Enzyme activity electrophoresis: development and applications

The development of rocket enzyme activity electrophoresis for the detection and quantification of various proteinases, lipases and pectinases is presented. Rocket enzyme activity electrophoresis is more sensitive than the radial diffusion assay and often enables distinction between qualitatively different enzymes present in the same samples, whereas the radial diffusion assay only provides information on the overall enzyme activity. However, calibration and optimization of the enzyme activity electrophoretic assay have to be performed for each new enzyme-substrate system to be analyzed. Some of the common pitfalls in the development of new enzyme activity electrophoretic assays are presented. Enzyme activity electrophoresis can be applied in combination with other electrophoretic assays. Particularly the combination of enzyme activity electrophoresis with various immunoelectrophoretic methods can provide detailed information on the enzymes studies.     

Creation of a synthetically useful lipase with higher than wild-type enantioselectivity and maintained catalytic activity

[reaction: see text] We have found that two Geotrichum candidum lipase isozymes have remarkably different abilities to differentiate between enantiomers of ethyl 2-methyldecanoate. By rational recombination of selected portions of the two isozymes, we have created a novel lipase with an enantioselectivity superior to that of the best wild-type parent isozyme. Site-directed mutagenesis identified two key amino acid residues responsible for the improved enantioselectivity without compromised total activity of the reengineered enzyme.     

Effect of counterions on the activity of lipase in cationic water-in-oil microemulsions

This paper delineates how the different counterions affect the physicochemical properties of the aqueous aggregates and thereby the lipase activities at the interface of cationic water-in-oil microemulsions. To this end, we have synthesized a series of cetyltrimethylammonium-based surfactants, 1-14, having aliphatic, aliphatic with aromatic substitution at the alpha position, and aromatic carboxylate anion as the counterion. The physicochemical characterizations of these aqueous aggregates were done by conductometric, tensiometric, fluorometric techniques to determine counterion binding (beta), critical micelle concentration (cmc), and micropolarity at the microenvironment. It has been found that the activity of lipase mainly increases with hydrophobicity (which is directly proportional to the counterion binding (beta) of the surfactant) of the counterion and reaches a maximum when the beta value is around 0.5. Increase in hydrophobicity as well as beta leads to the attachment of more counterions at interface resulting in enhancement of interfacial area. Consequently, the enzyme may attain flexible secondary conformation at the augmented surface area and also allow larger population of substrates and enzyme molecules at the interface leading to the enhancement in lipase activity. After an optimum value of beta, further increase probably produces a steric crowding at the interface, hindering the smooth occupancy of enzyme and the substrate in this region leading to decrease of enzyme activity, while molecular surface area of the counterion did not show any virtual influence on the lipase activity. Thus, the variation in the counterion structure and hydrophobicity plays a crucial role in modulating the lipase activity.     

Candida rugosa lipase Lip1–polyethyleneglycol interaction and the relation with its partition in aqueous two-phase systems

The interaction between a lipase from Candida rugosa (Lip1) and polyethyleneglycols of different molecular masses was studied using fluorescence and circular dichroism approaches in order to be applied to the analysis of the enzyme partition mechanism in aqueous two-phase systems of polyethyleneglycol–potassium phosphate. The decrease of the partition coefficients with the polyethyleneglycol molecular mass showed that the enzyme partition is driven by the excluded volume effect and not by the enzyme–polymer interaction. The polymer did not affect the secondary and tertiary structure of the enzyme nor its biological activity. The lipase from Candida rugosa lyophilizate was partitioned in favour of the polyethyleneglycol rich phase; PEG 2000 being the system which showed the better enzyme recovery (78.26%) with a purification factor of 2.3. This method could be applied as a first step to isolate the enzyme from a culture medium with good recovery and without modifying the enzymatic capacity and the molecular structure.     

Surfactant tail length-dependent lipase activity profile in cationic water-in-oil microemulsions

The catalytic activity of Chromobacterium viscosum lipase (CV-lipase) was estimated across varying surfactant tail lengths (C-10-C-18) in water-in-oil (w/o) microemulsions of cationic surfactants containing four different hydroxyethyl-substituted head groups. An attempt to find a correlation, if any, between the activity of interfacially solubilized lipase and the varying surfactant tails was made for the first time in micellar enzymology. The second-order rate constant, k2, in lipase-catalyzed hydrolysis of p-nitrophenyl-n-hexanoate at pH 6.0 and 25 degrees C shows an improvement in enzyme activity (approximately 30-140%) across different head groups of amphiphiles with increasing tail lengths in varying solution compositions. Improvement of enzyme activity is prominent in ascending from C-10 to C-14/C-16, depending on the nature of polar head group. The hydrolytic activity of lipase in different surfactant (50 mM)/water/isooctane/n-hexanol with varying z= [alcohol]/[surfactant] (6.4 or 4.8) was amplified by 25-250% with increment in surfactant tail length in comparison with widely used cationic w/o microemulsions having solution compositions (z=16). As a notable outcome of this research, we found w/o microemulsions of 25 mM tetradecyltrimethylammonium bromide/water/isooctane/n-hexanol (z=8) producing the highest ever activity of lipase in any w/o microemulsions.     

Separation of the isoenzymes of glyoxalase I from human red blood cells by electrophoresis and isoelectric focusing on polyacrylamide gel and by ion exchange chromatography.

Methods have been devised for the separation of the isoenzymes of glyoxalase I(S-lactoylglutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) from human red blood cells by electrophoresis and electrofocusing on polyacrylamide gel slabs. Three different staining methods were used for the location of the enzyme. Three electrophoretic phenotypes of the enzyme were resolved, the fast and slow types with one band and the intermediate type with three glyoxalase I activity bands. In gel electrofocusing (pH gradient 3.5-9.5) two glyoxalase I activity bands were found for all electrophoretic types. In electrofocusing on gel with a narrow pH gradient, at least four separate enzyme components were resolved for the fast and slow electrophoretic types and at least six components for the intermediate type. The phenotypes could be distinguished correspondingly to the electrophoretic results. Preparative separation of the isoenzymes were achieved by ion exchange chromatography on DEAE-Sephacel but gel chromatography on Sephadex G-100 gave the same elution volume for all enzyme phenotypes. This corresponds to an apparent molecular weight of about 47 000.     

Head-group size or hydrophilicity of surfactants: the major regulator of lipase activity in cationic water-in-oil microemulsions

To determine the crucial role of surfactant head-group size in micellar enzymology, the activity of Chromobacterium Viscosum (CV) lipase was estimated in cationic water-in-oil (w/o) microemulsions of three different series of surfactants with varied head-group size and hydrophilicity. The different series were prepared by subsequent replacement of three methyl groups of cetyltrimethylammonium bromide (CTAB) with hydroxyethyl (1-3, series I), methoxyethyl (4-6, series II), and n-propyl (7-9, series III) groups. The hydrophilicity at the polar head was gradually reduced from series I to series III. Interestingly, the lipase activity was found to be markedly higher for series II surfactants relative to their more hydrophilic analogues in series I. Moreover, the activity remained almost comparable for complementary analogues of both series I and III, though the hydrophilicity was drastically different. Noticeably, the head-group area per surfactant is almost similar for comparable surfactants of both series I and III, but distinctly higher in case of series II surfactants. Thus the lipase activity was largely regulated by the surfactant head-group size, which plays the dominant role over the hydrophilicity. The increase in head-group size presumably allows the enzyme to attain a flexible conformation as well as increase in the local concentration of enzyme and substrate, leading to the higher efficiency of lipase. The lipase showed its best activity in the microemulsion of 6 probably because of its highest head-group size. Furthermore, the observed activity in 6 is 2-3-fold and 8-fold higher than sodium bis(2-ethyl-1-hexyl)sulfosuccinate (AOT) and CTAB-based microemulsions, respectively, and in fact highest ever in any w/o microemulsions.     

表面活性剂对脂肪酶活性和选择性的影响

考察了几种表面活性剂对lipaseOF粗酶和纯化酶催化拆分酮基布洛芬的影响。除吐温-80,吐温-60和壬基酚聚氧乙烯醚外,大部分表面活性剂对酶活性有抑制作用,其中只有吐温-80能显著提高酶的立体选择性。酶的活性和选择性与表面活性剂浓度有关。在表面活性剂浓度为最佳(20mg/mL吐温-80或30mg/mL壬基酚聚氧乙烯醚)时lipaseOF粗酶的活性可分别提高13和15倍。加入80mg/mL吐温-80,粗酶和纯化酶的对映体选择率(E值)分别由1.1和8.0增至6.7和>100。     

Effect of Additives on Freeze-Drying and Storage of Yarrowia lipolytica Lipase

The extracellular lipase of Yarrowia lipolytica presents numerous potentialities for biotechnological applications. This work describes the development and storage of powders obtained from supernatants containing Y. lipolytica lipase by freeze-drying as downstream process that is important in obtaining a stable lipase powder with high enzymatic activity. Lipase was produced by Y. lipolytica U6 mutant strain in 20-L bioreactor. Non-concentrated cell-free culture supernatant samples were supplemented with different concentrations (0.5?C1?%) of maltodextrin and glycerol as additives to freeze-drying. Effects of additives, temperature, pH, and storage time on lipase powders were determined. After addition of additives, freeze-drying yield increased 3.5-fold compared to supernatant without additive. Maltodextrin with 0.5?% concentration gave the best protection of lipase during dehydration treatment and its freeze-drying yield (77?%) is better than other formulations. Lipase powders were stored at 4 and 25?°C for 46?weeks without loss of lipase activity. A common impediment to the production of commercial enzyme is their low-stability aqueous solutions. The present study shows that freeze-dried lipase powders of Y. lipolytica have good stability for storage and various applications.     

Expression and Characterization of a Novel Lipase from Aspergillus fumigatus with High Specific Activity

A novel lipase gene from Aspergillus fumigatus, afl1-1, was cloned and expressed with a molecular mass of 38 kDa in Escherichia coli for the first time. The recombinant lipase had a preference for short carbon chain p-nitrophenyl esters, especially toward C2 p-nitrophenyl ester and exhibited potent hydrolysis activity that had not been observed. The optimum pH and temperature of this new enzyme were 8.5 and 65 °C, respectively. The recombinant lipase (AFL1-1) is an alkaline enzyme which was stable in the pH range 6.0～8.5 for 16 h (at 4 °C) and at 30～50 °C for 1 h. It is an intracellular enzyme which was purified approximately 8.47-fold with an overall yield of 86.1% by single-step Ni-NTA affinity purification, with a very high specific activity of approximately 1.00?×?10(3) U mg(-1) on a standard substrate of p-nitrophenyl acetate. The Michaelis-Menten kinetic parameters V (max) and K (m) of the lipase were 1.37 mM mg(-1) min(-1) and 14.0 mM, respectively. Ca(2+) and other metal ions could not activate the lipase. According to the homology analysis and site-directed mutagenesis assay, the catalytic triad of the recombinant lipase was identified as Ser-165, Asp-260, and His-290 residues.     

Purification and partial characterization of a lipase from Bacillus coagulans ZJU318

An extracellular lipase was purified from the fermentation broth of Bacillus coagulans ZJU318 by CM-Sepharose chromatography, followed by Sephacryl S-200 chromatography. The lipase was purified 14.7-fold with 18% recovery and a specific activity of 141.1 U/mg. The molecular weight of the homogeneous enzyme was (32 kDa), determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The enzyme activity was maximum at pH 9.0 and was stable over a pH range of 7.0–10.0, and the optimum temperature for the enzyme reaction was 45°C. Little activity loss (6.2%) was observed after 1 h of incubation at 40°C. However, the stability of the lipase decreased sharply at 50 and 60°C. The enzyme activity was strongly inhibited by Ag⁺ and Cu²⁺, whereas EDTA caused no inhibition. SDS, Brij 30, and Tween-80 inhibited lipase, whereas Triton X-100 did not significantly inhibit lipase activity.     

Lipoprotein lipase activity of the fungusRhizopus microsporus

A method is described for the isolation and purification of lipoprotein lipase activity from the culture liquid of the fungusRhizopus microsporus. Some properties of the homogeneous enzyme have been studied: molecular weight 43,000, pI 3.7.     

Purification and partial characterization ofRhizomucor miehei lipase for ester synthesis

A commercialRhizomucor miehei lipase was purified by ammonium sulfate precipitation. Phenyl Sepharose 6 Fast Row hydrophobic interaction chromatography, and DEAE Sepharose Fast Flow anion-exchange chromatography. The recovery of lipase activity was 32% with a 42-fold purification. The molecular size of the purified enzyme was 31,600 Dalton and the pI 3.8. The enzyme was stable for at least 24 h within a pH range of 7.0-10.0, and 96.8% of the enzyme activity remained when kept at 30‡C for 24 h. Further, about 10–30% of the lipase activity was inhibited by K⁺, Li⁺, Ni⁺, Co²⁺, Zn²⁺, Mg²⁺, Sn²⁺, Cu²⁺, Ba²⁺, Ca²⁺, and Fe²⁺ ions and by SDS, but EDTA had no effect. Under the experimental conditions, the optimum temperature for the hydrolysis of olive oil was 50‡C (pH 8.0), and for the synthesis of 1-butyl oleate, 37‡C. It was concluded that hydrolytic activity of lipase alone is not a sufficient criterion for its synthetic potential. The optimal molar ratio of oleic acid and 1-butanol was 2:1 for 1-butyl oleate synthesis. The 1-butyl oleate yield was unaffected by purification of the enzyme after 12 h.     

Catalytic activity of lipase immobilized onto ultrathin films of cellulose esters

Ultrathin (approximately 2.0 nm) films of cellulose acetate (CA), cellulose acetate propionate (CAP), and cellulose acetate butyrate (CAB) supported on Si wafers have been prepared by adsorption and characterized by means of ellipsometry, atomic force microscopy (AFM), and contact angle measurements. CA, CAP, and CAB ultrathin films were characterized in air just after their formation and after annealing under reduced pressure at temperature higher than the corresponding melt temperature. Upon annealing, CA, CAP, and CAB ultrathin films became smoother and more hydrophobic, evidencing molecular reorientation at the solid-air interface. CA, CAP, and CAB films were used as supports for the immobilization of lipase. The adsorption of lipase onto annealed films was more pronounced than that onto untreated films, showing the strong affinity of lipase for the more hydrophobic substrates. Enzymatic activity was evaluated by a standard procedure, namely, (spectrophotometric) measurement of p-nitrophenol, the product formed from the hydrolysis of p-nitrophenyl dodecanoate (p-NPD). Lipase immobilized onto hydrophobic films exhibited higher activity than that of free lipase and could be recycled three times while retaining relatively high activity (loss of ca. 30% of original enzymatic activity). The effect of storing time on the activity of immobilized lipase was studied. Compared with free lipase, that immobilized onto more hydrophobic films retained 70% activity after 1 month. More importantly, the latter level of activity is similar to that of free lipase. However, lipase immobilized onto more hydrophilic films retained 50% and 30% activity after 20 and 30 days, respectively. These results are explained in terms of surface wettability and the contribution of the interactions between the polar residues of lipase and the glucopyranosyl moieties of cellulose ester to maintain the natural conformation of immobilized enzyme.     

A New Lipase Isolated from Oleaginous Seeds from <Emphasis Type="Italic">Pachira aquatica</Emphasis> (Bombacaceae)

A new lipase from seeds of Pachira aquatica was purified to homogeneity by SDS-PAGE obtaining an enzyme with a molecular weight of approximately 55 kDa. The purified lipase exhibited maximum activity at 40 degrees C and pH 8.0, for an incubation time of 90 min. Concerning temperature stability, at the range from 4 to 50 degrees C, it retained approximately 47% of its original activity for 3 h. The enzyme activity increased in the presence of Ca(++) and Mg(++), but was inhibited by Hg(++), Mn(++), Zn(++), Al(+++) and various oxidizing and reducing agents. The lipase was highly stable in the presence of organic solvents, and its activity was stimulated by methanol. The values of K(m) and V(max) were 1.65 mM and 37.3 micromol mL(-1) min(-1), respectively, using p-nitrophenylacetate as substrate. The enzyme showed preference for esters of long-chain fatty acids, but demonstrated significant activity against a wide range of substrates.     

Probing the relationship between interfacial concentrations and lipase activity in cationic W/O microemulsions: a quantitative study by chemical trapping

Interfacial concentrations and/or space: which one is the predominant factor in regulating lipase activity at the water-oil interface? This work is an endeavor toward probing the relationship between lipase activity and interfacial concentrations in cationic water-in-oil (W/O) microemulsions through quantitative study by a chemical trapping method. The interfacial concentrations of water ([H2Oi]), bromide ([Bri-]), and n-hexanol ([HexOHi]) were estimated in the W/O microemulsions of six surfactants with varying headgroup architecture and hydrophilicity across a wide W0 ([H2O]/[surfactant]) range. The surfactants were prepared by the replacement of methyl groups of cetyltrimethylammonium bromide (1) by n-propyl (2-4), one hydroxyethyl (5), and one methoxyethyl (6) group. The estimated [H2Oi] was found not to change much (30.0-36.7 M) with the variation in headgroup hydrophilicity or size from 1-5. However, [Bri-] was found to increase with a decrease in the degree of dissociation (alpha), being higher for 1 and 5 (2.4-3.3 M) and relatively lower (0.9-1.9 M) for others depending on W0. Interestingly, [H2Oi] was found to be little higher (41.5-42.2 M) in the case of 6. The present study elucidates the importance of interfacial water and counterion concentrations in modulating the lipase activity in reverse micelles. In our previous report, the lipase activity was found to increase from 1-4 and in 6, whereas that observed in 5 was comparable with 1, being largely regulated by the surfactant head group size (Das, D.; Roy, S.; Mitra, R. N.; Dasgupta, A.; Das, P. K. Chem.-Eur. J. 2005, 11, 4881). The only other parameter that increased distinctly with lipase activity is the headgroup size, not [H2Oi]. Thus, the role of [H2Oi] in comparison to the surfactant's headgroup size is not found to be that significant. Moreover, the lower [Bri-] in 2-4 and 6 perhaps enhances the probability of enzyme and substrate localization at the interface, leading to higher lipase activity.     

Physical immobilization of Rhizopus oryzae lipase onto cellulose substrate: activity and stability studies

Rhizopus oryzae lipase (ROL) was immobilized by adsorption onto oxidized cellulose fibers and regenerated films. The maximum adsorption level increases with the raise in the amount of carboxylic groups on cellulose surface confirming that adsorption is being governed mainly by electrostatic interaction between the enzyme and the substrate. This hypothesis was further confirmed by zeta-potential measurements showing a decrease in the zeta-potential of the fibers after enzyme adsorption. XPS analysis showed an intensification of the N 1s peak attesting the presence of the enzyme on the surface. The effect of temperature, pH and solvent polarity on the immobilized enzyme activity and stability was investigated. The catalytic esterification of oleic acid with n-butanol has been carried on using hexane as an organic solvent. A high conversion yield was obtained (about 80%) at 37 degrees C with a molar ratio of oleic acid to butanol 1:1 and 150IU immobilized lipase. The adsorption achieved two successive cycles with the same efficiency, and started to lose its activity during the third cycle.     

Determination of lipase activity in porcine pancreas and clinical analysis of lipase in human serum with surface acoustic wave enzyme sensor system

A new kind of surface acoustic wave (SAW) enzyme sensor system has been applied to the assay of extracted lipase activity based on triacetin solution as substrate. A linear relationship between the frequency change and the enzyme activity up to 500 U/L has been obtained, and the detection limit has been evaluated to 0.3 U/L. Kinetic parameters of the extracted lipase as well as that of the standard enzyme have been estimated. The effects of temperature, pH value and surfactants have been investigated. A comparison between SAW sensor system and the conductometric method has been carried out. The technique has been applied to the determination of lipase activity in serum samples. Received: 17 January 1995 / Revised: 9 March 1995 / Accepted: 16 March 1995 Correspondence to: Shouzhuo Yao     

Synthesis of functional polycarbonates by lipase-catalyzed ring-opening polymerization

Poly(5-benzyloxy-trimethylene carbonate) (PBTMC), a new functional polycarbonate was synthesized by enzymatic ring-opening polymerization in bulk at 150°C using Porcine pancreas lipase (PPL) or Candida rugosa lipase (CL) as catalyst. Influences of different polymerization conditions such as the source of enzyme, enzyme concentration and polymerization time on the molecular weight and yield were studied. The results showed that PPL exhibited higher activity than CL. Both higher molecular weight(Mn, 18953) and yield(98%) could be obtained by the use of PPL as catalyst. ¹H NMR spectrum showed no decarboxylation occurrence during the ring-opening polymerization.     

Nonionic surfactants: a key to enhance the enzyme activity at cationic reverse micellar interface

The primary objective of the present study is to understand how the different nonionic surfactants modify the anisotropic interface of cationic water-in-oil (W/O) microemulsions and thus influences the catalytic efficiency of surface-active enzymes. Activity of Chromobacterium viscosum lipase (CV-lipase) was estimated in several mixed reverse micelles prepared from CTAB and four different nonionic surfactants, Brij-30, Brij-92, Tween-20, and Tween-80/water/isooctane/n-hexanol at different z ([cosurfactant]/[surfactants]) values, pH 6 (20 mM phosphate), 25 degrees C across a varying range of W0 ([water]/[surfactants]) using p-nitrophenyl-n-octanoate as the substrate. Lipase activity in mixed reverse micelles improved maximum up to approximately 200% with increasing content of non-ionic surfactants compared to that in CTAB probably due to the reduced positive charge density as well as plummeted n-hexanol (competitive inhibitor of lipase) content at the interfacial region of cationic W/O microemulsions. The highest activity of lipase was observed in CTAB (10 mM) + Brij-30 (40 mM)/isooctane/n-hexanol)/water system, k2 = 913 +/- 5 cm3 g-1 s-1. Interestingly, this observed activity is even higher than that obtained in sodium bis (2-ethyl-1-hexyl) sulfosuccinate (AOT)/n-heptane reverse micelles, the most popular W/O microemulsion in micellar enzymology. To ascertain the influence of non-ionic surfactants in improving the activity of surface-active enzymes is not limited to lipase only, we have also investigated the catalytic activity of Horseradish peroxidase (HRP) in different mixed W/O microemulsions. Here also following the similar trend as observed for lipase, HRP activity enhanced up to 2.5 fold with increasing concentration of nonionic surfactants. Finally, the enzyme activity was correlated with the change in the microenvironment of mixed reverse micelles by steady-state fluorescence study using 8-anilino-1-napthalenesulphonic acid (ANS) as probe.