Lipase immobilization into porous chitoxan beads: activities in aqueous and organic media and lipase localization

Lipases were noncovalently immobilized in Chitoxan, a polyionic hydrogel obtained by complexation between chitosan and xanthan. The properties of free and immobilized lipases have been compared. In the aqueous medium, the activity was twice as high for immobilized lipases as for free lipases. Immobilized lipases in chitoxan were able to hydrolyze triacylglycerols in three distinct organic solvent media. At the microstructural level, lipases were not distributed uniformly in the chitoxan beads. Higher concentrations of lipase were found in the outer membrane-like layer of the beads, as compared with lower concentrations in the inner part of the beads.     

Application of photo-crosslinkable resin prepolymers to entrap microbial cells. Effects of increased cell-entrapping gel hydrophobicity on the hydrocortisone Δ1-Dehydrogenation

Summary Acetone-dried cells of Arthrobacter simplex, whose steroid ¹ activity had been previously induced, were entrapped by the use of photo-crosslinkable resin prepolymers. When the hydrophobicity of the cell-entrapping gel was increased by mixing a hydrophobic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polyethyleneglycol) (up to 30%), the hydrocortisone to prednisolone conversion rate of the immobilized cells increased significantly, attaining approximately 20% of that of the free cells. A 10% addition of organic solvents, such as methanol, to the aqueous reaction mixture enhanced the solubility of the substrate greatly and to a lesser degree the reaction rate of the immobilized cells. The presence of an electron acceptor, phenazine methosulfate or 2,6-dichlorophenolindophenol, stimulated the steroid conversion of the entrapped as well as the free cells. The stability of the entrapped cells over repeated reactions was improved by immobilization.     

酶固化技术最新研究进展(英文)

酶的本质是一种具有催化功能的蛋白质,能影响化学反应。然而,与传统的天然酶分子比较,固化酶相对更为脆弱,而传统的有机或无机催化剂其活性则比较固定。固化酶对于优化产业生产过程非常重要,近几十年来已开发出多种新型固化酶。本文在回顾酶固定化技术最新发展的同时。着重将其最新技术分别从吸附于载体,诱惑侦查及交联等三个方面进行综述。     

Efficient immobilization of lipases by entrapment in hydrophobic sol-gel materials

The commercial application of lipases as biocatalysts for organic synthesis requires simple but efficient methods to immobilize the enzyme, yielding highly stable and active biocatalysts which are easy to recover. In this study, we present a novel method to achieve lipase immobilization by entrapment in chemically inert hydrophobic silica gels which are prepared by hydrolysis of alkyl-substituted silanes in the presence of the enzyme. A typical immobilization procedure uses: an aqueous solution of lipase; sodium fluoride as a catalyst; and additives like polyvinyl alcohol or proteins and alkoxysilane derivatives like RSi-(OMe)(3) with R = alkyl, aryl, or alkoxy as gel precursors. The effect of various immobilization parameters like stoichiometric ratio of water, silane, type and amount of additive, type and amount of catalyst, and type of silane has been carefully studied. The new method is applicable for a wide variety of lipases, yielding immobilized lipases with esterification activities enhanced by a factor of up to 88, compared to the commercial enzyme powders under identical conditions. Studies on the stability of sol-gel immobilized lipases under reaction conditions or storage (dry, in aqueous or organic medium) revealed an excellent retention of enzymatic activity. The possible reasons for the increased enzyme activities are discussed. (c) 1996 John Wiley & Sons, Inc.     

Microbial lipases: at the interface of aqueous and non-aqueous media. A review

In recent times, biotechnological applications of microbial lipases in synthesis of many organic molecules have rapidly increased in non-aqueous media. Microbial lipases are the 'working horses' in biocatalysis and have been extensively studied when their exceptionally high stability in non-aqueous media has been discovered. Stability of lipases in organic solvents makes them commercially feasibile in the enzymatic esterification reactions. Their stability is affected by temperature, reaction medium, water concentration and by the biocatalyst's preparation. An optimization process for ester synthesis from pilot scale to industrial scale in the reaction medium is discussed. The water released during the esterification process can be controlled over a wide range and has a profound effect on the activity of the lipases. Approaches to lipase catalysis like protein engineering, directed evolution and metagenome approach were studied. This review reports the recent development in the field ofnon-aqueous microbial lipase catalysis and factors controlling the esterification/transesterification processes in organic media.     

Immobilized lipases from Candida antarctica for producing tyrosyl oleate in solvent-free medium

Abstract

Two immobilized lipases from Candida antarctica have been compared for the direct esterification of tyrosol with oleic acid in equimolar conditions and in the absence of organic solvent. Candida antarctica lipase B was immobilized on octyl-silica agglomerates and compared with commercial Novozym 435. Reduction of tyrosol particle size to 0.1 mm significantly increased the reaction rate with both immobilized lipases, and reduced pressure improved the final tyrosyl oleate yield up to 95% (w/w) in both cases. Immobilized lipases were recovered and reutilized in three consecutive trials with negligible inactivation. Under optimum conditions, a product mixture comprising more than 95% of tyrosyl oleate (w/w) was attained in less than 2 hours. Finally, the index of antioxidant activity obtained, according to the Rancimat method, indicated that tyrosyl oleate was slightly more effective than tyrosol as an antioxidant in a low polar matrix.     

Kinetics and mechanisms of reactions catalyzed by immobilized lipases*

This review focuses on the kinetics of several modes of immobilization of lipases, on the mechanisms of reactions of activation of immobilized lipases, and on the kinetics and mechanisms of reactions catalyzed by immobilized lipases. A comprehensive overview of the state of the art pertaining to structural features of lipases is provided as an aid to understand immobilization, interfacial activation, and catalytic performance. General rate expressions are duly derived; more frequent simplifying assumptions are stated and the results thereof listed. Physicochemical and statistical significance of parameters in rate expressions fitted to experimental data are also discussed whenever possible.     

Lipase-mediated hydrolysis of blackcurrant oil

Four commercially available lipases, both free and immobilized, were tested for their ability to catalyze hydrolysis of blackcurrant (Ribes nigrum) oil using two different approaches. The lipase from Mucor miehei was studied free and immobilized in two different ways. The former series of enzymic reactions were performed in tap water at 40 degrees C, but the latter series of enzymic processes were carried out in mixtures of isooctane and phosphate buffer (in a typical 2/1 ratio of the components) at 30 degrees C. These conditions were optimized to increase and/or to maximize the yields of the products, which were priority targets in this study. A rate of hydrolysis and a selective preference of the hydrolytic enzymes towards fatty acids, with a special focus on enrichment of alpha-linolenic acid and/or gamma-linolenic acid, were studied. Higher rates of hydrolysis of the blackcurrant oil in the former series of reactions were observed with the immobilized lipase from Pseudomonas cepacia used as biocatalyst. In the latter approach, the most favorable results of the rate of hydrolysis of the target blackcurrant oil were achieved with the immobilized lipase from Mucor miehei employed as biocatalyst. Only three lipases, selected from a series of lipases tested during this investigation, displayed specificity towards alpha-linolenic acid and gamma-linolenic acid, i.e. the immobilized lipase from P. cepacia, lipase from M. miehei and lipase from P. fluorescens.     

Technical methods to improve yield,activity and stability in the development of microbial lipases

Lipases are ubiquitous biocatalysts that catalyze various reactions in organic solvents or in solvent-free systems and are increasingly applied in various industrial fields. In view of the excellent catalytic activities and the huge application potential, more than 20 microbial lipases have been realized in large-scale commercial production. The potential for commercial exploitation of a microbial lipase is determined by its yield, activity, stability and other characteristics. This review will survey the various technical methods that have been developed to enhance yield, activity and stability of microbial lipases from four aspects, including improvements in lipase-producing strains, modification of lipase genes, fermentation engineering of lipases and downstream processing technology of lipase products.     

Immobilization of lipase in organic solvent in the presence of fatty acid additives

In this study porcine pancreatic lipase (PPL) was covalently immobilized on cross-linked polyvinyl alcohol (PVA) in organic media in the presence of fatty acid additives in order to improve its immobilized activity. The effects of fatty acid additions to the immobilization media were investigated choosing tributyrin hydrolysis in water and ester synthesis by immobilized PPL in n-hexane. Various fatty acids which are also the substrates of lipases in esterification reactions were used as active site protecting agents during the immobilization process in an organic solvent. The obtained results showed that covalent immobilization carried out in the presence of fatty acids as protective ligands improved the hydrolytic and esterification activity of immobilized enzyme. A remarkable increase in activity of the immobilized PPL was obtained when octanoic acid was used as an additive and the hydrolytic activity was increased from 5.2 to 19.2 μmol min⁻¹ mg⁻¹ as compared to the non-additive immobilization method. With the increase of hydrolytic activity of immobilized lipase in the presence of octanoic acid, in an analogous manner, the rate of esterification for the synthesis of butyl octanoate was also increased from 7.3 to 26.3 μmol min⁻¹ g⁻¹ immobilized protein using controlled thermodynamic water activities with saturated salt solutions. In addition, the immobilized PPL activity was maintained at levels representing 63% of its original activity value after 5 repeated uses. The proposed method could be adopted for a wide variety of other enzymes which have highly soluble substrates in organic solvent such as other lipases and esterases.     

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.     

Epoxidation of alkanes and cycloalkanes by microbial lipases immobilized with photo-crosslinkable resin prepolymer

Using commercial lipases immobilized in gels prepared from photo-crosslinkable prepolymers, n-alkenes (C 6 -C 12) and cyclic alkenes (C 5 -C 12 ) were converted to corresponding epoxides. Cyclooctene was converted most effectively with 54.5% of the maximum conversion by Candida cylindracea lipase immobilized in hydrophobic gel prepared from a photo-crosslinkable prepolymer, ENTP-4000. Substrates substituted with hydrophobic groups were epoxided, but the ones with hydrophilic groups were not epoxided.     

Comparing the effect of immobilization methods on the activity of lipase biocatalysts in ester hydrolysis

The activity of various lipases was compared, in both free and immobilized forms, using the kinetics of the hydrolysis reaction of p-nitrophenyl butyrate, which was followed with in situ UV/Vis diode array spectrophotometry. Several enzymes were used to catalyze the reaction, namely Candida antarctica lipase B and Fusarium solani pisi cutinase wildtype and three single-mutation variants. The enzymes were tested in three different forms: free, immobilized as cross-linked aggregates and supported on zeolite NaY. A simple kinetic model was used to allow a quantitative comparison of the behavior of the different catalysts. It was concluded that although immobilization reduces the activity of the enzyme, the zeolite offers a much higher specific activity when compared to the cross-linked aggregates, thus supplying a heterogeneous catalyst with promising catalytic properties.     

An overview on the recent advances in the transesterification of vegetable oils for biodiesel production using chemical and biocatalysts

Biodiesel, chemically defined as monoalkyl esters of long chain fatty acids, are derived from renewable feed stocks like vegetable oils and animal fats. It is produced by both batch and continuous transesterification processes in which, oil or fat is reacted with a monohydric alcohol in the presence of a catalyst. The conventional method of producing biodiesel involves acid and base catalysts to form fatty acid alkyl esters. Downstream processing costs and environmental problems associated with biodiesel production and byproducts recovery have led to the search for alternative production methods and alternative substrates. Enzymatic reactions involving lipases can be an excellent alternative to produce biodiesel through a process commonly referred to as alcoholysis, a form of transesterification reaction or through an interesterification reaction. In order to increase the cost effectiveness of the process, the enzymes are immobilized using a suitable matrix. The use of immobilized lipases and whole cells may lower the overall cost, while presenting less downstream processing problems. Main focus of this paper is to discuss the important parameters that affect the biodiesel yield, various immobilization techniques employed, mechanisms and kinetics of transesterification reaction and the recent advances in continuous transesterification processes.     

Enzymatic Separation and Resolution of Nucleophiles: A Predictive Kinetic Model

Analysis of nucleophile separation via lipase catalyzed reactions has been developed on the basis of competitive enzymatic kinetics. Ester synthesis as well as ester interchange reactions catalyzed by lipases in organic media have been analyzed according to a transfer reaction of the acyl group from/to the enzyme. The reversible reactions are conveniently simulated from the knowledge of the a competitive factor of the enzymatic system and of the final equilibrium conditions. The model which is proposed describes the reaction profile in a predictive way. Modelling of alcohol kinetic separation and resolution is given.     

Esterification of phenolic acids catalyzed by lipases immobilized in organogels

Lipases from Rhizomucor miehei and Candida antarctica B were immobilized in hydroxypropylmethyl cellulose organogels based on surfactant-free microemulsions consisting of n-hexane, 1-propanol and water. Both lipases kept their catalytic activity, catalyzing the esterification reactions of various phenolic acids including cinnamic acid derivatives. High reaction rates and yields (up to 94%) were obtained when lipase from C. antarctica was used. Kinetic studies have been performed and apparent kinetic constants were determined showing that ester synthesis catalyzed by immobilized lipases occurs via the Michaelis–Menten mechanism.     

Triglyceride interesterification by lipases. 3. Alcoholysis of pure triglycerides

Lipase-catalyzed alcoholysis of triolein dissolved in ethanol or isopropanol for the formation of ethyl and isopropyl esters was investigated. Of 16 lipases screened, Amano lipase from P. fluorescens was selected for investigation of the effects of basic reaction conditions on alcoholysis yields. Ethanolysis yields were only slightly affected by water additions to immobilized lipase preparations. Isopropyl ester yields decreased with water addition. Good operational stability was observed over 17 days. Changes in initial triolein concentration in the range 5–50 mM had very little effect on ester yields. The ionic strength of the phosphate buffer used in lipase immobilization affected ethanolysis and isopropanolysis yields in opposite ways. The highest ethanolysis yields were obtained with lipases immobilized from 250 mM buffer, while isopropyl ester yields were highest with lipases immobilized from water. In addition, the quantities and isomers of monoglyceride intermediates in ethanolysis were affected by the immobilization buffer strength. Larger quantities of 2-monoglycerides were formed in ethanolysis reactions with lipase preparations immobilized from water.     

Novel surfactant-coated enzymes immobilized in poly(ethylene glycol) microcapsules

Summary Novel surfactant-coated enzymes immobilized in poly(ethylene glycol) microcapsules have been developed for the re-use of an oil-soluble enzyme in organic media. The esterification rate of the surfactant-coated lipase immobilized in the microcapsules was thirty times that of the powder lipase. More than 90% of the enzymatic activity of the capsulated lipases has been maintained after recycling six times.     

Kinetics and mechanisms of reactions catalysed by immobilized lipases.

This review focuses on the kinetics and mechanisms of reactions catalysed by immobilized lipases. The effects of pH, temperature, and various substances on the catalytic properties of immobilized lipases and on the processes by which they are deactivated are reviewed and discussed.     

Enzyme Catalyzed Degradation and Formation of Peroxycarboxylic Acids

The ability of hydrolases to catalyze perhydrolysis, i.e. lysis of acyl substrates with hydrogen peroxide to form peroxycarboxylic acids, has been investigated. Lipases, esterases and cholinesterases were found to catalyze perhydrolysis but the preference of the enzymes for hydrogen peroxide relative to water as nucleophile was only 10-100 fold, even in the best cases. Hence, perhydrolysis proceeds with a very low efficiency in aqueous systems. Furthermore, all lipases, esterases and cholinesterases tested degrade peroxycarboxylic acids to the corresponding carboxylic acid and hydrogen peroxide. This reaction is most pronounced in the case of lipases while less so for cholinesterases. Consequently, cholinesterases are superior to the other hydrolases studied in catalyzing net formation of peracids in aqueous systems. In organic solvents, immobilized lipases efficiently catalyze formation of peracids from either triglycerides or the parent carboxylic acid. Proteases and phospholipase A-2 were found to neither degrade peracids nor catalyze perhydrolysis of carboxylic esters or phospholipids, respectively.