Crude Lipase Research Articles

A controlled fed-batch cultivation for the production of new crude lipases from Candida rugosa with improved properties in fine chemistry

A controlled constant feeding rate fed-batch strategy using oleic acid as inducer produced a crude lipase preparation from Candida rugosa (CRL-UAB) with higher protein content, carbohydrate content and lipase activity than commercial Sigma type VII CRL. CRL-UAB was partially characterised and tested in selective biotransformations of chiral compounds in aqueous (2-hydroxy 4-phenyl butanoic acid ethyl ester (HPBE)) and organic media (2-phenyl propionic acid and ketoprofen). CRL-UAB showed higher substrate specificity and enantioselectivity in aqueous media compared to Sigma CRL. Also, higher specific initial rates with 2-phenyl propionic acid and ketoprofen were observed in organic media. The influence of water on the esterification of ketoprofen was not relevant with CRL-UAB under the conditions tested, whereas a dramatic influence was observed in Sigma CRL. Different CRL-UAB batches obtained under the same cultivation controlled conditions were identical from the point of view of chromatographic behaviour, immobilisation rates and catalytic properties, indicating that a reproducible C. rugosa lipase extract had been obtained.

Production of lipase by soil fungi and partial characterization of lipase from a selected strain(Penicillium wortmanii)

Filamentous fungi from soil were screened for their ability to produce lipase. Among 56 filamentous fungi tested, one strain identified as Penicillium wortmanii was selected as the highest lipase producer. Maximum lipase production (12.5 U/ml) was obtained in 7-days cultures utilizing 5% (w/v) olive oil as the carbon source. Optimum pH and temperature for crude lipase were 7.0 and 45 degrees C, respectively. The enzyme was stable at 40 and 45 degrees C and it retained about 55% of its activity when heated at 50 degrees C for 1 hour.

Effect of fermentation conditions in the enzymatic activity and stereoselectivity of crude lipase from Candida rugosa.

Different fed-batch cultures of Candida rugosa were carried out using oleic acid as the only carbon source. The crude lipases obtained under several operational conditions and downstream processes showed different catalytic activity and isoenzymes ratio. This fact implied that the performance of the lipase produced could be modulated by using different operational fermentation conditions. These powders were compared with commercial lipase from Sigma (St. Louis, MO) in hydrolysis and synthesis reactions. Especially interesting was the fact that the enantioselectivity of a crude lipase was higher than that observed with commercial lipase in the resolution of racemic Ketoprofen. In addition, response of both lipases in the presence of water was different.

Composition of a crude lipase from Candida Cylindracea as studied by differential scanning calorimetry and thermogravimetry

Lipases are carboxyl-esterases (EC 3.1.1.3.) which, in vivo, catalyze the hydrolysis of triglycerides and isoluble substrates by a heterogeneous process. In vitro, they may also hydrolyze soluble esters, but the presence of a water–lipid interface significantly increases their lipolytic activity. The commercially available crude lipase contains ca. 30% of lactose as `extender'. In fact, the addition of sugars or polyols stabilizes the biological macromolecules, thereby preventing loss of enzyme activity and increasing resistance to denaturing conditions. By means of differential scanning calorimetry (DSC) and thermogravimetry (TG), it has been possible to evaluate the composition of a commercial lipase and also to demonstrate the addition of a monohydrate α-lactose, and the time of addition.

Production of extracellular alkaline lipase by a new thermophilicBacillus sp

A thermophilic soil isolate—Bacillus sp. RS-12, grew optimally at 50°C and not below 40°C. Production of an extracellular lipase by this organism was substantially enhanced when the type and concentration of carbon and nitrogen sources and initial pH of the culture medium were consecutively optimized. The lipase production was found to be growth-associated with maximum secretion in the late exponential growth phase,i.e. 15h of incubation. The enzyme activity as high as 0.98 nkat/mL was obtained under optimum conditions. Tween 80 (0.5%) and yeast extract (0.5%) were found to be the best carbon and nitrogen sources inducing maximum enzyme yield with initial pH 8.0 at 50°C. The kinetic characteristics of the crude lipase indicated the highest activity at 50–55°C and pH 8.0. It had a half life of 60, 18 and 15 min at 65, 70 and 75°C, respectively.

Enantioselective Adsorption of (+)-(S)-Naproxen in a Crosslinked Lipase in Organic Solvents

ABSTRACT Enantioselective adsorption of ( + )-(S)-naproxen from the racemate in cyclohexane, n-hexane, n-heptane, or isooctane, but not in a less hydrophobic solvent such as toluene, was obtained by using a crosslinked lipase (ChiroCLEC-CR) as the adsorbent. However, this behavior was not observed in cyclohexane when a crude lipase, a purified lipase, or the crosslinked lipase preexposed to the organic solvent for 24 hours was employed. Effects of racemic naproxen concentration and solvent hydrophobicity on the amount of enantiomer adsorbed to the adsorbent, the partition coefficient (i.e., the ratio of enantiomer concentration in the adsorbent to that in the organic solvent), and the selectivity factor [i.e., the ratio of partition coefficients for (+)-(S)- and (−)-(R)-naproxen] were determined. Moreover, desorption of the enantiomer from the absorbent was compared when a fresh organic solvent was substituted or a displacer such as steric acid was added to the solution after the adsorption operation.

Stability Of A Commercial Lipase From Mucor Jav Anicus: Kinetic Modelling Of Ph And Temperature Dependencies

The present communication reports experimental and modelling work pertaining to the independent roles of pH and temperature on deactivation of a crude lipase from Mucor juvanicus. Experimental data of lipolytic activities were generated by a classic pH-stat assay on a triolein emulsion following incubation at several pH values for a fixed time, or at several temperatures for various times; postulated models were then fitted by nonlinear fitting to such data. The pH-dependence data were best fit by assumption of three forms of enzyme with increasing states of protonation, with pKa values of 6.2 and 11.3, respectively, where only the intermediate form is stable within the time frame considered. The thermal-dependence data were best fit by assumption of parallel steps of deactivation and rearrangement, with activation energies of 228.8 and 221.7 kJ mol-1, respectively.

Treatment of Candida Rugosa Lipase with Short-Chain Polar Organic Solvents Enhances its Hydrolytic and Synthetic Activities

Following a simple and quick treatment based on dissolving the crude lipase from Candida rugosa in different percentages (v/v) of several polar organic solvents (methanol, ethanol, 1 and 2-propanol, 1 and 2-butanol and acetone) followed by dialysis, different preparations with enhanced activities were obtained. The opening of the lid covering the active site is proposed as the reason for explaining the activity enhancement, both in aqueous and anhydrous organic media.

Adsorption of lipase on polypropylene powder

Adsorption of different lipases by EP-100 polypropylene powder from crude and pure lipase preparations was studied. Langmuir isotherms described the adsorption equilibria well both for protein and lipase activity adsorption. Adsorption isotherms for five different proteins all gave a similar saturation level of 220 mg protein per g carrier. Twelve commercial lipase preparations were tested for selectivity in the adsorption of lipase. For all preparations the selectivity factor was larger than one. In a crude lipase preparation from Pseudomonas fluorescence, the specific activity in solution decreased by two orders of magnitude after adsorption. The adsorption was not significantly influenced by pH changes in the adsorption buffer, indicating that hydrophobic and not electrostatic interactions are the dominating adsorption forces. Adsorption of a crude lipase from Candida rugosa (Sigma) was fast and equilibrium was reached in 30 and 100 min for protein and lipase activity adsorption respectively. Desorption in aqueous solution was negligible. Investigations with seven different lipases showed no correlation between the specific lipolytic activity of dissolved enzyme in aqueous solution and the specific activity of adsorbed enzyme in an esterification reaction in organic solvent. © 1997 Elsevier Science B.V. All rights reserved.

A stable lipase fromCandida lipolytica

Although lipases have been intensively studied, some aspects of enzyme production like substrate uptake, catabolite repression, and enzyme stability under long storage periods are seldom discussed in the literature. This work deals with the production of lipase by a new selected strain of Candida lipolytica. Concerning nutrition, it was observed that inorganic nitrogen sources were not as effective as peptone, and that oleic acid or triacylglycerides (TAG) were essential carbon sources. Repression by glucose and stimulation by oleic acid and long chain TAG (triolein and olive oil) were observed. Extracellular lipase activity was only observed at high levels at late stationary phase, whereas intracellular lipase levels were constant and almost undetectable during the cultivation period, suggesting that the produced enzyme was attached to the cell wall, mainly at the beginning of cultivation. The crude lipase produced by this yeast strain shows the following optima conditions: pH 8.0-10.0, temperature of 55 degrees C. Moreover, this preparation maintains its full activity for at least 370 d at 5 degrees C.

Probing the substrate specificity for lipases. II. Kinetic and modeling studies on the molecular recognition of 2-arylpropionic esters by Candida rugosa and Rhizomucor miehei lipases

Racemic arylpropionic esters 1– 3, precursors of therapeutically important non-steroidal antiinflammatory drugs, were subjected to hydrolyses in the presence of either Candida rugosa or Rhizomucor miehei crude lipases. The hydrolyses of 1 and 2 proved to be highly enantioselective, whereas 3 was not transformed at all. Both the substrate specificity and the enantioselectivity of these lipases were explained through a molecular modeling study involving docking experiments between 1– 3 and the amino acids forming the enzymes active-sites, whose three-dimensional structures were obtained from X-ray crystallographic data, followed by extensive conformational analysis on their computer-generated complexes. The results of this study also account for the high enantioselective and good yielding hydrolysis of 3 (as the corresponding 2-chloroethyl ester) catalyzed by CRL pretreated with 2-propanol, recently reported in the literature, and lead to admit that such a treatment may operate very deep conformational changes on the amino acids of the enzyme active-site.

Optimization of the reaction medium for esterification catalyzed by A lipase from Pseudomonas fluorescens

The performance of a crude extract lipase from Pseudomonas fluorescensin esterification was evaluated in microaqueous, biphasic and surfactant-enriched biphasic systems containing various amounts of water (from almost no water to pure water). The results showed a strong negative influence of the water content on the thermodynamic equilibrium of the reaction in biphasic systems. From a kinetic point of view, the enzyme was more efficient in systems involving a water/organic solvent interface (4 times in the biphasic system, 12 times in the surfactant-enriched biphasic system).

Variations among four commercial lipases from Chromobacterium viscosum in ester synthesis

The properties of four commercial lipases from Chromobacterium viscosum, CvL 1–4, in ester synthesis were investigated. Three lipases showed a high synthetic activity in esterification, with conversions of oleic acid as high as 86–95% in 24 h, whereas one (CvL 1) gave a poor result of only 11% with the same quantity of 9 mg crude lipase preparation. The elution profiles of the four lipases from Sephacryl S-100 HR differed and SDS-PAGE suggested that while CvL 1 lipase had two equivalent protein bands of molecular size 33 and 27 kDa, respectively, the other three lipases showed only one main protein band of 33 kDa. Isoelectric focusing revealed that all of the lipases contained several isoforms, but the proportions of the isoforms varied. Furthermore, both aggregated and single lipase forms obtained after gel filtration were able to catalyse ester synthesis, but the two lipases from CvL 1 showed lower synthetic activities than the others.

Sugar ester‐modified lipase for the esterification of fatty acids and long‐chain alcohols

AbstractThe esterification reaction of a long‐chain fatty acid and a fatty alcohol with a surfactant‐modified lipase in a microaqueousn‐hexane system was studied. Various lipases from different sources were first modified with a surfactant of the sugar ester type to improve their dispersibility in apolar organic solvents. This enzyme modification technique converted inactive crude lipases to highly active biocatalysts for the esterification of long‐chain fatty acids and fatty alcohols in a microaqueous n‐hexane system. The hydrophilic‐lipophilic balance value and chainlength of the fatty acid residue of the fatty acid sugar ester, used for modifying the lipases, significantly influenced the amount of precipitated lipase that was dissolved in the aqueous solution, the protein content of the lipase‐surfactant complex and its esterification activity.

Preparation of lipase-surfactant complex for the catalysis of triglyceride hydrolysis in heterogeneous reaction systems

To facilitate lipase (E.C.3.1.1.3) catalyzed hydrolysis reaction of triglycerides in heterogeneous reaction systems, lipase-surfactant complex (LSC) was prepared by mixing an aqueous solution of lipase and surfactant in ethanol. The LSC prepared from lipase MF-30 derived from Pseudomonas sp. and Nonion SP-60R (sorbitan mono-stearate) showed a high LSC yield and activity recovery. About 30% of the protein in the crude lipase was recovered in the LSC precipitate, and further addition of surfactant to the supernatant was ineffective. Our preparation method for the selective separation of the catalytic active protein from a crude enzyme was suggested.

Lipase-catalyzed kinetic resolution of 3-hydroxy esters in organic solvents and supercritical carbon dioxide

The lipase-catalyzed kinetic resolution of different 3-hydroxy esters in organic solvents and supercritical carbon dioxide (SCCO 2) was studied. Similar enantiomeric excesses (ee, up to 99%) as found for transesterifications in organic solvents were achieved also in SCCO 2. The influence of various reaction parameters was investigated for the resolution of 3-hydroxy octanoic acid methyl esters with lipase from Pseudomonas cepacia in SCCO 2. Optimum conditions have been found at 110 bar, 40°C, and in the presence of 1.5 g molecular sieve by using vinyl acetate as acyl donor. The addition of different cosolvents had only a small effect on the reaction. Immobilization of the lipase on VA-epoxy resulted in similar optical purities as found for the crude lipase, but at halved reaction times. The effect of supercritical conditions on the stability of the lipase was also investigated.

An investigation of crude lipases for hydrolysis, esterification, and transesterification

Nine commerically available powdered lipases were investigated for their catalytic ability to hydrolyze olive oil and synthesize 1-butyl oleate by direct esterification and 2-ethyl-1-hexyl ester of rapeseed oil by transesterification. Under the experimental conditions used, a lipase from Candida rugosa exhibited the highest hydrolytic activity at 88 U mg −1 enzyme. Lipase from Pseudomonas fluorescens gave the highest conversion of oleic acid at about 95% in 24 h. Furthermore, lipases from C. rugosa and Rhizopus sp. resulted in the highest conversion of rapeseed oil at nearly 100%. Porcine pancreatic lipase showed the lowest hydrolytic activity at 1.5 U mg −1 enzyme and also the lowest synthetic activity with the conversions of oleic acid and rapeseed oil at only 50% and 19% respectively. For the lipase from Rhizomucor miehei, only esterification and transesterification activities were related. Finally, for the lipase from Chromobacterium viscosum, no relationship between the hydrolytic and synthetic activities was observed. The low multiple correlation coefficients in the order of R = 1 0.35–0.40 obtained from the regression analysis for the hydrolytic and synthetic activities for all lipases studied suggested little relationship between the hydrolytic and synthetic activities; however, the high multiple correlation coefficient of R = 0.97 ∗∗∗ for the conversion of oleic acid by esterification and rapeseed oil by transesterification by eight of the nine lipases studied suggested that there was a close relationship between esterification and transesterification. According to the results, the hydrolytic lipase activity may be of little value in predicting the synthetic activity, and in extreme cases, a lipase may exhibit no synthetic activity while possessing a high hydrolytic activity.

Thermotolerant strain of Bacillus licheniformis producing lipase

A thermotolerant variant of Bacillus licheniformis strain H1 (isolated from Jordan valley soil), was highly active in degrading macromolecules, and possessed a lipase activity with a half life of 30 min at 70°C. This activity was produced during exponential growth. The extracellular crude lipase showed maximal activity at pH 10, and retained 65% of its stability at pH 12.

Resolution of a tetrahydrofuran ester by Candida rugosa lipase (CRL) and an examination of CRL's stereochemical preference in organic media

Abstract Crude lipase from Candida rugosa (CRL) is able to resolve the C3-stereoisomers of the furo[2,3b]furan building block methyl 2-methoxytetrahydrofuran-3-carboxylate 6 by alcoholysis using n -butanol in octane. The reaction is not affected by the configuration at C2. The absolute configuration of the product 7 is 3S as determined by X-ray analysis of the crystalline derivative 14 . The stereochemical outcome of the reaction is compared to the active site model derived by the group of Kazlauskas (Ahmed et al., Biocatalysis 9 (1994), 209). Evidence is presented for the validity of this model for CRL-catalyzed alcoholysis, esterification and acidolysis reactions in organic media.

Studies on the enantioselectivity in the lipase-catalyzed synthesis of monoacylglycerols from isopropylidene glycerol

The regioselective lipase-catalyzed acylation of isopropylidene glycerol using different vinyl esters as acyl donors in toluene was studied. Reaction progress and enantioselectivity were monitored by gas chromatography using a permethylated β-cyclodextrin phase. All vinyl esters were completely converted after 20 to 24 h and it was found that the S-enantiomer reacted faster. Lower enantiomeric excess were found using e. g. vinyl palmitate (18 %ee) compared to e. g. vinyl butyrate (42 %ee) with crude lipase from Pseudomonas cepacia. Immobilization using the sol-gel method resulted in higher remaining activities (up to 69%) and increased enantioselectivity E (up to 8.5).