Immobilized Candida Antarctica Lipase Research Articles

Synthesis of dioctyl sebacate catalysed by cellulose-immobilized lipase

Dioctyl sebacate is a long carbon chain dibasic acid ester with methylene as the main body. It is widely used in plasticizers because of its good cold resistance and high viscosity. And it has excellent properties of non-toxicity and easy biodegradation, so it can be used as a potential new green bio-based plasticizer. At present, most of the preparation methods of dioctyl sebacate are chemical methods, which have many by-products and cause environmental pollution. In this article, cellulose microspheres were used as a carrier to immobilize Candida antarctica lipase B (CALB) to catalyse the esterification of sebacic acid and n-octanol to prepare dioctyl sebacate. Through single factor experiments, the lipase immobilized on cellulose microspheres was optimized to determine the best process conditions: add 500 µL CALB to the erlenmeyer flask, and then add 0.02 mol/L PBS buffer (pH 8.0). After being prepared into a 5 mL system, 0.1 g of cellulose microspheres were added as a carrier and reacted for 3 h at 35 °C and 180 r/min. Added 0.5 vt% polyethylene glycol diglycidyl ether (PEGDEG) to the system for crosslinking. The final immobilization rate of lipase immobilized on cellulose microspheres was 80.06%, and the enzyme activity was 288.07 U/g. Using the cellulose-immobilized lipase as a catalyst, in the toluene system, the molar ratio of n (sebacic acid)/n (n-octanol) substrate was 1:3.5, the amount of immobilized enzyme was 0.04 g, 4 Å molecular sieve 1.5 g, 40 °C, and 150 r/min for 30 h, the conversion rate of dioctyl sebacate was 76.45%.

Chemoenzymatic Synthesis of ABC-Type Enantiostructured Triacylglycerols by the Use of the p-Methoxybenzyl Protective Group.

This report demonstrates the first asymmetric synthesis of enantiopure structured triacylglycerols (TAGs) of the ABC type presenting three non-identical fatty acids, two of which are unsaturated. The unsaturated fatty acids included monounsaturated oleic acid (C18:1 n-9) and polyunsaturated linoleic acid (C18:2 n-6). This was accomplished by a six-step chemoenzymatic approach starting from (R)- and (S)-solketals. The highly regioselective immobilized Candida antarctica lipase (CAL-B) played a crucial role in the regiocontrol of the synthesis. The synthesis also benefited from the use of the p-methoxybenzyl (PMB) ether protective group, which enabled the incorporation of two different unsaturated fatty acids into the glycerol skeleton. The total of six such TAGs were prepared, four constituting the unsaturated fatty acids in the sn-1 and sn-2 positions, with a saturated fatty acid in the remaining sn-3 position of the glycerol backbone. In the two remaining TAGs, the different unsaturated fatty acids accommodated the sn-1 and sn-3 end positions, with the saturated fatty acid present in the sn-2 position. Enantiopure TAGs are urgently demanded as standards for the enantiospecific analysis of intact TAGs in fats and oils.

Relationship between the poly(glycerol adipate) prepolymers chain microstructure and linear viscoelastic properties of the elastomers

This paper investigates the influence of the time of enzymatic polymerization of glycerol and divinyl adipate on the chain microstructure and the thermal and linear viscoelastic properties (LVE) of the poly(glycerol adipate) prepolymer (pPGA). The immobilized Candida antarctica lipase B (CALB) was used as a regioselective catalyst in the polytransesterification reaction. A series of pPGA prepolymers were synthesized at different duration times of 8, 24, 48 and 72 h and the degree of polymerization (DP) and degree of branching (DB) were determined based on a quantitative NMR analysis. Due to lack in the literature of the described relationship between the properties of the pPGA prepolymers and the PGA elastomers, the main goal of the research was to correlate the microstructure of the prepolymers with linear viscoelastic properties of the elastomers obtained by thermal crosslinking of the respective prepolymers. The respective pPGA prepolymers were cured at 110 °C for 5 days to obtain PGA elastomers. The LVE properties of the PGA elastomers were investigated based on the master curves of the storage (G') and loss (G") modulus. The differences in DP and BD for the pPGA prepolymers translated into the PGA elastomer crosslinking densities and the rubbery plateau modulus (G0). It was shown for the first time that the minor differences in the structure of the pPGA prepolymers (DP of 13 and 21, and DB of 8.4% and 9.7% for the pPGA prepolymers synthesized for 8 and 24 h, respectively) translate into significant differences in the LVE of the PGA elastomers (G0 of 116 kPa and 50 kPa for the PGA elastomers derived from pPGA8 and pPGA24 prepolymers, respectively).

Clean production of isopropyl myristate: A cutting-edge enzymatic approach with a holistic techno-economic evaluation

This work aims to develop a simple, clean, and energy-efficient lipase-catalyzed method for the synthesis of isopropyl myristate (IPM). The enzymatic esterification between isopropyl alcohol and myristic acid was catalyzed using immobilized Candida Antarctica lipase. Response Surface Methodology (RSM) was applied to study the interactive effect of reaction conditions on IPM yield. The maximum experimental and predicted conversions were 92.4 % and 92.0 %, respectively. The optimized conditions were as follows: molar ratio of isopropyl alcohol to myristic acid molar ratio of 8:1, molecular sieves of 12.5 % w/w, a catalyst load of 4 % w/w, at a temperature of 60 °C and a reaction time of 2.5 h. Isopropyl myristate synthesized was isolated and fully characterized by GC–MS, FTIR, 1H and 13C NMR. Finally, to support the applicability perspective of this proposed method, a process diagram (PSD) was created using ASPEN PLUS software to simulate the production of IPM under the optimized conditions. The economic assessment of the whole process produced a positive net present value (NPV) of $44,797,732, return on investment (ROI) of 716.17 %, internal rate of return (IRR) of 110 %, payback period of 1.61, and a levelized cost of production (LCOP) of $1,777 per ton over a 14-year project lifespan. These results strongly suggest low-risk and high-profitability benefits to investing in this green route. Finally, the environmental impact was also assessed by calculating the quantity of CO2 generated from the proposed enzymatic process. The results showed a reduced emission rate of 0.25 ton CO2 eq. per ton of IPM produced. This underscores the lower environmental impact of this technology compared to traditional methods. Importantly, this study stands out as the first to conduct a comprehensive techno-economic assessment of the enzymatic synthesis of IPM, providing valuable insights into the economic viability and potential benefits of adopting this innovative and sustainable approach in the chemical manufacturing industry.

A paradigm shift for biocatalytic microreactors: Decoupling application from reactor design

Microreactors have been successfully applied to execute a broad range of biotransformations in flow. However, microreactors have typically been designed with a specific biotransformation or a specific biocatalyst immobilization method in mind, constraining their wider applicability. Furthermore, their design is typically either applicable for whole-cell or for enzyme biocatalysis, but not for both. We present a novel microreactor design which offers cartridge-like insertion of both immobilised enzymes and cells. A T-shaped lid opens and closes the reaction chamber (whilst leaving the rest of the microreactor unchanged), enables the easy insertion of immobilised biocatalysts, and thus allows the user to configure different reactor types. We demonstrated this novel concept showing three different reactor types: a hydrogel microreactor containing entrapped E. coli cells overexpressing transketolase (volumetric productivity of 2.23 ± 0.83 mmoll-ERY Lvoid1 min1), a packed-bed microreactor containing commercial beads with immobilised Candida antarctica lipase B (volumetric productivity of 317.69 ± 96.74 mmolBB Lvoid1 min1), and a micropillar microreactor containing surface-immobilised ω-transaminase (volumetric productivity of 0.08 ± 0.02 mmolACP Lvoid1 min1). The proposed design showed consistency and robustness for 10 consecutive T-shaped lid ‘open and close’ cycles and withstood the pressure of at least 4 bar. Design analysis further included Computational Fluid Dynamics models and Residence Time Distribution measurements. The presented design offers a standardised approach for multiple applications, underpinning process development and paving the way for off-the-shelf microreactor technology for biocatalysis.

Deacetylation of Mannosylerythritol Lipids in Hydrophobic Natural Deep Eutectic Solvents

AbstractMannosylerythritol lipids (MELs) are a promising group of biosurfactants due to their high fermentation yield, selfassembly and biological activity. During fermentation by Pseudozyma aphidis, a mixture of MELs with different levels of acylation is formed, of which the fully deacetylated form is the most valuable. In order to reduce the environmental impact of deacetylation, an enzymatic process using natural deep eutectic solvents (NADES) has been developed. We tested the deacetylation of a purified MELs mixture with immobilized Candida antarctica lipase B enzyme and 2‐ethylhexanol as co‐substrate in 140 h reactions with different NADES. We identified hydrophobic NADES systems with similar yields and kinetics as in pure 2‐ethylhexanol solvent. Our results indicate that deacetylation of MELs mixtures in NADES as a solvent is possible with yields comparable to pure co‐substrate and that hydrophobic NADES without carboxylic acid compounds facilitate the reaction to the greatest extent.

Regiospecificity of Immobilized Candida antarctica Lipase B (CAL-B) towards 2,3-Diacyl-1-O-alkyl Glyceryl Ether in Ethanol.

Highly pure 2,3-dioleoyl-1-O-alkyl glyceryl ether (DOGE), whose 1-position is a lipase-tolerant ether bond, was chemically synthesized and its detailed regioselectivity and acyl transfer were confirmed. During ethanolysis using immobilized Candida antarctica lipase B (CAL-B) with DOGE as the substrate, monooleoyl-1-O-alkyl glyceryl ethers (MOGEs) and a few 1-alkyl glyceryl ethers were formed upon consumption of the substrate. The structure of MOGE was confirmed using nuclear magnetic resonance spectroscopy and only the isomer of 2-MOGE was formed, indicating that CAL-B has complete α- regiospecificity. During ethanolysis, 3-MOGE was formed via acyl migration. These results indicate that the formation of 1-alkyl glyceryl ethers is not due to the imperfect regiospecificity of CAL-B, but rather due to ethanolysis of the formed 3-MOGE. The ethanolysis rate at the 3-α-position of DOGE was faster and the rate of acyl transfer was slightly slower for chain lengths greater than 14. These results show for the first time that both deacylation at the 3-position and acyl migration from the 2- to 3-position are affected by the structure of 1-position.

DIMENSIONING OF VINYLSULFONIC SUPPORTS FROM CASHEW APPLE BAGASSE BIOMASS IN THE IMMOBILIZATION OF LIPASES

In this work, the support, cashew apple bagasse (CAB), was chemically modified with divinyl sulfone (DVS), and it was evaluated to immobilize Candida antarctica lipase A (CAL-A). The best activation conditions of CAB support were defined by an advanced experimental design using the Taguchi method, assessing five factors at four levels (concentration of DVS, ionic strength, pH, temperature, and time). The support and biocatalyst (CAB-DVS-CAL-A) were characterized by Fourier transform infrared spectroscopy (FTIR), elemental analysis, thermogravimetry (TGA), scanning microscopy (SEM), fluorescence spectroscopy (XRF) and electrophoresis. The optimal conditions to activate the support were DVS concentration of 1.4 mol L-1 (3 mL of DVS in 20 mL of reaction volume), a concentration of sodium bicarbonate buffer at 5 mmol L-1, pH 3.0 at 30 °C for 12 h. The immobilization on CAB-DVS promoted increased thermal stability at 70 °C and different pHs of CAL-A. Therefore, the selected conditions allowed for a catalyst with a catalytic activity of 6.8 U g-1 and more stable than the free enzyme (CAL-A). This demonstrates that pretreated and DVS-activated CAB is a promising support for enzyme immobilization.

Effectiveness of different immobilized Candida antarctica lipase b (iCaLB) in the production of biobased oligoester derivatives from poly(butylene succinate) (PBS) and poly(butylene adipate) (PBA)

The use of lipases (Candida antarctica Lipase B, CaLB) immobilized on different solid supports for the preparation of biodegradable aliphatic polyesters poly(butylene adipate) (PBA) and poly(butylene succinate) (PBS) by esterification of unactivated dicarboxylic acids with 1,4-butanediol is presented. The immobilization of CaLB through absorption or a covalent attachment to different solid supports enhances their use as a catalyst for polycondensation reactions both in solution and in bulk. The results indicated that the different immobilized CaLBs were able to catalyze succesfully the polycondensation of both succinic and adipic acid with 1,4-butanediol, yielding the linear aliphatic polyesters PBS and PBA, respectively, by using a two-step polymerization process. Techniques such as nuclear magnetic resonance (NMR), relative molar mass obtained by gel permeation chromatography (GPC) and differential scanning calorimetry (DSC) were applied to compare the influence of the solid support on the obtention of PBA and PBS. Herein, it is shown that the nature of the solid support employed on each immobilized CaLB enzyme modifies the activity and effectivity towards polycondensation of PBA and PBS having a strong influence on the molecular weights and crystallinity.

Revisitation of the Preparation of (S,S)‐2,8‐Diazabicyclo[4.3.0]nonane Through Enzymatic Resolution.

AbstractA chemo‐enzymatic route for the preparation of (S,S)‐2,8‐diazabicyclo[4.3.0]nonane, a key chiral intermediate of Moxifloxacin, has been evaluated and improved. The activity of immobilized Candida antarctica lipase B was found to be increased four times for the selective hydrolysis of cis‐dimethyl 1‐acetylpiperidine‐2,3‐dicarboxylate at 50 °C as compared to 27 °C. The reaction has an optimum temperature of 50–60 °C and an optimum pH of 7.5. The reaction follows typical Michaelis‐Menten kinetics with Vmax,obsd=2472 units/g, Km,obsd=0.334 M, n=6, correlation coeff. r=0.995 at 50 °C. The reaction time and enzyme loading were reduced to 26 h at 15 % (w/w) as compared to 14 days at 50 % (w/w) loading. The enzyme was found to be recyclable up to 5 times. Further, the reduction of dimethyl pyridine‐2,3‐dicarboxylate was performed at a temperature of 27 °C under an H2‐filled balloon, in the presence of Pd/C and acetic acid, under atmospheric pressure to give cis‐(±)‐Dimethylpiperidine‐2,3‐dicarboxylate. We anticipate that our results will be beneficial to the pharmaceutical industry for the large‐scale production of (S,S)‐2,8‐diazabicyclo[4.3.0]nonane.

Enhancing lipases reactivity in benzylic acetates hydrolysis: impact of kosmotropic salts under non-conventional conditions

The effect of four kosmotropic salts additives, namely Na2CO3, Na2SO4, Na2HPO4, and NaCl, on the reactivity and selectivity of immobilized Candida antarctica lipase B (CAL-B) and a free lipase of Pseudomonas cepacia (PCL) during the hydrolysis of rac-4-(1-methoxyphenyl) ethyl and rac − 1-phenyl ethyl acetates (1a-2a) was investigated in this study. The study was carried out in two different mediums: non-aqueous conditions and in low water content media. The impact of the anionic counter-ions was examined in four solvents: nonpolar, semipolar, protic, and aprotic, with and without the aforementioned salts. In non-aqueous media, the addition of Na2CO3 significantly enhanced CAL-B hydrolysis rates, resulting in a conversion of 50% for 1a and 44% for 2a, with high enantioselectivities (E > 200). These effects were independent of the solvent hydrophobicity. In contrast, PCL required the presence of external water for effective hydrolysis of both acetates. The addition of salt additives had an adverse impact on PCL activity during the hydrolysis of 1a in hexane, toluene, and TBME solvents. However, when 2M2B was used, the inclusion of salt additives had a notable positive effect on the conversion rates, except for NaCl, which resulted in a lower conversion rate with a value of 15.5%. The presence of Na2CO3 improved the conversion rate during the hydrolysis of 2a in both hexane and TBME. Na2HPO4 further enhanced the conversion rate, reaching 44% in TBME. However, in 2M2B solvent, the addition of Na2HPO4 significantly reduced the enantioselectivity.

Scalable enzymatic polymerization and low-temperature post-polymerization of poly(butylene succinate): Process parameters and application

Poly(butylene succinate) is a semicrystalline bio-based and biodegradable polymer with good processability. However, using metal-based transesterification catalysts during chemical synthesis can be a drawback, especially for the biomedical and food packaging sector. An alternative approach was herein employed, using immobilized Candida antarctica Lipase B as a biocatalyst in a solvent-free process to synthesize PBS in line with green chemistry. The key process parameters (reaction temperature, pressure, time) were studied regarding the prepolymer’s molecular weight, thermal properties, and morphology. Thanks to the process's simplicity, scaling up followed, and a quantity of 20 g of PBS (Mn¯ 1700 g·mol−1) was received, thus filling the relevant gap in the open literature. The scaled-up grade was subsequently submitted to different sustainable post-polymerization methods (SSP, melt post-polymerization) to increase the molecular weight while maintaining the green character of the final product. The post-polymerized PBS (Mn¯ 3000 g·mol−1) was used as a carrier for the naturally occurring antioxidant flavonoid naringin. The release profile of naringin from the PBS nanocarrier was assessed, proving the enzymatically synthesized and upgraded PBS an effective matrix for high-purity encapsulation systems for the first time.

Chemoselective Lipase-Catalyzed Synthesis of Amido Derivatives from 5-Hydroxymethylfurfurylamine.

The acylations of furfurylamine and 5-hydroxymethylfurfurylamine (HMFA) have been studied finding immobilized Candida antarctica lipase B (CALB) as an ideal biocatalyst. CALB was used immobilized on two different supports (Novozyme 435 and EziG-CALB), with the polymer-coated controlled porosity glass carrier material from EnginZyme being an excellent carrier to yield an active and stable enzymatic preparation for the acylation of the primary amine group. The amount of the acyl donor in the reaction was a key factor to achieve the mono- and chemoselective N-protection of HMFA with large excess of ethyl acetate leading to the formation of the N,O-diacetylated product. Thus, a series of 16 nonactivated esters were used to selectively modify the amine group of HMFA, obtaining 9 hydroxy amides under mild reaction conditions and with quantitative yields through chromatography-free transformations. The influence of substrate concentration was studied, resulting in complete conversions in all cases after 22 h (100-1000 mM). Excellent results were observed at 100 and 200 mM of HMFA, while higher concentrations led to longer reaction times and, to some extent, the formation of the diacetylated product (up to 7% after 22 h at 1 M). After this optimization, a metric analysis was performed to confirm the high sustainability of the presented process (E-factor of 1.1 excluding solvents) upon intensification of the biotransformation to 1 g at 200 mM HMFA concentration. The possibility of obtaining orthogonally protected HMFA-derived amido esters has been achieved through a clean and sequential one-pot process using EziG-CALB, which involved the use of ethyl methoxy acetate as the nonactivated ester for N-acylation and the activated vinyl acetate for O-protection.

Transesterification of phosphatidylcholine with DHA-rich algal oil using immobilized Candida antarctica lipase B to produce DHA-phosphatidylcholine

Docosahexaenoic acid (DHA) enriched with phospholipids (PLs) (DHA-PLs) is a type of structured PL with good physicochemical and nutritional properties. Compared to PLs and DHA, DHA-PLs has higher bioavailability and structural stability and many nutritional benefits. To improve the enzymatic synthesis of DHA-PLs, this study investigated the preparation of phosphatidylcholine (PC) enriched with DHA (DHA-PC) via enzymatic transesterification of algal oil, which is rich in DHA-triglycerides, using immobilized Candida antarctica lipase B (CALB). The optimized reaction system incorporated 31.2% DHA into the acyl chain of PC and converted 43.6% PC to DHA-PC within 72 h at 50 °C, 1:8 PC: algal oil mass ratio, 25% enzyme load (based on total substrate mass), and 0.02 g/mL molecular sieve concentration. Consequently, the side reactions of PC hydrolysis were effectively suppressed and products with high PC content (74.8%) were produced. Molecular structure analysis showed that exogenous DHA was specifically incorporated into the sn-1 site of the PC by immobilized CALB. Furthermore, the evaluation of reusability with eight cycles showed that the immobilized CALB had good operational stability in the present reaction system. Collectively, this study demonstrated the applicability of immobilized CALB as a biocatalyst for synthesizing DHA-PC and provided an improved enzyme-catalyzed method for future DHA-PL synthesis.

Immobilized Candida antarctica lipase B as an sn-1,3 regiospecific biocatalyst for the interesterification of triacylglycerols with fatty acid ethyl esters.

Candida antarctica lipase B (CALB) is regarded as non-regiospecific. This study aimed to investigate the regiospecificity of CALB in the solvent-free interesterification of high-oleic sunflower oil with stearic acid ethyl ester for 1,3-distearoyl-2-oleoylglycerol (SOS)-rich fat preparation using a packed bed reactor. The content ratio of 1,2-distearoyl-3-oleoylglycerol (SSO) to SOS (denoted by SSO/SOS content) obtained using Lipozyme 435 (a commercially immobilized CALB; 0-4.1%), at residence times (1-32min) was similar to that obtained using Lipozyme RM IM (0-3.0%), but lower than that obtained using Lipozyme TL IM (6.0-39.4%). When immobilized on Lewatit VP OC 1600, Lipozyme CALB had an SSO/SOS content of 0-10.4%, which was greater than that of Palatase 20,000 L (0-1.1%) but was lower than that of Lipozyme TL 100 L (8.8-97.7%). Our findings suggest that immobilized CALB shows distinct sn-1,3 regiospecificity in the interesterification of triacylglycerol with fatty acid ethyl esters.

Performance evaluation studies of PEG esters as biolubricant base stocks derived from non-edible oil sources via enzymatic esterification

Polyethylene glycol esters (PEGEs) as, environment-friendly biolubricants were produced from different fatty acids derived from plant oils by enzymatic esterification reaction in a solvent-free system using commercially available immobilized Candida antarctica Lipase B (NS 435) biocatalyst. The raw materials used were a 1:1 ratio of oleic acid: linseed oil fatty acid, linseed oil fatty acid: ricinoleic acid, and oleic acid: ricinoleic acid as mixed fatty acids. Enzymatic esterification exhibited around 98.1%, 97.8%, and 98.5% maximum yield for three different PEGEs under the following optimum reaction conditions under vacuum: the molar ratio of mixed fatty acids to polyethylene glycol 200–2:1, reaction time - 4 h, catalyst loading - 10% (w/w), and reaction temperature - 60 °C. This study aimed to improve the thermo-oxidative stability and cold flow behavior of the esterified products. The synthesized esters were confirmed and characterized by acid value, Nuclear Magnetic Resonance Spectroscopy (NMR), and Fourier Transform Infrared Spectroscopy (FTIR). The fatty acid composition of PEGEs was determined by GC-FID (Gas Chromatography). Physicochemical properties, thermo-oxidative stability, and friction-reducing properties of the PEGEs were studied thoroughly and compared against conventional petroleum-based lubricants. Results revealed that the plant oil-based biolubricants had good lubricating properties like high flash point, low pour point, high viscosity index, high thermo-oxidative stability, and improved friction-reducing properties compared with petroleum-based hydraulic lube oil. Overall, the data indicated that enzymatically esterified biolubricants have significant potential as ISO VG-32, VG-46, and VG-68 grade biolubricant base stocks for various commercialized industrial applications.

An Evaluation of the Effect of Reaction Conditions in the Enzymatically Catalyzed Synthesis of Poly(ϵ‐Caprolactone)

AbstractPolycaprolactone (PCL) is a biocompatible polymer that degrades slowly in physiological environments. Properties, including flexibility and easy processability, endow PCL with a high potential for being used in biomedical and pharmaceutical applications. Typically, PCL is synthesized by high energy‐consuming ring‐opening polymerization processes catalyzed by metallic compounds. In addition, obtaining safe PCL requires tedious purification procedures that, in turn, reduce yield and generate large amounts of toxic emissions. As an alternative, PCL can be synthesized by employing lipases as enzymatic catalysts. However, the properties of the resulting polymer are highly dependent on the experimental conditions. In this paper, we studied the effect of synthesis variables, such as solvent, temperature, the concentration of monomer and catalyst, and lipase source, on the molecular weight of PCL and the reaction yield. According to the results, the highest molecular weight and monomer conversion were obtained when ϵ‐caprolactone was polymerized by 10 % of heterogeneous catalyst that contains Candida Antarctica lipase B in toluene at 70 °C. Under these conditions, polymerization exhibited controlled characteristics and allowed obtaining a polymer with a molecular weight of 14.12 kDa while the dispersion index was 1.34. The enzymatic catalyst was stable and allowed PCL formation over five usage cycles with minor activity loss. The reported findings suggest that immobilized Candida Antarctica lipase B is a suitable catalyst for the synthesis of PCL and could be employed at a large scale with minor energy consumption and shorter reaction times compared with the conventional processes.

Lipase-catalyzed production of pyridoxine monolaurate in solvent-free bioreactor system

This study demonstrated that solvent-free gas bubbling system enhanced production efficiency of pyridoxine monolaurate in the esterification catalyzed by immobilized Candida antarctica lipase B (Lipozyme 435). Volumetric productivity in solvent-free gas bubbling system (41.24 mmol/L/h) was 3.7 and 2.1-fold higher than those in conventional organic solvent system (11.10 mmol/L/h) and solvent-free system (19.86 mmol/L/h) using magnetic stirring, respectively. Among the three bioreactor systems, solvent-free gas bubbling system provided the best reusability of the lipase retaining 94.45 % of initial activity for six batch reactions. In the bioreactor system, 5-O-lauroyl-pyridoxine was regioselectively produced with maximum production of 371.17 mmol/L at 70 °C and 0.10 of substrate molar ratio ([pyridoxine]/[lauric acid]) for 9 h. Pyridoxine monolaurate exhibited interfacial activity at oil-water interfaces, suggesting it had emulsifying properties. Pyridoxine monolaurate is expected to be applied as a multi-functional emulsifier with nutritional values to replace both small molecule emulsifiers and pyridoxine hydrochloride in fortified beverages.

Continuous-flow biocatalysed kinetic resolution of 4-fluorophenyl-furan-2-yl methanol

Enantiomerically pure secondary alcohols are useful in the synthesis of several natural products and as active pharmaceutical intermediates (API). Due to the high demand for these chiral compounds, much progress has been made in the areas of asymmetric synthesis and catalysis. In this context, biocatalysis together with continuous flow technology can be a valuable tool for more versatile and sustainable methods, with lower cost, greater stereoselectivity and less environmental impact. This work aims to obtain an enantiomerically pure alcohol of industrial interest, (4 Fluorophenyl) (furan-2-yl) methanol (3), by performing a kinetic resolution using immobilized Candida antarctica lipase B (Novozyme 435, N435) under continuous-flow conditions. Initial study was carried out to optimize batch reaction conditions. The best results were obtained using isooctane as solvent, 37.7 mg of N435 and three equivalents of isopropenyl acetate as acyl donor at 60 °C for 24 h. Under these conditions, a conversion of 49% and 91 of enantiomeric ratio was obtained. Optimized batch conditions were translated to the continuous flow reactor leading to the desired product in 30 min of residence time, 47% conversion and an enantiomeric ratio of 61.

Biocatalytic Production of a Nylon 6 Precursor from Caprolactone in Continuous Flow.

6‐Aminocaproic acid (6ACA) is a key building block and an attractive precursor of caprolactam, which is used to synthesize nylon 6, one of the most common polymers manufactured nowadays. (Bio)‐production of platform chemicals from renewable feedstocks is instrumental to tackle climate change and decrease fossil fuel dependence. Here, the cell‐free biosynthesis of 6ACA from 6‐hydroxycaproic acid was achieved using a co‐immobilized multienzyme system based on horse liver alcohol dehydrogenase, Halomonas elongata transaminase, and Lactobacillus pentosus NADH oxidase for in‐situ cofactor recycling, with >90 % molar conversion (m.c.) The integration of a step to synthesize hydroxy‐acid from lactone by immobilized Candida antarctica lipase B resulted in >80 % m.c. of ϵ‐caprolactone to 6ACA, >20 % of δ‐valerolactone to 5‐aminovaleric acid, and 30 % of γ‐butyrolactone to γ‐aminobutyric acid in one‐pot batch reactions. Two serial packed‐bed reactors were set up using these biocatalysts and applied to the continuous‐flow synthesis of 6ACA from ϵ‐caprolactone, achieving a space‐time yield of up to 3.31 g6ACA h−1 L−1 with a segmented liquid/air flow for constant oxygen supply.