Rhizopus Delemar Research Articles

Activity and stability of lipase in the solid-phase glycerolysis of triolein

Lipase-catalyzed glycerolysis of triolein was studied in a solid-phase system. The activity and stability of three bacterial lipases in crude and purified forms were examined. Most of these lipases were suitable for the high-yield synthesis of monooleylglycerol. With crude lipase from Chromobacterium viscosum, 96% monooleylglycerol concentration was achieved. In the case of pure lipase from Pseudomonas cepacia, immobilization on Celite was necessary. This led to a strong increase in the long-term stability and final monooleylglycerol concentration. Immobilization on Sepharose™ led to less stable and active lipases from P. cepacia. In general, the highest loss in stability was observed in the first reaction hour, and purified lipases were less suitable. Furthermore, six yeast and mold lipases were employed as catalysts in the model reaction system. Most of them have been neither very stable nor very active in the glycerolysis reaction. Only purified lipase from Rhizopus delemar gave moderate monooleylglycerol concentrations. The addition of R. delemar at intervals influenced neither stability nor monooleylglycerol concentrations. The optimum cooling temperature for the solidification of the reaction mixture, which was necessary for high monooleylglycerol concentration, was determined to be 8°C.

Cloning and nucleotide sequence of cDNA encoding a lipase from Fusarium heterosporum.

Fusarium heterosporum produces a solvent-tolerant lipase. A 1.3-kbp lipase cDNA was isolated from the cDNA library by colony hybridization with an oligonucleotide probe corresponding to the N-terminal amino acid sequence. Nucleotide sequence analysis showed an open reading frame of 999 bp, and the deduced amino acid sequence contained the N-terminal sequence determined by Edman degradation and the consensus pentapeptide (-Gly-X-Ser-X-Gly-), which is conserved in lipase, esterase, and serine protease. The mature lipase consisted of 301 amino acid residues with a molecular mass of 32.7 kDa, preceded by the putative signal peptide or preprosequence. The enzyme was homologous to lipases from Humicola lanuginosa (39% homology), Rhizomucor miehei (32%), Rhizopus delemar (32%), and Rhizopus niveus (32%), and to mono- and diacylglycerol lipase from Penicillium camembertii (38%). Comparison of this lipase with these homologous enzymes suggested that the catalytic triad was composed of Ser144, Asp198, and His256, and that the oxyanion hole was formed with Ser 82.

Enzymatic Esterification of Diols in Reverse Micellar Media

AbstractWith the help of highly dynamic reverse micellar media, lipase‐catalyzed esterification reactions can be carried out at ambient conditions without any agitation. Having low water contents in reverse micellar media, these reversible reactions favor the esterification direction instead of the normal hydrolysis catalyzed by lipases. A proposed enzyme kinetic model has successfully predicted the reaction mechanism of lauric acid with 1,3‐propanediol in reverse micellar media. However, experiments conducted showed a likelihood of substrate inhibition at higher lauric acid concentrations (>70 mM) and a strong bonding effect among 1,3‐propanediol, water, and AOT head groups. The effect of water concentration in the reaction media was shown to be important. The optimal Wo's for achieving the greatest initial reaction rate and final conversion were 9 and 4 for Candida rugosa and Rhizopus delemar, respectively. For both lipases, reverse micellar media experiments showed a lipase localization possibility.

Enzymatic phosphatidylcholine hydrolysis in organic solvents: An examination of selected commercially available lipases

AbstractEight commercial lipase preparations were examined for the ability to hydrolyze phosphatidylcholine (PC) in hexane solutions. Only the enzymes fromHumicola lanuginosa, Rhizopus delemar andCandida rugosa displayed appreciable activity. Solvent polarity was the largest single factor affecting activity. TheH. lanuginosa sample was most active in polar solvents. TheR. delemar preparation was most active in polar (2‐hexamone) and nonpolar (decane) solvents and least active in solvents of intermediate polarity (hexane). The solvent dependence of the activity of theC. rugosa enzyme varied with the ratio of substrate to enzyme. Different degrees of activity were retained by the three enzymes after passive immobilization on Celite, controlled‐pore glass, polypropylene and Amberlite XAD‐7 resins. No single resin yielded the best retained activity for all three preparations. When examined in 2‐octanone, hexane and isooctane, the Celite‐immobilizedR. delemar andH. lanuginosa enzymes exhibited highest activity in 2‐octanone, while immobilizedC. rugosa was most active in isooctane. The water content at which maximum activity was observed was relatively independent of solvent polarity and the amount of catalyst but was proportional to the amount of PC in the reaction. The retention of activity by immobilizedRhizomucor miehei lipase (Lipozyme) during multiple hydrolytic cycles required a reduction in the water content of the system below that yielding optimal activity in a single cycle.

Microbial synthesis and properties of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in Comamonas acidovorans

Comamonas acidovorans DS-17 was isolated from activated sludge and found to produce copolymers of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) at 30°C under growth-limited conditions. When 1,4-butanediol or 4-hydroxybutyric acid was used as the sole carbon source, a P(4HB) homopolymer was produced. Random copolymers of 3HB and 4HB units were produced on the addition of glucose or 3-hydroxybutyric acid to the culture solution of 4-hydroxybutyric acid. The physical properties of P(3HB-co-4HB) copolyesters with high 4HB fractions (64–100 mol%) were investigated. The copolyester films with high 4HB fractions exhibited the characteristics of a thermoplastic elastomer, and the tensile strength increased from 17 to 104 MPa as the 4HB fraction was increased from 64 to 100 mol%. The biodegradabilities of P(3HB-co-4HB) films were studied in aqueous solutions of extracellular polyhydroxybutyrate (PHB) depolymerase from Alcaligenes faecalis or of lipase from Rhizopus delemer. The erosion rate of P(3HB-co-4HB) films by PHB depolymerase decreased as the 4HB fraction in copolyester was increased from 64 to 100 mol%. In contrast, the erosion rate of films by lipase increased with the 4HB fraction.

Conformational lability of lipases observed in the absence of an oil-water interface: crystallographic studies of enzymes from the fungi Humicola lanuginosa and Rhizopus delemar.

Conformational lability of lipases observed in the absence of an oil-water interface: crystallographic studies of enzymes from the fungi Humicola lanuginosa and Rhizopus delemar.

Improvement of Enzyme Activity and Stability for Reverse Micellar-Encapsulated Lipases in the Presence of Short-Chain and Polar Alcohols

In this report, we demonstrate that the initial activity of lipases from Rhizopus delemar and Candida rugosa encapsulated in Water /AOT/isooctane reverse micelles is greatly enhanced when short-chain n-alkanols (<C5) are present in the medium prior to lipase encapsulation and reduced when long-chain alcohols are present. In addition, enzyme stability for micellar encapsulated lipase was significantly increased when the medium contained both fatty acid and glycerol substrates (esterification reaction). For these cases, the substitution of tetradecane for isooctane as solvent further increased the lipase's half-life from ca. 1 to ca. 3 weeks. An explanation for these results is given which is based on the enhancement of intermicellar attractive interactions and micellar clustering by short-chain and polar alcohols and long-chain oils. Accompanying these changes is a disordering of the structure for the micellar interface and a reduction of the interfacial tension, both of which would reduce enzyme-surfactan...

ChemInform Abstract: Highly Asymmetric Enzymatic Hydrolysis and Transesterification of meso‐ Bis(acetoxymethyl)‐ and Bis(hydroxymethyl)cyclopentane Derivatives: An Insight into the Active Site Model of Rhizopus Delemar Lipase.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Esterification reactions catalyzed by lipases in microemulsions: The role of enzyme localization in relation to its selectivity

The activity of lipases from Rhizopus delemar, Rhizopus arrhizus, and Penicillium simplicissimum entrapped in microemulsions formulated by bis-(2-ethylhexyl)sulfo-succinate sodium salt (AOT) in isooctane has been studied in esterification reactions of various aliphatic alcohols with fatty acids. The effect of the nature of the fatty acids (chain length) and of the alcohols (primary, secondary, or tertiary; chain length; cyclic structures) on the lipase activities was investigated in relation to the reverse micellar structure. The lipases tested showed a selectivity regarding the structure of the substrates used when hosted in the AOT/isooctane microemulsion systems. Penicillium simplicissimum lipase showed higher reaction rates in the esterification of long chain alcohols as well as secondary alcohols. Primary alcohols had a low reaction rate and tertiary a very slow rate of esterification. Long chain fatty acids were better catalyzed as compared to the shorter ones. Rhizopus delemar and R. arrhizus lipases showed a preference for the esterification of short chain primary alcohols, while the secondary alcohols had a low rate of esterification and the tertiary ones could not be converted. The reaction of medium chain length fatty acids was also better catalyzed than in the case of the long ones. The observed lipase selectivity appeared to be related to the localization of the enzyme molecule within the micellar microstructure due to the hydrophobic/hydrophilic character of the protein. The reverse micellar structural characteristics, as well as the localization of the enzyme, were examined by fluorescence quenching measurements and spectroscopical studies.

Highly asymmetric enzymatic hydrolysis and transesterification of meso-biscacetoxymethyl)- and bis(hydroxymethyl)cyclopentane derivatives: an insight into the active site model of Rhizopus Delemar lipase

Rhizopus delemar lipase (RDL)-catalysed hydrolysis of meso-1,3-bis(acetoxymethyl) cyclopentane derivatives ( 7, 12) and Pseudomonas fluorescens lipase (PFL)-catalysed hydrolysis of 7 afforded the chiral monoacetates ( 15, 16) of >99 % ee. In explanation of these high enantioselectivities of RDL, the simple box-type active site model of enzyme was tentatively proposed. On the other hand, PFL-catalysed transesterification of meso-bis-(hydroxymethyl) cyclopentane ( 6) afforded (+)- 16 of >99 % ee. The obtained (+) and (−)- 16 were converted into the natural carbocyclic nucleoside (−)-aristeromycin ( 25), respectively.

Overexpression of aRhizopus delemar lipase gene inEscherichia coli

A cloned complementary deoxyribonucleic acid encoding the precursor polypeptide of an extracellular lipase from the fungus Rhizopus delemar was altered by site-directed mutagenesis to generate deoxyribonucleic acid fragments that specifically code for the polypeptides of the proenzyme and the mature form of the lipase. Attempts to produce these polypeptides in enzymatically active form in Escherichia coli revealed toxic effects toward the host. Therefore the polypeptides were expressed as inactive and insoluble forms in the cytoplasm of E. coli BL21 (DE3) cells using plasmid vector pET11-d. With this tightly regulated high-level expression system, lipase and prolipase polypeptides were produced to estimated levels of up to 21% and 15%, respectively, of total cellular protein. The insoluble polypeptides were solubilized in 8 M urea. Refolding into active forms was achieved by treatment with the redox system cystine/cysteine and dilution. Refolded mature lipase was purified to homogeneity by affinity and ion exchange chromatography. The enzyme had a specific activity comparable to that of lipase from the fungal culture. The quantities of pure enzyme obtained from a 1-L culture of E. coli exceeded those obtained from the fungal culture by a factor of at least 100. Refolded recombinant prolipase was purified essentially to homogeneity and had a specific activity similar to that of the mature enzyme. Its pH optimum was 7.5, rather than the pH 8 determined for recombinant mature lipase and for the enzyme purified from the fungal culture. Recombinant prolipase retained activity after 15 min incubation at 65 degrees C, while mature lipase retained activity only up to 45 degrees C.

Medium optimization by a fractional factorial design for lipase production by Rhizopus delemar

The aim of the present work is to improve production of extracellular lipase by Rhizopus delemar using industrial grade carbon and nitrogen sources and performing statistical optimization of the production medium. Soya paste and dextrin were selected in the first phase of this work as the best carbon and nitrogen sources and were used in the following experiments. Optimization of the medium composition was carried out with a fractional factorial design in two steps. Lipolytic activity at 48 h was 12-fold greater than with the initial medium. The results show that it is necessary to modify the carbon source-nitrogen source ratio, to reduce olive oil concentration and to add Tween 80 to the medium to achieve maximum production.

Enzymatic reaction in water-in-oil microemulsions. Part 1.—Rate of hydrolysis of a hydrophilic substrate: acetylsalicylic acid

The rates of catalytic hydrolysis of a hydrophilic substrate, acetylsalicylic acid (AA), have been measured both in aqueous solution and in water-in-oil (w/o) microemulsions formed by AOT in n-heptane. Catalysts used were lipase from Rhizopus delemar, α-chymotrypsin from Bovine pancreas and imidazole as an acid–base catalyst.

Enzymatic esterification of glycerol I. Lipase‐catalyzed synthesis of regioisomerically pure 1,3‐sn‐diacylglycerols

Regioisomerically pure 1,3‐sn‐diacylglycerols are conveniently prepared in high yields (&gt;80%) and in large quantities by enzymatic esterification of glycerol in the presence of various 1,3‐selective lipases(Chromobacterium viscosum, Rhizopus delemar, Rhizomucor miehei) and a variety of different acyl donors like free fatty acids, fatty acid alkyl esters and vinyl esters. All reactions are carried out in aprotic organic solvents of low water content, namelyn‐hexane, diethyl ether or tBuOMe. The creation of an artificial interphase between the solvent‐immiscible hydrophilic glycerol and the hydrophobic reaction media by the adsorption of glycerol onto a solid support prior to use was essential for the success of these transformations. The effects of reaction conditions and the regioselectivities of the lipases on the product yields are described in detail.

Enzymatic esterification of glycerol II. Lipase‐catalyzed synthesis of regioisomerically pure 1(3)‐rac‐monoacylglycerols

Regioisomerically pure 1(3)‐rac‐monoacylglycerols are conveniently prepared in high yields (&gt;75%) and in multigram quantities by enzymatic esterification of glycerol in the presence of various lipases(Chromobacterium viscosum, Rhizopus delemar, Rhizomucor miehei) with a variety of different acyl donors, such as free fatty acids, fatty acid alkyl esters, vinyl esters and triacylglycerols, as well as natural fats and oils. All reactions are carried out in aprotic organic solvents with low water content, namelyn‐hexane, diethyl ether, tBuOMe or mixtures of these solvents. Essential for the success of these transformations were the following two factors. First, the creation of an artificial interphase between the solvent‐immiscible hydrophilic glycerol and the hydrophobic reaction medium by its adsorption onto a solid support. Second, a facile system for the separation of the desired monoacylglycerol from the reaction mixture, coupled with the continuous recycling of acyl donor and undesirable by‐products.

Lipase-catalyzed hydrolysis of Crambe oil in AOT-isooctane reversed micelles

The hydrolysis by 1,3-specific lipases (Humicola lanuginosa, Mucor miehei, Rhizopus delemar andRhizopus javanicus) of the highly symmetric, high molecular weight triglycerides fromCrambe abyssinica (Crambe) seed oil is studied in an AOT-stabilized microemulsion system. Enzyme kinetic data shows that, of the lipases studied,Rhizopus javanicus lipases exerts the highest hydrolytic activity towards this new seed oil.

Preparation of a Lipid-Coated Lipase and Catalysis of Glyceride Ester Syntheses in Homogeneous Organic Solvents

Abstract A lipid-coated lipase was prepared by mixing aqueous solutions of lipase and dialkyl amphiphiles: A hydrophilic surface of the enzyme is covered with a lipid layer, of which lipophilic two-long-alkyl tails solubilize the enzyme in hydrophobic organic solvents. The lipid-coated lipase was prepared in various conditions by changing hydrophilic head groups of lipid molecules, a ratio of lipid/lipase, and an origin of lipase; the complex prepared from nonionic amphiphiles and lipase D from Rhizopus delemar showed a high stability and a high enzymatic activity in hydrophobic organic solutions. The lipid-coated lipase catalyzed di- and triacylglycerol syntheses from monoacylglycerols and aliphatic acids in the homogeneous and dry benzene solution in the presence of two pieces of molecular sieves (water: 80 ppm). The lipid-coated lipase could also catalyze ester exchange reactions in organic solvents with a small amount of water (250 ppm). Enzymatic activity was affected by a nature of organic solvents and a water content in organic solvents. The catalytic activity of the lipid-coated lipase was highly efficient compared with other enzyme systems such as the poly(ethylene glycol)-grafted lipase and the direct dispersion of lipase powder for glyceride syntheses in the dry organic solution.

Formation of polyol–fatty acid esters by lipases in reverse micellar media

The synthesis of polyol-fatty acid esters has strong implications in such industries as foods, cosmetics, and polymers. We have investigated these esterification reactions employing the polyols ethylene glycol, 2-monoglyceride, and sugars and their derivatives with the biocatalyst lipase in water/AOT/isooctane reverse micellar media. For the first reaction, 50-60% conversion was achieved and product selectivity toward the monoester over the diester shown possible by employing lipase from Rhizopus delemar. A simple kinetic model based on the formation of acyl-enzyme intermediate accurately predicted the effect of polyol concentration but not the effect of fatty acid or water concentration probably due to the model exclusion of partitioning effects. The success of this reaction in reverse micellar media is due greatly to its capacity to solubilize large quantities of glycol despite the media's overall hydrophobicity. The second reaction, investigated for its potential for production of "mixed" glycerides, also achieved about 50% conversion but had only a small portion of triglyceride in its product distribution. Also, isomerization of the 2-monoglyceride to 1-monoglyceride, followed by hydrolysis of the latter, unfortunately occurred to a significant extent. Attempts at esterification with hexoses and their derivatives such as glucose and mannitol produced no conversion.

Comparative Studies of Lipase fromRhizopus Delemarin Various Microemulsion Systems

Hydrolysis of triglycerides by lipase from Rhizopus delemar has been studied in three different types of microemulsion systems. Microemulsions were prepared by using anionic (AOT), cationic (CTAB) and nonionic (C12E4) surfactants. Various parameters affecting the reaction, such as temperature, pH optimum, water content (R = [H2O]/[surfactant]), as well as Km.app and Vapp, were determined using triolein and tributyrin as substrates. Maximum enzyme activity was obtained at R = 9, T = 30°C and pH = 6.5 in anionic surfactant systems, while in cationic, it was found at R = 7, T = 22.5°C and pH = 5.8. The stability of the enzyme was also studied in anionic and cationic systems under various conditions. The enzymatic reaction was also found to be very slow when it was studied in the C12E4 systems.

Enzymatic hydrolysis of triglycerides byRhizopus delemarimmobilized on biomass support particles

Abstract The best medium composition for production of intracellular Iipase by Rhizopus delemar contained oleic acid and no glucose. The technique of cell immobilization using polyurethane foam biomass support particles was studied as a means of further enhancing intracellular lipase activity. Fungal mycelia were found to strongly adhere to the polyurethane foam matrix during growth to form immobilized cell biocataiysts, which, after cell membrane permeabiiization and drying, exhibited a Iipase activity 3.3 times greater than that of cells grown in a freely suspended state. Upase within the Immobilized cells showed properties similar to purified extracellular Rhizopus delemar lipase but was more thermostable than the free enzyme. The biocatalyst was used for triglyceride hydrolysis in a continuous stirred‐tank reactor. In this format it was found that the degree of hydrolysis of olive oil and the flavor profile of lipolyzed milk fat could be easily controlled.