M. A. Taipa

Purification of lipases

Interest on lipases from different sources (microorganisms, animals and plants) has markedly increased in the last decade due to the potential applications of lipases in industry and in medicine. Microbial and mammalian lipases have been purified to homogeneity, allowing the successful determination of their primary aminoacid sequence and, more recently, of the three-dimensional structure. The X-ray studies of pure lipases will enable the establishment of the structure-function relationships and contribute for a better understanding of the kinetic mechanisms of lipase action on hydrolysis, synthesis and group exchange of esters. This article reviews the separation and purification techniques that were used in the recovery of microbial, mammalian and plant lipases. Several purification procedures are analysed taking into account the sequence of the methods and the number of times each method is used. Novel purification methods based on liquid-liquid extraction, membrane processes and immunopurification are also reviewed.

Activity, conformation and dynamics of cutinase adsorbed on poly(methyl methacrylate) latex particles

The adsorption of a recombinant cutinase from Fusarium solani pisi onto the surface of 100 nm diameter poly(methyl methacrylate) (PMMA) latex particles was evaluated. Adsorption of cutinase is a fast process since more than 70% of protein molecules are adsorbed onto PMMA at time zero of experiment, irrespective of the tested conditions. A Langmuir-type model fitted both protein and enzyme activity isotherms at 25 degrees C. Gamma(max) increased from 1.1 to 1.7 mg m(-2) and U(max) increased from 365 to 982 U m(-2) as the pH was raised from 4.5 to 9.2, respectively. A decrease (up to 50%) in specific activity retention was observed at acidic pH values (pH 4.5 and 5.2) while almost no inactivation (eta(act) congruent with 87-94%) was detected upon adsorption at pH 7.0 and 9.2. Concomitantly, far-UV circular dichroism (CD) spectra evidenced a reduction in the alpha-helical content of adsorbed protein at acidic pH values while at neutral and alkaline pH the secondary structure of adsorbed cutinase was similar to that of native protein. Fluorescence anisotropy decays showed the release of some constraints to the local motion of the Trp69 upon protein adsorption at pH 8.0, probably due to the disruption of the tryptophan-alanine hydrogen bond when the tryptophan interacts with the PMMA surface. Structural data associated with activity measurements at pH 7.0 and 9.2 showed that cutinase adsorbs onto PMMA particles in an end-on orientation with active site exposed to solvent and full integrity of cutinase secondary structure. Hydrophobic interactions are likely the major contribution to the adsorption mechanism at neutral and alkaline pH values, and a higher amount of protein is adsorbed to PMMA particles with increasing temperature at pH 9.2. The maximum adsorption increased from 88 to 140 mg cutinase per g PMMA with temperature raising from 25 to 50 degrees C, at pH 9.2.

Horseradish peroxidase immobilized through its carboxylic groups onto a polyacrylonitrile membrane: comparison of enzyme performances with inorganic beaded supports.

A hydrophilic polyacrylonitrile (PAN) flat sheet membrane was aminated (8.5 μmol of NH2/mg of dry support) for covalent binding of horseradish peroxidase (HRP), mediated by the soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Silica microbeads derivatized by silanization, to yield an aminated support, and commercial aminated glass microbeads were also coupled to HRP with EDC or activated with glutaraldehyde. The immobilized enzyme activities were determined in a batch enzyme reactor with an external loop, the highest specific immobilized HRP activity being obtained on the glass support (55.8U/mg of protein). Continuous operational stability studies showed that hydrophilic PAN membrane led to the highest retention of HRP activity after an overall period of 35 h, with a normalized productivity of 59.5 μmol of H2O2 reduced/(h·Uimmob HRP).

Fluorescence of the Single Tryptophan of Cutinase: Temperature and pH Effect on Protein Conformation and Dynamics {

The cutinase from Fusarium solani pisi is an enzyme with a single l‐tryptophan (Trp) involved in a hydrogen bond with an alanine (Ala) residue and located close to a cystine formed by a disulfide bridge between two cysteine (Cys) residues. The Cys strongly quenches the fluorescence of Trp by both static and dynamic quenching mechanisms. The Trp fluorescence intensity increases by about fourfold on protein melting because of the disruption of the Ala–Trp hydrogen bond that releases the Trp from the vicinity of the cystine residue. The Trp forms charge–transfer complexes with the disulfide bridge, which is disrupted by UV light irradiation of the protein. This results in a 10‐fold increase of the Trp fluorescence quantum yield because of the suppression of the static quenching by the cystine residue. The Trp fluorescence anisotropy decays are similar to those in other proteins and were interpreted in terms of the wobbling‐in‐cone model. The long relaxation time is attributed to the Brownian rotational correlation time of the protein as a whole below the protein‐melting temperature and to protein‐backbone dynamics above it. The short relaxation time is related to the local motion of the Trp, whose mobility increases on protein denaturation.

A spectroscopic analysis of thermal stability of the Chromobacterium viscosum lipase

The thermal stability of the lipase from Chromobacterium viscosum was assessed by deactivation (loss of activity), fluorescence, circular dichroism (CD) and static light scattering (SLS) measurements. Lipase fluorescence emission is dominated by the tryptophyl contribution. An increase in the tyrosyl contribution from 2 to 16% was only observed upon prolonged incubation at 60 degrees C. The effect of temperature on the tryptophyl quantum yield was studied and two activation energies were calculated. Tryptophan residues in the native structure have an activation energy of 1.9 kcal mol(-1) for temperature-dependent non-radiative deactivation of the excited state. A structural change occurs at approximately 66.7 degrees C and the activation energy increases to 10.2 kcal mol(-1). This structural change is not characterized by tryptophan exposure on the surface of the protein. The deactivation and the evolution of structural changes with time after lipase incubation at 60 degrees C were assessed by fluorescence, CD and SLS measurements. CD spectra show that both secondary and tertiary structures remain native-like after incubation at 60 degrees C in spite of the fluorescence changes observed (red-shift from 330 to 336 nm on the trytophyl emission). SLS measurements together with the CD data show that deactivation may be due to protein association between native molecules. Deactivation and the decrease on the fraction of non-associated native lipase evaluated by changes in fluorescence intensity with time, show apparent first order kinetics. According to the rate constants, fluorescence changes precede deactivation pointing to an underestimation of the deactivation. Reactivation upon dilution during the activity assay and substrate-induced reactivation due to lipase interfacial adsorption are possible causes for this underestimation.

Kinetic characterization ofpenicillium citrinum lipase in AOT/lsooctane-reversed micelles

A lipase from a wild strain ofPenicillium citrinum was encapsulated in AOT/isooctane-reversed micelles, and the kinetic parameters were studied relative to triolein hydrolysis. Lipolytic activity was strongly dependent on the water amount in the system (Wo) and presented a bell-shaped curve for this parameter, with a maximum in the range of Wo 10–15. Optimum conditions for enzyme activity were pH 8.0 and 45‡C. The influence of substrate concentration was also studied. The enzyme showed a Michaelis-Menten behavior and the apparent kinetics constants were calculated as beingVmax.app.- 120 U/mg and Kmapp = 49.2 mM.

Identification and characterization of Chromobacterium viscosum lipase

Separation of a commercial preparation of Chromobacterium viscosum by hydrophobic interaction chromatography yields two active fractions, one corresponding to a lipase of 33.0 ± 1.0 kDa by SDS-PAGE and the other to a high molecular weight aggregate (> 250 kDa) of the lipase with some impurities absorbing at 436 nm. Partial disaggregation of this complex occurs on gel filtration chromatography in the presence of 1% (w/v) CHAPS. On gel filtration under non reducing conditions the lipase behaves like a 17 kDa protein; in the presence of a strong denaturant and of a reducing agent a molecular size of 36 kDa is obtained, in accordance with SDS-PAGE results.

Purification of a Penicillium citrinum lipase by chromatographic processes

Abstract A lipase from a wild strain of Penicillium citrinum was purified by ammonium sulphate precipitation, gel filtration chromatography on a Superose 6 column and hydrophobic interaction chromatography (HIC) on a Phenyl Superose column. The yield and purification factor were 15.2% and 379 fold, respectively. The gel filtration step was efficiently scaled-up in a Superose 6 preparative grade column and after this step, the lipase was recovered in the form of a high molecular weight aggregate. The partial disaggregation of the complex was achieved by HIC and elution with 1.0% (w/v) CHAPS. The lipase produced by Penicillium citrinum forms a dimmer of 63 000 Da, as determined by SDS-PAGE, and it accumulates in the fermentation broth as high molecular weight aggregates (>2 00 000 Da). The analysis of the dimmer showed two subunits with similar molecular weights (31 000–33 000 Da) and isoelectric points (4.8–5.0).

Purification of lipases fromChromobacterium viscosum: Is there a high molecular weight lipase?

A commercial lipolytic preparation ofChromobacterium viscosum has been separated by gel filtration on a sephacryl 200 HR column. Two lipolytic active fractions were obtained. PAGE, SDS-PAGE and IEF analysis were performed on both the mixture and the two lipolytic fractions. The results do not completely agree with those reported in the literature, suggesting that the high molecular weight lipase is probably an aggregate of lipases contained in the low molecular weight peak.

A quantitative ELISA for monitoring the secretion of ZZ-fusion proteins using SpA domain as immunodetection reporter system

A sandwich-type enzyme-linked immunosorbent assay (ELISA) was established for monitoring the secretion of ZZ-fusion proteins. Two antibodies, a monoclonal mouse anti-human proinsulin and a rabbit antibovine IgG (strongly binding to the ZZ-domain), were used to quantify the secretion of recombinant human ZZ-proinsulin to the growth medium of Escherichia coli cultures. The method here reported conjugates the advantages of sandwich-type ELISA assays, namely, high sensitivity, specificity, and throughput, with the possibility of quantifying small protein molecules (e.g., peptides). A further advantage of gene fusion techniques integrating both downstream processing and product detection and quantitation is highlighted. The method is capable of detecting levels of 0.05 ng of ZZ-proinsulin.