catalytic roles.
Enzymes, as biological catalysts, enhance reaction rates by lowering activation energies through the stabilization of transition states They exhibit remarkable specificity for substrates due to their unique three-dimensional structures, which only accommodate certain substrates that match the active site's size, shape, and polarity The formation of a noncovalent enzyme-substrate complex is crucial for the high selectivity of enzyme catalysis, ensuring that the substrate is positioned accurately for optimal interaction with the enzyme's active site This specificity allows enzymes to be chemo-, regio-, and stereoselective, facilitating precise transformations among multiple functional groups, sites, and stereoisomers.
Enzymes are attractive catalysts for organic synthesis due to their renewable origins and biodegradability, making them ideal for sustainable chemistry They typically operate efficiently under mild conditions, which enhances their appeal Their application in the asymmetric synthesis of bioactive compounds has garnered significant interest in the pharmaceutical industry, thanks to their exceptional chiral selectivity The field of biocatalysis engineering encompasses the discovery, development, and optimization of biotransformation processes, covering various aspects such as substrate, medium, protein, biocatalyst formulation, biocatalytic cascade, and reactor engineering.
Enzymatic promiscuity challenges the traditional view of enzymes as highly specific catalysts limited to one substrate and reaction type Many enzymes exhibit a broader tolerance, enabling them to convert nonnatural substrates and catalyze multiple reaction types across diverse conditions This flexibility allows biocatalysis engineering to leverage enzymes that can accept various related substrates while maintaining selectivity for single products or stereoisomers Additionally, these enzymes can effectively catalyze alternative reactions and remain stable under harsh conditions, including non-aqueous environments.
Biocatalysis in non-aqueous solvents
The exploration of enzymatic reactions in organic solvents began with Sym's work in the 1930s, but it wasn't until the 1980s that non-aqueous biocatalysis gained significant attention, largely due to Zaks and Klibanov's groundbreaking studies They challenged the traditional view that enzymes require water to maintain their stability and catalytic activity, revealing novel enzymatic properties in anhydrous environments While enzymes are typically understood to function in aqueous conditions to preserve their active conformations, their catalytic activity is influenced by the amount of water bound to them.
Enzymes require only a few essential water molecules or a small hydrate coating for catalysis, highlighting the importance of water in maintaining their native conformation and providing molecular lubrication for flexibility Interestingly, enzymatic activity in hydrophobic solvents, which are immiscible with water, is significantly higher than in hydrophilic solvents like pyridine, acetone, and formamide This enhanced activity is due to the ability of hydrophobic solvents to supply more water to the enzymes, facilitating their function.
Some distinctive benefits of using enzymatic catalysis in non-aqueous (organic) solvents compared to aqueous solvents include: 13
Increased solubility of most organic compounds and certain apolar cofactors, as well as relative ease of product recovery from organic solvents compared to water
New reaction pathways that are unattainable in water due to kinetic or thermodynamic barriers can be explored, such as reversing hydrolysis to promote the synthesis of apolar esters.
pH significantly impacts enzymatic activity in aqueous solutions; however, it does not affect enzymes in anhydrous solvents Instead, the catalytic properties of enzymes are influenced by their "pH memory," which refers to the pH of the last aqueous solution they encountered.
The stability of enzymes significantly improves at higher temperatures when using non-aqueous solvents, as these solvents minimize harmful reactions like the deamidation of Asn/Gln residues and the hydrolysis of peptide bonds that typically occur in water.
Possible alternation of enzyme specificity such as substrate, enantiomeric, prochiral, regio- and chemoselectivities by switching from one solvent to another
Permanent enzyme complexes imprinted with ligand in organic solvents, which called
“molecular memory” effect; can increase enzyme’s activity and selectivity, whereas this effect is washed if placed in water
Enzymatic catalysis in non-aqueous solvents differs significantly from traditional aqueous conditions due to variations in molecular behavior This shift has broadened the landscape of biocatalysis, revealing unexpected outcomes and expanding the potential applications of enzymes beyond conventional settings.
The exploration and advancement of highly active enzymes in non-aqueous solvents, including traditional organic solvents and innovative alternatives like ionic liquids, deep eutectic solvents, and supercritical fluids, present significant opportunities for diverse applications.
Lipase catalysis in enantioselective synthesis
Nature is rich in biologically active and structurally complex compounds that have posed challenges to organic chemists for centuries A significant issue in modern chemistry is stereochemistry, particularly enantioselective synthesis, which is crucial in pharmaceuticals due to the differing biological activities of enantiomers and diastereomers Living organisms are made up of chiral biomolecules, with proteins consisting of L-amino acids and carbohydrates made of D-sugars, leading to physiological processes that often involve only one enantiomer The other enantiomer or stereoisomers may exhibit no activity or even toxicity Enzymes, known for their high enantioselectivity, have garnered interest as environmentally friendly methods for enantioselective synthesis.
Lipases, classified as triacylglycerol acyl hydrolases (EC 3.1.1.3), are the most widely utilized biocatalysts in organic synthesis due to their broad specificity, enhanced stability in non-aqueous environments, and ease of preparation without the need for cofactors They are readily available commercially and are distinguished from carboxyl esterases (EC 3.1.1.1) by their preference for triglycerides over water-soluble substrates Additionally, lipases can catalyze reversible hydrolytic reactions in non-aqueous systems, enabling them to facilitate (trans)esterifications and the formation of esters from acyl donors and alcohols, along with various other versatile transformations.
Lipases are utilized in the preparation of enriched enantiopure compounds through various methods, including the kinetic resolution (KR) and dynamic kinetic resolution (DKR) of racemates, as well as the desymmetrization of prochiral compounds These lipase-catalyzed techniques play a crucial role in enantioselective synthesis, facilitating the production of high-purity enantiomers.
Scheme 1.1 Lipase-catalyzed methods in enantioselective synthesis for the preparation of enriched enantiopure compounds
Kinetic resolution (KR) involves the differential rates of two enantiomers (k R and k S) during the conversion of racemic substrates (SR and SS) into products using an enzyme as a chiral catalyst The enantiomeric ratio, or E-value, is a crucial metric for assessing the efficiency of the KR process, reflecting the enzyme's enantioselectivity for one enantiomer of a racemate This ratio is defined by the relative rate constants of the enantiomers at the reaction's initial stage Figure 1.1 illustrates the changes in enantiopurities for both the substrate (ee S) and product (ee P) as a function of conversion for two KR processes with E-values of 5 and 50, highlighting that a non-selective reaction yields an E-value of 1.
An E-value exceeding 20 is essential for achieving an acceptable resolution, and it is important to note that the E-value remains constant over time The mathematical equations for calculating enantioselectivity, which are detailed in Section 6.7, rely on the enantiomeric excess of the product (ee P), the unreacted substrate (ee S), and the conversion (c).
Figure 1.1 Theoretical plots for enantiomeric excesses of product and substrate against conversion for kinetic resolution with E-values 5, 50 27 Reprinted with permission from
The ideal Kinetic Resolution (KR) process is limited to a maximum yield of 50% for enantiomerically pure materials To enhance this yield, the Dynamic Kinetic Resolution (DKR) method is introduced, which integrates KR with in-situ racemization of the unreacted enantiomer, theoretically achieving 100% yield of a single enantiomer Typically, the racemization in DKR is performed chemically rather than enzymatically, as natural racemization by racemases is infrequently required due to stereospecific interactions However, a significant challenge of DKR is the difficulty in controlling reaction conditions, as the incompatibility of enzymatic and chemical catalysts in a one-pot reactor can complicate the process.
Lipases can theoretically achieve 100% desymmetrization of meso and prochiral compounds, as illustrated in Scheme 1.1(C) While they are recognized as a traditional method for the enantioselective desymmetrization of meso-diols and similar compounds, this approach is infrequently utilized due to the limited availability of suitable meso or prochiral substrates.
Structure and catalytic mechanism of Candida antarctica lipase B
Candida antarctica lipase B (CALB) is recognized as a highly effective biocatalyst In the late 1960s, Japan initiated the Japanese Antarctic Research Expedition to study Antarctica, during which Antarctic soils were sampled for microbiological analysis Researchers discovered the yeast Candida antarctica in one of these samples, leading to the isolation of the enzyme by Novozymes, which subsequently commercialized it as Novozym 435®, immobilized on macroporous acrylic polymer resin.
CALB is a stable enzyme with a molecular weight of 33 kDa and an isoelectric point of 6.0, functioning effectively in an aqueous buffer with a pH range of 3.5 to 9.5 Its denaturation temperature ranges from 50 to 60 °C, and it features a three-dimensional structure measuring approximately 30 Å x 40 Å x 50 Å Composed of 317 amino acids, CALB's polypeptide chain exhibits a folding pattern similar to other globular α/β hydrolases, characterized by seven central β-sheets flanked by ten α-helices The catalytic triad consists of Ser105, Asp187, and His224, and unlike other lipases with a functional lid, CALB contains only a small flexible loop, which prevents the interfacial activation phenomenon The active site is accessible to external solvents through a narrow channel with a hydrophobic wall.
Figure 1.2 The overall structure of Candida antarctica lipase B (1TCA 38 - PDB code)
CALB, like other lipases, utilizes a reaction mechanism similar to that of serine proteases, characterized by a catalytic triad of Ser-His-Asp at its active site In the free enzyme, the aspartate residue coordinates with histidine, which acts as a charge relay, facilitating the withdrawal of a proton from the nucleophilic serine This activation enables serine to function as a nucleophile, attacking the ester carbonyl The resulting tetrahedral intermediate's oxyanion is stabilized by an oxyanion hole formed by Thr40 and Gln106.
Scheme 1.2 The catalytic mechanism of CALB based on Ser-His-Asp triad with an oxyanion hole (Thr40 and Gln106).
Substrate selectivity and chiral recognition by Candida antarctica lipase B
An almost flat hydrophobic wall (10) and a flexible pseudo-lid (5) host the opening of the active site, where the catalytically active Ser105 is situated at the bottom of a narrow and
The CALB enzyme features a deep funnel-like active site pocket measuring approximately 10 Å x 4 Å x 12 Å, with hydrophobic amino acids lining the inner walls and three hydrophilic residues near the catalytically active Ser105 The unique physical constraints and hydrophobic characteristics of this cavity, along with the stabilization of tetrahedral and acylated intermediates, significantly influence substrate selectivity Compared to other lipases, CALB's active site is relatively small, which contributes to its high substrate selectivity X-ray crystallographic analysis reveals that the active site contains two binding sites: one for acyl and another for alcohol moieties, with the acyl site being more spacious Consequently, CALB is anticipated to have broad specificity for acyl donors while demonstrating a much higher selectivity for alcohol substrates.
Figure 1.3 a) The active site of CALB bound with inhibitor (c), b) The site view of active site, of which Ser105 is coloured in green
This thesis explores the stereoselectivity of CALB-catalyzed transesterification for producing chiral compounds through the kinetic resolution of racemic alcohols CALB exhibits a broad substrate specificity, enabling the synthesis of both primary and secondary alcohols with various structural types, including aliphatic, aromatic, and allylic compounds The kinetic resolution of secondary alcohols typically yields significantly higher enantioselectivity compared to primary alcohols, as the hydroxyl group in secondary alcohols is directly attached to the chiral carbon, allowing the lipase to effectively differentiate between enantiomers In contrast, the lower enantioselectivity observed with primary alcohols can be attributed to specific factors.
10 remote position of chiral center from the reaction center The lipase show no activity towards tertiary alcohols due to their steric hindrance
CALB exhibits enantiopreference for sec-alcohols in accordance with Kazlauskas’s rule, where the R enantiomer is favored when the larger substituent holds higher priority in the CIP nomenclature The enzyme features a stereoselective pocket, formed by Thr42, Ser47, and Trp104, which enhances its selectivity for various sec-alcohols However, this pocket can only accommodate substituents up to the size of an ethyl group; significant reductions in both activity and selectivity occur when larger substituents are present.
Figure 1.4 Active site model for lipases derived from Kazlauskas’ rule 49 and applied to the resolution of racemic secondary alcohols.
Pressurized carbon dioxide as reaction solvents
Supercritical and liquid CO 2
Supercritical CO2 is an appealing green reaction medium for biocatalysis due to its low critical point of 7.4 MPa and 31°C, which aligns well with many natural enzymes Its gas-like low viscosity and high diffusivity enhance diffusion-limited reactions while maintaining liquid-like solvation properties Additionally, liquid CO2, sometimes called subcritical CO2, can be utilized under moderate conditions of 4.5 MPa and 10°C, offering low polarity advantages.
Figure 1.5 Phase diagram of CO2 and image of its density at different temperature and pressure 59 Reprinted with permission from Elsevier publisher
Supercritical and liquid CO2 serve as excellent solvents due to their low surface tension, enabling complete removal from reaction mixtures and facilitating product recovery without solvent contamination As a non-toxic, non-flammable, colorless, and tasteless solvent, CO2 is particularly appealing for extracting natural products in biotransformation processes within the food and pharmaceutical industries.
Liquid and supercritical carbon dioxide (scCO2) are promising alternative media; however, their low polarity significantly limits their ability to dissolve polar or high molecular-weight organic compounds As a result, most research utilizing solely liquid and scCO2 has focused on small hydrophobic molecules, restricting their application in various industrial settings.
CO 2 -expanded liquids
A CO2-expanded liquid (CXL), 60,61 is defined as the condensed phase of a mixture of compressed CO2 gas and a liquid (usually an organic liquid) that dissolves large amounts of
CO2 CO2-expanded liquids offer lower reaction pressures with wider temperature ranges than scCO2, with much better solvent power than liquid CO2 due to the componential liquid
Most commonly used liquids experience volume expansion under CO2 pressure, with the extent of this expansion largely dependent on the solubility of CO2 in the liquids Jessop and Subramanian have classified CXL systems into three categories based on their mutual solubility with CO2 Notably, Class II CXL systems can dissolve significant amounts of CO2 at high pressures, resulting in volume expansions several times greater than their initial volume This dissolved CO2 alters the physical properties of the liquid, making it less viscous and more diffusible, which enhances mass transport properties Additionally, the liquid component increases the solubility of polar compounds.
Figure 1.6 Green and sustainable use of carbon dioxide to tune the physical properties of solvents 63 Reprinted with permission from ACS publisher
CXLs offer a sustainable solution by addressing the high working pressure challenges of supercritical CO2, leading to lower equipment installation costs and reduced energy consumption Additionally, CXLs significantly minimize the environmental impact by replacing organic solvents with eco-friendly CO2, resulting in less organic waste.
Use of pressurized CO 2 solvents in biocatalysis to promote sustainable chemistry
Despite extensive research on supercritical CO2 for organic synthesis and biocatalysis, liquid CO2 has traditionally been viewed as a "poor" solvent, leading to limited studies on its practical application in biocatalysis One significant advantage of liquid CO2 is its ability to be maintained at relatively low pressures, which reduces the need for expensive high-pressure equipment required for supercritical CO2 reactions Additionally, its low temperature usage may enhance enantioselectivity, making it a promising alternative solvent Consequently, this thesis explores the potential of liquid CO2 as a viable solvent for biocatalysis in Chapter 2.
While CXL systems align with certain green chemistry and sustainability principles, like minimizing organic solvent waste and energy consumption, they typically rely on petroleum-derived volatile organic compounds, which are finite resources.
14 often harmful to the environment Therefore, classical CXLs cannot be adequately satisfactory as green solvents
The rising interest in bio-based solvents highlights their potential as renewable alternatives to petroleum-derived solvents These eco-friendly solvents offer significant environmental benefits, including sustainable production, enhanced biodegradability, and reduced toxicity One notable example is 2-methyltetrahydrofuran (MeTHF), which can be sourced from renewable materials like furfural or levulinic acid, and is increasingly being used as an alternative solvent in various organic synthesis processes, including biocatalysis.
Medina-Gonzalez et al introduced the idea of combining supercritical CO2 (scCO2) with a non-volatile bio-based liquid to create a biphasic system However, there have been no reports on the use of a bio-based liquid as a homogeneous medium for any reaction involving crosslinking (CXL) This thesis explores the potential of a CO2-expanded bio-based liquid as a sustainable and efficient solvent for enzymatic reactions, as detailed in Chapter 3.
The study highlights the importance of using pressurized CO2 as a solvent for biocatalysis, emphasizing its ability to enhance the biocatalytic activity of lipase, particularly with bulky substrates, compared to traditional organic solvents Additionally, CO2 can modify the solvent properties of bio-based liquids that are typically unfavorable for lipase catalysis, transforming them into effective solvents Therefore, pressurized CO2 emerges as a highly suitable and promising option for biocatalysis applications.
The main construction of this thesis is summarized as below:
Chapter 1 provides an overview of biocatalysis in non-aqueous solvents, focusing on the CALB-catalyzed kinetic resolution of sec-alcohols to produce enantiopure compounds It highlights the significance of using pressurized CO2 as an alternative solvent in the pursuit of sustainable chemistry.
Chapter 2 explores the use of liquid carbon dioxide as a reaction medium for lipase-catalyzed kinetic resolution of rac-1-phenylethanol, demonstrating its feasibility and compatibility in both batch and continuous-flow reactors.
In Chapter 3, we explore the use of compressed CO2 to enhance the solvent properties of various bio-based liquids, creating optimal conditions for lipase catalysis By modulating the physicochemical characteristics of these solvents, we can significantly improve lipase activity, leading to more efficient catalytic processes.
Chapter 4 Molecular behaviour of Candida antarctica lipase B in pressurized carbon dioxide
In this chapter, the underlying mechanism of crucial role of CO2 to induce the acceleration phenomenon was studied by both experimental investigation and molecular dynamics simulation
Broadening substrate scope of lipase catalysis
2 Liquid carbon dioxide: Broadening substrate scope of lipase catalysis
This chapter explores the kinetic resolution of rac-1-phenylethanol using immobilized lipase B from C antarctica (Novozym 435) with vinyl acetate as an acyl donor, focusing on a model reaction in liquid CO2 The study compares the effectiveness of this process in liquid CO2 to that in traditional organic solvents and evaluates the practical applications of liquid CO2 in continuous flow bioreactors.
The investigation revealed that the lipase expanded its substrate specificity to include bulky phenyl alkyl sec-alcohols, a notable finding since the enzyme previously exhibited no reactivity towards these substrates in conventional solvents.
CALB catalyzed KR of 1-phenylethanol in organic solvents and in liquid CO 2
The kinetic resolution (KR) of rac-1-phenylethanol catalyzed by CALB was conducted in liquid CO2 and various conventional organic solvents using a batch reactor Vinyl acetate served as the acylating agent for irreversible transesterification, as the resulting vinyl alcohol tautomerizes irreversibly to acetaldehyde To ensure accuracy, all reactions were carried out in identical reactors and were vigorously stirred with a magnetic bar to mitigate mass transfer effects.
Scheme 2.1 KR of rac-1-phenylethanol by CALB
The activity of lipase is significantly influenced by solvent hydrophobicity, as indicated by the log P value, which measures the solvent's ability to partition between an octanol-water mixture Generally, more hydrophobic solvents yield higher results Notably, CALB demonstrated the highest transesterification activity and exceptional enantioselectivity (ee p>99%) in liquid CO2, outperforming hexane and toluene.
The study investigates the impact of various solvents on the CALB-catalyzed kinetic resolution (KR) of 1-phenylethanol, using specific reaction conditions: 0.83 mmol of substrate, 5.4 mmol of vinyl acetate, 5 mg of Novozym 435®, and 10 ml of solvent at 20°C for 2 hours under a pressure of 6.5 MPa for liquid CO2 The results show that the enantiomeric excess of the (R)-acetate was consistently excellent, exceeding 99% in all tested media However, it was noted that no reaction occurred in certain conditions due to inadequate mixing of the enzyme and substrate, attributed to the significantly higher density of the solvent compared to the immobilized enzyme The dielectric constants of selected solvents were also evaluated, though specific data for some solvents was not available.
(20 o C, 6 MPa) 1.48, hexane 1.88, isooctane 1.94, toluene 2.38, i-Pr2O 3.88, CHCl3 4.81, THF
Liquid CO2 exhibits behavior akin to hydrocarbon solvents with low polarizability, though its log P value remains unreported To compare polarity, the dielectric constants of selected solvents were utilized The high activity of lipase in liquid CO2 and in solvents with elevated log P values may be attributed to the inability of hydrophobic solvents to remove essential water molecules from the enzyme's outer layers Despite being prepared in dry form, lipase requires residual water to maintain its catalytic activity.
The kinetic resolution (KR) of rac-1-phenylethanol by CALB was examined in various medium systems, including hexane, liquid CO2, and a solvent-free environment at 20°C Among these, hexane emerged as the most effective organic solvent for the CALB-catalyzed KR Notably, the lipase exhibited enhanced activity in liquid CO2, achieving a conversion rate of 50% after 12 hours This indicates that the enzyme maintains its activity in liquid CO2 over extended reaction periods under relatively high pressure.
18 demonstrated its robust activity when used in solvent free system, which afforded a relatively high conversion, and in hexane (about 30% and 35% after 6h, respectively)
Figure 2.2 The time courses of the KR of 1-phenylethanol in different nonaqueous media
Reaction conditions: Substrate 0.83 mmol, vinyl acetate 5.4 mmol, Novozym 435 5 mg, with or without 10 ml solvent, 20 o C, pressure for liquid CO2 6.5 MPa Enantiomeric excess of the
(R)-acetate was excellent (ee p > 99%) in all cases.
The impact of temperature on the CALB-catalyzed kinetic resolution of rac-1-phenylethanol was examined across various media, including hexane, liquid CO2, supercritical CO2 (scCO2), and a solvent-free system The results indicated that the reaction rate consistently increased with rising temperatures, while maintaining high enantioselectivity (ee p > 99%) Notably, transesterification in scCO2 at 40°C and 10 MPa demonstrated similar activity to that in hexane, achieving approximately 35% conversion after 2 hours However, a direct comparison of the effects of liquid CO2 and scCO2 on lipase activity is complicated due to the differing temperatures involved.
The effect of temperature on the CALB-catalyzed kinetic resolution (KR) of 1-phenylethanol was investigated under specific reaction conditions The experiment utilized 0.83 mmol of the substrate, 5.4 mmol of vinyl acetate, and 5 mg of Novozym 435, with or without 10 ml of solvent, at a temperature of 20°C for 2 hours, and maintained a liquid CO2 pressure of 6.5 MPa The study focused on measuring the enantiomeric excess resulting from these conditions.
(R)-acetate was excellent (ee p > 99%) in all cases.
On the other hand, pressure has negligible influence on reaction rate of the lipase catalyzed
The kinetic resolution (KR) of rac-1-phenylethanol demonstrated excellent enantioselectivity, with all enantiomeric excess (ee) values exceeding 99% Additionally, research by Csajagi et al indicated that varying pressure levels between 0.1 and 12 MPa did not significantly influence the productivity or enantioselectivity of CALB during the KR of rac-1-phenylpropan-2-ol in a mixed solvent system comprising hexane, THF, and vinyl acetate.
In liquid CO₂ Solvent free
Figure 2.4 Effect of the pressure on CALB catalyzed KR of 1-phenylethanol Reaction conditions: Substrate 0.83 mmol, vinyl acetate 5.4 mmol, Novozym 435 5 mg, 10 ml solvent,
20 o C, 2 h Enantiomeric excess of the (R)-acetate was excellent (ee p > 99%) in all cases.
The use of liquid CO 2 in continuous flow reactors for large-scale biosynthesis of
KR of 1-phenylethanol in a continuous packed – bed reactor using liquid
In an ideal continuous packed-bed reactor (CPBR), back-mixing is absent, allowing enzymes to interact with a decreasing substrate concentration gradient and an increasing product concentration gradient CPBRs are optimal for processes characterized by product inhibition, substrate activation, and reaction reversibility Additionally, the desired extent of reaction can be attained by utilizing an appropriately sized ideal CPBR.
To optimize the KR reaction using liquid CO2 in a CPBR, an experimental setup was established utilizing Novozym 435 The setup involved packing 1.4 g of immobilized CALB into a pressure-resistant stainless steel column measuring 3.94 mL in volume and 28.5 cm in length.
CO2 gas was introduced into the vessel using a CO2 pump until the target pressure was reached and maintained by a back pressure regulator Subsequently, a substrate solution was continuously pumped by an HPLC pump into a turbulent mixer, which consisted of a 0.5 mL tube filled with cotton, prior to its entry into the bioreactor The outflow from the reactor was collected and analyzed using gas chromatography (GC) to assess the enantiomeric excess of both the substrate (ee s) and the product (ee p).
Figure 2.8 Experimental apparatus of a CPBR using Novozym 435
The system operated successfully for three cycles, producing enantiopure acetate and alcohol products while achieving a low E-factor of less than 0.3, significantly minimizing waste This aligns with the green chemistry principle of waste prevention, where an ideal E-factor is zero, indicating no waste generation In contrast, many chemical processes, especially in fine chemicals and pharmaceuticals, typically have E-factors exceeding 25 By utilizing no organic solvents and an adequate amount of vinyl acetate, this approach demonstrates a commitment to reducing environmental impact in manufacturing processes.
Table 2.1 CALB catalyzed KR of rac-1-phenylethanol with continuous flow of liquid CO2 and substrates into a packed-column reactor a
Recovered d (g) ee s(%) ee p (%) c(%) E-value E- factor e
The reaction conditions involved using 1.6 g of immobilized enzyme with a CO2 flow rate of 1.0 mL/min and a substrate flow rate of 0.02 mL/min, maintaining a volume ratio of rac-1-phenylethanol to vinyl acetate at 2:1, at a temperature of 20°C and a pressure of 6.5 MPa Each cycle comprised four steps: pressurization and stabilization for 1 hour, sampling for 8 hours, washing with liquid CO2 for 1 hour, and depressurization to 0.1 MPa followed by storage for 14 hours under ambient conditions The influx was precisely monitored using a balance, measuring the substrate mixture over time, while the recovered outflow was collected during the stationary state over 8 hours The E-factor was calculated by dividing the kilograms of waste produced (acetaldehyde and a small excess of vinyl acetate) by the kilograms of products obtained (the enantiopure acetate and alcohol), highlighting the process's recyclability.
CO2 gas and re-used immobilized enzyme are not included in the calculation
Comparison of the productivity of continuous-flow and batch reactors using
To compare the productivity of continuous-flow and batch reactors, the specific reaction rate (r) is utilized, representing the product formation per minute per gram of enzyme The specific reaction rate for a continuous-flow system (r flow) is determined by the product concentration ([P] in mmol mL -1), the flow rate (f in mL min -1), and the mass of the applied enzyme (m e in grams), as outlined in Equation 2.1.
A stirred batch reaction is defined by its specific reaction rate (r batch), which is determined by the product amount (n p in mmol), reaction time (t in minutes), and the mass of the enzyme used (m e in grams), as outlined in Equation 2.2.
To accurately compare the productivity of continuous-flow reactions with batch mode processes, it is essential to evaluate both systems at the same conversion levels.
The productivity of reactions can be assessed using the space-time yield (Y), which measures the amount of product produced per unit time per reactor volume This metric is crucial when the reactor's installation costs exceed the cost of enzymes For continuous-flow systems, the space-time yield (Y flow) can be calculated using the product concentration ([P] in mmol mL -1), flow rate (f in mL min -1), and reactor volume (V r in mL), as outlined in Equation 2.3.
A space-time yield value of a batch reactor (Y batch) can be calculated from the amount of the product (n p (mmol)), the reaction time (t (min)), and the reactor volume (V r (mL)) according to
Table 2.2 presents the productivity metrics, specifically the reaction rate (r) and space-time yield (Y), for CSTR, CPBR, and BSTR utilizing immobilized lipase and liquid CO2 for the kinetic resolution of 1-phenylethanol The BSTR achieved high specific reaction rates but exhibited low space-time yields, while the CSTR showed that reducing substrate concentration could enhance conversion at the cost of productivity The CPBR achieved an optimal kinetic resolution with 50% conversion and excellent enantiomeric excess (ee p>99%), outperforming the CSTR, which only marginally increased conversion from 29% to 38% with a significant drop in productivity Notably, the CPBR consistently produced 36 μmol of product per minute per gram of enzyme, achieving a remarkable space-time yield of 12.5 μmol min^-1 mL^-1, surpassing the other reactor types.
Table 2.2 Comparison of the productivity of continuous-flow and batch reactions a
Enzyme loading (mg) (Reaction type)
Residence time (min) c b (%) r c (mol min -1 g -1 )
In a flow reaction conducted at a stationary state with a liquid CO2 flow rate of 1.5 mL/min and a substrate mixture flow rate of 0.02 mL/min, comprising 1-phenylethanol and vinyl acetate at 20°C and 7 MPa, samples were collected after 60 minutes The conversion (c) was assessed using gas chromatography (GC), revealing an excellent enantiomeric excess of (R)-acetate (ee p > 99%) across all experiments The specific reaction rate (r) was calculated for both the flow and batch systems using designated equations, while the space-time yield (Y) was determined for each system according to their respective formulas.
The batch system demonstrated significantly higher specific reaction rates—two to four times greater—compared to continuous modes across all conversion degrees This advantage is likely attributed to the inefficient design of continuous reactors, where inadequate mixing leads to increased diffusional resistance For optimal performance in a Continuous Stirred Tank Reactor (CSTR), effective stirring is essential for achieving complete mixing.
The viscosity of the medium and the volume of the immobilized enzyme should typically be ten times less than the reactor volume, which may restrict the capacity of Continuous Stirred Tank Reactors (CSTR) when reactor size is a limiting factor Despite a lower specific reaction rate in the Continuous Packed Bed Reactor (CPBR), this system demonstrates potential for higher transformation rates with increased substrate loading Consequently, optimizing operational conditions could enhance its overall productivity.
Expanding substrate scope of lipase-catalysed KR of sec-alcohols by using liquid
Substrate specificity of CALB catalyzed transesterification
The study examined the impact of liquid CO2 on CALB activity through the transesterification of various aromatic, aliphatic, and allylic primary and secondary alcohols in both liquid CO2 and hexane CALB demonstrated remarkable activity towards primary and secondary alcohols with methyl substituents at the hydroxymethine center, achieving excellent enantioselectivity in both solvents The enzyme's catalytic efficiency is influenced by the positioning of large substituents at the active site, which facilitates optimal orientation of medium substituents into the stereoselectivity pocket However, substituting the methyl group with an allylic or ethyl group significantly reduced activity, despite maintaining high enantioselectivity (ee p >99%) Notably, transesterification of 1-phenylpropanol occurred more rapidly in liquid CO2 compared to hexane, with conversion rates of 25% versus 4%, respectively.
Only with the odd substrate 1-phenyl-2-propanol (4a) did the lipase give a considerable drop in both activity and selectivity, when compared with other methyl alkyl sec-alcohols (3a-
The decreased activity and selectivity of CALB toward 1-phenylbut-3-en-2-ol, with 48% conversion over 36 hours and 85% enantiomeric excess, can be attributed to the unfavorable spatial orientation of the benzyl group at the enzyme's active site This effect parallels findings from another study, which highlighted a similar trend in CALB's performance with 1-phenylprop-2-en-1-ol, achieving 52% conversion.
12h, 96% ee p) Notably, the reaction was accelerated more rapidly in liquid CO2 than in hexane for catalysing resolution of 1-phenyl-2-propanol (4a), with higher selectivity at lower temperature (5 o C).
Expanding substrate scope of CALB using liquid CO 2
Table 2.3 highlights a significant acceleration effect of liquid CO2 on lipase-catalyzed transesterification using 1-phenyl-1-propanol (entry 8), unlike other substrates with smaller medium substituents when compared to hexane This raises questions about whether this effect can be applied to other substrates with bulky side chains, which may pose limitations for this robust enzyme.
Table 2.3 CALB catalyzed transesterification of various alcohols in hexane or liquid CO2 a
Liquid CO2 25 >99 >200 a Reaction conditions: alcohol (0.40 mmol) with vinyl acetate (2.2 mmol) and Novozym 435
(10 mg) in 10 ml hexane or liquid CO2 (6.5 MPa) at 20 o C N.d.: not determined due to low conversion observed b Novozym 435 ® (5 mg) c Temperature 5 o C
29 this question, the transesterification of a series of phenyl sec-alcohols containing different size chains by CALB were examined in liquid CO2 medium and hexane as a control (Table 2.4)
The enzyme demonstrated activity for bulky 1-phenylalkanols (9-15a) in liquid CO2, but showed minimal activity in hexane, with only α-cyclopropylbenzyl alcohol (14a) being converted in that medium The lipase's activity remained consistent regardless of the alkyl chain length, although the (R)-selectivity significantly decreased for substrates 9-13a Previous research by Hult and colleagues aimed to enhance the steroselectivity pocket of the robust wild-type CALB through the W104A mutation, which improved activity for bulky alcohols like 12a, favoring the (S)-enantiomers However, this (S)-selective variant exhibited low enantioselectivity for 3b, suggesting that the variant's cavity accommodates the unsubstituted phenyl group, leading to an inversion in enantiopreference.
Table 2.4 CALB catalyzed KR of bulky phenyl alkyl sec-alcohols in liquid CO2 a
Substrate Solvent Time (h) Product Conv (%) ee p (%) E-value
Liquid CO2 10 98 127 a Reaction conditions: alcohol (0.2 mmol) with vinyl acetate (1.1 mmol) and Novozym 435
A study was conducted using 100 mg of a substance dissolved in 10 ml of hexane or liquid CO2 at 6.5 MPa and 20°C The conversion rate was not determined due to low observed conversion Additionally, Novozym 435 was utilized at a concentration of 20 mg The resultant alcohol was analyzed for chirality through chiral GC after hydrolyzing the corresponding ester with 4 M NaOH in methanol.
The bulky phenyl alkyl substrates exhibited significant differences in reactivity when analyzed in hexane compared to liquid CO2, with a preference for all (R)-enantiomers This suggests that the alcohols effectively position the phenyl group towards the enzyme's active site entrance while directing the alkyl substituents into the stereoselective pocket of the lipase Notably, the lipase maintained consistently high conversion rates, independent of the alkyl substituent lengths.
Substrate specificity of Burkholderia cepacia lipase catalyzed
The substrate specificity of Burkholderia cepacia lipase (BCL, Amano Lipase PS-C) was examined in both liquid CO2 and hexane The reactions involving substrates 3a, 8-11a, and 14a showed similar outcomes in both solvents Notably, BCL exhibited an expanded substrate scope in liquid CO2 with 2-methyl-1-phenyl-1-propanol (15a), achieving 17% conversion over 38 hours, compared to a mere 4% conversion in hexane over 126 hours.
Figure 2.9 Substrate specificity of Burkholderia cepacia lipase in liquid CO2 Reaction conditions: alcohol (0.2 mmol), vinyl acetate (1.1 mmol), Amano PS-C (10-50 mg), in 10 mL hexane or liquid CO2 (7.0 MPa), at 20 o C
1-phenylethanol (𝟑𝐚) 1-phenylpropanol (𝟖𝐚) 1-phenylbutanol (𝟗𝐚) 1-phenylpentanol (𝟏𝟎𝐚) 1-phenylhexanol (𝟏𝟏𝐚) α-Cyclopropylbenzyl alcohol (𝟏𝟒𝐚)
Preparative scale synthesis of chiral compounds in liquid CO 2
Burkholderia cepacia lipase (BCL, Amano Lipase PS-C) was used for the kinetic resolution of six substrates (9–13a, 15a), and Candida antarctica lipase B (CALB, Novozym 435) for substrate
14a on a preparative scale in a 10 mL vessel containing liquid CO2 (Table 2.5) The corresponding chiral alcohols (S)-9–15a and acetates (R)-9–15b were successfully obtained in high yields and enantioselectivities.
Conclusion
This chapter highlights the advantages of using liquid CO2 as a solvent for lipase catalysis, demonstrating that lipases exhibit greater activity in liquid CO2 compared to traditional organic solvents Notably, liquid CO2 proves to be particularly effective for reactions involving bulky phenyl alkyl sec-alcohols, which lipases typically cannot react with in standard solvents, showcasing its unique potential in enhancing enzymatic reactions.
The study explored the practical applications of liquid CO2 in two continuous flow bioreactors, CSTR and CPBR Utilizing immobilized lipase in a CPBR enabled large-scale synthesis of enantiopure products while minimizing waste generation, evidenced by a low E-factor.
Table 2.5 Preparative scale KR of bulky phenyl ankyl sec-alcohols in liquid CO2
Reaction conditions: alcohols (1.5 mmol) with vinyl acetate (2.0 mmol), and Amano PS-C
(100 mg) in 10 mL liquid CO2 (7.0 MPa) at 20 o C a Isolated yields b Determined by chiral GC analysis of the resultant alcohol after hydrolyzing the corresponding ester by 4 M NaOH in MeOH c Novozym 435 (100 mg)
CO 2 -expanded bio-based liquids: Modulating lipase activity by controlling physicochemical properties of solvent
3 CO 2 -expanded bio-based liquids: Modulating lipase activity by controlling physicochemical properties of solvent
Bio-based liquids are emerging as alternative solvents, offering environmental benefits such as sustainable production, improved biodegradability, and reduced toxicity However, certain bio-based liquids, particularly those with high polarity like 2-methyltetrahydrofuran (MeTHF), may not be ideal for biocatalysis due to diminished biocatalytic activity This chapter explores the use of CO2 to modify solvent properties, resulting in "CO2-expanded bio-based liquids" that serve as excellent solvents for lipase catalysis, effectively enhancing lipase activity.
CO 2 -expanded bio-based liquids as novel solvents for KR of sec-alcohols by
Volumetric expansion of several bio-based liquids with CO 2
The volumetric expansion of MeTHF was investigated by varying CO2 pressure at 20°C, using an 11 mL autoclave with sapphire-glass windows for visual measurement A 1.0 mL sample of MeTHF was magnetically stirred while CO2 was introduced, and total volumes were recorded at different pressures Notably, the volume increased significantly when the pressure reached 5.3 MPa, resembling the behavior of THF, where CO2 solubility is attributed to intermolecular interactions Additionally, similar measurements were conducted for two other bio-based liquids, diethyl carbonate and propylene carbonate, as well as methanol, using the same methodology.
Figure 3.1 The visual inspection of CO2 – expanded MeTHF (A) Initial 1.0 mL of MeTHF at 0.1
MPa of CO2, (B) 8.0 mL of CO2-expanded MeTHF at 5.8 MPa of CO2
Figure 3.2 Volume expansion of bio-based liquids and MeOH with CO2.
KR of rac-1-phenylethanol by CALB in CO 2 -expanded MeTHF and other CO 2 -
The kinetic resolution of rac-1-phenylethanol using Candida antarctica lipase B was conducted in CO2-expanded MeTHF at varying CO2 pressures, revealing increased enzymatic activity with higher pressures Specifically, pressures up to 4 MPa enhanced conversions due to elevated CO2 composition, while a significant volume expansion from 5 MPa to 6 MPa resulted in only a marginal decrease in conversion rates (17.5% at 5 MPa vs 16.8% at 6 MPa) Notably, the lipase maintained exceptional enantioselectivity across all tested conditions, with E-values exceeding 200.
MeTHF Diethyl carbonate Propylene carbonate MeOH
The study investigates the impact of CO2 pressure on the CAL-B-catalyzed transesterification of rac-1-phenylethanol in CO2-expanded MeTHF The reaction was conducted under specific conditions: 0.20 mmol of rac-1-phenylethanol, 0.53 mmol of vinyl acetate, and 5.0 mg of Novozym 435® in 1.0 mL of MeTHF, with CO2 pressure varying from 0 to 6 MPa at a temperature of 20°C for 1 hour Remarkably, the enantiomeric excess of the resulting acetate was found to be excellent, exceeding 99% across all tested media.
Higher CO2 composition leads to increased conversion rates due to enhanced transport properties, improved hydrophobicity of solvent systems, and diminished solvent inhibition from polar MeTHF molecules.
The transport properties of CO2-expanded methanol (MeOH) show significant improvements in diffusivity and viscosity as CO2 solubility increases For instance, the diffusion of benzene can increase by over 300% when pure methanol is replaced with 90% CO2 at 40°C and 15 MPa Additionally, the viscosity of the solution decreases by approximately 75% when transitioning from pure methanol to CO2-expanded methanol at 25°C and 5.7 MPa.
A key consideration in lipase-catalyzed transesterification is the low hydrophobicity of MeTHF (log P = 1.0), which is enhanced by the dissociation of non-polar CO2 molecules This lower hydrophobicity may lead to reduced lipase activity in hydrophilic solvents, as these solvents can strip essential water from the enzymes Consequently, higher esterification rates are typically achieved with more hydrophobic solvents.
Research indicates that CO2-expanded methanol demonstrates tunability in its properties Roskar et al measured the dielectric constant and found that the polarity of CO2-expanded methanol decreases as CO2 pressure increases Conversely, observations by Wyatt et al provide additional insights into this phenomenon.
Recent studies on the mixed CO2/methanol solvent revealed significant changes in Kamlet-Tarf parameters, with basicity (β) and dipolarity/polarizability (π*) experiencing a notable decline near the critical region Additionally, Sih et al observed a decrease in molar volume (V m) as CO2 pressure increased Kamlet et al defined the log P relationship as log P = 0.24–3.38β + 0.0266V m − 0.96π*, highlighting the interplay between these parameters.
Put all these together, log P of methanol/CO2 solution at 35 o C increases from −0.54 at 0.1 MPa to 0.29 at 7.5 MPa
Higher activity in solvent systems with elevated CO2 compositions may be attributed to polar solvent molecules like MeTHF, which can interact strongly with the enzyme and compete with the substrate for the active site However, this solvent-inhibition effect of MeTHF is significant only at bulk volumes; at low concentrations (up to five times the substrate concentration), the impact is negligible.
Table 3.1 Effect of MeTHF amount on CAL-B-catalyzed transesterification of rac-1- phenylethanol
Reaction conditions: 0.20 mmol 1-phenylethanol, 0.22 mmol vinyl acetate, 5 mg Novozym
435, reaction volume 10 mL, 6 MPa of CO2, 20 o C *: equivalent to 1 mL
The study of CO2-expanded bio-based liquids reveals significant findings regarding their volumetric expansion properties Organic carbonates, recognized as non-VOC-producing solvents, were effectively utilized alongside pressurized CO2 Notably, diethylene carbonate exhibited volumetric expansion comparable to MeTHF and MeOH, indicating its classification as a class II liquid, which efficiently dissolves CO2 In contrast, propylene carbonate demonstrated a maximum volumetric expansion of only 200% at 5.3 MPa before separating into another phase, categorizing it as a class I liquid with limited CO2 solubility This highlights a gap in data concerning class I liquids, emphasizing the need for further research.
Recent studies have identified water, glycerol, and various polyols as part of a specific class of solvents Notably, propylene carbonate has emerged as a potential candidate for further research as a new model for Class I liquids In the context of CAL-B catalyzed transesterification of rac-1-phenylethanol, CXLs demonstrated superior performance compared to solely bio-based liquids Additionally, systems utilizing MeTHF (log P = 1.0) and diethylene carbonate (log P = 1.1) outperformed the propylene carbonate system (log P = -0.41).
The study investigates the CAL-B-catalyzed transesterification of rac-1-phenylethanol using various CO2-expanded bio-based systems and sole bio-based liquids The reaction was conducted under specific conditions: 0.20 mmol of rac-1-phenylethanol, 0.53 mmol of vinyl acetate, and 5 mg of Novozym 435 in a total volume of 10 mL, either in a CO2-expanded environment or solely with bio-based liquids at 20°C for 1 hour Notably, the reaction volume was maintained at 2 mL, highlighting the efficiency of the bio-based systems in catalyzing the transesterification process.
3.1.2.1 The substrate specificity of CALB towards secondary alcohols in neat MeTHF and in
The substrate specificity of CALB was examined for various aliphatic and aromatic secondary alcohols in a CO2-expanded MeTHF system, revealing that reactions with substrates 3-6a and 16-17a achieved 2 to 7 times higher conversion rates compared to sole MeTHF Notably, CALB exhibited significant activity (29% conversion) and excellent enantioselectivity (E-value > 200) for the bulky rac-1-adamantylethanol (18a) in CO2-expanded MeTHF, while showing minimal activity in MeTHF This trend was also observed with the free form of CALB The interest in sec-alcohols with bulky moieties at the stereogenic center is notable, as traditional enzyme-mediated reactions often yield low activity due to steric hindrance, necessitating the use of unusual or engineered enzymes.
The bulky substrate rac-1-adamantylethanol (18a) demonstrated impressive reactivity in CO2-expanded MeTHF, prompting further investigation of the reaction across various concentrations of MeTHF.
Table 3.2 Comparison of CALB catalyzed KR of sec-alcohols in MeTHF and in CO2-expanded MeTHF using various substrates
Reaction conditions: alcohol (1-6a: 0.40 mmol; 7a: 0.10 mmol), vinyl acetate (0.53 mmol),
Novozym 435 (10 mg) in 10 mL MeTHF or 10 mL CO2-expanded MeTHF (MeTHF concentration 10 % v/v, 6.0 MPa) at 20 o C N.d.: not determined due to low conversions observed
The effect of MeTHF concentration on the CALB-catalyzed kinetic resolution of rac-1-adamantylethanol (18a) was investigated under various conditions The reaction utilized 0.10 mmol of rac-1-adamantylethanol, 0.53 mmol of vinyl acetate, and 10 mg of Novozym 435 in a total volume of 10 mL MeTHF The experiments were conducted at two different CO2 pressures: 0.1 MPa with varying MeTHF volumes (3 mL, 2 mL, 1 mL, 0.5 mL, and 0 mL) and 6 MPa CO2, maintaining a temperature of 20°C for 24 hours The results highlight the influence of MeTHF concentration on the reaction efficiency under different CO2 pressures.
In CO2-expanded MeTHF systems, increased CO2 concentrations enhance performance, while pure liquid CO2 results in lower conversion rates due to reduced substrate solubility Additionally, the lipase exhibited minimal activity towards rac-1-adamantylethanol in various conventional organic solvents, with only a slight conversion of 3.1% observed in hexane.
Table 3.3 Effect of organic solvent on CALB-catalyzed kinetic resolution of rac-1- adamantylethanol
Reaction conditions: 0.10 mmol rac-1-adamatylethanol 18a, 0.53 mmol vinyl acetate, 10 mg Novozym 435, 10 mL solvent, 20 o C, 24 h
To explain the significant difference between reactivity of CALB towards bulky substrate rac-1-adamantylethanol 18a in CO2-expanded MeTHF (and liquid CO2) and conventional
Modulating lipase catalysis in CO 2 -expanded bio-based liquids by tuning the
Lipase-catalysed KR of rac-1-adamantylethanol
The transesterification of rac-1-adamatylethanol 18a was studied using vinyl acetate as an acyl donor in MeTHF and CO2-expanded MeTHF, employing various lipases Due to MeTHF's low hydrophobicity (log P = 1.0), which may hinder lipase-catalyzed reactions in organic solvents, similar reactions were conducted in hexane (log P = 3.5) and CO2-expanded hexane for comparison To reduce the solvent effect of vinyl acetate, a minimal volume of 50 µL was utilized, as larger quantities could act as a co-solvent.
Table 3.4 Lipase-catalyzed transesterification of rac-1-adamatylethanol 18a
MeTHF Neat hexane CO₂-expanded hexane Conv
(%) E-value CALB (Candida antarctica, acrylic resin)
TL (Pseudomonas stutzeri, beige powder)
PS-D (Burkholderia cepacia, diatomite) 200 6.9 >200 200 5.1 >200 12 >200 LIP 301
In a study examining the reaction conditions for the conversion of rac-1-adamatylethanol 18a using various lipases, it was found that under a CO2-expanded liquid solvent system (10% v/v concentration at 6 MPa CO2, 20°C for 24 hours), the lipases tested—AK from Pseudomonas fluorescens, AH from Burkholderia cepacia, and Lipozyme from Mucor miehei—yielded minimal conversion rates of less than 2% across all solvent systems.
Table 3.4 reveals that most enzymes tested displayed minimal to no catalytic activity for the reaction, despite their strong activity towards general secondary alcohols like rac-1-phenylethanol 3a However, CALB and lipase TL demonstrated significant conversions in CO2-expanded liquids, with CALB and PS-C performing notably better in these systems compared to traditional solvents like neat MeTHF or hexane.
3.2.2 KR of rac -1-adamantylethanol in different CO 2 -expanded bio-based liquids
The transesterification of rac-1-adamantylethanol 18a was catalyzed by CALB and conducted using various bio-based liquids, which were compared to their CO2-expanded counterparts This study utilized seven bio-based liquids with differing levels of hydrophobicity, as detailed in Table 3.5, alongside hexane and vinyl acetate.
Table 3.5 Hydrophobicity of some neat bio-based liquids, hexane and vinyl acetate
2-methylfuran 1.8 p-cymene 4.0 limonene 4.5 hexane 3.5 vinyl acetate 0.3
Recent studies have identified three lignocellulosic biomass derivatives—γ-valerolactone, MeTHF, and 2-methylfuran—along with diethyl carbonate and citrus-derived alkanes (p-cymene and (+)-limonene) as effective solvents for various reactions Although (−)-limonene is not a natural source, it was included for comparative analysis with (+)-limonene A recent solvent selection guide has evaluated and ranked the green credentials of these bio-based liquids, all of which are classified as non-hazardous These solvents demonstrate a remarkable ability to solubilize CO2, achieving up to tenfold volumetric expansion, which categorizes them as class II liquids according to Jessop and Subramaniam.
Figure 3.6 Volume expansion of bio-based liquids, hexane and vinyl acetate with CO2 at 20 oC *Data taken from Figure 3.2 for comparison
The conversion of CALB catalysed transesterification of rac-1-adamantylethanol 18a was very low in all neat bio-based solvents (Figure 3.7 Effect of bio-based liquids with and without
In a study on the CO2-expanded transesterification of rac-1-adamantylethanol catalyzed by CALB, reaction conditions included 0.10 mmol of rac-1-adamantylethanol, 0.53 mmol of vinyl acetate, 10 mg of Novozym 435, and 10 mL of CO2-expanded liquid (10% v/v bio-based concentration at 6 MPa) at 20°C for 24 hours The highest conversion achieved was less than 3% in neat (+)-limonene, indicating that the bulkiness of 1-adamantylethanol limited CALB's catalytic efficiency In contrast, the lipase demonstrated significantly higher conversion rates across all CO2-expanded liquid systems tested.
2-methylfuran γ-valerolactone(-)-limonene𝘱-cymene hexane vinyl acetate
The impact of bio-based liquids on the conversion rates of CALB-catalyzed transesterification of rac-1-adamantylethanol 18a is illustrated in Figure 3.7, highlighting the differences between reactions conducted with and without CO2-expansion The experimental conditions included a substrate concentration of 0.10 mmol rac-1-adamantylethanol, 0.53 mmol vinyl acetate, and 10 mg of Novozym 435, all within a CO2-expanded liquid environment at a concentration of 10% v/v and a pressure of 6 MPa, maintained at 20°C for 24 hours Comparative data can be found in Table 3.4.
The study revealed that CO2-expanded MeTHF achieved the highest conversion rate at 29%, followed closely by CO2-expanded diethylene carbonate at 25% Notably, there were no significant differences in conversion between (+) and (−)-limonene during the CALB-catalyzed reaction Additionally, CO2-expanded γ-valerolactone showed a conversion of 3.1%, which, although lower than the others, was still higher than that of neat γ-valerolactone These findings highlight the essential role of CO2 expansion in enhancing conversion rates across various systems.
CO2 pressure enhances the activity of lipases, which are known to perform better in hydrophobic solvents like p-cymene or hexane for (trans)esterification However, in the absence of CO2, lipases struggled to catalyze the reaction of 1-adamantylethanol in both bio-based solvents and hexane The significant conversion of CALB in CO2-expanded systems suggests that pressurized CO2 may induce crucial structural or flexibility changes in the enzyme.
3.2.3 Effect of substrate bulkiness on conversion of CALB catalysis in neat MeTHF and in
The study compared CALB-catalyzed transesterification of non-bulky and bulky alcohols in CO2-expanded MeTHF and neat MeTHF Simple primary alcohols without bulky substituents achieved high conversions in both solvents However, bulky substituents, such as anthracenyl, allowed the reaction to proceed only in CO2-expanded MeTHF, a finding corroborated by similar observations with other compounds.
Table 3.6 Effect of substrate bulkiness on CALB-catalyzed transesterification in MeTHF and
The reaction conditions involved the use of alcohols (2-3a at 0.40 mmol; 20-23a at 0.10 mmol; and 24a at 0.02 mmol), vinyl acetate (0.53 mmol), and Novozym 435® (10 mg) in a solvent system of either 10 mL MeTHF or 10 mL CO2-expanded MeTHF (with a MeTHF concentration of 10% v/v at 6 MPa) at a temperature of 20°C Notably, some conversions were not determined due to low conversion rates observed, and for certain reactions, a reduced amount of Novozym 435® (5 mg) was utilized.
45 sterically-hindered bulky sec-alcohols (21-22a) The bulkiness of 1-naphthyl (21a) was also observed as the main cause for the very low reactivity of CALB in a hydrolysis reaction 108
CALB demonstrated limitations in substrate scope, as it was unable to mediate the reaction of rac-9-(hydroxyethyl) anthracene 23a in a CO2-expanded system However, when investigating the reaction with the molecule 1-(7-phenyl-1,7-dicarba-closo-dodecaboran-1-yl)ethanol 24a, which features a bulky icosahedral boron cluster, CALB achieved very high conversion rates in CO2-expanded MeTHF, while exhibiting poor conversion in neat MeTHF.
Activity of CALB as a function of CO 2 mole fraction (X CO₂ ) and dipolarity/polarizability (π*)
The conversion efficiency of CALB-catalyzed transesterification of rac-1-adamantylethanol is significantly influenced by CO2 presence in CO2-expanded bio-based liquids Experimental conditions included varying MeTHF concentrations and CO2 pressures, revealing that CALB activity is positively correlated with CO2 mole fraction in the MeTHF system Studies demonstrated that enzymatic activity increased with higher CO2 concentrations, while adjustments were made to account for temperature-related activity variations.
The results illustrated in Figure 3.8(B) indicate that the relative activity of CALB in CO2-expanded MeTHF can be effectively adjusted by controlling the CO2 mole fraction, with this modulation occurring consistently across varying temperatures.
Hydrophobicity of solvents significantly influences lipase activity, with lipases typically showing enhanced performance in more hydrophobic environments A recent study explored the connection between reaction rates and various solvent characteristics, including polarity, but did not establish any statistically significant correlations with solvent dipolarity or polarizability (π*), which relates to the solvent's capacity to stabilize a charge.
In this study, we analyzed the activity of CALB in CO2-expanded MeTHF by adjusting the CO2 concentration, rather than using various solvents with differing π* values This approach minimizes the influence of solvent structure, including size and functional groups Our findings, illustrated in Figure 3.8(C), reveal that enzyme activity increases as the π* values of the solvent systems decrease across different temperatures To account for temperature-induced activity variations, we normalized the relative activities based on lipase activity at neat MeTHF's π* values, as shown in Figure 3.8(D) The results indicate a strong correlation between CALB activity and the π* parameter, providing the first evidence that enzyme performance is significantly influenced by the physicochemical properties of the solvents, which can be modulated by CO2 concentration.
Figure 3.8 Tuning CALB activity in CO2-expanded MeTHF as a function of CO2 mole fraction
The study investigates the influence of XCO₂ and dipolarity/polaribility on the reaction conditions involving 0.10 mmol rac-1-adamantylethanol, 0.53 mmol vinyl acetate, and 10 mg Novozym 435 in 1.0 mL MeTHF The experiments were conducted under varying pressures ranging from 0.1 MPa to 9 MPa The reaction rate, measured in micromoles of substrate per milligram of enzyme per hour, was calculated at conversions below 10% for both XCO₂ and dipolarity/polaribility The results were normalized to a value of 1 for the reaction at specific conditions.
XCO₂=0 for each temperature ((B) and (D)
Conclusion
This chapter explores the use of compressed CO2 to enhance the solvent properties of bio-based liquids, creating CO2-expanded bio-based liquids that serve as effective media for lipase catalysis The innovative application of CO2 in conjunction with these liquids demonstrated that lipase can achieve high activity and excellent enantioselectivity in this new solvent environment Additionally, the activity of CALB in CO2-expanded MeTHF at varying temperatures was closely linked to the dipolarity and polarizability of the solvent system, showcasing the tunability of these solvents for improved catalytic performance.
Chapter 4 Molecular behaviour of Candida antarctica lipase B in pressurized carbon dioxide
4 Molecular behaviour of Candida antarctica lipase B in pressurized carbon dioxide
Chapters 2 and 3 demonstrated that CALB exhibited enhanced catalytic activity for bulky substrates in the presence of CO2, whether in pure liquid CO2 or CO2-expanded liquids This chapter investigates the fundamental mechanism by which CO2 facilitates this phenomenon through experimental studies and molecular dynamics simulations.