Effects of substrate pretreatment and water activity on lipase-catalyzed cellulose acetylation in organic media

Lipase-catalyzed acetylation of cellulose solubilized in the dimethyl sulfoxide/paraformaldehyde organic solvent system was conducted with lipase A12 from Aspergillus niger. The accompanying side cellulase activity of the A. niger lipase partly accounted for the enhanced acetylation mediated by the enzyme, via facilitating the partial degradation of cellulose substrate as evidenced by high-performance size exclusion chromatograph analysis. The enzymatic cellulose acetylation was improved by substrate pretreatment with cellulase or ultrasound by 18 and 14%, respectively, as a result of the reduced substrate molecular size. Additionally, the ultrasound-pretreated cellulose as the starting substrate was beneficial for the cellulose solution preparation due to the increased accessible surface of cellulose as evidenced by its increased sedimentation volume and SEM micrographs. The effect of thermodynamic water activity (aw) on lipase catalytic activity in organic media was also investigated. The maximum acetylation extent (nearly 11 wt %) occurred at aw = 0.52, which was improved by 51% relative to the enzymatic reaction with no control of water activity. The much larger extent to which the lipase-catalyzed cellulose acetylation was enhanced by water activity optimization than by substrate pretreatment further supported the predominant role played by the major lipase activity of the A. niger lipase over its side cellulase activity in catalyzing cellulose ester synthesis in organic media.     

Polar organic solvent added to an aqueous solution changes hydrolytic property of lipase

For developing further uses of lipase as a biocatalyst, its hydrolytic activity toward some esters was investigated in a miscible solution composed of a buffer and a polar organic solvent. Twenty percent dimethylformamide, 35% dimethylsulfoxide, 15% 1,4-dioxane, 15% dimethoxyethane, and 2% diethoxyethane promoted the hydrolysis by a lipase from Rhizomucor miehei toward some hydrophobic substrates, 4-methylumbelliferyl oleate, 4-methylumbelliferyl palmitate, and monoolein. While hydrolysis by this lipase toward the substrates with a relatively weak hydrophobicity (4-metylumbelliferyl heptanoate and 4-methylumbelliferyl nanoate) was suppressed by these solvents. A fluorometric analysis showed that the polar organic solvent in the buffer induced some conformational change around a tryptophan residue of R. miehei lipase. In addition to the influence of the miscible solvent on the solubility of the substrates, the conformational change of the protein induced by the miscible solvent would also affect the reactive properties of the lipase. Adding a polar organic solvent to an aqueous solution will be an efficient method for changing hydrolytic performance of lipases.     

Lipases as practical biocatalysts

Lipases are the most used enzymes in synthetic organic chemistry, catalyzing the hydrolysis of carboxylic acid esters in aqueous medium or the reverse reaction in organic solvents. Recent methodological advancements regarding practical factors affecting lipase activity and enantioselectivity are reviewed. Select practical examples concerning the use of lipases in the production of chiral intermediates are also highlighted.     

On the issue of interfacial activation of lipase in nonaqueous media

The question of whether lipases can be activated by adsorption onto an interface in organic solvents was addressed using Rhizomucor miehei lipase as a model. In aqueous solution, this enzyme was shown to undergo a marked interfacial activation. However, lipase (either lyophilized or precipitated from water with acetone) suspended in ethanol or 2-(2-ethoxyethoxy)ethanol containing triolein exhibited no jump in catalytic activity when the concentration of triolein exceeded its solubility in these solvents, thereby resulting in formation of an interface. To test whether the lack of interfacial activation was due to the insolubility of the enzyme in organic media, lipase was covalently modified with poly(ethylene glycol). The modified lipase, although soluble in nonaqueous media, was still unable to undergo interfacial activation, regardless of the hydrophobicity of the interface. This inability was found to be caused by the absence of adsorption of lipase onto interfaces in organic solvents, presumably because of the absence of the hydrophobic effect (the driving force of lipase adsorption onto hydrophobic interfaces in water) in such media. The uncovered lack of interfacial adsorption and activation suggests that the short alpha-helical "lid" covering the active center of the lipase remains predominantly closed in nonaqueous media, thus contributing to diminished enzymatic activity. (c) 1996 John Wiley & Sons, Inc.     

Purification, characterization, and molecular cloning of lactonizing lipase from Pseudomonas species

An extracellular lipase catalyzing the synthesis of macrocyclic lactones in anhydrous organic solvents was purified to homogeneity from Pseudomonas nov. sp. 109, and characterized. The lipase showed a pI of 5.3 on isoelectric focusing and a Mr of 29,000 +/- 1,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With respect to substrate specificity, optimum chain length for acyl moiety varied depending on the type of reaction catalyzed: C18 in monomer lactone formation, C11 or shorter in dimer lactone formation, and C8 in ester hydrolysis. The amino-terminal 19 amino acid residues of the purified lipase were determined as Ser-Thr-Tyr-Thr-Gln-Thr-Lys-Tyr-Pro-Ile-Val-Leu-Ala-His-Gly-Met-Leu-Gly- Phe, and the gene encoding the lipase was identified by hybridization to a synthetic 20-nucleotide probe, cloned, and sequenced. Nucleotide sequence analysis predicted a 311-amino acid open reading frame, a putative ribosome-binding site, and a 26-amino acid sequence at the amino terminus of the sequence that is not found in the mature protein. This 26-amino acid sequence has many of the characteristics common to known signal peptides. The lipase gene encoded a sequence of Val-Asn-Leu-Ile-Gly-His-Ser-His-Gly-Gly which is very well conserved among lipases, and showed 38-40% overall homology to the amino acid sequences of lipases from Pseudomonas fragie and Pseudomonas cepacia, but showed little homology to those of other lipases, suggesting that some structural features are required for catalyzing macrocyclic lactone synthesis in organic solvents and are restricted to lipases of the Pseudomonas origin.     

Enzymatic enrichment of polyunsaturated fatty acids using novel lipase preparations modified by combination of immobilization and fish oil treatment

Novel modification methods for lipase biocatalysts effective in hydrolysis of fish oil for enrichment of polyunsaturated fatty acids (PUFAs) were described. Based on conventional immobilization in single aqueous medium, immobilization of lipase in two phase medium composed of buffer and octane was employed. Furthermore, immobilization (in single aqueous or in two phase medium) coupled to fish oil treatment was integrated. Among these, lipase immobilized in two phase medium coupled to fish oil treatment (IMLAOF) had advantages over other modified lipases in initial reaction rate and hydrolysis degree. The hydrolysis degree increased from 12% with the free lipase to 40% with IMLAOF. Strong polar and hydrophobic solvents had negative impact on immobilization-fish oil treatment lipases, while low polar solvents were helpful to maintain the modification effect of immobilization-fish oil treatment. After five cycles of usage, the immobilization-fish oil treatment lipases still maintained more than 80% of relative hydrolysis degree.     

An active-site titration method for lipases

A method for active-site titration of lipases has been developed based on irreversible inhibition by methyl p-nitrophenyl n-hexylphosphonate. This method was applied to five lipases displaying from minor to pronounced interfacial activation. Soluble and immobilized lipases were successfully titrated in aqueous media. A low concentration of sodium dodecyl sulfate was needed for lipases displaying pronounced interfacial activation. The carrier of some of the immobilized preparations adsorbed part of the produced p-nitrophenolate. This problem could be solved by extracting the p-nitrophenolate after inhibition. The method was extended to apolar organic solvents in the case of immobilized lipase preparations.     

Influence of the organic solvents on the activity in water and the conformation of Candida rugosa lipase: Description of a lipase-activating pretreatment

The influence on lipase activity in water of a pretreatment on Candida rugosa lipase using water miscible and immiscible solvents was studied. The lipase activity in the hydrolysis of esteric substrates in aqueous media increases when the lipase was previously treated with various nearly anhydrous organic media. This activation, which was irreversible, was higher for longer pretreatment times. It was dependent on the pretreatment medium (water activity and solvent used). A relation between variations in the emission intensity and the activities of treated and untreated lipases was found. Activating pretreatment did not shift the peak of fluorescence emission but gave rise to variations in the secondary protein structure by increasing the helical nature. A similar increment in the hydrolysis rate in water can be obtained with the addition of an appropriate amount of solvent (acetonitrile or n-heptane) to the aqueous reaction medium.     

Water-in-ionic liquid microemulsion-based organogels as novel matrices for enzyme immobilization

The use of water-in-ionic liquid microemulsion-based organogels (w/IL MBGs) as novel supports for the immobilization of lipase B from Candida antarctica and lipase from Chromobacterium viscosum was investigated. These novel lipase-containing w/IL MBGs can be effectively used as solid phase biocatalysts in various polar and non-polar organic solvents or ILs, exhibiting up to 4.4-fold higher esterification activity compared to water-in-oil microemulsion-based organogels. The immobilized lipases retain their activity for several hours at 70°C, while their half life time is up to 25-fold higher compared to that observed in w/IL microemulsions. Fourier-transform infrared spectroscopy data indicate that immobilized lipases adopt a more rigid structure, referring to the structure in aqueous solution, which is in correlation with their enhanced catalytic behavior observed.     

Enzymatic properties of polyethylene glycol-modified cholesterol esterase in organic solvents.

The solvent dependency and substrate specificity of polyethylene glycol (PEG)-modified cholesterol esterase (CEH) catalyzing cholesterol ester synthesis in organic solvents were studied. When cholesterol and linoleic acid were used as the substrates, PEG-modified CEH synthesized cholesterol linoleate only in water-immiscible organic solvents. Among some solvents capable of solubilizing all of the reaction components (PEG-modified CEH, cholesterol, and linoleic acid), chloroform was most suitable for enzymatic cholesterol linoleate synthesis, and the synthetic activity for cholesterol linoleate decreased in the order chloroform, benzene, toluene, and cyclohexane. PEG-modified CEH synthesized various cholesterol esters with significant substrate specificity. The substrate specificity for cholesterol ester synthesis in benzene was analogous to that for cholesterol ester hydrolysis in aqueous solution.     

Comparison of hydrolytic activity in water and heptane for thirty-two commercial lipase preparations

The protein content and the rates of hydrolysis of p-nitrophenyl palmitate (pNPP) in water (soluble enzyme and emulsified substrate) and in heptane (soluble substrate and insoluble enzyme) were measured for thirty-two commercial lipase preparations. The protein content of the powders varied in a wide range as well as the activity on emulsified pNPP showing the high heterogeneity of the commercial samples. Activity in heptane also varied but to a lesser extent than that in water. There was no direct correlation between activities in water and in heptane as assayed with the same hydrolytic reaction. The ratio of activity in heptane to that in water, R(O/A) ratio, was introduced to characterize activity in organic media. Six lipases showed R(O/A) values higher than 1 demonstrating a higher activity in organic solvent than in water. A linear correlation of R(O/A) with activity in water (log plot) suggested the strong influence of diffusional limitations on activity of solid enzyme suspended in organic solvents.     

Lipase-catalyzed irreversible transesterification of 1-(2-furyl)ethanol using isopropenyl acetate

Asymmetric acetylation of racemic 1-(2-furyl)ethanol with the innocuous acyl donor isopropenyl acetate catalyzed by lipases in organic media afforded the chiral alcohol and acetate in high enantiomeric excess (up to 99%). The effect of molecular sieves as well as organic solvents on the kinetic resolution were studied. An effective separation of the enantiomers of both substrate and product was performed using gas chromatography on the chiral stationary phase heptakis-(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin.     

Catalytic properties of lipases immobilized on various mesoporous silicates

Lipases SP525, AK, LIP, and PS were immobilized on three kinds of mesoporous silicates (FMS, PESO, and SBA) with diameters of 27 to 92 A. The amount of lipase activity adsorbed on these supports was related to the pore size of the silicate. Enantioselectivities of immobilized lipases were similar to those of free lipases, and recycling could be done in both aqueous and organic solvents.     

华根霉脂肪酶有机相合成酶活的研究

通过比较7种微生物脂肪酶的有机相合成酶活、水相水解酶活及在正庚烷中催化己酸乙酯合成的能力,证明了合成酶活与水解酶活相关性不高,合成酶活比水解酶活更能反映脂肪酶的合成能力。通过比较两株华根霉(Rhizopus chinensis)脂肪酶酶活,发现合成酶活相差较大,表明相同种属微生物的脂肪酶合成酶活存在不同。对．Rhizopus chinensis-2液态发酵产脂肪酶进程研究发现,水解酶活高峰先于合成酶活高峰大约12h。将不同培养时间的Rhizopus chinensis-2全细胞脂肪酶用于催化己酸乙酯合成,具有高合成酶活的全细胞脂肪酶催化己酸乙酯合成反应较快。因此,全细胞脂肪酶用于催化有机相酯合成反应时,具有高脂肪酶合成酶活的菌体具有较好的催化酯合成能力。     

Direct solubilization of enzyme aggregates with enhanced activity in nonaqueous media

A protein solubilization method has been developed to directly solubilize protein clusters into organic solvents containing small quantities of surfactant and trace amounts of water. Termed "direct solubilization," this technique was shown to solubilize three distinct proteins - subtilisin Carlsberg, lipase B from Candida antarctica, and soybean peroxidase - with much greater efficiencies than extraction of the protein from aqueous solution into surfactant-containing organic solvents (referred to as extraction). More significant, however, was the dramatic increase in directly solubilized enzyme activity relative to extracted enzyme activity, particularly for subtilisin and lipase in polar organic solvents. For example, in THF the initial rate towards bergenin transesterification was ca. 70 times higher for directly solubilized subtilisin than for the extracted enzyme. Furthermore, unlike their extracted counterparts, the directly solubilized enzymes yielded high product conversions across a spectrum of non-polar and polar solvents. Structural characterization of the solubilized enzymes via light scattering and atomic force microscopy revealed soluble proteins consisting of active enzyme aggregates containing approximately 60 and 100 protein molecules, respectively, for subtilisin and lipase. Formation of such clusters appears to provide a microenvironment conducive to catalysis and, in polar organic solvents at least, may protect the enzyme from solvent-induced inactivation.     

脱脂棉固定化脂肪酶的非水活性与稳定性

脂肪酶具有非水催化作用,但其非水催化活性和稳定性需进一步提高,这是非水酶学的瓶颈问题之一。理想的策略是模拟脂肪酶的界面活化机制,以大分子代替水,优化、稳定化和有效分散酶蛋白,阻止其在有机相中变性。因此,选用多羟基、比表面积大、惰性、且与酶蛋白能兼容的大分子--脱脂棉纤维,作为固定化载体,以1∶0.9的质量比,通过物理吸附,将假单胞菌脂肪酶(Pseudomonas cepacia lipase)固定在脱脂棉纤维上。在催化己醇与乙酸乙烯酯的转酯反应中,反应1 h,脱脂棉固定化脂肪酶转化底物的能力是酶粉的3.7倍。在每次6 h共6次的循环催化中,固定化酶和酶粉转化底物的能力分别平均每次降低约0.3%和2.4%。表明脱脂棉固定化脂肪酶的非水活性,尤其是稳定性明显提高。这为通过固定化有效提高脂肪酶的非水催化作用,以满足工业应用的需要,提供了一种有效的途径和重要参考。     

An improved method of lipase preparation incorporating both solvent treatment and immobilization onto matrix

A simple and effective preparation of lipases for use in organic solvents is hereby proposed. Lipases in aqueous solution were treated with isopropanol, immediately followed by immobilization onto a commercially available macroporous resin CRBO2 (crosslinked polystyrene with N-methylglucamine as a functional group). The dual modification of lipases by (1) isopropanol treatment and (2) immobilization improved the activity and stability of lipases more significantly than either of the two treatments alone. The degree of lipase activation was dependent on isopropanol–buffer (v/v) ratio and the source of lipase used. Among the lipases tested, Rhizopus oryzae lipase was more significantly activated. The maximum specific activity of R. oryzae lipase after dual modification was 94.9 mmol h⁻¹ g⁻¹, which was, respectively, 3.3-, 2.5- and 1.5-fold of untreated free, untreated immobilized and treated free lipases. The conformations of the treated and untreated free lipases were investigated by circular dichroism (CD) measurement. Changes in the far- and near-UV CD spectra of lipase indicate that lipase activation is accompanied by changes in secondary and tertiary structures of lipases. The increase in negative molar elipticity at 222 nm suggests that the α-helical content of lipase increase after pretreatment.     

A novel thermostable lipase from Basidiomycete <Emphasis Type="Italic">Bjerkandera adusta </Emphasis>R59: characterisation and esterification studies

Microbial lipases are widely diversified in their enzymatic properties and substrate specificities, which make them very attractive for industrial application. Partially purified lipase from Bjerkandera adusta R59 was immobilized on controlled porous glass (CPG) and its properties were compared with those of the free enzyme. The free and immobilized lipases showed optimal activities at 45 and 50°C, respectively. Both enzyme forms were highly thermostable up to 60°C. The enzymes were stable at pH from 6.0 to 9.0 and their optimal pH for activity was 7.0. The free lipase was more thermostable in n-hexane than in aqueous environment. Both lipase preparations had good stabilities in non-polar solvents and were capable of hydrolysing a variety of synthetic and natural fats. Non-immobilized lipase activity was inhibited by disulphide bond reagents, serine and thiol inhibitors, while EDTA and eserine had no effect on enzyme activity. All anionic detergents tested in experiments inhibited lipase activity. The free lipase showed good stability in the presence of commercial detergents at laundry pH and temperatures. Applications of free and immobilized lipases for esterification were also presented.     

Stability of a Pseudomonas Sp. Lipase:Comparison Between Solubilized Enzyme in Reverse Micelles and Suspended Lipase in Dry Solvents

The stability of a relatively hydrophobic lipase from Pseudomonas sp., solubilized in reverse micellar media or suspended in dry solvents, was studied and compared. Factors such as the enzyme-solvent interaction, enzyme environment, hydration degree of the system, interphase quality, droplet size, and water activity were studied. A mixed micellar system which stabilized the lipase is reported. In the case of simple AOT micelles, lipase destabilization with respect to water in small droplet sizes and stabilization in the biggest micelles was observed. These effects resulted from lipase penetration into the interphase of the smaller nanodroplets, and the restriction of its conformational mobility in the region of structured water of the largest micelles, respectively. Mixed micelles increased lipase stability, which was mainly related to increased droplet size. Modification with polyethylene glycol decreased lipase stability in reverse micelles, due to the greater interaction with the micellar interphase. The preparation of nanodroplets, in which native and modified lipases were 5.4 and 9.4 times, respectively, more stable than in water, is reported. In contrast to the micellar media, low water contents (low A_w values) stabilized the solid lipase suspended in organic solvent systems. Under the hydration conditions studied here, lipase stability increased when more polar solvents were used. Two alternatives were necessary to obtain similar stabilities in n-heptane as compared with polar solvents: reduction of the water content or use of a low aquaphilic support.     

器壁固定化脂肪酶Pseudomonas cepacia lipase的非水活性与稳定性

许多脂肪酶在有机体系中表现出催化作用,可用于绿色有机合成. 但其催化活性和稳定性明显低于水/油（有机相）界面上的表现. 为了提高脂肪酶在有机反应体系中的活性和稳定性,依据脂肪酶的界面活化机制,以水为酶蛋白构象优化剂、羧甲基纤维素为赋形剂,通过物理吸附的方式,将典型的假单胞菌脂肪酶（Pseudomonas cepacia lipase）固定在锥形瓶的内壁上,形成简易的生物反应器. 为方便检测器壁固定化酶促反应动力学,选择特征吸收为640 nm的生化指示剂2,6-二氯靛酚为反应底物,乙酸乙烯酯为酰化试剂,丙酮为溶剂. 光谱检测表明,催化反应0.5 h后,器壁固定化脂肪酶转化底物的能力是脂肪酶粉的6倍. 在每次催化5 h共10次的循环催化中,器壁固定化脂肪酶的催化活性平均每次仅降低3.2%,而酶粉降低11.8%. 结果表明,该器壁固定化脂肪酶的活性和稳定性相对于酶粉明显提高,这将为通过固定化有效提高脂肪酶的非水催化作用提供重要的参考.