Showing papers in "Journal of Molecular Catalysis B-enzymatic in 2003"
TL;DR: The specific activity and enantioselectivity for the Celite-immobilised enzyme was compared to conventional organic solvents ( n -hexane, MTBE and vinyl acetate) and two lipases, Candida antarctica lipase B and Pseudomonas sp.
Abstract: Three different ionic liquids, 1-butyl-3-methyl imidazolium bis[(trifluoromethyl)sulfonyl]amide [BMIM][BTA], 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIM][PF 6 ] and 1-butyl-3-methyl imidazolium tetrafluoroborate [BMIM][BF 4 ] were used as reaction media for the transesterification of 1-phenylethanol catalysed by esterases from Bacillus subtilis and Bacillus stearothermophilus . No transesterification activity could be detected in the ionic liquids when the lyophilised powder of the esterases was used as biocatalysts. By immobilising the esterases onto Celite it was possible to obtain activity in the ionic liquids. The specific activity and enantioselectivity for the Celite-immobilised enzyme was compared to conventional organic solvents ( n -hexane, MTBE and vinyl acetate). Highest specific activity was obtained in n -hexane for both enzymes while the specific activity was similar in MTBE, vinyl acetate and in the ionic liquids. The enantioselectivity ( E ∼ 7.0 and 3.0 for B. subtilis and B. stearothermophilus esterase, respectively) was however independent of the solvent. The kinetic resolution was also performed with two lipases, Candida antarctica lipase B and Pseudomonas sp. lipase (PS-D). Also for these two enzymes, the enantioselectivity was not affected by the solvent and high enantioselectivity E >100 was obtained in all solvents investigated. The stability of the esterase from B. stearothermophilus at 40 °C was considerably increased in the ionic liquids [BMIM][BF 4 ] and [BMIM][PF 6 ] as compared to n -hexane and MTBE. A half-life of >240 h was obtained in [BMIM][PF 6 ], which was >30- and >3-fold higher as compared to n -hexane and MTBE.
178 citations
TL;DR: Under optimum conditions, the binary method of immobilization yielded a higher protein loading and an activity of 13.8 U/g-chitosan as compared to the immobilized lipase prepared by activation and by cross-linking.
Abstract: Industrial application of lipase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to free lipase. In the present study, lipase was immobilized to chitosan beads utilizing its amino and the hydroxyl groups, which is called “binary immobilization”. Lipase was first immobilized to chitosan beads by activating its hydroxyl groups with carbodiimide followed by cross-linking more lipase to the amino groups with glutaraldehyde. Under optimum conditions, the binary method of immobilization yielded a higher protein loading of 287.2 μg/g-chitosan and an activity of 13.8 U/g-chitosan as compared to the immobilized lipase prepared by activation and by cross-linking. Broader pH tolerance and higher heat stability could be achieved by this method. Immobilized lipase retained 74% residual activity after ten hydrolysis cycles and 67% after 7 days of storage. Kinetic parameters V max and K m and the energy of activation ( E a ) were determined for the immobilized lipase.
175 citations
TL;DR: The major human lipases include the gastric, pancreatic and bile-salt-stimulated lipase that aid in the digestion and assimilation of dietary fats, and the hepatic, lipoprotein and endothelial lipases that function in the metabolism of lipoproteins.
Abstract: The major human lipases include the gastric, pancreatic and bile-salt-stimulated lipase that aid in the digestion and assimilation of dietary fats, and the hepatic, lipoprotein and endothelial lipase that function in the metabolism of lipoproteins. The triacylglycerol and phopholipase activities of these enzymes enable these varied functions. The lipase enzymes exhibit a high degree of sequence homology not only within but also across species. This and the diverse chromosomal location of their genes point to a multigenic family of enzymes involved in absorption and transport of lipids. Inactivation of lipolytic activity of microorganisms to control infection, inhibition of digestive lipase to control obesity, stimulation of metabolic lipase to reduce hyperlipidemia or procoagulant state, or use of pancreatic lipase supplement in the management of cystic fibrosis are examples of how lipase activity modulation can impact human health.
175 citations
TL;DR: This review outlines the recent progress during the last 25 years concerning the biotransformation of exogenous substrates by plant cultured cells.
Abstract: This review outlines the recent progress during the last 25 years concerning the biotransformation of exogenous substrates by plant cultured cells. The plant cultured cells have abilities of the regio- and stereoselective hydroxylation, oxido-reduction, hydrogenation, glycosylation, and hydrolysis for various organic compounds as well as microorganisms. The reaction types and the stereochemistry of the products involved in the biotransformations are described. The development of techniques using immobilized plant cells are also delineated.
152 citations
TL;DR: The gene cloning, primary structure, and 3D-structure of glutaminase are illustrated and the mechanisms of action of salt-tolerant glutaminases are briefly discussed.
Abstract: Glutaminase is widely distributed in microorganisms including bacteria, yeast and fungi. The enzyme mainly catalyzes the hydrolysis of γ-amido bond of l -glutamine. In addition, some enzymes also catalyze γ-glutamyl transfer reaction. A highly savory amino acid, l -glutamic acid and a taste-enhancing amino acid of infused green tea, theanine can be synthesized by employing hydrolytic or transfer reaction catalyzed by glutaminase. Therefore, glutaminase is one of the most important flavor-enhancing enzymes in food industries. In this review, subsequent to a discussion on the definition of glutaminase, the enzymatic properties, applications of glutaminase in the food industry, and occurrence and distribution of the enzyme are described. We then illustrate the gene cloning, primary structure, and 3D-structure of glutaminase. Finally, to facilitate the future applications of glutaminase in food fermentations, the mechanisms of action of salt-tolerant glutaminase are briefly discussed.
140 citations
TL;DR: In this paper, six ionic liquids based on dialkylimidazolium or quaternary ammonium cations associated with perfluorinated or bis(trifluoromethyl)sulfonyl amide anions were used as reaction media for ester synthesis catalyzed by both free Candida antarctica lipase B and α-chymotrypsin at 2% (v/v) water content and 50°C.
Abstract: Six different ionic liquids based on dialkylimidazolium or quaternary ammonium cations associated with perfluorinated or bis(trifluoromethyl)sulfonyl amide anions were used as reaction media for ester synthesis catalyzed by both free Candida antarctica lipase B and α-chymotrypsin at 2% (v/v) water content and 50 °C. All the assayed ionic liquids proved adequate media for enzyme-catalyzed transesterification, and in the case of lipase, the synthetic activity was clearly enhanced with respect to that obtained with organic solvents of similar polarity. In general, all the ionic liquids increased the thermal stability of both the enzymes. As for example, 1-ethyl-3-methylimidazolium tetrafluoroborate enhanced 5- and 4-times the synthetic activity and half-life time, respectively, of lipase in comparison to 1-butanol.
121 citations
TL;DR: The enzyme was able to hydrolyze both soluble and insoluble emulsified substrates and was classified as a lipase, expressing some esterase activity as well, and the most advantageous method for immobilization was found to be ionic binding to DEAE Cellulose.
Abstract: Extracellular thermostable lipase produced by the thermophilic Bacillus stearothermophilus MC 7 was purified to 19.25-fold with 10.2% recovery. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was shown to be 62 500 Da. The purified enzyme expressed maximum activity at 75-80 OC and its half life was 30min at 70 OC. The K,,, and V,,,,, were calculated to be, respectively, 0.33 mM and 188 pMmin-' mg-' withp-nitrophenyl palmitate @NPP) as a substrate. Enzyme activity was inhibited by divalent ions of heavy metals, thiol and serine inhibitors, whereas calcium ion stimulated its activity. The most advantageous method for immobilization was found to be ionic binding to DEAE Cellulose. The enzyme was able to hydrolyze both soluble and insoluble emulsified substrates and was classified as a lipase, expressing some esterase activity as well. 0 2003 Elsevier Science B.V. All rights reserved.
120 citations
TL;DR: In this paper, the authors explored the enzymatic selective acylation of carbohydrates in ionic liquids and found that the reactions in ionics took place more rapidly and more selectively than those in conventional organic solvents.
Abstract: The enzymatic selective acylations of carbohydrates in ionic liquids were explored in both organic solvents and ionic liquids to see any significant differences in terms of reactivity and regioselectivity between two different classes of reaction media. Monoprotected glycosides (methyl-6-O-trityl-glucosides and galactosides) were chosen as the substrates with Candida rugosa lipase as an acylation enzyme. Two organic solvents, THF and chloroform, and two ionic liquids, [BMIM]+PF6− ([BMIM]+ = 1-butyl-3-methylimidazolium) and [MOEMIM]+PF6− ([MOEMIM]+ = 1-methoxyethyl-3-methylimidazolium), were employed as reaction media. The enzymatic reactions were performed in the presence of vinyl acetate at room temperature. It was observed that the reactions in ionic liquids took place more rapidly and more selectively than those in conventional organic solvents.
118 citations
TL;DR: In this article, the mechanism and extent of these mediated oxidations of Adlerol to Adlerone (α-carbonyl) have been investigated and compared with chemical oxidations.
Abstract: Laccase oxidises the phenolic but not the non-phenolic subunits of lignin. Redox mediators drive laccase towards the oxidation of non-phenolic subunits, particularly the benzyl alcohol groups. In search for non-polluting delignification strategies of wood pulp for paper making, oxidations by laccase/mediator systems have been performed on Adlerol, a non-phenolic α-hydroxyl-β-arylether model of lignin. The mechanism and extent of these mediated oxidations of Adlerol to Adlerone (α-carbonyl) have been investigated and compared with chemical oxidations. The more valuable mediator of laccase results to be violuric acid, which performs a radical oxidation of Adlerol. Earlier studies had revealed that Adlerone is efficiently degraded under, for example, kraft cooking conditions, whereas Adlerol resists degradation. Thus, a pre-oxidation of the α-hydroxyl-β-arylether subunits in wood pulp by the laccase/VLA system appears promising for weakening the network of lignin, thereby activating it towards subsequent oxydelignification treatments.
112 citations
TL;DR: The extreme thermophilic cyclodextrin glucanotransferase from Thermoanaerobacter sp.
Abstract: The extreme thermophilic cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. was covalently attached to Eupergit C. Different immobilization parameters (incubation time, ionic strength, pH, ratio enzyme/support, etc.) were optimized. The maximum yield of bound protein was around 80% (8.1 mg/g support), although the recovery of β-cyclodextrin cyclization activity was not higher than 11%. The catalytic efficiency was lower than 15%. Results were compared with previous studies on covalent immobilization of CGTase. The enzymatic properties of immobilized CGTase were investigated and compared with those of the soluble enzyme. Soluble and immobilized CGTases showed similar optimum temperature (80–85 °C) and pH (5.5) values, but the pH profile of the immobilized CGTase was broader at higher pH values. The thermoinactivation of the CGTase coupled to Eupergit C was slower than the observed with the native enzyme. The half-life of the immobilized enzyme at 95 °C was five times higher than that of the soluble enzyme. The immobilized CGTase maintained 40% of its initial activity after 10 cycles of 24 h each. After immobilization, the selectivity of CGTase (determined by the ratio CDs/oligosaccharides) was notably shifted towards oligosaccharide production.
108 citations
TL;DR: In this article, 1-Butyl-2,3-dimethylimidazolium tetrafluoroborate ([bdmim]BF 4 ) was found to be an excellent solvent to realize a lipase-recycling system using vinyl acetate as acyl donor.
Abstract: 1-Butyl-2,3-dimethylimidazolium tetrafluoroborate ([bdmim]BF 4 ) was found to be an excellent solvent to realize a lipase-recycling system using vinyl acetate as acyl donor. No accumulation of an acetaldehyde oligomer was observed in this solvent system and it was possible to use the lipase repeatedly 10 times while still maintaining perfect enantioselectivity and high reactivity.
TL;DR: In this article, the relative efficiency of four >NOH-type mediators (HBT, HPI, VLA, NHA) has been evaluated, and compared with that of the structurally different mediator ABTS.
Abstract: The oxidation of CH bonds in alkylarenes can take place by dioxygen, under catalysis by the phenol-oxidase enzyme laccase, provided that suitable mediator compounds are added. The actual oxidation of the substrate is carried out by the oxidised form of the mediator, in a non-enzymatic step. The relative efficiency of four >NOH-type mediators (HBT, HPI, VLA, NHA) has been evaluated, and compared with that of the structurally different mediator ABTS. Laccase/mediator catalysed oxidations of non-phenolic substrates can proceed via two different mechanisms. Either on monoelectronic oxidation, by the oxidised form of mediator ABTS, or, by abstraction of hydrogen atom, by a >NO radical species derived from the >NOH-type mediators. The former mechanism requires substrates with a low oxidation potential; the latter mechanism requires substrates with relatively weak CH bonds. Electrochemical and thermochemical evidence is provided (i) to explain the failure in the oxidation of specific alkylarenes and (ii) in support to the rationalisation of the experimental findings. Particular emphasis is given to discuss the effect of the mediator-to-substrate molar ratio upon the efficiency of the oxidation procedure. In order to explain why, in previous literature studies, better results may have been obtained by using the mediator in more that stoichiometric amounts, we propose the concurrent formation of degradation products from the mediator, which could be responsible for the onset of alternative oxidation pathways. A better understanding of the natural role of laccase in the oxygen-dependent degradation of lignin in wood emerges from this study.
TL;DR: It is proposed that AAT-catalyzed acyl transfer proceeds with a general acid/base mechanism, where the enzyme and both acyl donor and acyl acceptor form a ternary complex before catalysis can occur.
Abstract: Anthocyanins are responsible for the orange to blue coloration of flowers, fruits, and leaves. They are beneficial to human health and widely used as food colorants. Anthocyanin acyltransferases (AATs) are the plant enzymes that catalyze the regiospecific acyl transfer from acyl-CoA to the sugar moiety of anthocyanins. AATs are classified on the basis of their acyl-donor specificity into two categories; i.e. aliphatic and aromatic acyltransferases. However, the acyl-acceptor specificity of AAT differs greatly with the enzyme. Primary structural analyses of several AATs revealed that AATs form a subfamily within the versatile acyltransferase family and share highly conserved sequences such as motif 1 (-His-Xaa3-Asp-) and motif 3 (-Asp-Phe-Gly-Trp-Gly-) with each other. It is proposed that AAT-catalyzed acyl transfer proceeds with a general acid/base mechanism, where the enzyme and both acyl donor and acyl acceptor form a ternary complex before catalysis can occur. The histidine and aspartic acid residues located at motifs 1 and 3, respectively, appear to play very important roles during the proposed general acid/base catalysis. AAT cDNAs have been expressed in heterologous systems, providing a basis for applications of AATs in biotechnology, such as flower color modification and food colorant production by metabolic engineering of anthocyanin biosynthesis in plants.
TL;DR: This review will focus on the virulent traits of these secreted enzymes from bacteria and fungi, and special emphasis will be laid on Candida albicans research.
Abstract: Up to now, lipases of microbial origin are known to be very useful in a palette of industrial applications. But it becomes more and more obvious that extracellular lipases also play a role during microbial infections. This review will focus on the virulent traits of these secreted enzymes from bacteria and fungi. Special emphasis will be laid on Candida albicans research. This human pathogenic fungus possesses a lipase gene family, which is expressed and differentially regulated under a variety of in vitro conditions. First results show that this isoenzyme family is also expressed during an experimental infection. In addition, putative functions of extracellular lipases of pathogenic micro-organisms will be discussed.
TL;DR: In all cases studied, only flavonoid monoester is identified as the product, which indicates that this lipase-catalyzed esterification is regioselective and the enzymatic acylation of flavonoids seems to follow Michaelis–Menten kinetics.
Abstract: Flavonoids rutin and naringin were acylated with fatty acids of medium carbon chain (with 8–12 carbon atoms on their molecule) in a reaction catalyzed by immobilized lipase from Candida antarctica (Novozyme) in various solvent systems. The reaction parameters affecting the acylation rate and the conversion of the enzymatic process, such as the nature of the organic solvent and acyl donor used, the water activity ( a w ) of the system, as well as the kinetic of the reaction have been investigated. In all cases studied, only flavonoid monoester is identified as the product, which indicates that this lipase-catalyzed esterification is regioselective. The enzymatic acylation of flavonoids seems to follow Michaelis–Menten kinetics.
TL;DR: In this article, the octadecyl-MML preparation was the only enzyme derivative which exhibited enantioselectivity towards R isomer (with E values ranging from 5 at 4°C and pH 7 to 12 at pH 5 and 25°C).
Abstract: Purified lipase from Mucor miehei (MML) has been covalently immobilized on different epoxy resins (standard hydrophobic epoxy resins, epoxy-ethylenediamine, epoxy-iminodiacetic acid, epoxy-copper chelates) and adsorbed via interfacial activation on octadecyl-Sepabeads support (fully coated with very hydrophobic octadecyl groups) These immobilized enzyme preparations were used under slightly different conditions (temperature ranging from 4 to 25 °C and pH values from 5 to 7) in the hydrolytic resolution of ( R , S )-2-butyroyl-2-phenylacetic acid Different catalytic properties (activity, specificity, enantioselectivity) were found depending on the particular support used For example, the epoxy-iminodiacetic acid-Sepabeads gave the most active preparation at pH 7 while, at pH 5, the ethylenediamine-Sepabeads was superior More interestingly, the enantiomeric ratio ( E ) also depends strongly on the immobilized preparation and the conditions employed Thus, the octadecyl-MML preparation was the only immobilized enzyme derivative which exhibited enantioselectivity towards R isomer (with E values ranging from 5 at 4 °C and pH 7 to 12 at pH 5 and 25 °C) The other immobilized preparations, in contrast, were S selective Immobilization on iminodiacetic acid-Sepabeads afforded the catalyst with the highest enantioselectivity ( E =59 under optimum conditions)
TL;DR: It could be demonstrated, that this hyperthermophilic hydrogenase exhibits a high stability under reaction conditions and generation as well as regeneration of NADPH were performed in batch and repetitive batch experiments with recycling of the biocatalyst.
Abstract: The soluble hydrogenase I (H-2:NADP(+) oxidoreductase, EC 1.18.99.1) from the marine hyperthermophilic strain of the archaeon Pyrococcus furiosus was partially purified by anion-exchange chromatography. This P furiosus hydrogenase I preparation (PF H(2)ase I) has been used as biocatalyst in the enzymatic production and regeneration of beta-1,4-nicotinamide adenindinucleotide phosphate, reduced form (NADPH), utilizing cheap molecular hydrogen and forming protons as the only side-product. Any excess of dihydrogen can be removed easily. It could be demonstrated, that this hyperthermophilic hydrogenase exhibits a high stability under reaction conditions. Generation as well as regeneration of NADPH were performed in batch and repetitive batch experiments with recyclisation of the biocatalyst. In two repetitive batch-series 6.2 g l(-1) NADPH could be produced with a total turnover number (ttn: mol produced NADPH/mol consumed enzyme) of 10,000. Utilizing the thermophilic NADPH-dependent alcohol dehydrogenase from Thermoanaerobium spec. (ADH M) coupled to the PF H(2)ase I in situ NADPH-regenerating system, two prochiral model substrates, acetophenone and (2S)-hydroxy-1-phenyl-propanone (HPP), were quantitatively reduced to the corresponding (S)-alcohol and (1R,2S)-diol. An e.e. >99.5% and d.e. >98%, respectively, with total turnover numbers (ttn: mol product/mol consumed cofactor NADP(+)) of 100 and 160 could be reached. (C) 2003 Elsevier B.V. All rights reserved.
TL;DR: In this article, the effect of mean pore diameter and surface chemistry on enzyme activity in the hydrolysis of casein has been examined, and the optimum pH, optimum temperature, thermal stability, operational stability, and storage stability of the immobilized and freely soluble enzymes were compared.
Abstract: Alcalase 2T, a commercial preparation of Subtilisin Carlsberg, was covalent immobilized onto physiochemically characterized silica supports. The effect of mean pore diameter and surface chemistry on enzyme activity in the hydrolysis of casein has been examined. Two sets of chemically distinct silica supports were used presenting terminal amino (SAPTES) or hydroxyl groups (STESPM-pHEMA). The percentage of immobilized protein was smaller in SAPTES (31–39%) than in STESPM-pHEMA (62–71%), but presented higher total and specific activity. Silicas with large pores (S1000, 130/1200 A) presented higher specific activities relative to those with smaller pore sizes (S300, 130/550 A). The influence of glutaraldehyde concentration and the time of enzyme coupling to the S1000SAPTES supports was examined. The apparent Km value for the S1000SAPTES immobilized enzyme is lower than the soluble one which may be explained by the partitioning effects of the substrate. No intraparticle diffusion limitations were observed for the immobilized enzyme and therefore the substrate diffusion does not influence the observable kinetics. Finally, the optimum pH, optimum temperature, thermal stability, operational stability, and storage stability of the immobilized and freely soluble enzymes were compared.
TL;DR: The immobilisation on Accurel MP1004 improves the lipase performance both in terms of activity and of substrate conversion.
Abstract: Accurel MP1004, a porous polypropylene powder, was characterised for lipase immobilisation. The particle size ranges between 40–80 mesh, corresponding to particle diameters of 177–420 μm. The pore distribution falls between macroporous and mesoporous domains. A crude lipase preparation from Mucor javanicus was immobilised on Accurel MP1004 by adsorption. During the adsorption process, by measuring the variation of pore volume and of pore size distribution of mesopores as a function of enzyme loading, a significant penetration of the enzyme molecules into the pores was found to occur. The various proteins in the crude lipase preparation are quickly adsorbed by the Accurel MP1004. However, they are progressively displaced by the lipase which shows a greater affinity for the support. A transesterification reaction, between glycerol tricaprylate and 1-butanol in solvent-free conditions, was catalysed by the lipase before and after the immobilisation process. The immobilisation on Accurel MP1004 improves the lipase performance both in terms of activity and of substrate conversion.
TL;DR: The 4-methylumbelliferone-derivative substrates method has shown to be a useful tool for the characterization of a variety of lipases from microbial origin, including those cloned in heterologous hosts.
Abstract: Lipases and esterases have been recognized as very useful biocatalysts because of their wide-ranging versatility in industrial applications, their stability, low cost, and non-requirement for added cofactors. The physical properties of lipidic substrates, typically water insoluble, have determined a great difficulty in studying lipolytic enzymes. A method for fast and simple detection of lipolytic activity, based on the use of 4-methylumbelliferone (MUF)-derivative substrates was developed. The system has been used for the detection of lipase activity either from microbial colonies, cell culture suspensions, or from proteins separated on SDS-polyacrylamide or isoelectric focusing gels. The use of MUF-derivative substrates has also been extended to the quantitative determination of lipolytic activity from a variety of assays including optimum pH and temperature determination, growth dependency, kinetics or stability studies, or residual activity quantification after treatment with potential inhibitors. The method has shown to be a useful tool for the characterization of a variety of lipases from microbial origin, including those cloned in heterologous hosts.
TL;DR: An interactive kinetic model as a function of catechol and dissolved oxygen concentrations was developed for enzymatic polymerization and corresponding biokinetic parameters have been evaluated for the first time.
Abstract: Oxidative polymerization of catechol catalyzed by laccase enzyme from Trametes versicolor (ATCC 200801) in aqueous solution containing acetone was investigated in a batch system. The effects of initial catechol and dissolved oxygen concentrations on the initial reaction rate of oxidative polymerization were experimented. An interactive kinetic model as a function of catechol and dissolved oxygen concentrations was developed for enzymatic polymerization and corresponding biokinetic parameters have been evaluated for the first time. The parameters of the model, Vmax, Kmm, KmO2 and Ki were found 0.247 g O2 m−3 min−1, 70.75 g m−3, 10.4 g m−3 and 48.15 g m−3, respectively. The activation energy and reaction rate constant of oxidative catechol polymerization were calculated as 66.8 kJ mol−1 and 216.3 s−1, respectively. The estimated activation energy and rate constant of the reaction are of the order of magnitude with previously reported values for laccase-catalyzed reactions of different monomers.
TL;DR: Chimeric chitinases that were more thermostable than the parental enzyme were successfully obtained by RETT-based recombination.
Abstract: We describe a new approach to in vitro DNA recombination technique termed recombined extension on truncated templates (RETT). RETT generates random recombinant gene library by template-switching of unidirectionally growing polynucleotides from primers in the presence of unidirectional single-stranded DNA fragments used as templates. RETT was applied to the recombination of two homologous chitinase genes from S. marcescens ATCC 21074 and S. liquefaciens GM1403. When the shuffled genes were examined by restriction mapping and sequence analysis, it was found that chimeric genes were produced at a high frequency (more than 70%) between two chitinase genes with 83% of sequence identity. The number of crossovers within each chimeric gene ranged from one to four, and the recombination points were randomly distributed along entire DNA sequence. We also applied RETT to directed evolution of a chitinase variant for enhancing thermostability. Chimeric chitinases that were more thermostable than the parental enzyme were successfully obtained by RETT-based recombination.
TL;DR: Based on pure MCM-22 precursor, MCM36 was produced by swelling and pillaring with SiO2 pillars, and the resulting material contained a mesoporous region between the microporous layers.
Abstract: Based on pure MCM-22 precursor, MCM-36 was produced by swelling and pillaring with SiO2 pillars. These materials were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), thermogravimetric analysis/differential scanning calorimetry (TGA/DSC), MAS NMR, and N2 adsorption isotherms. Compared with the MCM-22 sample, the resulting MCM-36 material contains a mesoporous region between the microporous layers, with a mesopore volume of 0.472 cm3/g, and the surface area increased up to 671 m2/g. Lipase from Candida antarctica B (CALB) was immobilized on both supports by physical adsorption at equilibrium. Twenty milligrams of CALB/g of MCM-22 was adsorbed on the external surface of crystals, while only 4 mg CALB/g support was adsorbed onto the mesoporous hybrid material MCM-36. The mechanism of adsorption was also discussed. The acylation of alcohols (1-butanol and 1-octanol) by vinyl esters (vinyl acetate and vinyl stearate) was used as a test reaction in order to evaluate the catalytic activity of MCM-36-immobilized lipase. The test reaction indicated MCM-36-immobilized enzyme as an active catalyst for the acylation and its activity was approximately two times higher than that of the free lipase (for the vinyl acetate/1-butanol system).
TL;DR: The glycogen phosphorylase gene of Aquifex aeolicus, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli and the characteristics of the expressed enzyme were examined and a maximum degree of specificity was observed toward substrates in the phosphorolytic direction.
Abstract: The glycogen phosphorylase gene ( glgP ) of Aquifex aeolicus ( Aae ), a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli and the characteristics of the expressed enzyme were examined. The recombinant enzyme was purified to homogeneity by heat-treatment at 70 °C for 15 min to denature the contaminating E. coli proteins, followed by Ni–NTA agarose column chromatography to selectively trap the His-tagged enzyme. The purified enzyme gave a single band on SDS–PAGE with a molecular mass of approximately 80 kDa. The enzyme displayed optimal activity at pH 6.5 and was stable in the pH range from 4.0 to 10.0. The temperature at which optimal enzyme activity was observed was 100 °C and the enzyme retained 66% of its original activity after heating at 100 °C for 30 min. Kinetic studies using the purified enzyme demonstrated that the smallest primer molecule accepted for catalysis in the synthetic direction was maltotriose (G3) and that the smallest effective substrate for the reverse process, phosphorolysis, was maltotetraose (G4). The K m and k cat values were determined for various oligosaccharides (G3–G7) in both synthetic and phosphorolytic reactions and, remarkably, a maximum degree of specificity was observed toward substrates in the phosphorolytic direction.
TL;DR: The recombinant protein cloned into Escherichia coli showed both esterase activity and acylamino acid-releasing enzyme (AARE) activities and was extremely stable and protein concentration-dependent.
Abstract: The gene APE1547 of the aerobic thermophilic Aeropyrum pernix K1 encoding 582 amino acid residues was cloned into Escherichia coli. BL21 (DE3) by using vector pET11a with a T7 promoter. An alignment of similarity analysis of APE1547 with protein sequences from A. pernix K1 databank revealed that it showed a lipase motif and low homology with the known thermophilic esterases. However, it had a high degree homology with several acyl amino acid-releasing enzymes. After purified by ion exchange chromatography and gel filtration chromatography, the recombinant protein showed both esterase activity and acylamino acid-releasing enzyme (AARE) activities. The optimum of temperature and pH of the esterase activity are 90 °C and 8.0, respectively. The recombinant protein showed the hydrolytic activity for a wide range of substrates, such as p-nitrophenyl alkanoate esters of varying alkyl chain lengths, pNA-labelled amino acid and peptide. The highest activity was observed for the substrate p-nitrophenyl caprylate. The recombinant enzyme was extremely stable and protein concentration-dependent. Its half-life at 90 °C was over 160 h. at the concentration of 2.14 mg/ml, which renders this new esterase very attractive for biotechnological applications.
TL;DR: The Baeyer–Villiger oxidation of various substituted 1-indanones is described, leading to the corresponding lactones with good to excellent yields depending on the aromatic ring substituents.
Abstract: The microbiological Baeyer–Villiger oxidation of various substituted 1-indanones is described. Three bacterial strains have been explored: an E. coli TOP10 [pQR 239] constructed to overexpress the cyclohexanone monoxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871, an E. coli TOP10 [hapE] strain recently constructed to overexpress 4-hydroxyacetophenone monoxygenase (HAPMO) of Pseudomonas fluorescens ACB and the wild type Pseudomonas sp. NCIMB 9872 strain known to metabolise cyclopentanone. This last strain oxidised some of the proposed substrates, leading to the corresponding lactones with good to excellent yields depending on the aromatic ring substituents.
TL;DR: In this paper, a peroxygenase enzyme from oat (Avena sativa) seeds was used to find a replacement for the acid catalyst, and a method for immobilization of this enzyme using a hydrophobic membrane support was devised.
Abstract: Fatty epoxides are used as plasticizers and plastic stabilizers and are intermediates for the production of other chemical substances. The currently used industrial procedure for fatty epoxide synthesis requires a strong acid catalyst which can cause oxirane ring opening and side product formation. To find a replacement for the acid catalyst, we have been conducting research on a peroxygenase enzyme from oat (Avena sativa) seeds and have devised a method for immobilization of this enzyme using a hydrophobic membrane support. In this study, fatty acids and fatty methyl esters commonly encountered in commercial vegetable oils were tested as substrates for immobilized peroxygenase, and the epoxide products were characterized. The epoxidation time course of linoleic acid showed two distinct phases with nearly complete conversion to monoepoxide before diepoxide was produced. The diepoxide formed from linolenic acid was found to be 9,10-15,16-diepoxy-12-octadecenoic acid, and only a trace of triepoxide was obtained. Additionally it was discovered that acyclic alkenes with internal double bonds, a cyclic alkene, and an alkene with an aromatic substituent were substrates of peroxygenase. However, alkenes with terminal unsaturation were unreactive. With every substrate examined, oat seed peroxygenase exhibited specificity for epoxidation, producing no other products, and oxirane ring opening did not occur.
TL;DR: In this paper, an acyl group was added to isoquercitrin p-coumarate, which improved its thermostability and light-resistivity, and was the most stable of all the acylated isquercitrins tested.
Abstract: The enzymatic synthesis of acylated isoquercitrins was accomplished by the lipase-catalyzed transesterification with carboxylic acid vinyl esters as acyl donors in an organic solvent. The introduction of an acyl group into isoquercitrin improved its thermostability and light-resistivity. In particular, isoquercitrin p-coumarate was the most stable of all the acylated isoquercitrins tested.
TL;DR: In this paper, the behavior of the catalytic membranes has been studied in a bioreactor operating under non-isothermal conditions as a function of the applied temperature difference ΔT.
Abstract: β-Galactosidase has been immobilised through spacers of different length on nylon membranes grafted with glycidyl methacrylate. Hexamethylendiamine, ethylendiamine or hydrazine have been separately used as spacers.
The behaviour of the catalytic membranes has been studied in a bioreactor operating under non-isothermal conditions as a function of the applied temperature difference ΔT.
Comparison of the enzyme reaction rates under isothermal and non-isothermal conditions resulted in percentage activity increases (PAI) and reduction of the production time (τr) proportional to the size of the applied ΔT. Both these parameters increased with the increase of the spacer length.
Results have been discussed in the frame of reference of the process of thermodialysis which reduces the limitations to the diffusion of substrate and reaction products across the catalytic membrane, limitations introduced by the grafting and immobilisation process.
The advantages of employing non-isothermal bioreactors in biotechnological productive processes have been outlined.
TL;DR: Rhodococcus erythropolis NCIMB 11540 was found to have a highly active nitrile hydratase/amidase enzyme system present which accepts theNitrile function of α-hydroxynitriles (cyanohydrins) as substrates, which leads to α-Hydroxy carboxylic acids which are much valued chiral building blocks in organic synthesis.
Abstract: Rhodococcus erythropolis NCIMB 11540 was found to have a highly active nitrile hydratase/amidase enzyme system present which accepts the nitrile function of α-hydroxynitriles (cyanohydrins) as substrates. This biocatalytic hydrolysis using whole bacterial cells leads to α-hydroxy carboxylic acids which are much valued chiral building blocks in organic synthesis. Employing enantiopure cyanohydrins, which are easy available using ( R )- or ( S )-hydroxynitrile lyases, the products were obtained in high yield without racemization, decomposition or side reactions. Herein, the application of this biotransformation for preparative scale applications is described. To clarify the substrate acceptance of the nitrile hydrolyzing enzymes of R. erythropolis NCIMB 11540, several selected model compounds were subjected to biocatalytic hydrolysis. Reaction conditions were optimized to enable preparative scale conversions. In this manner, ( R )-2-chloromandelic acid and ( R )-2-hydroxy-4-phenylbutyric acid, two important pharmaceutical intermediates, were prepared in a gram scale. The substrate concentrations used were 9.3 and 13 g/l, respectively. The process yielded both acids in high optical (ee>99 and 98%) and chemical (98%) yield after short reaction times (3 and 1.5 h).