Alcoholysis and reverse hydrolysis reactions in organic one-phase system with a hyperthermophilic beta-glycosidase

Alcoholysis and reverse hydrolysis reactions were performed enzymatically in one-phase water-saturated 1-heptanol systems. Lactose or glucose was used as substrate to produce heptyl-beta-galactoside and/or heptyl-beta-glucoside, respectively. When alcoholysis of lactose was performed at 37 degrees C with beta-galactosidase from Escherichia coli, the initial rate was 14 nmol/mL min, and the limiting factors were the poor solubility of the substrate in 1-heptanol and low thermal stability of the enzyme. When a hyperthermophilic beta-glycosidase was used at 90 degrees C, the rate was 3.14-fold higher; in this case a higher concentration of soluble lactose in the water-saturated heptanol was available to the enzyme due to the higher temperature. The hyperthermophilic beta-glycosidase was also able to use glucose and galactose as substrates to achieve the reverse hydrolysis reaction. As a consequence, when lactose was used as substrate, heptyl-beta-galactoside was formed by alcoholysis, while the released glucose moiety was used in a secondary reverse hydrolysis reaction to produce heptyl-beta-glucoside. Both reactions followed Michaelis-Menten kinetics behavior. Neither lactose nor heptyl glycosides were hydrolyzed by this enzyme in water-saturated heptanol. However, the conversion was limited by a strong product inhibition and the formation of oligosaccharides, especially at high substrate concentrations, reducing the final glycoside yield.     

Enzymatic resolution to (-)-ormeloxifene intermediates from their racemates using immobilized Candida rugosa lipase

In the synthesis of (-)-ormeloxifene, a drug candidate recently under development, enzymatic resolution of potential intermediates can be carried out using a simple, practical method. Five commercially available lipases, Candida rugosa lipase, Candida antarctica lipase B, Mucor miehei lipase, Pseudomonas cepacia lipase, and Humicola lanuginosa lipase, all immobilized on Accurel(R), were initially screened in combination with four different substrates belonging to the class of phenyl esters. Excellent stereoselectivity was observed using C. rugosa lipase with an acetate as substrate, but low reaction rates were observed in scale-up experiments. However, by changing the acyl part of the ester into a hexanoyl moiety and subjecting this substrate to enzymatic hydrolysis in aqueous acetonitrile at room temperature by C. rugosa lipase, it became possible to run the reaction to a 50% conversion on a 10 g scale within a period of 4 h, obtaining a phenolic product of more than 95% ee that could be converted to the target molecule, (-)-ormeloxifene, in two synthetic steps. Simple recovery of the immobilized enzyme by filtration allowed multiple recycling of the catalyst without significant loss of enzymatic activity. Capillary electrophoresis with sulfobutyl ether beta-cyclodextrin as a chiral buffer additive and acetonitrile as an organic modifier was demonstrated to provide an excellent chiral analytical tool for monitoring the enzymatic reactions.     

Lipase-catalysed hydrolysis of short-chain substrates in solution and in emulsion: a kinetic study.

We have studied the enzymatic hydrolysis of solutions and emulsions of vinyl propionate, vinyl butyrate and tripropionin by lipases of various origin and specificity. Kinetic studies of the hydrolysis of short-chain substrates by microbial triacylglycerol lipases from Rhizopus oryzae, Mucor miehei, Candida rugosa, Candida antarctica A and by (phospho)lipase from guinea-pig pancreas show that these lipolytic enzymes follow the Michaelis-Menten model. Surprisingly, the activity against solutions of tripropionin and vinyl esters ranges from 70% to 90% of that determined against emulsions. In contrast, a non-hyperbolic (sigmoidal) dependence of enzyme activity on ester concentration is found with human pancreatic lipase, triacylglycerol lipase from Humicola lanuginosa (Thermomyces lanuginosa) and partial acylglycerol lipase from Penicillium camembertii and the same substrates. In all cases, no abrupt jump in activity (interfacial activation) is observed at substrate concentration corresponding to the solubility limit of the esters. Maximal lipolytic activity is always obtained in the presence of emulsified ester. Despite progress in the understanding of structure-function of lipases, interpretation of the mode of action of lipases active against solutions of short-chain substrates remains difficult. Actually, it is not known whether these enzymes, which possess a lid structure, are in open or/and closed conformation in the bulk phase and whether the opening of the lid that gives access to the catalytic triad is triggered by interaction of the enzyme molecule with monomeric substrates or/and multimolecular aggregates (micelles) both present in the bulk phase. From the comparison of the behaviour of lipases used in this study which, in some cases, follow the Michaelis-Menten model and, in others, deviate from classical kinetics, it appears that the activity of classical lipases against soluble short-chain vinyl esters and tripropionin depends not only on specific interaction with single substrate molecules at the catalytic site of the enzyme but also on physico-chemical parameters related to the state of association of the substrate dispersed in the aqueous phase. It is assumed that the interaction of lipase with soluble multimolecular aggregates of tripropionin or short-chain vinyl esters or the formation of enzyme-substrate mixed micelles with ester bound to lipase, might represent a crucial step that triggers the structural transition to the open enzyme conformation by displacement of the lid.     

Lipase-catalysed hydrolysis of short-chain substrates in solution and in emulsion: a kinetic study

We have studied the enzymatic hydrolysis of solutions and emulsions of vinyl propionate, vinyl butyrate and tripropionin by lipases of various origin and specificity. Kinetic studies of the hydrolysis of short-chain substrates by microbial triacylglycerol lipases from Rhizopus oryzae, Mucor miehei, Candida rugosa, Candida antarctica A and by (phospho)lipase from guinea-pig pancreas show that these lipolytic enzymes follow the Michaelis–Menten model. Surprisingly, the activity against solutions of tripropionin and vinyl esters ranges from 70% to 90% of that determined against emulsions. In contrast, a non-hyperbolic (sigmoidal) dependence of enzyme activity on ester concentration is found with human pancreatic lipase, triacylglycerol lipase from Humicola lanuginosa (Thermomyces lanuginosa) and partial acylglycerol lipase from Penicillium camembertii and the same substrates. In all cases, no abrupt jump in activity (interfacial activation) is observed at substrate concentration corresponding to the solubility limit of the esters. Maximal lipolytic activity is always obtained in the presence of emulsified ester. Despite progress in the understanding of structure–function of lipases, interpretation of the mode of action of lipases active against solutions of short-chain substrates remains difficult. Actually, it is not known whether these enzymes, which possess a lid structure, are in open or/and closed conformation in the bulk phase and whether the opening of the lid that gives access to the catalytic triad is triggered by interaction of the enzyme molecule with monomeric substrates or/and multimolecular aggregates (micelles) both present in the bulk phase. From the comparison of the behaviour of lipases used in this study which, in some cases, follow the Michaelis–Menten model and, in others, deviate from classical kinetics, it appears that the activity of classical lipases against soluble short-chain vinyl esters and tripropionin depends not only on specific interaction with single substrate molecules at the catalytic site of the enzyme but also on physico-chemical parameters related to the state of association of the substrate dispersed in the aqueous phase. It is assumed that the interaction of lipase with soluble multimolecular aggregates of tripropionin or short-chain vinyl esters or the formation of enzyme–substrate mixed micelles with ester bound to lipase, might represent a crucial step that triggers the structural transition to the open enzyme conformation by displacement of the lid.     

Enhancement of cellulose accessibility and enzymatic hydrolysis by simultaneous wet milling

It has been shown that the rate of enzymatic saccharification of cellulosic materials including “pure” cellulose (Whatman CF?11 cellulose), newsprint, lignocellulose (prehydrolyzed to remove hemicelluloses), and wood can be substantially increased by simultaneous wet milling. An enhanced hydrolysis rate was sustained above that observed for ball milling: providing a more extensive saccharification. The cellulosic substrates were wet milled with a variety of grinding elements, such as sand, glass beads, and stainless-steel beads, agitated in a shaker bath. Simultaneous hydrolysis was achieved with a 2% substrate slurry in a 0.1M acetate buffer at 45°C and pH 5. The effectiveness of this process was dependent upon the lignified matrix of the cellulose microfibrils, the grinding elements, and the oscillation frequency of the shaker bath. Wet milling “pure” cellulose for 48 hr, with 3.5 mm glass beads and 200 oscillations/min (opm), yielded 1031 mg reducing sugar/g substrates (93% saccharification) as compared to 483 mg (44%) for the ball-milled sample and 253 mg (23%) for the unmilled material. With the lignified substrates stainless-steel beads (3.5 mm) were more effective than glass. For lignocellulose 529 mg sugar/g substrate (93% saccharification) could be obtained by wet milling with cellulase for 24 hr. This was about three times greater than that of the ball milled (169 mg, 30%) and 10 times greater than that of the unmilled (52 mg, 9%) substrates. The method was also effective for wood particles (60 mesh) giving 143 mg sugar/g wood (approximately 38% saccharification) in 48 hr, whereas the ball-milled sample gave only 79 mg (21%) and the unmlilled substrate 38 mg (10%). These observations can be explained on the basis of the current crystalline theory for the morphology of the cellulosic microfibrils. The advantage of wet milling and simultaneous hydrolysis apparently depends on a continuous generation of accessible sites and sustained rapid hydrolysis rate as the saccharification proceeds, where in the pretreated substrates the hydrolysis rate slow down as the active sites are reduced.     

Synthesis of glycerides containing n-3 fatty acids and conjugated linoleic acid by solvent-free acidolysis of fish oil

Menhaden oil, a rich source of n-3 fatty acids, was interesterified with conjugated linoleic acid (CLA) in a reaction medium composed solely of substrates and either free or immobilized commercial lipase preparations. Of five lipases tested, an immobilized preparation from Mucor miehei provided the fastest rate of incorporation of CLA into fish oil acylglycerols; however, and as observed with most of the lipases utilized, a significant proportion of the n-3 fatty acid residues were liberated in the process. A soluble lipase from Candida rugosa converted free CLA to acylglycerol residues while leaving the n-3 fatty acid residues virtually untouched. Even though the reaction rate was slower for this enzyme than for the other four lipase preparations, the specificity of the free C. rugosa lipase gives it the greatest potential for commercial use in preparing fish oils enriched in CLA residues but still retaining their original n-3 fatty acid residues.     

New Evidence for Dimerization-Dependence of the Activity of Candida Rugosa Lipase

The specific activity of the lipase of Cundidu rugosu decreases with increasing enzyme concentration even in the presence of soluble substrates. Data about the hydrolysis of 2-chloropropionic acid ethyl ester (CPEE) had suggested that this phenomenon may be caused either by dimerization of the lipase or by adsorption onto the reactor wall. In order to distinguish between both models, experiments were carried out by changing not only the enzyme concentration but also the wetted surface area of the reaction vessel. These novel data reveal that wetted glass surfaces are of only minor importance - if any. Thus, the decrease of activity seems to be caused by some kind of dimerization of the lipase. In addition, it is shown that adsorption onto hydrophobic surfaces can have a dramatic effect on the specific activity. In the presence of large hydrophobic surface areas the specific activity is found to be almost as high as that observed in the presence of insoluble substrate. The analysis of a commonly used test system for lipase activity measurements based on triacetin hydrolysis exhibits a similar activity-enzyme concentration dependence.     

Lipase immobilization on differently functionalized vinyl-based amphiphilic polymers: influence of phase segregation on the enzyme hydrolytic activity

Microbial lipase from Candida rugosa was immobilized by physical adsorption onto an ethylene-vinyl alcohol polymer (EVAL) functionalized with acyl chlorides. To evaluate the influence of the reagent chain-length on the amount and activity of immobilized lipase, three differently long aliphatic fatty acids were employed (C8, C12, C18), obtaining EVAL functionalization degrees ranging from 5% to 65%. The enzyme-polymer affinity increased with both the length of the alkyl chain and the matrix hydrophobicity. In particular, the esterified polymers showed a tendency to give segregated hydrophilic and hydrophobic domains. It was observed the formation of an enzyme multilayer at both low and high protein concentrations. Desorption experiments showed that Candida rugosa lipase may be adsorbed in a closed form on the polymer hydrophilic domains and in an open, active structure on the hydrophobic ones. The best results were found for the EVAL-C18 13% matrix that showed hyperactivation with both the soluble and unsoluble substrate after enzyme desorption. In addition, this supported biocatalyst retained its activity for repetitive cycles.     

Candida rugosa lipase-catalysed kinetic resolution of 2-substituted-aryloxyacetic esters with dimethylsulfoxide and isopropanol as additives

Candida rugosa lipase-catalysed hydrolysis of three different 2-substituted-aryloxyacetic esters was performed in aqueous buffer containing dimethyl sulphoxide and isopropanol from 0 to 80% v/v as additives, in order to obtain an enhancement of the enantioselectivity. For 2-(p-chlorophenoxy)acetic acid and 2-n-butyl-2-(p-chlorophenoxy)acetic acid ethyl esters, DMSO enhanced enzyme enantioselectivity more than IPA with an opposite enzymatic enantiopreference. The cosolvents moderately improved Candida rugosa lipase enantioselectivity for 2-phenyl-2-(p-chlorophenoxy)acetic acid ethyl ester.     

Kinetics, mechanism, and time course analysis of lipase-catalyzed hydrolysis of high concentration olive oil in AOT-isooctane reversed micelles

Candida rugosa lipase has been used to investigate the hydrolysis of high concentration olive oil in the AOT-isooctane reversed micellar system at W(o) = 10, pH 7.1, and 37 degrees C. Results from this work show the hydrolytic reaction obeys Michaelis-Menten kinetics up to the initial substrate concentration of 1.37M, with turnover number k(cat) and Michaelis constant K(M) of 67.1 mumol/min mg enzyme and 0.717M, respectively. A competitive inhibition by the main product, oleic acid, has been found with a dissociation constant K(I) for the complex EP* of 0.089M. The rate equation was further analyzed in the time course reaction and was found in agreement with the experimental results for lower substrate concentrations, up to 0.341M. Large deviation occurred at high substrate concentrations, which may be due to the effects of large consumption of water on kinetics, on the formation of glycerol, and on the deactivation of lipase in the hydrolysis reaction as well.     

Irreversible inhibition of pancreatic lipase by bis-p-nitrophenyl methylphosphonate

The reaction of porcine pancreatic lipase with an organophosphorus compound bis-p-nitrophenyl methylphosphonate (BNMP) resulted in the complete and irreversible inhibition of lipase activity on tributyrin emulsion (25 degrees C, pH 7.5, 40 mM Na-veronal-HCl buffer) whereas the activity of the enzyme on p-nitrophenyl acetate solution remained unchanged. The BNMP-modified enzyme did not bind on hydrophobic interfaces (siliconized glass beads). Tyr 49 was presumed to be the modification site, and the conclusion has been made that this residue is implicated in the interface recognition site of pancreatic lipase.     

Activity and enantioselectivity of wildtype and lid mutated Candida rugosa lipase isoform 1 in organic solvents

The activity and enantioselectivity of lipase 1 from Candida rugosa and of a chimera enzyme obtained by replacing the lid of isoform 1 with the lid of isoform 3 were compared in organic solvents. The alcoholysis of chloro ethyl 2-hydroxy hexanoate with methanol and of vinyl acetate with 6-methyl-5-hepten-2-ol were used as model reactions in different reaction conditions. The chimera enzyme was less active and enantioselective than the wildtype in all the conditions tested. A rationale for such decreases could be that the chimera lipase has a lower proportion of enzyme molecules in the open form. This might lead to a hindered access to the enzyme active site, thus affecting the catalytic activity.     

Immobilization of Candida rugosa lipase on a pH-sensitive support for enantioselective hydrolysis of ketoprofen ester

Candida rugosa lipase (Lipase OF) was immobilized by covalent binding to a pH-sensitive support showing reversibly soluble-insoluble characteristics with pH change. The immobilized lipase could carry out the enantioselective hydrolysis of ketoprofen ester in a soluble form yet be recovered after precipitation by simply adjusting pH. Its activity and enantioselectivity for hydrolysis of 2-chloroethyl ester of ketoprofen were enhanced 1.5-fold and 8.7-fold compared with those of free lipase. After eight catalytic cycles, the immobilized enzyme was still 46% active and its enantioselectivity remained unchanged.     

Diacylglycerol lipase and kinase activities in rabbit aorta and coronary microvessels

Diacylglycerol lipase and kinase activities were measured in particulate and soluble fractions from rabbit aorta (intima-media) and coronary microvessels. With rabbit aorta, the hydrolysis at the sn-1 position of 1-palmitoyl-2-oleoyl-sn-glycerol had a pH optimum of 5-6 and was greater than hydrolysis at the sn-2 position (pH optimum of 6.5). Only the 2-monoacylglycerol accumulated during incubations at pH 5 and 6.5. These results are consistent with an ordered two-step reaction sequence where the fatty acid at the sn-1 position is released first, followed by the hydrolysis of the fatty acid from the 2-monoacylglycerol by a monoacylglycerol lipase with a neutral pH optimum. Lipase activity (sn-2 hydrolysis) at pH 6.5 was greater than kinase activity at all substrate concentrations. The presence of arachidonate at the sn-2 position of the diacylglycerol increased kinase activity but had little effect on lipase activity. Kinase activity was mainly particulate, whereas 50-60% of diacylglycerol lipase and 50% of monoacylglycerol lipase activity were soluble. Diacylglycerol lipase and kinase were also present in coronary microvessel preparations. Diacylglycerol lipase (sn-2 hydrolysis) activity in coronary microvessels was not enhanced by preincubation of the enzyme preparation with cAMP-dependent protein kinase.     

A new kinetic approach for studying phospholipase C (Clostridium perfringens alpha toxin) activity on phospholipid monolayers

The enzymatic activity of purified phospholipase C (alpha toxin) from Clostridium perfringens was investigated with various phospholipid monolayers. A two-step reaction was used. Enzymatic hydrolysis of insoluble lecithin films by phospholipase C, generating 1,2-diacylglycerol and water-soluble phosphocholine, was coupled with the action of pancreatic lipase in order to give rise to fatty acid and 2-monoacylglycerol, which are rapidly desorbed from the interface. With this new procedure, it is possible to obtain continuous and accurate kinetic measurements of the phospholipase C catalyzed reaction with phospholipid monolayers as the substrate. It is thus possible to avoid the use of radiolabeled substrates as necessary in previous studies, and the difficulties caused by diacylglycerol accumulation in the lipid film are minimized. No hydrolysis was detected when either phosphatidylethanolamine, phosphatidylserine, or phosphatidylglycerol films were used as substrates. By means of a film transfer technique, Ca2+ and Zn2+ ions were found to play a specific and critical role. The present study demonstrates clearly for the first time that Ca2+ is essential for enzyme binding to lipid films, whereas Zn2+ is specifically involved in the catalytic hydrolysis of the substrate.     

Purification and Properties of a Glycerol Ester Hydrolase (Lipase) from Propionibacterium shermanii

An intracellular glycerol ester hydrolase (lipase) from Propionibacterium shermanii was recovered from cell-free extracts and purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography on diethylaminoethylcellulose. Maximum enzyme activity was observed at pH 7.2 and 47 C when an emulsion of tributyrin was used as substrate. The enzyme was stable between pH 5.5 and 8. Heating the enzyme solution at 45 C for 10 min resulted in a 75% decrease in activity. Maximum rate of hydrolysis of triglycerides was observed on tripropionin, followed in order by tributyrin, tricaproin, and tricaprylin. The lipase was strongly inhibited by mercury and arsenicals, but specific sulfhydryl reagents had little or no inhibiting effect on the enzyme activity. The enzyme also showed some esterase activity, but the hydrolysis of substrates in solution was small as compared to the hydrolysis of substrates in emulsion.     

Fabrication and application of enzyme-incorporated peptide nanotubes

Enzyme engineering is a fast-growing field in the pharmaceutical and food markets. For those applications, various substrates have been examined to immobilize and stabilize enzymes. In this report, we examined peptide nanotubes as supports for enzymes. When a model enzyme, Candida rugosa lipase, was encapsulated in peptide nanotubes, the catalytic activity of nanotube-bound lipases was increased 33% as compared to free-standing lipases at room temperature. At an elevated temperature, 65 degrees C, the activity of lipases inside the nanotubes was 70% higher than free-standing lipases. The activity enhancement of lipases in the peptide nanotubes is likely induced by the conformation change of lipases to the open form (the enzymatically active structure) as lipases are adsorbed on the inner surfaces of peptide nanotubes.     

Langmuir-Blodgett based lipase nanofilms of unique structure-function relationship

Proteins represent versatile building blocks for realization of nanostructured materials of unique structure-function relationship to be applied in nanobiotechnology. Following a recent work [Bruzzese, D., Pastorino, L., Pechkova, E., Sivozhelezov, Nicolini, C., Increase of catalytic activity of lipase towards olive oil by Langmuir-Film Immobilization of Lipase, Enzyme and Microbial Technology, submitted for publication.], the Langmuir-Blodgett technique was utilized to develop nanostructured crystal materials based on enzymes interfacially activated with olive oil as substrate. Particularly, thin films of lipase from both Mucor miehei and Candida rugosa were fabricated and characterized by UV-vis spectroscopy, Atomic force microscopy and biochemical assays. As the first step the M. miehei protein films were studied at the air-water interface and then transferred onto a solid support for further characterization of the enzymatic activity also versus surface pressure, proving that Langmuir-Blodgett film provides a better catalytic effect in lipase than a mere oil-water boundary. Moreover, improvement of lipase catalytic performance was achieved for the M. miehei versus the C. rugosa, despite its almost random distribution of hydrophobic patches and the low purity of its preparation.     

Enzymatic resolution of (S)-(+)-naproxen in a trapped aqueous-organic solvent biphase continuous reactor

A trapped aqueous-organic biphase system for the continuous production of (S)-(+)-2-(6-methoxy-2-naphthyl) propionic acid (Naproxen) has been developed. The process consists of a stereoselective hydrolysis of the racemic Naproxen methyl ester by Candida rugosa lipase in a trapped aqueous-organic biphase system. The reaction has been carried out in a laboratory-scale continuous-flow stirred tank reactor (CSTR). The staring material has been supplied in and remaining substrate recovered by organic phase. YWG-C(6)H(5), a poorly polar synthetic support, has been employed to immobilize the lipase and to restrict the aqueous phase. Lipase immobilized on YWG-C(6)H(5) containing aqueous phase has been added into the CSTR to catalyze the hydrolysis. A dialysis membrane tube containing a continuous flow closed-loop buffer has been applied in the CSTR for the extraction of product and recruiting of the aqueous part consumed. Various reaction conditions have been studied. The activity of immobilized enzyme was effected by the polarity of support, the substrate concentration, logP value of organic phase and the product inhibition. At steady-state operating conditions, an initial conversion of 35% has been obtained. The CSTR was allowed to operate continuously for 60 days at 30 degrees C with a 30% loss of activity. The hydrolysis reaction yielded (S)-(+)-Naproxen with >90% enantiomeric excess and overall conversion of 30%.     

The lipase/acyltransferase from Candida parapsilosis: molecular cloning and characterization of purified recombinant enzymes.

Candida parapsilosis has been previously shown to produce a lipase (i.e. able to catalyze efficiently the hydrolysis of insoluble lipid esters such as triacylglycerols) that preferentially catalyses transfer reactions such as alcoholysis in the presence of suitable nucleophiles other than water, even in aqueous media with high (> 0.9) water thermodynamic activity. The present work describes the cloning and the overexpression of the gene coding for this enzyme. Two ORFs (CpLIP1 and CpLIP2) were isolated. The deduced 465-amino-acid protein sequences contained the consensus motif (G-X-S-X-G) which is conserved among lipolytic enzymes. Only one of the two deduced proteins (CpLIP2) contained peptide sequences obtained from the purified lipase/acyltransferase. Homology investigations showed that CpLIP2 has similarities principally with 11 lipases produced by C. albicans (42-61%) and the lipase A from Candida antarctica (31%) but not with the other lipases sequenced so far. Both CpLIP1 and CpLIP2 were expressed in Saccharomyces cerevisiae, but only CpLIP2 coded for an active protein. The substrate specificity and the catalytic behavior of purified recombinant CpLIP2, with or without a C-terminal histidine tag, were not changed compared to those of the native lipase.