Pseudomonas Fluorescens Lipase Research Articles

Improved performance of Pseudomonas fluorescens lipase immobilized on a magnetic mesoporous solvothermal hybrid-nanocarrier and its application in ultrasonication assisted estolide synthesis

The exponential growth of the biotechnological market necessitates the exploring novel, economically viable materials for enzyme immobilization to enhance the biocatalyst performance. This study involves preparing a hybrid carrier with a magnetic mesoporous nature, using milled rice husk (MRH) and multi-walled carbon nanotubes (MWCNTs) via a one-pot solvothermal process at 200 °C for 6 h. The carrier, rich in-NH2 groups, was chemically bonded to Pseudomonas fluorescens lipase (PFL) using EDC/NHS cross-linkers. The synthesized support achieved a maximum PFL loading of 94.92 mg of protein per gram of support. Moreover, the maximum activity recovery was achieved at 1196 %, under the optimal conditions: immobilization temperature 25℃, pH 9.0, immobilization time 2 h and PFL mass concentration 4 mg/mL. Subsequently, the derived enzyme was used to catalyze ricinoleic acid esterification to synthesize estolide under ultrasound-assisted conditions. The optimized principal parameters for reaction temperature, reaction time, and immobilized lipase loading were respectively determined to be 48.99 °C, 1.60 h, and 3.09 % via response surface methodology. Ricinoleic acid conversion reached 65.23 % under the optimal conditions with the assistance of 40 % power and 40 kHz ultrasonic frequency. Furthermore, remarkable operational stability was also observed, with a retention rate of 49.28 %, even after 10 consecutive batches. This study demonstrates promising prospects for industrial-scale application using ultrasound as a fast and ecologically friendly procedure.

Simultaneous geraniol and citronellol transesterification using Pseudomonas fluorescens lipase for the production of fragrance and flavour esters: A kinetic study

This study aims to develop, for the very first time, a kinetic model for the simultaneous transesterification of geraniol and citronellol using Pseudomonas fluorescens lipase to produce their respective esters. This allows us to test the feasibility of the bioprocess and comprehend the enzyme behaviour, fine-tune process efficiency, and predict process performance. Geranyl and citronellyl esters are key ingredients in fragrance and flavour formulations in the food sector, as well as bioactive compounds for pest and insect repellents in the agricultural sector. Unlocking a greener and sustainable production of these terpene esters directly from plant-derived essential oil is crucial to meet the 2050 net zero targets to produce bio-based chemicals. The simultaneous transesterification of geraniol and citronellol in a batch reactor successfully achieved conversions of 66 % for citronellol and 94 % for geraniol within 5 h. Key optimum conditions were 1:8 molar ratio (geraniol:vinyl acetate), an enzyme loading of 10 wt % of geraniol, temperature of 40 °C, and a stirring speed of 250 RPM. The results indicated that simultaneous transesterification followed a ternary complex mechanism with competitive inhibition by alcohol. Initial testing of rose geranium oil under optimised conditions yielded 32 % citronellol and 50 % geraniol conversion after 30 h. Overall, this study shows promise as a green pathway utilising enzyme catalysis to selectively produce esters from diverse terpene mixtures and can be extended to terpene alcohols derived from flower essential oils.

Immobilization of Pseudomonas fluorescens Lipase on Hollow Poly(o-phenylenediamine) Microspheres and Its Application in the Preparation of Citronellyl Acetate

In order to address the challenges associated with free lipase in organic solvents, including aggregation, poor stability, and low catalytic efficiency, this study developed two types of poly(o-phenylenediamine) microspheres (solid and hollow) as supports for immobilizing lipase. The immobilization process utilized an adsorption method, with the poly(o-phenylenediamine) hollow microspheres being identified as the optimal support in a 2:5 enzyme-to-support ratio. On this basis, the lipase was immobilized by the covalent binding method. The immobilization conditions consisted of treating the support with 2% glutaraldehyde and immobilization at 40 °C for 2 h in pH 7.0 buffer. The specific activity of the immobilized enzyme was 5.3 times higher than that of the free enzyme. Covalent-binding immobilized lipase was also used for the preparation of citronellyl acetate by transesterification reaction, and, in optimized reaction conditions where the amount of immobilized enzyme was 0.1 g/mL, the reaction temperature was 50 °C and the shaking speed was 200 r/min during the reaction. Under these conditions, the citronellyl acetate yields can exceed 99% after 2 h. Furthermore, the stability of the immobilized lipase was investigated, and the residual activity of the immobilized enzyme was 95% after seven repetitions, while that of the free enzyme was only 70%. After 56 days of storage at room temperature, the immobilized enzyme retained 60% of its original viability, while the free enzyme retained only 31%.

Enantiodivergent Chemoenzymatic Dynamic Kinetic Resolution: Conversion of Racemic Propargyl Alcohols into Both Enantiomers.

Natural lipases typically recognize enantiomers of alcohols based on the size differences of substituents near the carbinol moiety and selectively react with the R enantiomers of secondary alcohols. Therefore, lipase-catalyzed dynamic kinetic resolution (DKR) of racemic secondary alcohols produces only R enantiomers. We report herein a method for obtaining S enantiomers by DKR of secondary 3-(trialkylsilyl)propargyl alcohols by using a well-known R-selective Pseudomonas fluorescens lipase in combination with a racemization catalyst VMPS4, in which the silyl group reverses the size relationship of substituents near the carbinol moiety. We have already reported R-selective DKR of the corresponding propargyl alcohols without substituents on the ethynyl terminal carbon, and the presence of an easily removable silyl group has enabled us to produce both enantiomers of propargyl alcohols in high chemical yields and with high enantiomeric excess. In addition, immobilization of the lipase on Celite was found to be important for achieving a high efficiency of the DKR.

Co-immobilization of lipase and laccase on agarose-based supports via layer-by-layer strategy: Effect of diffusional limitations

Co-immobilized enzymes present desirable advantages to performing cascade reactions when employed in one-pot processes, mainly because of the shorter lag times owing to better reagent diffusion across the active sites of the different enzymes. But high product yields require high enzyme loads, usually achieved with high surface area and porous supports, where diffusional limitations may be introduced or increased. Heterogeneous biocatalysts were prepared by co-immobilizing Pseudomonas fluorescens lipase (PFL) and Aspergillus sp. Laccase (Novozymes 51003) on two agarose-based supports, DEAE-Sepharose and Octyl-agarose. A layer-by-layer strategy was used by adding polyethyleneimine (PEI) to allow the attachment of the upper enzyme layer onto the lower adjacent layer. A final step, crosslinking with glutaraldehyde, was performed to prevent enzyme leaching under harsh environmental conditions. The heterogeneous biocatalysts showed lower temperature dependence and a significant increase in thermal stability at 50 ºC. No internal mass transfer effects were observed when a low protein load was used during co-immobilization (2 mgprotein/gsupport of each enzyme). However, increasing the enzyme load (more than 10 mgprotein/gsupport) promoted internal diffusion limitations. The layer-by-layer strategy defined in this work enabled the preparation of a multi-enzymatic heterogeneous biocatalyst with high activity, thermal stability, and no diffusional limitations, depending on enzyme concentration.

Rice straw enhancing catalysis of Pseudomonas fluorescens lipase for synthesis of citronellyl acetate.

Citronellyl acetate as an important flavor, can be effectively synthesized by lipase catalysis in nonaqueous system. But lipases usually behave low catalytic activity due to aggregation and denaturation of them in organic phase. To enhance the nonaqueous catalysis, based on the mechanism of lipases activated at water/oil (organic phase) interface, the inexpensive race straw was processed into powder and filaments on which Pseudomonas fluorescens lipase was immobilized by physical adsorption, used for synthesis of citronellyl acetate via transesterification of citronellol and vinyl acetate. Results showed that the desired loading was 10mg lipase immobilized on 30mg rice straw filaments or 25mg rice straw powder. When the two immobilized lipases were employed in the reaction system consisted of 1-mL citronellol and 2-mL vinyl acetate at 37℃ and 160rpm, the conversions all reached 99.8% after 12h. Under the reaction condition, the conversion catalyzed by 10mg native lipase was 85.1%. Undergoing six times of 8-h reuses in the organic system, the filament and power immobilized lipases had weak activity attenuation rates 0.36 and 0.32% h-1, lower than 1.52% h-1 of native lipase. Even at the room temperature and the static state without shaking and stirring, the rice straw filaments immobilized lipase could brought conversion 62.9% after 10h but the native lipase only gave 37.0%. Obviously, the rice straw, especially its filaments, is an inexpensive and available natural material to prepare immobilized lipase with desired catalysis in organic phase, meant significant potential in flavor industry.

Melia azedarach leaf powder stabilizing Pseudomonas fluorescens lipase to catalyze synthesis of geranyl acetate

To improve the stability of lipases in organic phases and promote the enzymatic synthesis, the inexpensive Melia azedarach leaf powder was taken as carrier for lipase immobilization, which was considered as solid “water” that could stabilize enzyme proteins. On the leaf powder, Pseudomonas fluorescens lipase was immobilized by physic adsorption in a column glass bottle and used for synthesis geranyl acetate via transesterification of geraniol and vinyl acetate. It is found that the immobilized lipase prepared in a mass ratio of 10 : 25 (enzyme: leaf powder, mg: mg) not only has a fine catalytic activation but also has an excellent nonaqueous stability in solvent-free nonaqueous reaction system composed of 1 mL geraniol and 2 mL vinyl acetate. Namely, the best loading of this immobilized lipase was 11.7 mg mL −1 in the reaction system in which, at 37°C and 160 rpm, the reaction conversion reached 90.1% after 10 h and achieved at 99.3% after 16 h. And after undergoing six times of 10-h reuses, the immobilized lipase had no decrease in the catalytic ability of converting geraniol except a little fluctuation 0.09% h −1 in conversion during reuse. Differently, the native lipase showed an activity attenuation 0.61% h −1 in the organic system in the reuse. Obviously, this immobilized lipase has been significantly improved in the nonaqueous stability, meant great potential in practical application. • Melia azedarach leaf powder is a useful carrier for lipase immobilization. • Immobilized Pseudomonas fluorescens lipase is desired to prepare citronellyl acetate. • Reuses show that the immobilized lipase has little decrease in nonaqueous activity. • The bottle with immobilized lipase is a simple bioreactor with great potential.

Preparation of mesoporous nanofilm constructed millimeter-sized macroporous SiO2 for lipase immobilization

A novel mesoporous nanofilm constructed millimeter-sized macroporous SiO 2 (MNCMMS) was prepared through a dual-templating method, using an epoxy resin macroporous polymer with three-dimensional skeleton structure as the shape and macropore structure directing agent and polyethylene glycol 20000 (PEG20000) as the mesopore porogen. MNCMMS was used as the support for immobilization of Pseudomonas fluorescens lipase (PFL) in order to overcome the limitations of the immobilized enzyme on mesoporous nanomaterials and to improve the performance of PFL. The immobilization conditions and the properties of the immobilized PFL were investigated. The structures and properties of the as-prepared materials were investigated by various characterization techniques. The results revealed that MNCMMS possessed two sets of three-dimensional continuous pass-through macroporous channels with uniform pore size (700 nm), large specific surface area (317 m 2 /g) and pore volume (89.2%), high mechanical strength (10.4 MPa), and that the macropore wall was a continuous mesoporous (pore size of 10 nm) nanofilm with the thickness ranging from 40 to 50 nm. Under the optimum conditions, the activity of PFL@MNCMMS reached to 4897 U/g and enzyme activity recovery achieved to 255%. Compared with free lipase, the stability and reusability of the immobilized lipase were significantly improved. Moreover, the immobilized lipase can be easily recovered from the reaction system for reuse. • A novel hierarchically structured porous SiO 2 (MNCMMS) has been prepared. • The activity of PFL immobilized on MNCMMS was significantly enhanced. • PFL@MNCMMS possesses high storage, operational and thermal stabilities. • The macroscopic size of PFL@MNCMMS significantly improves the recyclability.

Biodiesel production from alternative raw materials using a heterogeneous low ordered biosilicified enzyme as biocatalyst

BackgroundCumulative reported evidence has indicated that renewable feedstocks are a promising alternative source to fossil platforms for the production of fuels and chemicals. In that regard, the development of new, highly active, selective, and easy to recover and reuse catalysts for biomass conversions is urgently needed. The combination of enzymatic and inorganic heterogeneous catalysis generates an unprecedented platform that combines the advantages of both, the catalytic efficiency and selectivity of enzymes with the ordered structure, high porosity, mechanical, thermal and chemical resistance of mesoporous materials to obtain enzymatic heterogeneous catalysts. Enzymatic mineralization with an organic silicon precursor (biosilicification) is a promising and emerging approach for the generation of solid hybrid biocatalysts with exceptional stability under severe use conditions. Herein, we assessed the putative advantages of the biosilicification technology for developing an improved efficient and stable biocatalyst for sustainable biofuel production.ResultsA series of solid enzymatic catalysts denominated LOBE (low ordered biosilicified enzyme) were synthesized from Pseudomonas fluorescens lipase and tetraethyl orthosilicate. The microscopic structure and physicochemical properties characterization revealed that the enzyme formed aggregates that were contained in the heart of silicon-covered micelles, providing active sites with the ability to process different raw materials (commercial sunflower and soybean oils, Jatropha excisa oil, waste frying oil, acid oil from soybean soapstock, and pork fat) to produce first- and second-generation biodiesel. Ester content ranged from 81 to 93% wt depending on the raw material used for biodiesel synthesis.ConclusionsA heterogeneous enzymatic biocatalyst, LOBE4, for efficient biodiesel production was successfully developed in a single-step synthesis reaction using biosilicification technology. LOBE4 showed to be highly efficient in converting refined, non-edible and residual oils (with high water and free fatty acid contents) and ethanol into biodiesel. Thus, LOBE4 emerges as a promising tool to produce second-generation biofuels, with significant implications for establishing a circular economy and reducing the carbon footprint.

Enzymatic biodiesel production by hydroesterification using waste cooking oil as feedstock

Biodiesel production was investigated by hydroesterification using enzymatic catalysis in both hydrolysis and esterification reactions. Soybean oil resulting from food frying processes (WCO) was used as feedstock. Lipase from Geotrichum candidum was produced by submerged fermentation and used without prior purification as biocatalyst in WCO hydrolysis, which was optimized using a factorial design. A complete hydrolysis of WCO (44.1% mass fraction) was obtained after 80 min of reaction at 40 °C, performed in an emulsifier-free system with no buffer (40 °C and 900 rpm). In a subsequent step, FFA obtained from the hydrolysis reaction were esterified (40 °C, 200 rpm) using ethanol as acyl-acceptor (1:1.5) and Pseudomonas fluorescens lipase (PFL) immobilized on styrene-divinylbenzene (SDB) as biocatalyst (15% m/v). This biocatalyst was recycled for 6 cycles with no significant activity losses. The biodiesel composition was found by gas chromatography, and proton nuclear magnetic resonance (1H NMR) spectra and Fourier transform infrared spectroscopy (FTIR) were used to verify the quality and purity of biodiesel. Enzymatic biodiesel achieved FAEE content of 94.1 ± 0.5% and no traces of monoacyglycerol (MAG), diacylglycerol (DAG), triacylglycerol (TAG) or free glycerol were observed, indicating that the hydroesterification route produced high-quality biodiesel, despite the fact that WCO was used as feedstock.

Directed Evolution of Pseudomonas fluorescens Lipase Variants With Improved Thermostability Using Error-Prone PCR

Lipases catalyze the hydrolysis of fats and oils, and have been widely used in various industrial fields. However, bacterial lipases have a lower thermostability in industrial processes, which was a limiting factor in their industrial application. In this study, we obtained an improve variant of Pseudomonas fluorescens lipase (PFL) with enhanced thermostability using classical error-prone PCR. Wild-type PFL showed an optimal temperature and pH of 50°C and pH 7.5, respectively. Due to the low thermostability of PFL, a library containing over 3000 individual mutants as constructed using error-prone PCR. Screening for thermotolerance yielded the mutants L218P and P184C/M243C with Tm values of 62.5 and 66.0°C, which was 2.5 and 6°C higher than that of the WT, respectively. The combination of the two mutants (P184C/M243C/L218P) resulted in an approximately additive effect with a Tm value of 68.0°C. Although the increase of Tm was not substantial, the mutant also had dramatically increased methanol tolerance. Structural analysis revealed that the introduction of a disulfide bond between P184C and M243C and the substitution of Pro to reduce the flexibility of a loop increased the thermostability of PFL, which provides a theoretical foundation for improving the thermostability and methanol tolerance of lipase family I.1 to resist the harsh conditions of industrial processes.

Immobilization of lipase onto metal–organic frameworks for enantioselective hydrolysis and transesterification

AbstractEnzyme immobilization enhances the catalytic activity and stability of the enzyme, and also improves reusability. Metal–organic frameworks (MOFs), which possess diversified structures and porosity, have been used as excellent carriers for enzyme immobilization. Pseudomonas fluorescens lipase (PFL) has been successfully immobilized onto MOFs by covalent cross‐linking to obtain a series of immobilized lipase (PFL@MOFs). PFL@MOFs are used for catalytic enantioselective hydrolysis of 2‐(4‐hydroxyphenyl) propionic acid ethyl ester enantiomers (2‐HPPAEE) in aqueous medium and transesterification of 4‐methoxymandelic acid enantiomers (4‐MMA) in organic medium. The experimental results indicated that PFL@Uio‐66(Zr) exhibits excellent enzymatic catalysis performances and high enantioselectives. In addition, to improve catalytic activity and reusability, PFL is modified by the polyethylene glycol (PEG) to prepare PEG‐modified lipase (PFL‐PEG), then PFL‐PEG is immobilized onto Uio‐66(Zr) to prepare PFL‐PEG@Uio‐66(Zr), demonstrating better reusability and catalytic activity compared with PFL@Uio‐66(Zr).

Synthesis of Chiral 1,3‐keto‐acetates through Enzymatic Kinetic Resolution with Amano Lipase from Pseudomonas fluorescens

AbstractThis study provides an enzymatic kinetic resolution (EKR) method for the biocatalytic enantioselective synthesis of chiral β‐keto acetates. For the first time, the attitude of several lipase enzymes has been examined under different conditions for the EKR of the racemic 1,3‐keto alcohols. We found that the Amano lipase from Pseudomonas fluorescens (AL‐PF) lipase showed the best results for the resolution of the racemic 1,3‐keto alcohols. We developed a highly effective EKR method by changing the enzyme, temperature, solvent, and enzyme amount in order to synthesize the chiral 1,3‐keto esters, which are useful intermediates in organic synthesis. This study presents an efficient method for the synthesis of 1,3‐keto acetates with a good yield and high enantioselectivity with the R configuration.

Improvement of extraction and concentration method for polyunsaturated fatty acid production from Nile tilapia processing waste

In this current study, the economical and environmentally friendly method of extraction and concentration of polyunsaturated fatty acid (PUFA) from Nile tilapia waste was improved. First, Nile tilapia waste was collected and characterized. Lyophilized Nile tilapia had a 30.3% lipid content, which was suitable for oil extraction and PUFA concentration. After extraction and concentration, Nile tilapia waste oil (TWO) concentrated with Pseudomonas fluorescens lipase gave an acceptable peroxide and acid value of fish oils for human consumption. Fatty acid composition was also evaluated. After enzymatic concentration, PUFA increased dramatically. A high percentage of PUFA (59.0%) and the ratio of fatty acids n-6/n-3 (1.2) indicates good nutritional values of enzymatically concentrated TWO. Moreover, the optimization study of enzymatic concentration was evaluated to increase quality of TWO. A concentration of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was determined. A high concentration of EPA+DHA was obtained when P. fluorescens (3.6%) and Thermomyces lanuginosa (5.3%) lipases were applied. The highest EPA and DHA content (6.0%) was observed with 0.1 g of immobilized T. lanuginosa lipase per 2 g oil after an 18-h reaction time. Similar conditions were also preferred when using P. fluorescens lipase; however, only 4.0% EPA and DHA were observed. This is the first report on PUFA concentration from Nile tilapia waste using a one-step extraction and saponification in combination with enzymatic concentration. Notably, this method is expected to be a more economically possible technique for large-scale production and allows the possibility of using Nile tilapia waste as substrate for PUFA concentration.

Mathematical modeling of enzymatic syntheses of biosurfactants catalyzed by immobilized lipases

This work aimed at the kinetic modeling of enzymatic reactions for the production of sugar fatty esters type biosurfactants. The production of biosurfactants by esterification (72 h, 250 rpm stirring and 1.5 g of molecular sieve) of oleic and lauric acids with fructose and lactose, in the presence of tert-butanol and 2-methyl-2-butanol, has already been evaluated experimentally using Candida antarctica lipase B immobilized (CALB-IM-T2-350) and Pseudomonas fluorescens lipase (PFL) immobilized on octyl-silica. Acid conversion against time assays were carried out changing the following parameters: acid:sugar molar ratio (1:1 and 1:2), sugar concentration (25 and 50 mM), temperature (45 and 55 °C) and mass of biocatalyst (0.25 and 0.5 g). The Ping Pong Bi Bi kinetic model was fitted to data selected from the above-cited assays, assuming the production of only monoesters (relating the consumption of sugar and acid by an equimolar relation). The values for Vmax, Ksugar and Kacid were estimated. When CALB-IM-T2-350 was used, at 55 °C, Vmax = 1133 ± 221 mmol/Lh, Ksugar = 1378 ± 1696 mmol/L and Kacid = 3298 ± 1015 mmol/L. In the syntheses with PFL-octyl-silica, the results indicated lower conversions, with Vmax varying between 124 ± 13 and 221 ± 34 mmol/Lh (depending on reaction conditions) and K = 10,241 ± 1526 mmol/L (in average). A very good fit of the proposed model to the experimental data was obtained. For validation purpose, a different set of experimental data was used. The validation concentration profile showed an excellent prediction capability of the model.

P(GMA‐HEMA)/SiO2 Nanofilm Constructed Macroporous Monolith for Immobilization of Pseudomonas Fluorescens Lipase

AbstractMonolithic macroporous composites constructed by poly(glycidyl methacrylate‐2‐hydroxyethyl methacrylate)/SiO2 nanofilm (P(GMA‐HEMA)/SiO2) were prepared and then used as the supports for immobilization of Pseudomonas fluorescens lipase (PFL). The as‐prepared macroporous materials were characterized by scanning electron microscope (SEM), N2 adsorption‐desorption experiments, Fourier transform infrared (FT‐IR) spectroscopy and thermogravimetric (TG) analysis. The immobilization conditions and the properties of the immobilized PFL were investigated. The characterization results revealed that the macroporous SiO2 has a 3D continuous pass‐through macropore structure, large pore volume and high specific surface area. After modification with P(GMA‐HEMA) and the subsequent immobilization of PFL, the structural characteristics were preserved. Under the optimum conditions, the activity of the immobilized PFL reached to 3773 U g−1 and enzyme activity recovery achieved to 232 %. Compared with free lipase, the thermal and pH stabilities of the immobilized lipase were significantly enhanced, and it also exhibited an outstanding reusability.

Novel Bacterial Surface Display System Based on the Escherichia coli Protein MipA.

Bacterial surface display systems have been developed for various applications in biotechnology and industry. Particularly, the discovery and design of anchoring motifs is highly important for the successful display of a target protein or peptide on the surface of bacteria. In this study, an efficient display system on Escherichia coli was developed using novel anchoring motifs designed from the E. coli mipA gene. Using the C-terminal fusion system of an industrial enzyme, Pseudomonas fluorescens lipase, six possible fusion sites, V140, V176, K179, V226, V232, and K234, which were truncated from the C-terminal end of the mipA gene (MV140, MV176, MV179, MV226, MV232, and MV234) were examined. The whole-cell lipase activities showed that MV140 was the best among the six anchoring motifs. Furthermore, the lipase activity obtained using MV140 as the anchoring motif was approximately 20-fold higher than that of the previous anchoring motifs FadL and OprF but slightly higher than that of YiaTR232. Western blotting and confocal microscopy further confirmed the localization of the fusion lipase displayed on the E. coli surface using the truncated MV140. Additionally the MV140 motif could be used for successfully displaying another industrial enzyme, α-amylase from Bacillus subtilis. These results showed that the fusion proteins using the MV140 motif had notably high enzyme activities and did not exert any adverse effects on either cell growth or outer membrane integrity. Thus, this study shows that MipA can be used as a novel anchoring motif for more efficient bacterial surface display in the biotechnological and industrial fields.

Lipase Immobilized on a Novel Rigid–Flexible Dendrimer-Grafted Hierarchically Porous Magnetic Microspheres for Effective Resolution of (R,S)-1-Phenylethanol

With the rapid development of biotechnological industry, there is an urgent need for exploiting new materials to immobilize enzymes to improve the performance of biocatalysts. In this paper, hierarchically porous magnetic microspheres (PFMMs) were prepared through solvothermal method and rapidly grafted with a novel rigid-flexible dendrimer first synthesized from monomers of trimesoyl chloride (TMC) and 1,6-hexanediamine (HDA) via interfacial polymerization process for covalent immobilization of Pseudomonas fluorescens lipase (PFL). The maximum PFL loading of the synthesized support reaches 87.5 mgprotein/gsupport, and 864% activity recovery of PFMMs-G3.0-PFL can be achieved at pH 9.0. Then, it was used to catalyze the resolution of (R,S)-1-phenylethanol with vinyl acetate. Under the optimized conditions, 50.0% conversion with 99.0% ees can be reached within 1.5 h. In addition, a conversion of 49.2% and ees of 96.9% can be retained after 10 batches of running, displaying an excellent operational stability. Importantly, a further investigation shows that the obviously improved reusability of the immobilized PFL is ascribed to the increased rigidity in comparison to fully flexible dendrimer. Thus, the newly constructed protocol for lipase immobilization exhibits a great prospect in biochemical engineering.

Lipase-Catalyzed Chemoselective Ester Hydrolysis of Biomimetically Coupled Aryls for the Synthesis of Unsymmetric Biphenyl Esters.

Lipases are among the most frequently used biocatalysts in organic synthesis, allowing numerous environmentally friendly and inexpensive chemical transformations. Here, we present a biomimetic strategy based on iron(III)-catalyzed oxidative coupling and selective ester monohydrolysis using lipases for the synthesis of unsymmetric biphenyl-based esters under mild conditions. The diverse class of biphenyl esters is of pharmaceutical and technical relevance. We explored the potency of a series of nine different lipases of bacterial, fungal, and mammalian origin on their catalytic activities to cleave biphenyl esters, and optimized the reaction conditions, in terms of reaction time, temperature, pH, organic solvent, and water–organic solvent ratios, to improve the chemoselectivity, and hence control the ratio of unsymmetric versus symmetric products. Elevated temperature and increased DMSO content led to an almost exclusive monohydrolysis by the four lipases Candida rugosa lipase (CRL), Mucor miehei lipase (MML), Rhizopus niveus lipase (RNL), and Pseudomonas fluorescens lipase (PFL). The study was complemented by in silico binding predictions to rationalize the observed differences in efficacies of the lipases to convert biphenyl esters. The optimized reaction conditions were transferred to the preparative scale with high yields, underlining the potential of the presented biomimetic approach as an alternative strategy to the commonly used transition metal-based strategies for the synthesis of diverse biphenyl esters.

Lipase‐Catalyzed tert‐Butoxycarbonylation of Alcohols Using Boc2O

AbstractLipase‐catalyzed tert‐butoxycarbonylation of alcohols using di‐tert‐butyl dicarbonate (Boc2O) was developed. Aspergillus niger lipase, Burkholderia cepacia lipase, Pseudomonas fluorescens lipase, Candida rugosa lipase, and Candida antarctica lipase B were examined. The reaction of benzyl alcohol with Boc2O proceeded smoothly in the presence of Candida rugosa lipase in hexane to produce benzyl tert‐butyl carbonate in a good yield. Similarly, the tert‐butoxycarbonylation reactions of 4‐methylbenzyl alcohol, 4‐methoxybenzyl alcohol, 4‐chlorobenzyl alcohol, 4‐bromobenzyl alcohol, 4‐nitrobenzyl alcohol, 2‐phenylethanol, 3‐phenyl‐1‐propanol, cinnamyl alcohol, and 2‐naphthalene methanol using Boc2O in the presence of Candida rugosa lipase in hexane resulted in very good to‐excellent yields of the corresponding tert‐butoxycarbonylated alcohols.