Immobilization of Lipase from Rhizopus Niveus: A Way to Enhance its Synthetic Activity in Organic Solvent

Lipase (EC 3.1.1.3) from Rhizopus niveus was immobilized by physical adsorption on various carriers, including different types of Celite, Spherosil and Duolite. After the enzyme immobilization, the recovered hydrolytic and synthetic activities on the different carriers were then determined. The results showed that the highest synthetic activity was obtained when Duolite XAD 761 was used as the carrier. However the recovered hydrolytic activity after the immobilization on this resin was relatively low although this carrier showed the best protein loading capacity. The highest recovered hydrolytic activity was observed when the lipase was immobilized on Celite Hyflo-Supercel using an immobilization buffer adjusted to pH 4. The comparison of the free and immobilized lipase specific activities suggest that the immobilization on Celite Hyflo-Supercel, Spherosil XOA 200 and silica has enhanced the lipase hydrolytic activity. On the other hand, the use of the lipase immobilized on Duolite XAD 761 as biocatalyst of synthetic reaction, compared to that of the free enzyme, allows the reaction initial velocity to be increased 12.2-fold. In addition, the synthetic activity of the lipase immobilized on Duolite XAD 761 was shown to be maximum at a water activity in the range of 0.32-0.52.     

Mass transfer effects in solvent-free fat interesterification reactions: influences on catalyst design

The use of solvent-free systems in the oil and fats industry is commonplace. Initial studies on interesterification were carried out in solvent systems because the lipase was immobilized solely by adsorption onto particles of diatomaceous earth. In this study, the mass transfer characteristics associated with the continuous interesterification of olive oil in a solvent-free system have been examined, for lipase immobilized on the three ion-exchange materials: Duolite ES562, Duolite ES568, and Spheroil DEA. The process of immobilization is influenced by the internal structure of the material and this in turn influences the interesterification activity of the catalyst. Individually prepared catalysts for the three support materials have shown that external mass transfer limitations are unlikely even at low flowrates.In the case of Spherosil DEA, with a mean pore diameter of 1480 A, the wide pores would be expected to reduce internal mass transfer limitations; however, it is more likely that the reduction in activity with increased catalyst loading is due to the lipase molecules being immobilized in a tightly packed monolayer. In such a situation, some active sites of the lipase molecules would become inaccessible to substrate molecules leading to an observed reduction in activity. For Duolite ES568, the observed results are very similar to those seen for Spherosil DEA, however, the pore structure of this support material indicate that some internal mass transfer limitations may also be occurring. Yet the contribution of the individual effects cannot be determined. The results observed for the support Duolite ES562 are different than those observed for the other materials and reflect the heterogeneity of Duolite ES562. The large proportion of narrow pores in the support mean that, for the catalysts examined, immobilization is most likely to have occurred in the external pores of the particles, and as such no internal mass transfer limitation is observed.It is clear that for interesterification the material chosen for enzyme immobilization will have an important role in determining the catalyst efficiency. External mass transfer limitations are very minor and observed internal mass transfer limitations may be caused by both internal mass transfer and the manner in which the immobilization process occurs. (c) 1994 John Wiley & Sons, Inc.     

Improvement of activity and stability of Rhizomucor miehei lipase by immobilization on nanoporous aluminium oxide and potassium sulfate microcrystals and their applications in the synthesis of aroma esters

In this study, Rhizomucor miehei lipase (RML) was immobilized on the hexagonally-ordered nanoporous aluminium oxide membranes (RML-Al₂O₃-NP) by adsorption and as protein-coated microcrystals (RML-PCMCs) by simultaneously precipitating RML on micron-sized potassium sulfate crystals (K₂SO₄) in pre-chilled acetone. The hydrolytic activities of immobilized lipase preparations were investigated in terms of p-nitrophenyl palmitate hydrolysis and their esterification activities were examined for the synthesis of some aroma esters such as butyl acetate, isoamyl acetate, hexyl acetate, heptyl acetate, and geranyl acetate. The immobilization yields were 33.8 and 25.1%, respectively for RML immobilized on Al₂O₃-NP membranes and potassium sulfate crystals. The catalytic efficiency ratios of RML-Al₂O₃-NP and RML-PCMCs were 2.3- and 3.9-fold higher than that of the free lipase, respectively in terms of hydrolytic activity. The free lipase was stabilized as 4.1- and 10.5-fold, respectively at 40 and 50?°C when immobilized on Al₂O₃-NP. The corresponding stabilization factors were 4.6- and 12.8-fold higher for RML-PCMCs. RML-Al₂O₃-NP and RML-PCMCs maintained 84 and 86% of their initial hydrolytic activities, respectively after 10 reuses. Of the synthesized aroma esters, the highest yield was obtained for the geranyl acetate. After 4?h reaction time, no geraniol was detected in the preparative-scale (196?g/L) synthesis of geranyl acetate for both the immobilized lipases when the initial geraniol amount, vinyl acetate amount, RML-PCMCs amount, and reaction temperature values were 1?mmol, 3?mmol, 100?mg (or 300?mg RML-Al₂O₃-NP), and 50?°C, respectively. These results show that the immobilization of R. miehei lipase by adsorption on nanoporous aluminium oxide and as protein-coated microcrystals leads to the obtention of highly stable, catalytically more active, and reusable lipase preparations.     

Enzymic Interesterification of fats: Immobilization and Immunogold Localization of Lipase on Ion-Exchange Resins

A 1,3 specific lipase from Mucor miehei has been immobilized to two phenolformaldehyde resins with active tertiary amine groups and different porous structures (Duolite ES562 and ES568N). Duolite ES568N has a more uniform pore structure, allowing more rapid uptake of lipase. Immobilized lipase particles were treated by washing, dehydration with glycolmethacrylate and embedding in polymer. Following immunogold staining of thin sections, examined by electron or light microscopy showed that the location of the lipase was dependent on the pore structure of the support material and the immobilization time.     

Regioselective Enzymatic Hydrolysis of 3',5'-DI-O-Acetylthymidine and Observation of a Surface Effect Due to Polystyrene

The selective enzymatic hydrolysis of 3',5'-di-O-acetylthyidine (1) was studied. The lipases from porcine pancreas and Aspergillus niger, and pig liver esterase, all catalysed selective hydrolysis of the 5'O-acetyl group, but the lipase from Candida cylindracea catalysed selective hydrolysis of the 3'-O-acetyl group. Highest selectivity, leading to essentially pure 3'-O-acetylthymidine, was achieved using porcine pancreatic lipase in dilute solution at pH 7.5. Provision of an artificial interface in the form of polystyrene beads led to a significant increase in the rate of hydrolysis, accompanied by a marked fall in selectivity. Other changes in the hydrolysis conditions, such as raising the concentration of substrate or adding cosolvent, also led to a fall in selectivity.     

Immobilization of Rhizomucor miehei lipase onto montmorillonite K-10 and polyvinyl alcohol gel

Abstract

Immobilization of enzymes from different sources on various supports in designed systems increases enzymes’ stability by protecting the active site of it from undesired effect of reaction environment. Also, immobilization decreases the cost of separation and facilities the reuse of the enzymes. Therefore, the design of new immobilization enzyme preparations has been an inevitable area of modern biotechnology. Herein, Rhizomucor miehei lipase (RML) was immobilized on montmorillonite K-10 (MMT-RML) by adsorption and in polyvinyl alcohol (PVA-RML) by entrapment to obtain a more stable and active lipase preparation. The free and immobilized lipase preparations were characterized for p-nitrophenyl palmitate hydrolysis. The apparent Michaelis–Menten (K_mapp) constant was almost the same for the free RML and PVA-RML, whereas the corresponding value was 17.7-fold lower for MMT-RML. PVA-RML and MMT-RML have shown a 1.1 and 23.8 folds higher catalytic efficiency, respectively, than that of the free RML. The half-lives of PVA-RML and MMT-RML were found to be 7.4 and 3.4 times longer than the free RML at 35?°C, respectively. PVA-RML and MMT-RML maintained 65% and 87% of their initial activities after four reuses. These results showed that the catalytic performance of RML has improved significantly by immobilization.     

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。     

Structural basis for the cold adaptation of psychrophilic M37 lipase from Photobacterium lipolyticum

The M37 lipase from Photobacterium lipolyticum shows an extremely low activation energy and strong activity at low temperatures, with optimum activity seen at 298 K and more than 75% of the optimum activity retained down to 278 K. Though the M37 lipase is most closely related to the filamentous fungal lipase, Rhizomucor miehei lipase (RML) at the primary structure level, their activity characteristics are completely different. In an effort to identify structural components of cold adaptation in lipases, we determined the crystal structure of the M37 lipase at 2.2 A resolution and compared it to that of nonadapted RML. Structural analysis revealed that M37 lipase adopted a folding pattern similar to that observed for other lipase structures. However, comparison with RML revealed that the region beneath the lid of the M37 lipase included a significant and unique cavity that would be occupied by a lid helix upon substrate binding. In addition, the oxyanion hole was much wider in M37 lipase than RML. We propose that these distinct structural characteristics of M37 lipase may facilitate the lateral movement of the helical lid and subsequent substrate hydrolysis, which might explain its low activation energy and high activity at low temperatures.     

Biocatalytic, Enantioselective Preparations of (R)- and (S)-Ethyl 4-Chloro-3-Hydroxybutanoate, a Useful Chiral Synthon

The synthesis of optically active ethyl 4-chloro-3-X-butanoate derivatives la-d (X = OH, a; OCOCH₃, b; OCOC₃H₇, c; OCH₂C₆H₅, d) was realized using various biocatalytic approaches such as microbiological reduction of ethyl 4-chloro-3-oxobutanoate 2 with lactic acid bacteria, hydrolysis of lb-d by the hydrolytic enzymes PLE and BChE and the transesterification of la catalyzed by a lipase from Pseudomonas fluorescens (PFL).     

Enzymatic Resolution of Zeaxanthin

Enzymatic esterification of optically inactive zeaxanthin with propanoic or palmitic acid in hexane with Candida cylindracea lipase gave the corresponding (3R,3'R)-diesters in 20% and 50% ee, respectively. When using the optically pure enantiomers the enzymatic esterification rate of (3R,3'R)-zeaxanthin was higher than for the enantiomer.     

Hydrolysis and Esterification with Lipase from Candida Cylindracea. Influence of the Reaction Conditions and Acid Moiety on the Enantiomeric Excess

The enantiomeric ratio for hydrolysis and synthesis of 1-phenyl ethanol esters of straight chain aliphatic carboxylic acids catalyzed by Candida cylindracea lipase was determined. A distinct maximum in enantiomeric ratio was observed for valeric and caproic acid in the hydrolytic direction. No significant maximum could be determined in the esterification reaction. Even though the enzyme provided larger enantiomeric ratios in the synthetic direction the enantiomeric excess of the alcohol was not higher. The enantiomeric excess was depressed by racemization reactions in the esterification as the reaction approached thermodynamic equilibrium at an insufficient conversion. While choosing the optimal chain length of the acyl donor is important in hydrolytic reactions it seems to be of greater value to raise the equilibrium conversion in the esterification reactions.     

Enzymic Interesterification of fats: Immobilization and Immunogold Localization of Lipase on Ion-Exchange Resins

A 1,3 specific lipase from Mucor miehei has been immobilized to two phenolformaldehyde resins with active tertiary amine groups and different porous structures (Duolite ES562 and ES568N). Duolite ES568N has a more uniform pore structure, allowing more rapid uptake of lipase. Immobilized lipase particles were treated by washing, dehydration with glycolmethacrylate and embedding in polymer. Following immunogold staining of thin sections, examined by electron or light microscopy showed that the location of the lipase was dependent on the pore structure of the support material and the immobilization time.     

Esterification In Organic Solvents: Selection Of Hydrolases And Effects Of Reaction Conditions

Fifty different hydrolases were screened for retention of high esterification activity in an organic solvent with citronellol as substrate. Although 22 hydrolases were very active as catalysts in the organic solvent, lipase from Candida cylindracea (lipase OF 360) was selected for further examination of the effects of reaction conditions on enzyme activity, with regard to catalyst availability and activity retention after immobilization. When the enzyme was entrapped in hydrophobic polyurethane gels, water-saturated isooctane was found to be the most suitable solvent, whereas polar solvents caused reversible catalyst inactivation. Entrapment significantly enhanced the operational stability of the lipase in the organic solvent.     

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.     

Optimization of invertase immobilization by adsorption in ionic exchange resin for sucrose hydrolysis

This work presents as a main objective to study the immobilization process of yeast invertase by adsorption in the ion exchanging resin Duolite A-568 for invert sugar production. Initially, a kinetic study of the soluble form of the enzyme was carried out. At the sequence was studied the immobilization process of yeast invertase in the weakly exchanging anionic resin Duolite A-568. The influences of the pH, enzyme concentration and temperature in the enzyme immobilization were analyzed through a central composite design (CCD). The results indicated that the retention of the catalytic activity in immobilization was strongly dependent of these variables, being maximum in a pH value of 5.0, with an enzyme concentration of 12.5 g/L (1.875 g of protein per liter) and temperature of 30 °C. The simultaneous influence of pH and temperature on the free and immobilized invertase activity was also studied through a CCD.     

Effects of the Reaction Conditions on the Enantio-Selective Esterification Using Candiada Cylindracea Lipase

Parameters influencing the synthetic activity and enantioselectivlty of the Candida cylindracea lipase were investigated. The test reaction was presented through the enantioselective esterification of 2—substituted propionic acids in organic solvents. It was found that both the activity and the enantioselectivity of the lipase could be most effectively influenced by solvent engineering. Since the solvent has a different effect on activity and enantioselectivity of the lipase, selection of the most suitable solvent or solvent mixture is an important optimization question.     

Transesterification with Candida Cylindracea Lipase in a Biphasic Aqueous-Organic System. Dependence of the Enantiomeric Ratio and the Reaction Rate on the Proportions of Water and Cyclohexane

18 September 1989

A transesterification reaction between 1-phenyl ethyl butyrate and 1-heptanol was carried out with Candida cylindracea lipase. The reaction was studied, with respect to the reaction rate and the enantiomeric ratio at different proportions of water and cyclohexane. A significantly lower reaction rate was observed for incubations with less than 0.5% water compared with those at a higher water content. Transesterification dominated over hydrolysis as the main reaction even at 70% water and a dramatic increase of the enantioselectivity was observed at this high water content. It is proposed that these effects depend on the mechanism of lipase catalysis at the interface of emulsion droplets. This interface binding gives 1-heptanol a positional preference compared with water for nucleophilic attack of the acyl enzyme and may, in addition, induce an optimal conformation for high enantioselectivity of the enzyme.     

Activity and stability of chemically modified Candida antarctica lipase B adsorbed on solid supports

The effect of various covalent chemical modifications on the transesterification activity and stability of adsorbed lipase B from Candida antarctica (CALB) was studied in 2-butanone and o-xylene. CALB species modified with either polyethylene glycol 2000 monomethyl ether (MPEG), polyethylene glycol 300 mono-octyl ether (OPEG) or n-octanol (OCT) were used in combination with a hydrophobic (Accurel) and a hydrophilic (Duolite) support. The thermostabilities of adsorbed CALB in both solvents, and that of free CALB in o-xylene were not influenced by the modifications. In contrast, the thermostability of free CALB in 2-butanone decreased 2.5-fold after MPEG modification and increased 1.5-fold after modification with OPEG and n-octanol, compared to that of native CALB. The activities of the native and modified CALB species were up to 9-fold higher after adsorption onto Accurel than those of the corresponding free enzymes. Adsorption of these enzyme species onto Duolite only resulted in a 2- to 3-fold increase in the activity of OPEG- and OCT-modified CALB. The modified CALB species adsorbed onto Accurel show similar or up to 2-fold lower activities than do native adsorbed CALB species, while 1.5- to 6-fold higher activities were found for modified CALB species adsorbed onto Duolite. We propose that hydrophobic modifiers induce conformational changes of CALB during adsorption on a hydrophobic support whereas all three modifiers protect CALB from structural alterations during adsorption onto a hydrophilic support. Received: 18 March 1999 / Received revision: 21 June 1999 / Accepted: 27 June 1999     

Overexpression of Serratia marcescens lipase in Escherichia coli for efficient bioresolution of racemic ketoprofen

Lipase from Serratia marcescens ECU1010 was cloned and overexpressed in E. coli. After optimization, the maximum lipase activities reached 5000–6000 U/l and this recombinant lipase could enantioselectively hydrolyze (S)-ketoprofen esters into (S)-ketoprofen. Among six alkyl esters of racemic ketoprofen investigated, this lipase showed the best enantioselectivity for the kinetic resolution of ketoprofen ethyl ester, with an ee_p (enantiomeric excess of product) of 91.6% and E-value of 63 obtained at 48.2% conversion. Twelve nonionic surfactants were tested for enhancing the enantioselectivity of this lipase in the bioresolution of ketoprofen ethyl ester. A very high E-value of 1084 was achieved, with an optical purity of >99% ee_p and a yield of 42.6% in the presence of 3% Brij 92V. Further studies showed that the selectivity of the lipase was improved with the increase of Brij 92V concentration. The substrate (ketoprofen ethyl ester) does not inhibit the lipase activity, while the product (S)-ketoprofen inhibits the lipase activity to some extent. These results indicate that the S. marcescens lipase is very useful for biocatalytic production of chiral profens such as (S)-ketoprofen.     

Studies on the Stability of Lipase from Candida Cylindracea, Free and Incorporated in Polymerisable Zwitterionic Surfactant Vesicles

Lipase from Candida cylindracea (CCL) was incorporated into vesicles of a polymerisable zwitterionic surfactant: bis[2-(pentacosa-10,12-diynoyloxy)ethyl]-2-aminoethanesulfonic acid (BPAS). Vesicle systems of BPAS were characterised in terms of morphology (Electron Microscopy) and stability. Polymerisation of BPAS vesicles did not alter the morphology and polymeric vesicles were considerably more stable than the monomeric analogues. CCL was incorporated into the vesicle membrane by spontaneous insertion. The enzyme remained fully active after incorporation into the vesicle bilayer; especially in homogeneous assay mixtures the vesicle incorporated enzyme showed an increased activity when compared to the free lipase. The stability of free and incorporated lipase was determined by measuring the residual activity of the various systems when mixed with ethanol (50% v/v) or 2-(n-butoxy)ethanol (37.5% v/v), at 50°C and 60°C and in the presence of the proteolytic enzyme trypsin. In all cases the vesicle incorporated enzyme showed an increased stability against the denaturating conditions.