Short chain flavour esters synthesis by microbial lipases

Summary The peparative synthesis of 35 short chain flavour esters by lipases fromMucor miehi, Aspergillus sp.,Candida rugosa andRhizopus arrhizus was investigated in organic media. Acetic, propionic, butyric, valeric and caproic acids, as well as methanol, ethanol, butanol, i-pentanol, hexanol, citronellol and geraniol were used as substrates. Most of the esters were synthesized in good yield by at least one of the lipase preparations tested. Different conversion yields were observed according to the lipase specificity toward the acid or the alcohol moiety of the ester. Methyl- and ethyl acetates were also produced by changing the organic solvent. Enzymatic catalysis in organic solvent is thought to be a valuable method for preparative synthesis of flavour esters.     

Novel preparation method for surfactant-lipase complexes utilizing water in oil emulsions

A novel preparation method for surfactant-lipase complexes has been developed utilizing water in oil emulsions. In order to optimize the preparation conditions, we have investigated the effects of several operational parameters on the enzymatic activity of the surfactant-lipase complexes in organic media. When a nonionic surfactant was employed under optimal preparation conditions [alkaline pH 8-10, organic/aqueous = 90/10 (v/v), concentration of surfactant, 10 mM[, the surfactant-lipase complex efficiently catalyzed the esterification of benzyl alcohol with lauric acid in organic media. The esterification rate of the surfactant-lipase complex was increased over 16-fold relative to the native powder lipase. Furthermore, the lipase complex showed high storage stability. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 455-460, 1997.     

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.     

Regioselective Enzymatic Synthesis of Non-Steroidal Anti-Inflammatory Drugs Containing Glucose in Organic Media

Enzymatic transesterification of glucose with the vinyl ester of non-steroidal anti-inflammatory drugs (NSAIDs) was in organic media performed for synthesis of novel NSAIDs-glucose conjugates. Glucose was regioselectively acylated at the 6-hydroxyl group. The indomethacin-glucose conjugate and ketoprofen-glucose conjugate were produced by the catalysis of alkaline protease from Bacillus subtilis in the respective yields of 42% (over 48 h) and 63% (over 40 h). The etodolac-glucose conjugate was obtained in 26% yield (over 144 h) by lipase from Candida antarctica.     

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。     

Extracellular and cell-bound lipase activity in relation to growth of Geotrichum candidum

Summary The imperfect fungus Geotrichum candidum produced extracellular lipase in a basic peptone-salt medium. By adding olive oil or Tween 80 to the basic medium the lipase yields could be enhanced and the maximal yields were found with Tween 80, which resulted in a sixfold increase in extracellular lipase activity as compared with basic medium. During the early phase of growth in medium with olive oil the proportion of cell-bound activity was higher than that of extracellular activity, and a delay in the secretion of extracellular lipase was found. The proportion of cell-bound activity from growth in basic medium and in basic medium with Tween 80 was lower than that of extracellular activity during the entire growth phase. Analyses by polyacrylamide gel electrophoresis showed that the lipase activity from growth in all three media could be ascribed to equivalent protein bands at 57 000 and 61 000 daltons. Immunodiffusion showed that the cell-bound preparation contained lipase that was immunologically identical with purified extracellular lipase from G. candidum.     

A water activity control system for enzymatic reactions in organic media

A water activity control system for enzymatic synthesis in organic media, for litre-scale reactors has been constructed. Water activity, a(w), is a key factor when using enzymes in non-conventional media and the optimum value varies for different enzymes. The control system consists of a water activity sensor in the headspace of a jacketed glass reactor (equipped with narrow steel tubes to introduce air), gas-washing bottles containing blue silica gel (a(w)=0) and water (a(w)=1), a PC to monitor water activity and a programmable logic controller (PLC) to control the water activity. The system was evaluated by adjusting water activity in the medium, with a deviation from the set point of less than +/-0.05. Synthesis of cetyl palmitate, under controlled water activity and catalysed by two different lipase preparations, namely, Novozym 435 (immobilised Candida antarctica lipase B) and immobilised Candida rugosa lipase, were also performed. Novozym 435 catalyses reactions very well at extremely low water activity while C. rugosa lipase shows low activity for a(w)<0.5.     

Stability and structure of Penicillium chrysogenum lipase in the presence of organic solvents

Abstract

The present work describes the enzymatic properties of Penicillium chrysogenum lipase and its behavior in the presence of organic solvents. The temperature and pH optima of the purified lipase was found to be 55?°C and pH 8.0 respectively. The lipase displayed remarkable stability in both polar and non-polar solvents upto 50% (v/v) concentrations for 72?h. A structural perspective of the purified lipase in different organic solvents was gained by using circular dichroism and intrinsic fluorescence spectroscopy. The native lipase consisted of a predominant α-helix structure which was maintained in both polar and non-polar solvents with the exception of ethyl butyrate where the activity was decreased and the structure was disrupted. The quenching of fluorescence intensity in the presence of organic solvents indicated the transformation of the lipase microenviroment P. chrysogenum lipase offers an interesting system for understanding the solvent stability mechanisms which could be used for rationale designing of engineered lipase biocatalysts for application in organic synthesis in non-aqueous media.     

An extracellular solvent stable alkaline lipase from Pseudomonas sp. DMVR46: Partial purification,characterization and application in non-aqueous environment

The biosynthesis of esters is currently of much commercial interest because of the increasing popularity and demand for natural products among consumers. Biotransformation and enzymatic methods of ester synthesis are more effective when performed in non-aqueous media. In present study, an organic solvent stable Pseudomonas sp. DMVR46 lipase was partially purified by acetone precipitation and ion exchange chromatography with 28.95-fold purification. The molecular mass of the lipase was found to be ∼32 kDa. The partially purified lipase was optimally active at 37 °C and pH 8.5. The enzyme showed greater stability toward organic solvents such as isooctane, cyclohexane and n-hexane retaining more than 70% of its initial activity. The metal ions such as Ca²⁺, Ba²⁺ and Mg²⁺ had stimulatory effects on lipase activity, whereas Co²⁺ and Zn²⁺ strongly inhibited the activity. Also lipase exhibited variable specificity/hydrolytic activity toward different 4-nitrophenyl esters. DMVR46 lipase was further immobilized into AOT-based organogels used for the synthesis of flavor ester pentyl valerate in presence of organic solvents. The organogels showed repeated use of enzyme with meager loss of activity even upto 10 cycles. The solvent-stable lipase DMVR46 thus proved to be an efficient catalyst showing an attractive potency for application in biocatalysis under non-aqueous environment.     

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.     

Comparative study of the enzymatic synthesis of cephalexin at high substrate concentration in aqueous and organic media using statistical model

Synthesis of cephalexin with immobilized penicillin acylase at high substrates concentration at an acyl donor to nucleophile molar ratio of 3 was comparatively evaluated in aqueous and ethylene glycol media using a statistical model. Variables under study were temperature, pH and enzyme to substrate ratio and their effects were evaluated on cephalexin yield, ratio of initial rates of cephalexin synthesis to phenylglycine methyl ester hydrolysis, volumetric and specific productivity of cephalexin synthesis, that were used as response parameters. Results obtained in both reaction media were modeled using surface of response methodology and optimal operation conditions were determined in terms of an objective function based on the above parameters. At very high substrates concentrations the use of organic co-solvents was not required to attain high yields and actually almost stoichiometric yields were obtained in a fully aqueous media with the advantages of higher productivities than in an organic co-solvent media and compliance with the principles of green chemistry.     

Understanding the effect of tert-butanol on Candida antarctica lipase B using molecular dynamics simulations

The use of organic solvents as reaction media for enzymatic reactions has many advantages. Several organic solvents have been proposed as reaction media, especially for transesterifications using Candida antarctica lipase B (CalB). Among organic solvents, tert-butanol is associated with an enhanced conversion rate in bio-diesel production. Thus, it is necessary to understand the effect of tert-butanol on CalB to explain the high-catalytic efficiency compared with the reaction in other hydrophilic organic solvents. In this study, the effects of tert-butanol on the structure of CalB were investigated by MD simulations. The overall flexibility was increased in the presence of tert-butanol. The substrate entrance and the binding pocket size of CalB in tert-butanol were maintained as in TIP3P water. The distance between the catalytic residues of CalB in tert-butanol indicated a higher likelihood of forming hydrogen bonds. These structural analyses could be useful for understanding the effect of tert-butanol on lipase transesterification.     

Lipase from solvent tolerant Pseudomonas aeruginosa strain: production optimization by response surface methodology and application

Solvent tolerant Pseudomonas aeruginosa strain PseA has been studied for lipase activity. This strain has earlier been reported to be secreting alkaline and solvent stable protease. It produced an extra cellular lipase with suitable properties for detergent applications viz. (i) alkaline in nature, (ii) stability and compatibility towards bleach oxidants, surfactants and detergent formulations and (iii) resistant to proteolysis. Since the culture supernatant contains both protease and lipase which are together required in detergent formulations, enzymes from P. aeruginosa seem ideal for use as detergent additive. P. aeruginosa lipase exhibited remarkable stability in wide range of organic solvents at 25% (v/v) concentration. This property can be useful for solvent bioremediation and biotransformations in non-aqueous media. Media optimization for cost effective production of lipase was carried out by response surface methodology which led to 5.58-fold increase in lipase production (4580 IU/ml) over un-optimized media.     

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.     

Promiscuous enzyme-catalyzed cascade reaction: Synthesis of xanthone derivatives

Based on the screening of biocatalysts and reaction conditions including organic solvent, water content, lipase loading, reaction temperature and time, lipase TLIM exhibited the prominent promiscuity for the Knoevenagel-Michael cascade reactions of 1, 3-diketones with aromatic aldehydes to synthesize xanthone derivatives. This procedure provides satisfactory advantages such as environmental begin, simple work-up, generality, obtaining in excellent yields (80–97%), and potential for recycling of biocatalyst.     

Effect of the immobilization protocol on the properties of lipase B from Candida antarctica in organic media: Enantiospecifc production of atenolol acetate

In this work, we intend to check the effect of the immobilization protocol on the performance of lipase B from Candida antarctica (CalB) in organic medium. To this purpose, CalB has been immobilized on Eupergit C (EC) under different conditions and on EC partially modified with ethylenediamine (EDA), iminodiacetic acid (IDA) or metal chelate (IDA-Cu²⁺) and used for kinetic resolution of (R/S) 4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]benzeneacetamide (rac-atenolol). Enantiomeric resolution of atenolol was carried out by a transesterification reaction using vinyl acetate as acylant agent and an organic solvent as reaction medium. After a preliminary optimization of the reaction to obtain satisfactory yields, toluene was selected as the optimal solvent and the performances of the different CalB biocatalysts were compared. The R enantiomer was preferred in all cases but their performances were substantially different, with high differences in reaction rates, reaction yields in this kinetically controlled synthesis (EC-CalB gave a conversion of 76% while EC-IDA-Cu²⁺-CalB gave just a 27%), and enantiospecificities (EC-CalB gave an E value of 65 while EC-IDA-Cu²⁺-CalB gave a value of 13). Replacing toluene with hexane caused a decrease in enzyme activity, reaction yields and enantiospecificity of the reaction. It was remarkable that some preparations were much more sensitive to this solvent change than others. Considering that the activity decreased by less than 10% per reaction cycle, these differences are likely associated with the differences in the enzyme catalytic properties caused by the different immobilization protocols and not by inactivation of immobilized enzyme preparations during the reaction. These results confirmed that use of different immobilization protocols may be a powerful tool for altering enzyme properties when used in organic media.     

Effect of cultivation conditions on cell-surface display of Flo1 fusion protein using sake yeast

The cell-surface display of the Flo1p anchor system with a flocculation functional domain was examined under various cultivation conditions. As a model system, lipase from Rhizopus oryzae with the pro sequence was genetically fused to the Flo1 short (FS) anchor (FSProROL) and displayed on the sake yeast cell-surface under the control of the SED800 promoter (pSED800). The nutrients and carbon source in the culture media affected the display of the fusion protein FSProROL on the sake yeast cell-surface. The lipase activity in whole cells cultivated in poor media, without peptone and/or yeast extracts, were higher than those cultivated in rich media. In addition, glucose and maltose were effective carbon sources for increasing the lipase activity in whole cells, and the addition of di- or tri-saccharide as the carbon source reduced the release of the lipase activity into the culture supernatants. The initial glucose concentration was found to influence the total lipase activity and it mainly affected the lipase activity in whole cells. Under the optimum condition, sake yeast was found to show high cell density and high lipase activity in short time cultivation.     

Is induction of the exocellular lipase of Xanthomonas maltophila NK7 by fats and detergents simply the result of continual detachment from the cell surface?

The synthesis and release of the extracellular lipase of Xanthomonas maltophila NK7 grown in rich media was induced by non-substrates of the lipase: hexadecane, the non-hydrolysable detergents, Brij-35 and Triton X-100, and by oleic acid just as efficiently as by the triacylglycerols and hydrolysable detergents normally used to increase lipase yields. It is suggested that induction is the response to physical stripping by absorption of the lipase to micelles and emulsified droplets rather than to a metabolic effect. This view is supported by the following observations: (i) the longer chain triacylglycerols were better inducers than short chain triacylglycerols although they are hydrolysed more slowly by X. maltophila lipase; (ii) the yield of lipase increased with the concentration of olive oil and oleic acid well beyond the concentration which saturated the uptake system, as judged by the disappearance of oil from the medium.     

Lipase catalysed synthesis of sugar ester in organic solvents

Summary The synthesis of sugar esters catalysed by lipase in organic solvents was studied. Immobilized Candida and Mucor miehei lipase catalysed the synthesis of fructose and glucose esters of stearic acid in tertiary butyl alcohol with yields of 10 to 24 %. In the presence of phenyl or butyl boronic acid synthesis of glucose ester was achieved in hexane, heptane, benzene and toluene. The only positive reaction on disaccharides was found with palatinose.     

Enzymatic polymerization catalyzed by surfactant-coated lipases in organic media

Structural ring-opening of lactones driven by enzymatic polymerization has been performed using low concentration dosages of surfactant-coated lipases in organic media. By comparison, enzymatic polymerization rate with coated lipase proceeded at a rate 100-fold better than native powder. Similarly a higher polymeric molecular weight (21,300), narrow dispersity (Mw/Mn=1.9) and better conversion (100%) were obtained following polyesterification tests with surfactant-coated lipase.