Acetyl esterase from mediterranean oranges: partial purification,immobilisation and biotransformations

Acetyl esterase (acetic-ester acetylhydrolase, EC 3.1.1.6) from citrus peel, whose natural role is not well known, catalyses, in vitro, the hydrolysis of acetyl groups from a wide range of substrates. This enzyme was extracted from Mediterranean orange peel, largely available in Italy, and purified 190-fold by a single chromatographic step on Sepabeads FP-HG. SDS polyacrylamide gel electrophoresis of the purified enzyme showed a major protein band, corresponding to a molecular mass of 45 kDa. Both free and immobilised enzyme were used in biotransformations. The enzyme removed the acetyl group in the 3 position of β-lactamic antibiotics, such as cephalosporin C and the intermediate 7-aminocephalosporanic acid with ≥98% conversion and 91–93% product yield.     

Trichoderma reesei acetyl esterase catalyzes transesterification in water

Partially purified Trichoderma reesei RUT-C30 acetyl esterase preparation was found to catalyze acyl transfer reactions in organic solvents, mixtures of organic solvents with water and even in water. Using different acyl donors, the best results for acetyl transfer in water were obtained using vinyl acetate. As acetyl acceptors, a variety of hydroxyl bearing compounds in aqueous solutions were used. Degree of conversion and the number of newly formed acetates varied according to the acceptor used. Conversions over 50% were observed for the majority of several common monosaccharides, their methyl and deoxy derivatives and oligosaccharides. In several cases, the transesterification reaction exhibited strict regioselectivity, leading to only one acetyl derivative. Preparative potential of the transesterification in water was demonstrated by acetylation of methyl β- -glucopyranoside, 4-nitrophenyl β- -glucopyranoside and kojic acid, yielding 56.4% of methyl 3-O-acetyl β- -glucopyranoside, 70.2% of 4-nitrophenyl 3-O-acetyl β- -glucopyranoside and 30.9% of 7-O-acetyl-kojic acid as the only reaction products.

This enzymatically catalyzed transacetylation in water, which is applied to transformation of saccharides for the first time, opens a new area in chemoenzymatic synthesis. Its major advantages are simplicity, highly regioselective esterification of polar compounds, high yields, low enzyme consumption and elimination of the need to use toxic organic solvents.     

A xylose-tolerant beta-xylosidase from Paecilomyces thermophila: characterization and its co-action with the endogenous xylanase

An extracellular β-xylosidase from the thermophilic fungus Paecilomyces thermophila J18 was purified 31.9-fold to homogeneity with a recovery yield of 2.27% from the cell-free culture supernatant. It appeared as a single protein band on SDS–PAGE with a molecular mass of approx 53.5 kDa. The molecular mass of β-xylosidase was 51.8 kDa determined by Superdex 75 gel filtration. The enzyme was a glycoprotein with a carbohydrate content of 61.5%. It exhibited an optimal activity at 55 °C and pH 6.5, respectively. The enzyme was stable in the range of pH 6.0–9.0 and at 55 °C. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It released xylose from xylooligosaccharides with a degree of polymerization ranging between 2 and 5. The rate of xylose released from xylooligosaccharides by the purified enzyme increased with increasing chain length. It had a K_m of 4.3 mM for p-nitrophenol-β-d-xylopyranoside and was competitively inhibited by xylose with a K_i value of 139 mM. Release of reducing sugars from xylans by a purified xylanase produced by the same organism increased markedly in the presence of β-xylosidase. During 24-hour hydrolysis, the amounts of reducing sugar released in the presence of added β-xylosidase were about 1.5–1.73 times that of the reaction employing the xylanase alone. This is the first report on the purification and characterization of a β-xylosidase from Paecilomyces thermophila.     

S-acetyl phosphopantetheine: deacetyl citrate lyase S-acetyl transferase from Klebsiella aerogenes

An enzyme has been partially purified from Klebsiella aerogenes which transfers an acetyl group from S-acetyl phosphopantetheine to deacetyl citrate lyase. This converts the deacetyl citrate lyase which has no enzyme activity, to citrate lyase, the active enzyme. A variety of other acetyl thioesters including acetyl CoA did not serve as acetyl donors.     

Acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase. Evidence for a transmembrane acetylation mechanism

The lysosomal membrane enzyme acetyl-CoA: alpha-glucosaminide N-acetyltransferase catalyzes the transfer of an acetyl group from acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate. The reaction mechanism was examined using highly purified lysosomal membranes from rat liver. The reaction was followed by measuring the acetylation of a monosaccharide acetyl acceptor, glucosamine. The enzyme reaction was optimal above pH 5.5, and a 2-3-fold stimulation of activity was observed when the membranes were assayed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicated that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. Further evidence to support this mechanism was provided by characterization of the enzyme half-reactions. Membranes incubated with acetyl-CoA and [3H]CoA were found to produce acetyl-[3H]CoA. This exchange was optimal at pH values above 7.0. Treating membranes with [3H] acetyl-CoA resulted in the formation of an acetyl-enzyme intermediate. The acetyl group could then be transferred to glucosamine, forming [3H]N-acetylglucosamine. The transfer of the acetyl group from the enzyme to glucosamine was optimal between pH 4 and 5. The results suggest that acetyl-CoA does not cross the lysosomal membrane. Instead, the enzyme is acetylated on the cytoplasmic side of the lysosome and the acetyl group is then transferred to the inside where it is used to acetylate heparan sulfate.     

Fat metabolism in higher plants. Further characterization of wheat germ acetyl coenzyme A carboxylase.

Wheat germ acetyl CoA carboxylase was purified 600-fold over the crude homogenate. The purified enzyme gave rise to complex electrophoretic patterns in dissociating gels. As isolated, the activity of wheat germ acetyl CoA carboxylase exhibited profound dependence on the composition of the reaction mixture. In addition to the substrates MgATP, HCO₃, and acetyl CoA, the enzyme required both free Mg²⁺ and K⁺ for optimal activity. The effects of the two ions were additive. At pH 8.5, Mg²⁺ activated the carboxylase by adding to the enzyme prior to the other reactants in an equilibrium ordered reaction mechanism.     

Molecular characterization and enzymatic hydrolysis of naringin extracted from kinnow peel waste

Kinnow peel, a waste rich in glycosylated phenolic substances, is the principal by-product of the citrus fruit processing industry and its disposal is becoming a major problem. This peel is rich in naringin and may be used for rhamnose production by utilizing α-L-rhamnosidase (EC 3.2.1.40), an enzyme that catalyzes the cleavage of terminal rhamnosyl groups from naringin to yield prunin and rhamnose. In this work, infrared (IR) spectroscopy confirmed molecular characteristics of naringin extracted from kinnow peel waste. Further, recombinant α-L-rhamnosidase purified from Escherichia coli cells using immobilized metal-chelate affinity chromatography (IMAC) was used for naringin hydrolysis. The purified enzyme was inhibited by Hg2+ (1 mM), 4-hydroxymercuribenzoate (0.1 mM) and cyanamide (0.1 mM). The purified enzyme established hydrolysis of naringin extracted from kinnow peel and thus endorses its industrial applicability for producing rhamnose.     

Deacetylation of xylans by acetyl esterases of Trichoderma reesei

Summary Two previously purified esterases of Trichoderma reesei were used to study the deacetylation of polymeric, oligomeric and dimeric acetylated xylan fragments. For the first time nearly complete enzymatic deacetylation of polymeric xylan with purified acetyl xylan esterase was demonstrated, resulting in precipitation of the remaining polymer structure. The esterases had very different substrate specifities, one having a preference for high molecular weight substrates and the other showing high activity only towards acetyl xylobiose. The latter enzyme was also regioselective, cleaving off the acetyl substituent only from the C-3 position of the xylopyranose ring. The highest xylose yield from acetylated xylan was obtained by the synergistic action of xylanase, \-xylosidase and acetyl xylan esterase. Offprint requests to: M. Sundberg     

Establishment of the enzymatic protein acetylation independent of acetyl CoA: recombinant glutathione S-transferase 3-3 is acetylated by a novel membrane-bound transacetylase using 7,8-diacetoxy-4-methyl coumarin as the acetyl donor

The current knowledge on biological protein acetylation is confined to acetyl CoA-dependent acetylation of protein catalyzed by specific acetyl transferases and the non-enzymatic acetylation of protein by acetylated xenobiotics such as aspirin. We have discovered a membrane-bound enzyme catalyzing the transfer of acetyl groups from the acetyl donor 7,8-diacetoxy-4-methyl coumarin (DAMC) to glutathione S-transferase 3-3 (GST3-3), termed DAMC:protein transacetylase (TAase). The purified enzyme was incubated with recombinant GST3-3 subunit and DAMC, the modified protein was isolated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) in gel digested with trypsin and the tryptic digest was analyzed by mass spectrometry. The N-terminus and six lysines, Lys-51, -82, -124, -181, -191 and -210, were found to be acetylated. The acetylation of GST3-3 described above was not observed in the absence of either DAMC or TAase. These results clearly establish the phenomenon of protein acetylation independent of acetyl CoA catalyzed by a hitherto unknown enzyme (TAase) utilizing a certain xenobiotic acetate (DAMC) as the active acetyl donor.     

Production, purification and properties of γ-glutamyltranspeptidase from a newly isolated Bacillus subtilis NX-2

Production, purification and properties of γ-glutamyltranspeptidase from a newly isolated Bacillus subtilis NX-2 was investigated. At the optimum conditions for enzyme formation, a high level, 3.2 U/ml of γ-GTP was obtained. The extracellular γ-GTP from this strain was purified 111.15-fold to homogeneity from the culture supernatant by acetone precipitation, hydrophobic interaction chromatography and ion exchange chromatography. The purified enzyme was a heterodimer consisting of one large subunit (43 kDa) and one small subunit (32 kDa), and exhibited high activity at 40–60 °C, pH 8.0. It preferred basic amino acids as γ-glutamyl acceptor in transpeptidation, and the stereochemistry of the γ-glutamyl acceptor had no influence on the enzyme activity, which was different from other γ-GTPs reported. Furthermore, it was proved that γ-GTP of this strain could catalyze the transfer of l-glutamine to glycylglycine to synthesize Gln–Gly–Gly, which was promising for the synthesis of valuable γ-glutamyl peptides.     

Effect of Drying Methods on Quality of Leaf Protein Concentrate

Glutathione thiol esterase activity in cell extracts of a yeast: Saccharomyces cerevisiae was separated into three peaks when filtered on a Sephadex G-150 gel column. One of the enzymes in these peaks was purified. The enzyme was a single polypeptide chain with a molecular weight of 28,000 and catalyzed the complete hydrolysis of S-acetylglutathione and S-lactoylglutathione. S- Methyl-, S-hexyl-, S-glyceryl-, S-succinylglutathiones, and acetyl CoA were not hydrolyzed. In addition to the hydrolytic activity, the purified enzyme showed a group transfer activity and catalyzed the formation of acetyl CoA from S-acetylglutathione and CoA. The purified enzyme was not identical with glyoxalase II in molecular weight, substrate specificity, or behaviors toward inhibitors.     

Purification and characterization of an esterase involved in cellulose acetate degradation by Neisseria sicca SB

An esterase catalyzing the hydrolysis of acetyl ester moieties in cellulose acetate was purified 1,110-fold to electrophoretic homogeneity from the culture supernatant of Neisseria sicca SB, which can assimilate cellulose acetate as the sole carbon and energy source. The purified enzyme was a monomeric protein with a molecular mass of 40 kDa and the isoelectric point was 5.3. The pH and temperature optima of the enzyme were 8.0-8.5 and 45 degrees C. The enzyme catalyzed the hydrolysis of acetyl saccharides, p-nitrophenyl esters of short-chain fatty acids, and was slightly active toward aliphatic and aromatic esters. The K(m) and Vmax for cellulose acetate (degree of substitution, 0.88) and p-nitrophenyl acetate were 0.0162% (716 microM as acetyl content in the polymer) and 36.0 microM, and 66.8 and 39.1 mumol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate, which indicated that the enzyme was a serine esterase.     

Purification and characterization from rat kidney membranes of a novel platelet-activating factor (PAF)-dependent transacetylase that catalyzes the hydrolysis of PAF, formation of PAF analogs, and C2-ceramide

We have previously identified two enzyme activities that transfer the acetyl group from platelet-activating factor (PAF) in a CoA-independent manner to lysoplasmalogen or sphingosine in HL-60 cells, endothelial cells, and a variety of rat tissues. These were termed as PAF:lysoplasmalogen (lysophospholipid) transacetylase and PAF:sphingosine transacetylase, respectively. In the present study, we have solubilized and purified this PAF-dependent transacetylase 13,700-fold from rat kidney membranes (mitochondrial plus microsomal membranes) based on the PAF:lysoplasmalogen transacetylase activity. The mitochondria and microsomes were prepared and washed three times, then solubilized with 0.04% Tween 20 at a detergent/protein (w/w) ratio of 0.1. The solubilized fractions from mitochondria and microsomes were combined and subjected to sequential column chromatographies on DEAE-Sepharose, hydroxyapatite, phenyl-Sepharose, and chromatofocusing. The enzyme was further purified by native-polyacrylamide gel electrophoresis (PAGE) and affinity gel matrix in which the competitive inhibitor of the enzyme, 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphoethanolamine was covalently attached to the CH-Sepharose. On SDS-PAGE, the purified enzyme showed a single homogeneous band with an apparent molecular mass of 40 kDa. The purified enzyme catalyzed transacetylation of the acetyl group not only from PAF to lysoplasmalogen forming plasmalogen analogs of PAF, but also to sphingosine producing N-acetylsphingosine (C2-ceramide). In addition, this enzyme acted as a PAF-acetylhydrolase in the absence of lipid acceptor molecules. These results suggest that PAF-dependent transacetylase is an enzyme that modifies the cellular functions of PAF through generation of other diverse lipid mediators.     

Purification and characterization of an extracellular beta-glucosidase produced by Phoma sp. KCTC11825BP isolated from rotten mandarin peel

A beta-glucosidase from Phoma sp. KCTC11825BP isolated from rotten mandarin peel was purified 8.5-fold with a specific activity of 84.5 U/mg protein. The purified enzyme had a molecular mass of 440 kDa with a subunit of 110 kDa. The partial amino acid sequence of the purified beta-glucosidase evidenced high homology with the fungal beta- glucosidases belonging to glycosyl hydrolase family 3. Its optimal activity was detected at pH 4.5 and 60 degrees C, and the enzyme had a half-life of 53 h at 60 degrees C. The Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose were 0.3 mM and 3.2 mM, respectively. The enzyme was competitively inhibited by both glucose (Ki=1.7 mM) and glucono-delta-lactone (Ki=0.1 mM) when pNPG was used as the substrate. Its activity was inhibited by 41% by 10 mM Cu2+ and stimulated by 20% by 10 mM Mg2+.     

Rhamnogalacturonan acetylesterase: a novel enzyme from Aspergillus aculeatus,specific for the deacetylation of hairy (ramified) regions of pectins

Rhamnogalacturonan acetylesterase, able to specifically hydrolyse the acetyl asters present in modified hairy (ramified) regions (MHR) of apple pectin, was identified. The enzyme removed about 70% of the total acetyl groups in MHR. This acetylesterase did not cause the release of acetyl groups from a range of other acetylated substrates, either synthetic or extracted from plants, including the acetylated smooth regions present in beet pectin. Pretreatment of pectic polysaccharides in order to remove arabinose side chains had no effect on the acetyl release, wor was an effect found on the rate or degree of acetyl release, when the purified acetylesterase was combined with pectolytic enzymes, pectin methylesterase or arabinanases. Correspondence to: A. G. J. Voragen     

Purification and Characterization of Polyphenol Oxidase From Waste Potato Peel by Aqueous Two-Phase Extraction

Potato peel from food industrial waste is a good source of polyphenol oxidase (PPO). This work illustrates the application of an aqueous two-phase system (ATPS) for the extraction and purification of PPO from potato peel. ATPS was composed of polyethylene glycol (PEG) and potassium phosphate buffer. Effect of different process parameters, namely, PEG, potassium phosphate buffer, NaCl concentration, and pH of the system, on partition coefficient, purification factor, and yield of PPO enzyme were evaluated. Response surface methodology (RSM) was utilized as a statistical tool for the optimization of ATPS. Optimized experimental conditions were found to be PEG1500 17.62% (w/w), potassium phosphate buffer 15.11% (w/w), and NaCl 2.08 mM at pH 7. At optimized condition, maximum partition coefficient, purification factor, and yield were found to be 3.7, 4.5, and 77.8%, respectively. After partial purification of PPO from ATPS, further purification was done by gel chromatography where its purity was increased up to 12.6-fold. The purified PPO enzyme was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by K_m value 3.3 mM, and V_max value 3333 U/mL, and enzyme stable ranges for temperature and pH of PPO were determined. These results revealed that ATPS would be an attractive option for obtaining purified PPO from waste potato peel.     

Isolation and characterization of a (1 → 3)-β-glucan endohydrolase from germinating barley (Hordeum vulgare): amino acid sequence similarity with barley (1 → 3, 1 → 4)-β-glucanases

A (1 → 3)-β-glucan 3-glucanohydrolase (EC 3.2.1.39) has been purified approx. 190-fold from extracts of germinating barley. The enzyme has an apparent M_r 32 000, a pI of 8.6, and a pH optimum of 5.6. Analysis of hydrolysis products released from the (1 → 3)-β-glucan, laminarin, shows that the enzyme is an endohydrolase. Sequence analysis of the 46 NH₂-terminal amino acids of the (1 → 3)-β-glucanase reveals 54% positional identity with barley (1 → 3,1 → 4)-β-glucanases (EC 3.2.1.73) and suggests a common evolutionary origin for these two classes of β-glucan endohydrolases. The barley (1 → 3)-β-glucanase also exhibits significant similarity with a (1 → 3)-β-glucanase from tobacco.     

Site-directed Mutagenesis Identifies Amino Acid Residues Associated with the Dehydrogenase and Isomerase Activities of Human Type I (Placental) 3β-Hydroxysteroid Dehydrogenase/Isomerase

3β-Hydroxysteroid dehydrogenase/steroid Δ^{5 → 4}-isomerase (3β-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding human wild-type I (placental) and the human type I mutants - H261R, Y253F and Y253,254F. Western blots of SDS-polyacrylamide gels showed that the baculovirus-infected Sf9 cells expressed the immunoreactive wild-type, H261R, Y253F or Y253,254F protein that co-migrated with purified placental 3β-HSD/isomerase (monomeric M_r=42,000 Da). The wild-type, H261R and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells (5.01). In kinetic studies with purified enzyme, the H261R mutant enzyme had no 3β-HSD activity, whereas the K_m and V_max values of the isomerase substrate were similar to the values obtained with the wild-type and native enzymes. The V_max (88 nmol/min/mg) for the conversion of 5-androstene-3,17-dione to androstenedione by the Y253F isomerase activity was 7.0-fold less than the mean V_max (620 nmol/min/mg) measured for the isomerase activity of the wild-type and native placental enzymes. In microsomal preparations, isomerase activity was completely abolished in the Y253,254F mutant enzyme, but Y253,254F had 45% of the 3β-HSD activity of the wild-type enzyme. In contrast, the purified Y253F, wild-type and native enzymes had similar V_max values for substrate oxidation by the 3β-HSD activity. The 3β-HSD activities of the Y253F, Y253,254F and wild-type enzymes reduced NAD⁺ with similar kinetic values. Although NADH activated the isomerase activities of the H261R and wild-type enzymes with similar kinetics, the activation of the isomerase activity of H261R by NAD⁺ was dramatically decreased. Based on these kinetic measurements, His²⁶¹ appears to be a critical amino acid residue for the 3β-HSD activity, and Tyr²⁵³ or Tyr²⁵⁴ participates in the isomerase activity of human type I (placental) enzyme.     

A novel cephalosporin deacetylating acetyl xylan esterase from Bacillus subtilis with high activity toward cephalosporin C and 7-aminocephalosporanic acid

A cephalosporin deacetylating acetyl xylan esterase was cloned from the genomic DNA of Bacillus subtilis CICC 20034 and functionally expressed in Escherichia coli. Its gene contained an open reading frame of 957 bp encoding 318 amino acids with a calculated mass of 35,607 Da, and it displayed significant identity to acetyl xylan esterases from Bacillus sp. 916, B. subtilis 168, and Bacillus pumilus Cect5072. The enzyme was a native homohexamer but a trimer under the condition of 1 % sodium dodecyl sulfate (SDS); both forms were active and could transit to each other by incubating in or removing SDS. The enzyme belongs to carbohydrate esterase family 7 and had a double specificity on both the acetylated oligosaccharide and cephalosporin C (CPC) and 7-aminocephalosporanic acid (7-ACA). The activity of this purified enzyme toward CPC and 7-ACA was highest among all the acetyl xylan esterase from CE family 7, which were 484 and 888 U/mg, respectively, and endowed itself with great industrial interest on semi-synthetic β-lactam antibiotics. The optimum pH of the purified enzyme was 8.0, and the optimum temperature was 50 °C, and the enzyme had high thermal stability, broad range of pH tolerance, and extremely organic solvent tolerance.