Distribution of acetyl esterase in wood-rotting fungi

The distribution of acetyl esterase was studied in 30 strains of wood-rotting fungi. A screening test on agar plates using glucose β-d-pentaacetate as a substrate indicated that all tested fungi produced acetyl esterase to form a clear zone on the culture. All fungi also showed positive responses in an agar test using carboxymethyl cellulose acetate. Enzyme assay showed that extracellular acetylxylan esterase activity was present in the filtrates of wood-meal culture of all these fungi. The ratio of fungal acetylxylan esterase activity to 4-nitrophenyl acetyl esterase activity were higher than that of porcine liver esterase, indicating that fungal esterases have high affinity for acetylated carbohydrates. Acetyl esterase is suggested to be distributed widely in wood-rotting fungi for degradation of native acetylated hemicelluloses.     

Regulation of the activity of choline acetyl transferase by lipoic acid

The observations reported in this article demonstrate that lipoic acid strongly influences the activity of a purified preparation of choline acetyl transferase. The reduced form, dihydrolipoic acid, is a powerful activator of the enzyme while lipoic acid itself has an inhibitory effect and counteracts the stimulatory effect of dihydrolipoic acid. It is proposed that dihydrolipoic acid serves an essential function in the action of this enzyme and that the ratio of reduced to oxidized lipoic acid in the cell may play an important role in the regulation of the activity of the enzyme. The implications of these findings for cell function and acetyl choline formation are discussed.Affiliation     

The capacity of plastids from developing pea cotyledons to synthesise acetyl CoA

In order to determine whether the enzymes required to convert triose phosphate to acetyl CoA were present in pea (Pisum sativum L.) seed plastids, a rapid, mechanical technique was used to isolate plastids from developing cotyledons. The plastids were intact and the extraplastidial contamination was low. The following glycolytic enzymes, though predominantly cytosolic, were found to be present in plastids: glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), and pyruvate kinase(EC 2.7.1.40). Evidence is presented which indicates that plastids also contained low activities of enolase (EC 4.2.1.11) and phosphoglycerate mutase (EC 2.7.5.3). Pyruvate dehydrogenase, although predominantly mitochondrial, was also present in plastids. The plastidial activities of the above enzymes were high enough to account for the rate of lipid synthesis observed in vivo.Abbreviations FPLC fast protein liquid chromatography - PPi pyrophosphate     

Studies on wheat acetyl CoA carboxylase and the cloning of a partial cDNA

Wheat germ acetyl CoA carboxylase (ACCase) was purified by liquid chromatography and electroelution. During purification bovine serum albumin (BSA) was used to coat Amicon membranes used to concentrate partially pure ACCase. Despite further SDS-PAGE/electroelution and microbore HPLC steps BSA remained associated. This presented serious protein sequencing artefacts which may reflect the affinity of BSA for fatty acids bound to ACCase. To avoid these artefacts the enzyme was digested in gel with Endoproteinase LysC protease without the presence of BSA, and the resulting peptides blotted and sequenced.A partial cDNA (1.85 kb) encoding ACCase from a wheat embryo library was cloned, which hybridised to a 7.5 kb RNA species on northern blot of wheat leaf poly(A)⁺ RNA. The partial cDNA therefore represents about 0.25 of the full-length cDNA. The clone was authenticated by ACCase peptide sequencing and immuno cross-reactivity of the overexpressed clone. The derived amino acid sequence showed homology with both rat and yeast ACCase sequences (62%).Antibodies raised against wheat acetyl CoA carboxylase were specific for a 220 kDa protein from both wheat embryo and leaf. In addition, by using a novel quick assay for ACCase that utilised ¹²⁵I-streptavidin, we showed the major biotin containing protein to be 220 kDa in both leaf and germ. This is in marked contrast to the previously published molecular mass of 75 kDa allocated to wheat leaf ACCase.     

枯草芽胞杆菌乙酰木聚糖酯酶的鉴定及诱导剂对酯酶活力的影响

以枯草芽胞杆菌CICC 20034为研究对象,对其分泌的高相对分子质量酯酶进行鉴定,并考察诱导剂对其活力的影响。结果表明:枯草芽胞杆菌CICC 20034可分泌一种相对分子质量为1.07×105的酯酶,经蛋白质质谱鉴定为乙酰木聚糖酯酶,单体分相对子质量为3.56×104。在发酵培养基中添加乙酸乙酯和木糖可以显著的促进乙酰木聚糖酯酶的活力,而三丁酸甘油酯和大分子诱导剂——木聚糖、玉米芯粉和壳聚糖对酯酶的活力几乎无促进作用。枯草芽胞杆菌CICC 20034以乙酸乙酯为诱导剂时最高比酶活为0.62 U/mL,为已知报道的野生细菌乙酰木聚糖酯酶的最高酯酶活力。     

Molecular cloning, and characterization of a modular acetyl xylan esterase from the edible straw mushroom Volvariella volvacea

A new Volvariella volvacea gene encoding an acetyl xylan esterase (designated as Vvaxe1) was cloned and expressed in Pichia pastoris. The cDNA contained an ORF of 1047 bp encoding 349 amino acids with a calculated mass of 39 990 Da. VvAXE1 is a modular enzyme consisting of an N-terminal signal peptide, a catalytic domain, and a cellulose-binding domain. The amino acid sequence of the enzyme exhibited a high degree of similarity to cinnamoyl esterase B from Penicillium funiculosum, and acetyl xylan esterases from Aspergillus oryzae, Penicillium purpurogenum, and Aspergillus ficuum. Recombinant acetyl xylan esterase released acetate from several acetylated substrates including beta-d-xylose tetraacetate and acetylated xylan. No activity was detectable on p-nitrophenyl acetate. Enzyme-catalyzed hydrolysis of 4-methylumbelliferyl acetate was maximal at pH 8.0 and 60 degrees C, and reciprocal plots revealed an apparent K(m) value of 307.7 microM and a V(max) value of 24 733 IU micromol(-1) protein. ReAXE1 also exhibited a capacity to bind to Avicel and H(3)PO(4) acid-swollen cellulose.     

Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations

Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity.     

Purification and properties of hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase from potatoes

A rapid method for the purification of hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (CQT) from potato tubers which had been stored at low temperatures is described. The method involves affinity chromatography on Blue Sepharose with biospecific desorption of CQT with its substrate, CoA. Elution of the Blue Sepharose column with a gradient of CoA leads to the resolution of CQT, a protein with MW of ca 41500, into 3 peaks of activity; the largest peak elutes first. This fraction is purified × 1440 and gives a single band of protein after PAGE which suggests a high degree of purity. The properties of the 3 fractions of CQT, with respect to substrates and to a number of inhibitors, are described. The first and last eluting CQT fractions are specific for quinate and show no activity towards shikimate. The second peak, however, shows a small activity towards shikimate but this is thought to be due to an underlying peak of a shikimate specific enzyme. The major peak of CQT activity found in potatoes stored at 0° is absent from those stored at 10° throughout the period after harvest.     

Activity of glutathione S‐transferase and esterase enzymes in Helicoverpa armigera (Hübner) after exposure to entomopathogenic fungi

Helicoverpa armigera, a polyphagous insect of crops and vegetables, is acquiring resistance against many commercial insecticides. The present study shows variations in the activity of two detoxification enzymes, namely esterase and glutathione S‐transferase (GST), in H. armigera after exposure to different isolates of entomopathogenic fungi. After treatment of larvae with the different isolates (Day 0), samples were collected on three days (Days 3, 5 and 7) for enzyme analysis. High GST activity was found in samples of hemolymph, intestine and fat bodies of H. armigera following treatment with Beauveria bassiana (isolate Bb‐08), Metarhizium anisopliae (isolates Ma‐11.1 and Ma‐4.1), and Isaria fumosorosea (isolates If‐02 and If‐2.3). High esterase activity was recorded in samples of the intestine and fat bodies on various days after treatment, whereas increased esterase activity in hemolymph was noted only in samples from Day 5 after treatment with M. anisopliae (Ma‐4.1). The detection of high GST and esterase activity demonstrates the possibility of the development of resistance against these microbial control agents in H. armigera.     

Biosynthesis of polylactic acid and its copolymers using evolved propionate CoA transferase and PHA synthase

For the synthesis of polylactic acid (PLA) and its copolymers by one‐step fermentation process, heterologous pathways involving Clostridium propionicum propionate CoA transferase (Pct_Cp) and Pseudomonas sp. MBEL 6‐19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1_Ps6‐19) were introduced into Escherichia coli for the generation of lactyl‐CoA endogenously and incorporation of lactyl‐CoA into the polymer, respectively. Since the wild‐type PhaC1_Ps6‐19 did not efficiently accept lactyl‐CoA as a substrate, site directed mutagenesis as well as saturation mutagenesis were performed to improve the enzyme. The wild‐type Pct_Cp was not able to efficiently convert lactate to lactyl‐CoA and was found to exert inhibitory effect on cell growth, random mutagenesis by error‐prone PCR was carried out. By employing engineered PhaC1_Ps6‐19 and Pct_Cp, poly(3‐hydroxybutyrate‐co‐lactate), P(3HB‐co‐LA), containing 20–49 mol% lactate could be produced up to 62 wt% from glucose and 3HB. By controlling the 3HB concentration in the medium, PLA homopolymer and P(3HB‐co‐LA) containing lactate as a major monomer unit could be synthesized. Also, P(3HB‐co‐LA) copolymers containing various lactate fractions could be produced from glucose alone by introducing the Cupriavidus necator β‐ketothiolase and acetoacetyl‐CoA reductase genes. Fed‐batch cultures were performed to produce P(3HB‐co‐LA) copolymers having 9–64 mol% of lactate, and their molecular weights, thermal properties, and melt flow properties were determined. Biotechnol. Bioeng. 2010; 105: 150–160. © 2009 Wiley Periodicals, Inc.     

Structure and function of an acetyl xylan esterase (Est2A) from the rumen bacterium Butyrivibrio proteoclasticus

Butyrivibrio proteoclasticus is a significant component of the microbial population of the rumen of dairy cattle. It is a xylan‐degrading organism whose genome encodes a large number of open reading frames annotated as fiber‐degrading enzymes. We have determined the three‐dimensional structure of Est2A, an acetyl xylan esterase from B. proteoclasticus, at 2.1 Å resolution, along with the structure of an inactive mutant (H351A) at 2.0 Å resolution. The structure reveals two domains—a C‐terminal SGNH domain and an N‐terminal jelly‐roll domain typical of CE2 family structures. The structures are accompanied by experimentally determined enzymatic parameters against two model substrates, para‐nitrophenyl acetate and para‐nitrophenyl butyrate. The suite of fiber‐degrading enzymes produced by B. proteoclasticus provides a rich source of new enzymes of potential use in industrial settings. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Assessment of virus production and chloramphenicol acetyl transferase expression by insect cells in serum-free and serum-supplemented media using a temperature-sensitive baculovirus

Spodoptera frugiperda insect cells were grown in Sf-900 serum-free medium and two kinds of serum-supplemented media (IPL -41 and Grace's). The specific growth rates of uninfected cells were found to be 0.024, 0.35, and 0.034 h(-1) respectively, at 33 degrees C. The IPL -41 medium supported to highest maximum cell density (10.6 x 10(6) cells/mL) compared to 3.5 x 10(6) and 8.7 x 10(6) cells/mL with the Grace's and serum-free media, respectively. In temperature shifdown experiments with a temperature-sensitive baculo-virus (acts10YM1CAT), virus titer and chloramphenicol acetyl transferase (CAT) expression were highest in the IPL -41 (5.1 x 10(7) PFU/mL and 20000 U/mL). Use of Grace's medium gave higher virus titers than the serum-free medium (4.4 x 10(6) vs 4.1 x 10(5) PFU/mL) as well as higher CAT titers (7050 vs 1980 U/mL). Interestingly, in the three media used, the highest virus and CAT titers were obtained at MOI (multiplicity of infection) of 0.02 At MOI of 2.0 virtually no increase in virus of CAT titer was observed. This result is contrary to those obtained at constant-temperature (27 degrees C) infection and cell culture, in which higher virus titers and recombinant protein expression and obtained at higher MOI.     

Quantitation of chloramphenicol acetyl transferase in transgenic tobacco plants by ELISA and correlation with gene copy number

A monoclonal antibody to chloramphenicol acetyl transferase (CAT) was used in an indirect competitive enzyme immunoassay (ELISA) for the quantitation of CAT in leaf extracts of eighteen transgenic tobacco plants containing the CAT gene fused to the cauliflower mosaic virus 35S promoter. The ELISA could be used to quantify CAT when present in extracts at 20 ng/ml. Enzymatic activity and electrophoretic mobility of CAT in these extracts was not different from CAT from Escherichia coli. Concentrations of CAT in these transgenic plants ranged from 79 to 732 ng CAT/mg protein. The average coefficient of variation among three replicate samples was 15%. All plants were sampled on two separate occasions. The CAT concentrations often varied between the two sampling dates. We determined the CAT gene copy number and the number of independently segregating loci in each plant by Southern blot analysis and progeny testing. We found no significant differences in CAT expression among all ten plants with a single CAT gene. We also found a significant correlation between CAT gene copy number and the level of CAT expressed in each plant, although plants with one gene copy sometimes had more CAT than plants with more than one gene copy. In this population, therefore, gene copy number contributed more to the variation in CAT expression than did position effects.     

Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose

Cell wall hemicelluloses and pectins are O‐acetylated at specific positions, but the significance of these substitutions is poorly understood. Using a transgenic approach, we investigated how reducing the extent of O‐acetylation in xylan affects cell wall chemistry, plant performance and the recalcitrance of lignocellulose to saccharification. The Aspergillus niger acetyl xylan esterase AnAXE1 was expressed in Arabidopsis under the control of either the constitutively expressed 35S CAMV promoter or a woody‐tissue‐specific GT43B aspen promoter, and the protein was targeted to the apoplast by its native signal peptide, resulting in elevated acetyl esterase activity in soluble and wall‐bound protein extracts and reduced xylan acetylation. No significant alterations in cell wall composition were observed in the transgenic lines, but their xylans were more easily digested by a β‐1,4‐endoxylanase, and more readily extracted by hot water, acids or alkali. Enzymatic saccharification of lignocellulose after hot water and alkali pretreatments produced up to 20% more reducing sugars in several lines. Fermentation by Trametes versicolor of tissue hydrolysates from the line with a 30% reduction in acetyl content yielded ~70% more ethanol compared with wild type. Plants expressing 35S:AnAXE1 and pGT43B:AnAXE1 developed normally and showed increased resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis, probably due to constitutive activation of defence pathways. However, unintended changes in xyloglucan and pectin acetylation were only observed in 35S:AnAXE1‐expressing plants. This study demonstrates that postsynthetic xylan deacetylation in woody tissues is a promising strategy for optimizing lignocellulosic biomass for biofuel production.     

Liposome-mediated introduction of the chloramphenicol acetyl transferase (CAT) gene and its expression in tobacco protoplasts

The expression plasmid vector pUC8CaMVCAT, containing the chloramphenicol acetyl transferase (CAT) gene, was encapsulated in large unilamellar vesicles (LUV) and introduced into tobacco protoplasts derived from either cell suspension culture or leaf mesophyll. Treatment with liposomes took place in a buffer containing either NaCl or CaCl₂, but no polyethylene glycol. The presence of polylysine in the incubation buffer increased the adsorption of liposomes to protoplasts but decreased the efficiency of CAT gene expression.The expression of the introduced CAT gene could be monitored for at least seven days, following the treatment (about 25% acetylation at day 3 as well as at day 7). Plasmid DNA sequences could be detected, apparently unmodified, for at least nine days in the plant cells, though unintegrated in the host genome.     

Distinct roles of carbohydrate esterase family CE16 acetyl esterases and polymer-acting acetyl xylan esterases in xylan deacetylation

Mass spectrometric analysis was used to compare the roles of two acetyl esterases (AE, carbohydrate esterase family CE16) and three acetyl xylan esterases (AXE, families CE1 and CE5) in deacetylation of natural substrates, neutral (linear) and 4-O-methyl glucuronic acid (MeGlcA) substituted xylooligosaccharides (XOS). AEs were similarly restricted in their action and apparently removed in most cases only one acetyl group from the non-reducing end of XOS, acting as exo-deacetylases. In contrast, AXEs completely deacetylated longer neutral XOS but had difficulties with the shorter ones. Complete deacetylation of neutral XOS was obtained after the combined action of AEs and AXEs. MeGlcA substituents partially restricted the action of both types of esterases and the remaining acidic XOS were mainly substituted with one MeGlcA and one acetyl group, supposedly on the same xylopyranosyl residue. These resisting structures were degraded to great extent only after inclusion of α-glucuronidase, which acted with the esterases in a synergistic manner. When used together with xylan backbone degrading endoxylanase and β-xylosidase, both AE and AXE enhanced the hydrolysis of complex XOS equally.     

CH/pi hydrogen bonds as evidenced in the substrate specificity of acetylcholine esterase

The crystal structure of acetylcholine esterase (AchE) in complex with various inhibitors, investigated as drugs for improvement of the cognitive ability of early stage Alzheimer's disease, has been analyzed with the use of our program CHPI. A number of CH/pi hydrogen bonds have been disclosed in the binding of the inhibitors with Torpedo californica AchE. It has been demonstrated that, in order to be effective in the binding with AchE, C-H bonds in the inhibitor need not be polarized.