Production, Characterization, and Application of an Organic Solvent-Tolerant Lipase Present in Active Inclusion Bodies

An organic solvent-tolerant lipase from Serratia marcescens ECU1010 (rSML) was overproduced in Escherichia coli in an insoluble form. High concentrations of both biomass (50 g cell wet weight/L culture broth) and inclusion bodies (10.5 g/L) were obtained by applying a high-cell-density cultivation procedure. Activity assays indicated that the enzymatic activity of rSML reached 600 U/L. After treatment with isopropyl ether for 12 h, the maximum lipase activity reached 6,000 U/L. Scanning electron microscopy and Fourier transform infrared microspectroscopy revealed the activation mechanism of rSML in the presence of organic solvents. rSML was stable in broad ranges of temperatures and pH values, as well as in a series of organic solvents. Besides, rSML showed the best enantioselectivity for the kinetic resolution of (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester. These features render the S. marcescens ECU1010 lipase attractive for biotechnological applications in the field of organic synthesis and pharmaceutical industry.     

A Calcium-Ion-Stabilized Lipase from <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> ZS04 and its Application in Resolution of Chiral Aryl Alcohols

An extracellular organic solvent-tolerant lipase-producing bacterium was isolated from oil-contaminated soil samples and was identified taxonomically as Pseudomonas stutzeri, from which the lipase was purified and exhibited maximal activity at temperature of 50 °C and pH of 9.0. Meanwhile, the lipase was stable below or at 30 °C and over an alkaline pH range (7.5–11.0). Ca²⁺ could significantly improve the lipase thermal stability which prompts a promising application in biocatalysis through convenient medium engineering. The lipase demonstrated striking features such as distinct stability to the most tested hydrophilic and hydrophobic solvents (25 %, v/v), and DMSO could activate the lipase dramatically. In the enzyme-catalyzed resolution, lipase ZS04 manifested excellent enantioselective esterification toward the (R)-1-(4-methoxyphenyl)-ethanol (MOPE), a crucial chiral intermediate in pharmaceuticals as well as in other analogs with strict substrate specificity and theoretical highest conversion yield. This strong advantage over other related schemes made lipase ZS04 a promising biocatalyst in organic synthesis and pharmaceutical applications.     

Chemoenzymatic synthesis of optically active Mugetanol isomers: use of lipases and oxidoreductases in fragrance chemistry

Straightforward synthetic strategies for the preparation of optically active Mugetanol isomers have been developed through different independent chemoenzymatic routes implying the use of either alcohol dehydrogenases in aqueous media or lipases in organic solvents coupled with a catalytic hydrogenation process. Among the alcohol dehydrogenases tested, ADH RS1 showed the best activities in the bioreduction of 4-isopropylacetophenone. The lipase from Pseudomonas cepacia (PSL-C I) reached high activity values in the lipase-catalyzed transesterification of 4-isopropylphenyl)ethanol, while Candida antarctica lipase B showed the best stereopreference in the acetylation of 4-isopropylcyclohexylethanol.     

A novel lipase enzyme panel exhibiting superior activity and selectivity over lipase B from Candida antarctica for the kinetic resolution of secondary alcohols

A novel, commercially available lipase enzyme panel performing kinetic bioresolutions of a number of secondary alcohols is reported. The secondary alcohols that have been chosen are known from the literature to be particularly challenging substrates to resolve. Following initial screening, four co-solvents were investigated for each lead enzyme in an effort to assess their tolerance to common organic solvents. The superiority of these novel enzymes over lipase B from Candida antarctica (CALB) has been demonstrated.     

Improved synthesis of disaccharides with Escherichia coli β-galactosidase using bio-solvents derived from glycerol

A noticeably increase in activity, keeping total regioselectivity was found in the synthetic behaviour of Escherichia coli β-galactosidase in glycerol-based solvents using a 1:7 molar ratio of donor (pNP-β-Gal): acceptor (GlcNAc). Yields of up to 97% of β(1→6) with different solvents were found. These reactions take place without noticeable hydrolytic activity and with total regioselectivity, representing a considerable improvement over the use of aqueous buffer or conventional organic solvents. There is a clear dependence of the catalytic results on the solvent structure, which is analysed in terms of polarity and hydrophobicity.     

Biocatalytic promiscuity: lipase-catalyzed asymmetric aldol reaction of heterocyclic ketones with aldehydes

The new promiscuous activity of lipase from porcine pancreas, type II (PPL II), has been observed to catalyze the direct asymmetric aldol reaction of heterocyclic ketones with aromatic aldehydes. PPL II showed favorable catalytic activity and had a good adaptability to different substrates in the reaction. The enantioselectivities of up to 87% ee and diastereoselectivities of up to 83:17 (anti/syn) were achieved. It is interesting that PPL II possesses the function of aldolase in organic solvents.     

Production and Biotechnological Application of Extracellular Alkalophilic Lipase from Marine Macroalga-Associated <Emphasis Type="Italic">Shewanella algae</Emphasis> to Produce Enriched C<Subscript>20-22</Subscript><Emphasis Type="Italic">n</Emphasis>-3 Polyunsaturated Fatty Acid Concentrate

An extracellular alkalophilic lipase was partially purified from heterotrophic Shewanella algae (KX 272637) associated with marine macroalgae Padina gymnospora. The enzyme possessed a molecular mass of 20 kD, and was purified 60-fold with a specific activity of 36.33 U/mg. The enzyme exhibited V_max and K_m of 1000 mM/mg/min and 157 mM, respectively, with an optimum activity at 55 °C and pH 10.0. The catalytic activity of the enzyme was improved by Ca²⁺ and Mg²⁺ ions, and the enzyme showed a good tolerance towards organic solvents, such as methanol, isopropanol, and ethanol. The purified lipase hydrolyzed the refined liver oil from leafscale gulper shark Centrophorus squamosus, yielding a total C_20-22 n-3 PUFA concentration of 34.99% with EPA + DHA accounting the major share (34% TFA), after 3 h of hydrolysis. This study recognized the industrial applicability of the thermostable and alkalophilic lipase from marine macroalga-associated bacterium Shewanella algae to produce enriched C_20-22 n-3 polyunsaturated fatty acid concentrate.     

Characterization of a New Cold-adapted Lipase from Pseudomonas sp. TK-3

A psychrotrophic Pseudomonas sp. TK-3 was isolated from dirty and cool stream water in Toyama, Japan from which we cloned and characterized the bacterial lipase LipTK-3. The sequenced DNA fragment contains an open reading frame of 1,428?bp that encoded a protein of 476 amino acids with an estimated molecular mass of 50,132?Da. The lipase showed high sequence similarity to those of subfamily ??.3 lipase and had a conserved GXSXG motif around the catalytic Ser residue. Its optimal temperature was 20?C25?°C, lower than in most other subfamily ??.3 lipases. The lipase exhibited about 30?% of maximal activity at 5?°C. The optimal pH value was 8.0. The activity was strongly inhibited by EDTA and was highly dependent on Ca²⁺. Tricaprylin and p-nitrophenyl caprylate were the most favorable substrates among the triglycerides and p-nitrophenyl esters, respectively. LipTK-3 also showed high activity towards natural substrates including edible vegetable oils and animal fats. Furthermore, LipTK-3 was very active and stable in the presence of several detergents, metal ions, and organic solvents. This cold-adapted lipase may prove useful for future applications.     

CO2-expanded bio-based liquids as novel solvents for enantioselective biocatalysis

For the first time, CO₂-expanded bio-based liquids were reported as novel and sustainable solvents for biocatalysis. Herein, it was found that by expansion with CO₂, 2-methyltetrahydrofuran (MeTHF), and other bio-based liquids, which were not favorable solvents for immobilized Candida antarctica lipase B (Novozym 435) catalyzed transesterification, were tuned into excellent reaction media. Especially, for the kinetic resolution of challenging bulky secondary substrates such as rac-1-adamantylethanol, the lipase displayed very high activity with excellent enantioselectivity (E value > 200) in CO₂-expanded MeTHF (MeTHF concentration 10% v/v, 6 MPa), whereas there was almost no activity observed in conventional organic solvents.     

A Cold-Adapted and Organic Solvent-Tolerant Lipase from a Psychrotrophic Bacterium Pseudomonas sp. Strain YY31: Identification, Cloning, and Characterization

A novel cold-adapted lipase (designated as LipYY31) was obtained from a psychrotrophic Pseudomonas sp. YY31. The strain YY31 was gram-negative, rod shaped, motile by means of one polar flagellum, and exhibited chemotaxis toward oil droplets under a microscope. The strain displayed remarkable degradation of edible oil and fat even at 5 °C. The LipYY31 DNA fragment contains an open reading frame of 1,410 bp which encoded a protein of 470 amino acids with an estimated molecular mass of 49,584 Da. LipYY31 showed high sequence similarity to those of subfamily Ι.3 lipase and had a conserved GXSXG motif around the catalytic Ser residue. Its optimal temperature was 25–30 °C, and it retained 20–40 % of its activity at 0–5 °C. The optimal pH value was 8.0. The activity was strongly inhibited by Cd²⁺, Zn²⁺, EDTA and was highly dependent on Ca²⁺. Tricaprin and p-nitrophenyl caprate were the most favorable substrates among the triglycerides and p-nitrophenyl esters, respectively. LipYY31 also had high activity towards natural substrates including edible vegetable oils and animal fat. Furthermore, LipYY31 was very active and stable in the presence of several detergents and organic solvents. In particular, the lipase exhibited high stability against organic solvents such as methanol, ethanol, and isopropanol.     

Isolation,Purification and Characterisation of an Organic Solvent-Tolerant Ca2+-Dependent Protease from Bacillus megaterium AU02

A new organic solvent-tolerant strain Bacillus megaterium AU02 which secretes an organic solvent-tolerant protease was isolated from milk industry waste. Statistical methods were employed to achieve optimum protease production of 43.6 U/ml in shake flask cultures. The productivity of the protease was increased to 53 U/ml when cultivated under controlled conditions in a 7-L fermentor. The protease was purified to homogeneity by a three-step process with 24 % yield and specific activity of 5,375 U/mg. The molecular mass of the protease was found to be 59 kDa. The enzyme was active over a wide range of pH (6.0–9.0), with an optimum activity at pH 7.0 and temperature from 40 to 70 °C having an optimum activity at 50 °C. The thermal stability of the enzyme increased significantly in the presence of CaCl₂, and it retained 90 % activity at 50 °C for 3 h. The K _m and V _max values were determined as 0.722 mg/ml and 0.018 U/mg respectively. The metalloprotease exhibited significant stability in the presence of organic solvents with log P values more than 2.5, nonionic detergents and oxidising agent. An attempt was made to test the synthesis of aspartame precursor (Cbz-Asp-Phe-NH₂) which was catalysed by AU02 protease in the presence of 50 % DMSO. These properties of AU02 protease make it an ideal choice for enzymatic peptide synthesis in organic media.     

Separation and Immobilization of Lipase from Penicillium simplicissimum by Selective Adsorption on Hydrophobic Supports

Lipases are an enzyme class of a great importance as biocatalysts applied to organic chemistry. However, it is still necessary to search for new enzymes with special characteristics such as good stability towards high temperatures, organic solvents, and high stereoselectivity presence. The present work’s aim was to immobilize the lipases pool produced by Penicillium simplissicimum, a filamentous fungi strain isolated from Brazilian babassu cake residue. P. simplissicimum lipases were separated into three different fractions using selective adsorption method on different hydrophobic supports (butyl-, phenyl-, and octyl-agarose) at low ionic strength. After immobilization, it was observed that these fractions’ hyperactivation is in the range of 131% to 1133%. This phenomenon probably occurs due to enzyme open form stabilization when immobilized onto hydrophobic supports. Those fractions showed different thermal stability, specificity, and enantioselectivity towards some substrates. Enantiomeric ratio for the hydrolysis of (R,S) 2-O-butyryl-2-phenylacetic acid ranged from 1 to 7.9 for different immobilized P. simplissicimum lipase fractions. Asymmetry factor for diethyl 2-phenylmalonate hydrolysis ranged from 11.8 to 16.4 according to the immobilized P. simplissicimum lipase fractions. Those results showed that sequential adsorption methodology was an efficient strategy to obtain new biocatalysts with different enantioselectivity degrees, thermostability, and specificity prepared with a crude extract produced by a simple and low-cost technology.     

Esterification and transesterification with immobilized lipase in organic solvent

In this paper, we report the reactions of esterification and transesterification catalyzed by the following lipase adducts in organic solvents: (1) glass-adsorbed; (2) acetone precipitated on porous glass, kieselghur-adsorbed; (3) Al₂O₃-adsorbed; and (4) agar bead-adsorbed. The optimal water content varied for different forms of the enzymes. Under the most favorable conditions, kieselguhr-adsorbed and agar bead-adsorbed lipases, which have higher catalytic activities in organic solvents, are the best of all forms of lipases.     

有机相酶催化拆分制备(S)-2-氯-1-(2-噻吩)-乙醇

首次在有机相中对酶催化条件下的2-氯-1-(2-噻吩)-乙醇的反应进行了研究. 通过对不同来源酶的筛选, 找到了Novozym 435和Alcaligenes sp两种选择性较好的酶, 它们均对该反应具有较高的选择性和较快的反应速度, 在此基础上进一步通过对溶剂、温度、摇床转速以及酶用量的筛选, 确定了能够有效拆分2-氯-1-(2-噻吩)-乙醇的较佳反应条件. 当温度35 ℃, 酶量10 mg/mL, 反应72.5 h, 产物的ee值为98.5%时收率为48.6%.     

Design and synthesis of useful intermediates for novel lipoxygenase substrates through enzymatic resolution

Unnatural lipoxygenase substrates carrying spacing modifiers with a non-ionic hydroxy terminus and with methylene (flanked by the cis,cis diene moiety) pro-(S)-hydrogen can be synthesized from the intermediates 1a–1c. These intermediates are conveniently synthesized via enzymatic resolution with lipase in organic solvents.     

Peracetylated β-cyclodextrin as additive in enzymatic reactions: enhanced reaction rate and enantiomeric ratio in lipase-catalyzed transesterifications in organic solvents

Peracetylated β-cyclodextrin has been employed as a macrocyclic additive to enhance the enantiomeric ratio E and reaction rate in Pseudomonas cepacia lipase (PSL)-catalyzed enantioselective transesterification of 1-(2-furyl)ethanol in organic solvents. The beneficial action of the cyclodextrin used as a regulator of lipase was tentatively interpreted as increasing the conformational flexibility of the enzyme and undergoing host–guest complexation with the product, thereby preventing product inhibition and leading to an enhancement of the enantiomeric ratio E and the reaction rate. The effect of the organic solvent on the present cyclodextrin-mediated enzymatic transesterification has been studied.     

A facile preparation of (±)-β-hydroxy nitriles and their enzymatic resolution with lipases

A simple and efficient method for the preparation of racemic 4-aryloxy-3-hydroxybutanenitriles is described. Lipase mediated kinetic resolution in organic media was then utilised to effect enantioseparation. Lipases from different sources were screened in the resolution reaction using a number of organic solvents. Enantiomeric excesses of up to 99% were obtained by employing lipase from Pseudomonas cepacia in di-iso-propyl ether medium.     

Essential Role of Gly33 in a Novel Organic Solvent-Tolerant Lipase from Serratia marcescens ECU1010 as Determined by Site-Directed Mutagenesis

A novel lipase lipB from Serratia marcescens ECU1010 is highly stable in the presence of organic solvents. By sequence and structure comparison with homologous lipase lipA, three amino acid residues were found to be different between them. To identify the residues which increase the organic solvent stability of lipB, residues that potentially provide this stability were mutated to the ones of lipA at equivalent positions. The replacement of Gly at position 33 by Asp obviously decreased its stability in organic solvents. Molecular modeling and structural analysis also suggested that the Gly33 residue is important for the organic solvent stability of lipB.     

Characterization of a Novel Organic Solvent Tolerant Protease from a Moderately Halophilic Bacterium and Its Behavior in Ionic Liquids

An extracellular protease was purified from a novel moderately halophilic bacterium Salinivibrio sp. strain MS-7 by the combination of an acetone precipitation (40–80 %) step and a DEAE-cellulose anion exchange column chromatography. Kinetic parameters of the enzyme exhibited V _max and K _m of 130 U/mg and 1.14 mg/ml, respectively, using casein as a substrate. The biochemical properties of the enzyme revealed that the 21-kDa protease had a temperature and pH optimum of 50 °C and 8.0, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, Pefabloc SC, chymostatin, and also EDTA, indicating that it belongs to the class of serine metalloproteases. Interestingly, Ba²⁺ and Ca²⁺ (2 mM) strongly enhanced the enzyme activity, while Fe²⁺ and Mg²⁺ activated moderately and Zn²⁺, Ni²⁺, and Hg²⁺ decreased the enzyme activity. The effect of organic solvents with different logP on the purified protease revealed complete stability in toluene, ethyl acetate, chloroform, and n-hexane at 10 and 50 % (v/v) and moderate stability even in 50 % of DMSO and ethanol. The behavior of the MS-7 protease in three imidazolium-based ionic liquids exhibited suitable activity in these green solvent systems, especially in 1-hexyl-3-methylimidazolium hexafluorophosphate ([C₆MIM][PF₆]). Comparison of the purified protease with other previously reported proteases suggests that strain MS-7 secrets a novel organic solvent-tolerant protease with outstanding activity in organic solvents and imidazolium-based ionic liquids, which could be applied in low water synthetic section of industrial biotechnology.     

Production and Characterization of Cross-Linked Aggregates of Geobacillus thermoleovorans CCR11 Thermoalkaliphilic Recombinant Lipase

Immobilization of enzymes has many advantages for their application in biotechnological processes. In particular, the cross-linked enzyme aggregates (CLEAs) allow the production of solid biocatalysts with a high enzymatic loading and the advantage of obtaining derivatives with high stability at low cost. The purpose of this study was to produce cross-linked enzymatic aggregates (CLEAs) of LipMatCCR11, a 43 kDa recombinant solvent-tolerant thermoalkaliphilic lipase from Geobacillus thermoleovorans CCR11. LipMatCCR11-CLEAs were prepared using (NH₄)₂SO₄ (40% w/v) as precipitant agent and glutaraldehyde (40 mM) as cross-linker, at pH 9, 20 °C. A U₁₀(5⁶) uniform design was used to optimize CLEA production, varying protein concentration, ammonium sulfate %, pH, glutaraldehyde concentration, temperature, and incubation time. The synthesized CLEAs were also analyzed using scanning electron microscopy (SEM) that showed individual particles of <1 µm grouped to form a superstructure. The cross-linked aggregates showed a maximum mass activity of 7750 U/g at 40 °C and pH 8 and retained more than 20% activity at 100 °C. Greater thermostability, resistance to alkaline conditions and the presence of organic solvents, and better durability during storage were observed for LipMatCCR11-CLEAs in comparison with the soluble enzyme. LipMatCCR11-CLEAs presented good reusability by conserving 40% of their initial activity after 9 cycles of reuse.