Showing papers in "Biocatalysis and Biotransformation in 2003"
TL;DR: In this paper, linear or branched glucan was produced from glucose-1-phosphate by glucan phosphorylase alone or together with bracnhing enzyme.
Abstract: Glucan phosphorylase, branching enzyme, and 4-α-glucanotransferase were employed to produce glucose polymers with controlled molecular size and structures. Linear or branched glucan was produced from glucose-1-phosphate by glucan phosphorylase alone or together with bracnhing enzyme, where the molecular weight of linear glucan was strictly controlled by the glucose-1-phosphate/primer molar ratio, and the branching pattern by the relative branching enzyme/glucan phosphorylase activity ratio. Cyclic glucans were produced by the cyclization reaction of 5-αglucanotransferases and branching enzyme on amylose and amylopectin. Molecular size and structure of cyclic glucan was controlled by the type of enyzyme and substrate chosen and by the reaction conditions. This in vitro approach can be used to manufacture novel glucose polymers with applicable value.
110 citations
TL;DR: X-ray structural data is used to simulate the Bacillus subtilis lipase A-catalyzed substrate hydrolysis by using quantum mechanical and molecular mechanical calculations to devise more efficient strategies for the directed evolution of enantioselective enzymes.
Abstract: Chiral compounds are of steadily increasing importance to the chemical industry, in particular for the production of pharmaceuticals. Where do these compounds come from? Apart from natural resources, two synthetic strategies are available: asymmetric chemical catalysis using transition metal catalysts and biocatalysis using enzymes. In the latter case, screening programs have identified a number of enzymes. However, their enantioselectivity is often not high enough for a desired reaction. This problem can be solved by applying directed evolution to create enantioselective enzymes as shown here for a lipase from Bacillus subtilis. The reaction studied was the asymmetric hydrolysis of meso-1,4-diacetoxy-2-cyclopentene with the formation of chiral alcohols which were detected by electrospray ionization mass spectrometry. Iterative cycles of random mutagenesis and screening allowed the identification of several variants with improved enantioselectivities. In parallel, we have started to use X-ray structural d...
59 citations
TL;DR: Using efficient screening techniques, extracellular glucosyltransferase (GTF) enzyme activity, resulting in α-glucan synthesis from sucrose, was detected in various Lactobacilli strains.
Abstract: Limited information is available about homopolysaccharide synthesis in the genus Lactobacillus. Using efficient screening techniques, extracellular glucosyltransferase (GTF) enzyme activity, resulting in α-glucan synthesis from sucrose, was detected in various lactobacilli. PCR with degenerate primers based on homologous boxes of known glucosyltransferase (gtf) genes of lactic acid bacteria strains allowed cloning of fragments of 10 putative gtf genes from eight different glucan producing Lactobacillus strains (five Lactobacillus reuteri strains, one Lactobacillus fermentum strain, one Lactobacillus sake strain and one Lactobacillus parabuchneri strain). Sequence analysis revealed that these lactobacilli possess a large variation of (putative) gtf genes, similar to what has been observed for Leuconostoc and Streptococcus strains. Homologs of GTFA of Lb. reuteri 121 (synthesizing reuteran, a unique glucan with α(1 → 4) and α-(1 → 6) glycosidic bonds) (Kralj et al., 2002) were found in three of the four other Lb. reuteri strains tested. The other Lactobacillus GTF fragments showed the highest similarity with GTF enzymes of Leuconostoc spp.
45 citations
TL;DR: The glucan branching enzyme of the hyperthermophile Aquifex aeolicus is the most common enzyme for the synthesis of α(1→6) glycosidic bonds in glycogen and amylopectin this paper.
Abstract: Glucan branching enzymes are responsible for the synthesis of α(1→6) glycosidic bonds in glycogen and amylopectin. The glucan branching enzyme of the hyperthermophile Aquifex aeolicus is the most t...
41 citations
TL;DR: An overview of the applications of engineered glycosynthases and thioglycoligases for the enzymatic synthesis of O- and S-glycosidic linkages in oligosaccharides is presented.
Abstract: An overview of the applications of engineered glycosynthases and thioglycoligases for the enzymatic synthesis of O- and S-glycosidic linkages in oligosaccharides is presented. Glycosynthases lack the catalytic nucleophile of retaining glycosidases and use glycosyl fluorides with inverted anomeric stereochemistry as glycosyl donors. To date, nine enzymes from seven different glycosyl hydrolase families have been engineered to perform the glycosynthase reaction. Thioglycoligases lack the catalytic acid/base residue of retaining glycosidases and use dinitrophenyl glycosides as donors and deoxy-thiosugars as acceptors. The regioselectivity of the transglycosylation reaction is entirely controlled by the position of the thiol in the acceptor. To date, two retaining exo glycosidases and one endo glycanase, all from different glycosyl hydrolase families, have been engineered in this fashion.
39 citations
TL;DR: An efficient and genetically stable expression system for the directed evolution of epoxide hydrolase from Aspergillus niger (ANEH) has been constructed and a screening assay for determining the enantioselectivity in the kinetic resolution of styrene oxide has been established using mass spectrometry.
Abstract: An efficient and genetically stable expression system for the directed evolution of epoxide hydrolase from Aspergillus niger (ANEH) has been constructed. Error prone polymerase chain reaction (PCR) with defined mutation rates was used to create biodiversity in two libraries of mutants. Screening for activity allowed the isolation of clones with improved properties. One of these clones shows an expression level 3.4 higher than the original wild type clone in E. coli SG13009 and a 3.3 fold increased catalytic efficiency on 4-(p-nitrophenoxy)-1,2-epoxybutane. In addition, a screening assay for determining the enantioselectivity in the kinetic resolution of styrene oxide has been established using mass spectrometry.
36 citations
TL;DR: Acarbose has a dual role for the producer: it inhibits α-glucosidic enzymes of competitors and functions as a carbophor for the uptake of glucose or starch molecules.
Abstract: The α-glucosidase inhibitor acarbose produced by Actinoplanes sp. SE50/110 is a pseudotetrasaccharide, which consists of an unsaturated cyclitol (carba-sugar), 4-amino-4,6-dideoxyglucose and maltose. The cyclitol (valienol) and the 4-amino-4,6-dideoxyglucose are linked via an N-glycosidic (imino) bond, forming the so-called acarviosyl moiety, which is primarily responsible for the inhibitory effect on α-glucosidases. The gene cluster encoding the biosynthetic genes for the synthesis of acarbose (acb-genes) was sequenced and 25 open reading frames belonging to the acb-gene cluster were identified. Based on the analysis of the enzymes encoded by the acb-cluster, the biosynthesis and ecological role of acarbose is described. The gene cluster includes genes which encode: proteins for the synthesis of the cyclitol; the enzymes for the synthesis of dTDP-4-amino-4,6-dideoxyglucose; glycosyltransferases for the condensation reactions; ATP-dependent exporters and importers; extracellular starch degrading e...
33 citations
TL;DR: An α-galactosidase gene from Pyrococcus furiosus was identified, cloned and functionally expressed in Escherichia coli and it was concluded that glutamate 117 is the catalytic nucleophile, whereas the acid/base catalyst remains to be identified.
Abstract: An α-galactosidase gene from Pyrococcus furiosus was identified, cloned and functionally expressed in Escherichia coli. It is the first α-galactosidase from a hyperthermophilic archaeon described to date. The gene encodes a unique amino acid sequence compared to other α-galactosidases. Highest homology was found with α-amylases classified in family 57 of glycoside hydrolases. The 364 amino acid protein had a calculated mass of 41.6 kDa. The recombinant α-galactosidase specifically catalyzed the hydrolysis of para-nitrophenyl-α-galactopyranoside, and to some extent that of melibiose and raffinose. The enzyme proved to be an extremely thermo-active and thermostable α-galactosidase with a temperature optimum of 115°C and a half-life time of 15 hours at 100°C. The pH optimum is between 5.0 and 5.5. Sequence analysis showed four conserved carboxylic residues. Site-directed mutagenesis was applied to identify the potential catalytic residues. Glu117Ala showed decreased enzyme activity, which could be rescued by...
33 citations
TL;DR: The conversion to oligosaccharides using Lentikat-d Extransucrase was higher than that obtained for alginate-dextransucase, probably due to the reduction of diffusional limitations derived from its lenticular shape.
Abstract: Insoluble (cell-bound) dextransucrase from Leuconostoc mesenteroides B-1299 was encapsulated in highly elastic and stable hydrogels formed by polyvinyl alcohol. The gelation was carried out by controlled partial drying at room temperature, resulting in lens-shaped particles, called LentiKats. A similar recovery of activity (approximately 55%) was achieved when compared with entrapment in calcium alginate gels. Under reaction conditions, the protein leakage in LentiKats was reduced from 18% to 4% by pre-treatment of the dextransucrase with glutaraldehyde. The immobilized dextransucrases were tested in the acceptor reaction with methyl α-D-glucopyranoside. The conversion to oligosaccharides using Lentikat-dextransucrase was higher than that obtained for alginate-dextransucrase, probably due to the reduction of diffusional limitations derived from its lenticular shape. In addition, a shift of selectivity towards the synthesis of oligosaccharides containing α(1→2) bonds was observed for the Lentikat-biocataly...
30 citations
TL;DR: Principal components analysis (PCA) was used as a tool to explain the differences in the observations of the multidimensional data set obtained from the multiple conditions, and components seem to represent solvent toxicity and a protective effect of the solvent from carveol toxicity.
Abstract: The effect of some solvents, present in different amounts, upon whole cells of Rhodococcus erythropolis DCL14 carrying out the biotransformation of (−)-carveol to (−)-carvone was studied. The solvents tested were ethyl butyrate, n-hexane, cyclohexane, iso-octane, n-dodecane, dimethyl sulfoxide, bis(2-ethylhexyl) phthalate and FC-70. The volumes of each solvent corresponded to organic:aqueous phase ratios of 0.0005, 0.0025, 0.005, 0.025 and 0.2. To assess any potential solvent protection towards substrate toxicity, assays were carried out at two initial carveol concentrations (15 and 50 mM). Carvone accumulation was followed by gas chromatography. Cell viability, several aspects of cell morphology and the ability to form clusters were monitored by fluorescence microscopy. Principal components analysis (PCA) was used as a tool to explain the differences in the observations of the multidimensional data set obtained from the multiple conditions. PCA using the different volumes of each solvent as variable sugg...
25 citations
TL;DR: This paper describes how a multi-enzyme extract from Agrobacterium radiobacter, rich in d-hydantoinase and N-carbamyl-d-amino acid amidohydrolase was immobilized on chitin for its application on the synthesis of p-hydroxyphenylglycine and the adsorption derivative showed a higher activity than the covalent one.
Abstract: The immobilization of a multi-enzyme extract is an excellent method for multi-step biotransformations. This paper describes how a multi-enzyme extract from Agrobacterium radiobacter, rich in d-hydantoinase and N-carbamyl-d-amino acid amidohydrolase was immobilized on chitin for its application on the synthesis of p-hydroxyphenylglycine. The adsorption derivative showed a higher activity than the covalent one. Compared to the soluble multi-enzymatic extract, the adsorption derivative showed greater pH-stability in the pH range under study. Its optimum pH ranged from 7–8. Furthermore, it showed high activity at low temperature.
TL;DR: A method was developed to make xylogalacturonans (XGAs) with different degrees of xyloslyation from gum tragacanth using Aspergillus tubingensis, and XGH produces a distinctive set of oligosaccharides from XGA, which is different from the hydrolysis products of PG action.
Abstract: A method was developed to make xylogalacturonans (XGAs) with different degrees of xyloslyation from gum tragacanth (XGA-25, XGA-29, XGA-35 and XGA-47), using alkali treatment at 4°C and acid treatment at 100°C. Ester linkages as well as fucose and arabinose substituents could selectively be removed by this procedure. Galactosyl- and xylosyl-linkages appeared to be more stable, while some backbone degradation of the galacturonan took place upon prolonged acid treatment. Using XGA-35, endoxylogalacturonan hydrolase (XGH) from Aspergillus tubingensis, expressed in Kluyveromyces lactis, was characterised with respect to kinetic parameters, temperature and pH effects.XGA-25 and XGA-47 were degraded with endopolygalacturonases (PGs) from Aspergillus niger (PG1, PG2), from A. tubingensis (PF-arf), from Kluyveromyces fragilis (PG-kluyv) and XGH from A. tubingensis. The activity of the different PGs decreased with increasing degrees of xylosylation. However, for each PG a different tolerance for the presen...
TL;DR: In this article, three D-glucopyranosyl analogues, C-(1-azido-α-D-GLU-glU-poly(1-acetamido)-α,D-glUCU-DGLU(1)-formamide, and C-( 1-hydroxy-β-DglU-, β-DG-glugopyranoyl) formamide, were recognized as moderate competitive inhibitors of muscle GPb with Ki values of 1.80 (± 0.2) mM, 0.31
Abstract: The catalytic site of glycogen phosphorylase (GP) is currently under investigation as a target for inhibition of hepatic glycogenolysis under high glucose conditions. Three D-glucopyranosyl analogues, C-(1-azido-α-D-glucopyranosyl) formamide, C-(1-acetamido-α-D-glucopyranosyl) formamide, and C-(1-hydroxy-β-D-glucopyranosyl) formamide, were recognised as moderate competitive inhibitors of muscle glycogen phosphorylase b (GPb) [with respect to α-D-glucose 1-phosphate (Glc-1-P)] with Ki values of 1.80 (±0.2) mM, 0.31 (±0.01) mM, and 0.88 (±0.04) mM, respectively. In order to elucidate the structural basis of inhibition, we determined the structure of muscle GPb complexed with the three compounds at 2.1, 2.06 and 2.0 A resolution, respectively. The complex structures revealed that the inhibitors can be accommodated in the catalytic site of T-state GPb with very little change of the tertiary structure, and provide a rationalisation for understanding potency of the inhibitors. The glucopyranose moiety m...
TL;DR: In this article, a three-phase system where immobilized Candida antarctica lipase B (Novozyme 435) was used to catalyze the formation of peroxyoctanoic acid from the parent carboxylic acid and hydrogen peroxide in toluene was studied.
Abstract: The synthesis of α-pinene oxide was studied in a three-phase system where immobilized Candida antarctica lipase B (Novozyme 435) was used to catalyze the formation of peroxyoctanoic acid from the parent carboxylic acid and hydrogen peroxide in toluene. The peroxycarboxylic acid formed was then used in situ for the oxidation of α-pinene to the corresponding epoxide. When hydrogen peroxide was added in the reaction mixture gradually over 6 h, conversions increased up to 31.6%. Initial rates of α-pinene oxidation increased from 85 to 708 mmol L−1 h−1 when the amount of H2O2 increased from 5 to 60 mmol. When the lipase was exposed to 75 mmol H2O2 for 0.5 h before its addition in the reaction mixture, its activity decreased to about 50%. The reusability of lipase was studied in five reaction cycles and was found to depend on the concentration of the hydrogen peroxide used.
TL;DR: In this article, a kinetic model for the deactivation of partially purified pyruvate decarboxylase (PDC) by benzaldehyde (0−200 mM) in MOPS buffer (2.5 M) has been developed.
Abstract: Based on experimental data, a kinetic model for the deactivation of partially purified pyruvate decarboxylase (PDC) by benzaldehyde (0–200 mM) in MOPS buffer (2.5 M) has been developed. An initial lag period prior to deactivation was found to occur. With first order dependencies of PDC deactivation on exposure time and on benzaldehyde concentration, a reaction time deactivation constant of 2.64×10−3 h−1 and a benzaldehyde deactivation coefficient of 1.98×10−4 mM−1 h−1 were determined for benzaldehyde concentrations up to 200 mM. The PDC deactivation kinetic equations established in this study are an essential component in an overall model being developed to describe the enzymatic biotransformation of benzaldehyde and pyruvate to produce the pharmaceutical intermediate (R)-phenylacetylcarbinol (R-PAC).
TL;DR: The results of functional screening using high-throughput tools for the characterization of membrane P450-catalyzed activities, suggest that the 204–214 sequence segment of human CYP1A1 is not critical for polycyclic aromatic hydrocarbon recognition, as was hypothesized from previous data.
Abstract: Two complementary methods are described that associate in vitro and in vivo steps to generate sequence diversity by segment directed saturated mutagenesis and family shuffling. A high-throughput DNA chip-based procedure for the characterization and potentially the equalization of combinatorial libraries is also presented. Using these approaches, two combinatorial libraries of cytochrome P450 variants derived from the CYP1A subfamily were constructed and their sequence diversity characterized. The results of functional screening using high-throughput tools for the characterization of membrane P450-catalyzed activities, suggest that the 204–214 sequence segment of human CYP1A1 is not critical for polycyclic aromatic hydrocarbon recognition, as was hypothesized from previous data. Moreover, mutations in this segment do not alter the discrimination between alkoxyresorufins, which, for all tested mutants, remained similar to that of wild-type CYP1A1. In contrast, the constructed CYP1A1–CYP1A2 mosaic st...
TL;DR: In this paper, the authors investigated the partitioning behavior of the reactants 1-butanol, propionic acid and butyl propionate in an aqueous-organic two-phase system consisting of alginate beads suspended in hexane.
Abstract: The partitioning behavior of the reactants 1-butanol, propionic acid and butyl propionate in an aqueous-organic two-phase system consisting of alginate beads suspended in hexane was investigated. Partitioning experiments with a single reactant showed that, even in the dilute region, the equilibrium concentrations of 1-butanol and propionic acid cannot be described by constant partition coefficients as is normally done in the field of biocatalysis. Besides the aqueous alginate beads, two other aqueous phases with different compositions (solutions with and without electrolytes) were also used for partitioning experiments. The equilibrium concentrations of the reactants obtained from the systems with the three different aqueous phases (water, water plus electrolytes, alginate beads) demonstrated that the partitioning behavior of the reactants is scarcely influenced by the presence of the electrolytes or by the alginate matrix, at least up to reactant concentrations of 80 mmol/l in the organic phase. The comp...
TL;DR: Directed evolution was used to enhance the catalytic activity of E. coli alkaline phosphatase (EAP), and several improved variants were obtained through two rounds of error-prone PCR and one round of DNA shuffling followed by a rapid, sensitive screening procedure.
Abstract: Directed evolution was used to enhance the catalytic activity of E. coli alkaline phosphatase (EAP). Through two rounds of error-prone PCR and one round of DNA shuffling followed by a rapid, sensitive screening procedure, several improved variants were obtained. Their enzymatic kinetic properties, thermal stabilities and possible mechanism for the improvement were investigated. In 1.0 M Tris buffer, the specific activity of the most active EAP variant S2163 was 1500 units/mg protein, showing it to be 3.6 times more active than the D101S parent enzyme and ∼40 times more active than the wild-type EAP. At the same time, the Km value of the S2163 variant decreased to 1491 μM from the 2384 μM of the D101S. As a result, the kcat/Km ratio of this variant showed a 5.8-fold enhancement over that of D101S parent enzyme. Three activating amino acid substitutions, K167R, G180S and S374C, which were located far away from the center of the catalytic pocket, were identified by sequencing the genes encoding evolved enzym...
TL;DR: The capacity of CGTase for using βCD as glucosyl donor and transferring it to sucrose molecules was investigated and it was shown that this enzyme was able to produce polyglucosyl-fructosides (GnF) by a coupling reaction between βCD and sucrose.
Abstract: The capacity of CGTase for using βCD (beta cyclodextrin) as glucosyl donor and transferring it to sucrose molecules was investigated We showed that this enzyme was able to produce polyglucosyl-fructosides (GnF) by a coupling reaction between βCD and sucrose Maltooligosaccharides were also synthesised but in lesser amounts The degree of polymerisation (DP) of the different products was limited to a value of 8 and this allowed us to purify all of them by size exclusion chromatography Mass spectrometry and NMR analysis of the unknown products revealed that they consisted of linear maltooligosaccharides of various DP bound to the glucose moiety of a sucrose molecule by a α(1→4) linkage
TL;DR: Optimization of cultivation conditions and co-expression of molecular chaperones resulted in a further increase in activity and a reduction of the inclusion bodies formation, respectively, and the Eph1-catalysed kinetic resolution of a new Eph 1 substrate, 1-oxaspiro[2.5]octane-2-carbonitrile was demonstrated.
Abstract: The Rhodotorula glutinis epoxide hydrolase, Eph1, was produced in the heterologous host Escherichia coli BL21(DE3) in order to develop a highly effective epoxide hydrolysis system. A 138-fold increase in Eph1 activity was found in cell extracts of the recombinant E. coli when compared to cell extracts of Rhodotorula glutinis, despite the formation of Eph1 inclusion bodies. Optimization of cultivation conditions and co-expression of molecular chaperones resulted in a further increase in activity and a reduction of the inclusion bodies formation, respectively. Compared to Rhodotorula glutinis cells and cell extracts, a total increase in Eph1 activity of over 200 times was found for both Escherichia coli cells and crude enzyme preparations of these cells. The improved conditions for recombinant Eph1 production were used to demonstrate the Eph1-catalysed kinetic resolution of a new Eph1 substrate, 1-oxaspiro[2.5]octane-2-carbonitrile.
TL;DR: It is proposed that it might be advantageous to select improved molecular starting points before embarking on the arduous walk through sequence space towards optimized performance.
Abstract: The search for novel enzymes with biotechnological potential in the fine chemical, food and feed, detergent and cosmetics industries is driven by the need to improve existing processes and applications, to design novel processes for innovative products or intermediates or to avoid intellectual property related operative restrictions. Strategies for obtaining novel biocatalysts will be based on screening natural biodiversity or a combination of nature derived scaffolds and optimization by directed evolution technology. Considering the enormous potential of in vitro mutational and recombinatorial strategies to alter genes and improve enzyme properties, we propose that it might be advantageous to select improved molecular starting points before embarking on the arduous walk through sequence space towards optimized performance
TL;DR: Information has been used to design a new chromogenic substrate [1,2-di-O-octyl-sn-glycerol-3-O-(4-nitrophenyl) glutarate] for the determination of the lipolytic activity of PCL.
Abstract: Evaluation of Pseudomonas cepacia lipase (PCL) activity by a titrimetric method with triacylglycerols (TAG) and synthetic dialkylglycerol esters (DAGE) established the chain length selectivity of the enzyme and this information has been used to design a new chromogenic substrate [1,2-di-O-octyl-sn-glycerol-3-O-(4-nitrophenyl) glutarate] for the determination of the lipolytic activity of PCL.
TL;DR: The superiority of the carbamoylmethyl ester to conventional esters, for example the methyl ester, was once again established and some segment condensations were also achieved by the same procedure.
Abstract: We examined Aspergillus melleus protease (Amano protease P) and A. oryzae protease (Amano protease A) as catalysts for peptide bond formation via the kinetically controlled approach. As the coupling efficiency was only moderate, even with a good amino acid substrate as the carboxyl component, in acetonitrile as a solvent (with or without a small amount of added water) that we had mainly employed previously in α-chymotrypsin catalyzed couplings, other solvent systems were sought. In 1,1,1,3,3,3-hexafluoro-2-propanol-DMF (1:1) without added water, these Aspergillus proteases were found to remain active for a long period of time and to be utilizable for peptide synthesis when the carbamoylmethyl ester was employed as the acyl donor, though the coupling efficiencies were dependent rather largely on the combination of the amino acid residues at the coupling site. The superiority of the carbamoylmethyl ester to conventional esters, for example the methyl ester, was once again established. Furthermore, some segm...
TL;DR: Deletion mutants of the Xyn10A enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme.
Abstract: The thermophilic marine bacterium Rhodothermus marinus produces a modular family 10 xylanase (Xyn10A). It consists of two N-terminal family 4 carbohydrate binding modules (CBMs) followed by a domain of unknown function (D3), and a catalytic module (CM) flanked by a small fifth domain (D5) at its C-terminus. Several truncated mutants of the enzyme have been produced and characterised with respect to biochemical properties and stability. Multiple calcium binding sites are shown to be present in the two N-terminal CBMs and recent evidence suggests that the third domain of the enzyme also has the ability to bind the same metal ligand. The specific binding of Ca2+ was demonstrated to have a pronounced effect on thermostability as shown by differential scanning calorimetry and thermal inactivation studies. Furthermore, deletion mutants of the enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme. Finally, recent evidence indicates that...
TL;DR: Aspergillus niger pectinase, together with κ-carrageenan, could be precipitated in the presence of 0.2% KCl and re-dissolved by tenfold dilution of the salt as mentioned in this paper.
Abstract: Aspergillus niger pectinase, together with κ-carrageenan, could be precipitated in the presence of 0.2% KCl and re-dissolved by ten-fold dilution of the salt. The free as well as this reversibly-soluble (rs) enzyme were evaluated for hydrolysis of polygalacturonic acid, chitosan and chitin. The rs-enzyme showed 92%, 80% and 74% activity (as compared to the corresponding amount of enzyme when present as a free enzyme) towards the three substrates, respectively. There was no significant change in the pH and temperature optima of the rs-enzyme. This preparation could be reused six times without loss of any detectable polygalacturonase activity. This biocatalyst design was found to be efficient for the hydrolysis of polygalacturonic acid, chitosan and chitin.
TL;DR: An overview of recent developments in the undersanding and engineering of transglycosylation and hydrolysis specificity in CGTase, which is one of the best-studied α-amylase family enzymes.
Abstract: Cyclodextrin glycosyltransferase (CGTase) is a member of the α-amylase family, a large group of enzymes that act on α-glycosidic bonds in starch and related compounds. Over twenty different reaction and product specificities have been found in this family. Although three-dimensional structure elucidation and the biochemical characterization of site-directed mutants have yielded a detailed insight into the mechanism of bond cleavage, the variation in reaction and product specificity is far from understood. This article gives an overview of recent developments in the undersanding and engineering of transglycosylation and hydrolysis specificity in CGTase, which is one of the best-studied α-amylase family enzymes.
TL;DR: The Populus tremula x tremuloides Mich. expressed sequence tag (EST) database provides a rich and largely unexplored source of enzymes which can be developed into tools for wood fibre engineering, either by way of post-harvest processing or by transgenic technology.
Abstract: The Populus tremula x tremuloides Mich. expressed sequence tag (EST) database provides a rich and largely unexplored source of enzymes which can be developed into tools for wood fibre engineering, either by way of post-harvest processing or by transgenic technology. Enzyme discovery through functional genomics techniques, such as cDNA microarray analysis, is spotlighting particular carbohydrate-active enzymes which play a role in cell wall growth and development in wood-forming tissues. The cloning and heterologous expression of these enzymes is facilitating their study on a fundamental level, which lays the groundwork for advanced biotechnological applications.
TL;DR: This is the first report quantifying the enrichment over iterative selection rounds for the isolation of rare catalysts displayed on phage, and it shows that this selection method can be in principle generalized to most chemical reactions.
Abstract: Isolation of genes encoding catalysts for defined chemical reactions should be facilitated by selection of proteins for catalysis from molecular repertoires. Display of proteins on phage allows the coupling between a protein and its gene. Furthermore, if the reaction product can be linked to the phage enzyme catalyzing the reaction, affinity chromatography for the product yields the protein catalyzing the reaction and its gene. One main advantage of this selection method is that it can be in principle generalized to most chemical reactions.Here, iterative in vitro selections for polymerase activity were used to isolate a single phage-polymerase among more than 108 phage particles. Three to five selection cycles were required depending on the fraction of infectious phage-polymerases in the initial phage population. This is the first report quantifying the enrichment over iterative selection rounds for the isolation of rare catalysts displayed on phage.
TL;DR: In this paper, normal roots of Capsicum frutescens were excised from tissue-cultured plants into half strength Murashige and Skoog's medium with 2.23 μM naphthalene acetic acid.
Abstract: Normal roots of Capsicum frutescens were excised from tissue-cultured plants into half strength Murashige and Skoog's medium with 2.23 μM naphthalene acetic acid. Maximum growth of cultured roots was 6.5 g fresh weight 40 ml-1, as recorded on day 20. Even though normal roots were unable to accumulate capsaicin, they contained other phenylpropanoid intermediates and vanillylamine, as detected by HPLC analysis. Normal roots of Capsicum frutescens were treated with ferulic acid and protocatechuic aldehyde in order to study their biotransformation ability. Ferulic acid, which is the nearest precursor to vanillin, when fed at concentrations of 1 and 2 mM led to the accumulation of vanilla flavour metabolites, vanillin being the major one. In cultures treated with 1 and 2 mM ferulic acid, maximum vanillin accumulation of 12.3 and 16.4 μM was observed, on day 6 after precursor addition, respectively. Feeding of ferulic acid and β-cyclodextrin complex (2 mM each) enhanced the accumulation of biotransformed produc...
TL;DR: In this article, the reactivity of rhizopus delemar lipase immobilized in sodium bis(2-ethylhexyl) sulfosuccinate microemulsion-based organogels (MBGs) was investigated as a function of the water content in the gel.
Abstract: The reactivity of Rhizopus delemar lipase immobilized in sodium bis(2-ethylhexyl) sulfosuccinate microemulsion-based organogels (MBGs) was investigated as a function of the water content in the gel. Gelatin was used as the gelling component of the MBGs. The maximal reaction rate of 6.5 μM s−1 was obtained at a WG (molar ratio of water to AOT in the MBG phase) value of 100. In the experimental WG conditions, the reaction proceeded under an effectiveness factor of 0.02 to 0.04 giving the reaction-limiting regime.