Purification and characterisation of an NAD+-dependent secondary alcohol dehydrogenase from Pseudomonas maltophilia MB11L

A constitutive NAD(+)-linked alcohol dehydrogenase was purified 338-fold from cells of Pseudomonas maltophilia MB11L grown on glucose. Maximum activity was observed with cyclic and linear secondary alcohols, with little activity seen against primary or aromatic alcohols. Substrate oxidation activity was maximal at pH 10.0, while substrate reduction was optimal at pH 4.5. The Km values for propan-2-ol, NAD+ and acetone were 87, 413 and 143 microM respectively. The enzyme is a tetramer with subunit Mr of approximately 44,000. It has an isoelectric point of 4.75, and was inhibited by chelating agents, thiol reagents and certain metal ions.     

Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp. strain MB1

A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an M(r) of approximately 65,000. The apparent K(m) of the enzyme (mean +/- standard deviation) for cocaine was measured as 1.33 +/- 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.     

Immunoelectron microscopic demonstration of an esterase on the outer membrane of Xanthomonas maltophilia.

Xanthomonas maltophilia (later synonym of Pseudomonas maltophilia), an ubiquitous species, is known to show proteolytic and lipolytic activities. A cell-bound esterase which hydrolyzes beta-naphthyl acetate during growth has been extracted from a strain isolated from soil. Because of its strongly hydrophobic character, the enzyme could be efficiently solubilized only by Triton X-100. This nonionic detergent must be added in polyacrylamide gels to permit migration. Polyclonal rabbit antibodies raised against the Triton-soluble esterase complex were used to localize the enzyme at the ultrastructural level. Electron microscopy of cell sections of this organism and immunogold labeling demonstrated that the enzyme was located on the outer membrane. Such an envelope-bound esterase may produce assimilable substrates for X. maltophilia which can grow in various environments.     

Immunoelectron microscopic demonstration of an esterase on the outer membrane of Xanthomonas maltophilia

Xanthomonas maltophilia (later synonym of Pseudomonas maltophilia), an ubiquitous species, is known to show proteolytic and lipolytic activities. A cell-bound esterase which hydrolyzes beta-naphthyl acetate during growth has been extracted from a strain isolated from soil. Because of its strongly hydrophobic character, the enzyme could be efficiently solubilized only by Triton X-100. This nonionic detergent must be added in polyacrylamide gels to permit migration. Polyclonal rabbit antibodies raised against the Triton-soluble esterase complex were used to localize the enzyme at the ultrastructural level. Electron microscopy of cell sections of this organism and immunogold labeling demonstrated that the enzyme was located on the outer membrane. Such an envelope-bound esterase may produce assimilable substrates for X. maltophilia which can grow in various environments.     

Gene Cloning and Nucleotide Sequencing and Properties of a Cocaine Esterase from Rhodococcus sp. Strain MB1

A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an M_r of approximately 65,000. The apparent K_m of the enzyme (mean ± standard deviation) for cocaine was measured as 1.33 ± 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.     

Decreasing the level of ethyl acetate in ethanolic fermentation broths of Escherichia coli KO11 by expression of Pseudomonas putida estZ esterase

During the fermentation of sugars to ethanol relatively high levels of an undesirable coproduct, ethyl acetate, are also produced. With ethanologenic Escherichia coli strain KO11 as the biocatalyst, the level of ethyl acetate in beer containing 4.8% ethanol was 192 mg liter(-1). Although the E. coli genome encodes several proteins with esterase activity, neither wild-type strains nor KO11 contained significant ethyl acetate esterase activity. A simple method was developed to rapidly screen bacterial colonies for the presence of esterases which hydrolyze ethyl acetate based on pH change. This method allowed identification of Pseudomonas putida NRRL B-18435 as a source of this activity and the cloning of a new esterase gene, estZ. Recombinant EstZ esterase was purified to near homogeneity and characterized. It belongs to family IV of lipolytic enzymes and contains the conserved catalytic triad of serine, aspartic acid, and histidine. As expected, this serine esterase was inhibited by phenylmethylsulfonyl fluoride and the histidine reagent diethylpyrocarbonate. The native and subunit molecular weights of the recombinant protein were 36,000, indicating that the enzyme exists as a monomer. By using alpha-naphthyl acetate as a model substrate, optimal activity was observed at pH 7.5 and 40 degrees C. The Km and Vmax for alpha-naphthyl acetate were 18 microM and 48.1 micromol. min(-1). mg of protein(-1), respectively. Among the aliphatic esters tested, the highest activity was obtained with propyl acetate (96 micromol. min(-1). mg of protein(-1)), followed by ethyl acetate (66 micromol. min(-1). mg of protein(-1)). Expression of estZ in E. coli KO11 reduced the concentration of ethyl acetate in fermentation broth (4.8% ethanol) to less than 20 mg liter(-1).     

The esterase activity of a pigmented bacterial group belonging to the Pseudomonas genus

Bacteria belonging to the Pseudomonas genus and isolated from zonal soils in different geographical zones of the USSR as well as from the rhizosphere of cultivated and wild plants were tested for their esterase activity. The studied collection of cultures included 205 strains of different pigmented Pseudomonas species which, according to the conventional taxonomy, were assigned to the so-called "Pseudomonas fluorescens complex". As was shown in this study, many Pseudomonas species are potential producers of nonspecific esterases. P. maltophilia and P. geniculata synthesizing pyomelanin have the highest activity of esterase. The activity of esterase correlates with the formation of a melanin-like pigment in Pseudomonas cultures. It also correlates with the species to which a culture belongs, which makes it possible to use this property as an additional criterion for the identification of Pseudomonas species.     

Human liver cocaine esterases: ethanol-mediated formation of ethylcocaine

A new, pharmacologically active metabolite of cocaine, ethylcocaine, has been reported in individuals after concurrent use of cocaine and ethanol. Formation of ethylcocaine may contribute to the common coabuse of these two drugs and the apparent danger of this practice. We have identified a nonspecific carboxyl-esterase that catalyzes the ethyl transesterification of cocaine to ethylcocaine in the presence of ethanol. In the absence of ethanol, this human liver esterase catalyzes the hydrolysis of cocaine to benzoylecgonine, a metabolite that is inactive as a psychomotor stimulant. A second human liver esterase is also described. This enzyme catalyzes hydrolysis of cocaine to ecgonine methyl ester, also inactive as a stimulant. These two liver esterases may play important roles in regulating the metabolic inactivation of cocaine.     

Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones

The directed evolution of an esterase from Pseudomonas fluorescens using the mutator strain Epicurian coli XL1-Red was investigated. Mutants were assayed for their ability to hydrolyze a sterically hindered 3-hydroxy ester, which can serve as a building block in the synthesis of epothilones. Screening was performed by plating esterase producing colonies derived from mutation cycles onto minimal media agar plates containing indicator substances (neutral red and crystal violet). Esterase-catalyzed hydrolysis of the 3-hydroxy ester (ethyl or glycerol ester) was detected by the formation of a red color due to a pH decrease caused by the released acid. Esterases isolated from positive clones were used in preparative biotransformations of the ethyl ester. One variant containing two mutations (A209D and L181V) stereoselectively hydrolyzed the ethyl ester resulting in 25% ee for the remaining ester.     

Directed evolution of an esterase: screening of enzyme libraries based on pH-indicators and a growth assay

In order to resolve a sterically hindered 3-hydroxy ethyl ester, which was not accepted as substrate by 20 wild-type hydrolases, a directed evolution of an esterase from Pseudomonas fluorescens (PFE) was performed. Mutations were introduced using the mutator strain Epicurian coli XL1-Red. Enzyme libraries derived from seven mutation cycles were assayed on minimal media agar plates supplemented with pH indicators (neutral red and crystal violet), thus allowing the identification of active esterase variants by the formation of a red color caused by a pH decrease due to the released acid. A further selection criteria was introduced by using the corresponding glycerol estar, because release of the carbon source glycerol facilitates growth on minimal media. By this strategy, one double mutant (A209D and L181V) of PFE was identified, which hydrolyzed the 3-hydroxy ethyl ester in a stereoselective manner (25% ee for the remaining ester, E approximate to 5).     

Quaternary structure and composition of squash NADH:nitrate reductase

NADH:nitrate reductase (EC 1.6.6.1) was isolated from squash cotyledons (Cucurbita maxima L.) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on Bio-Gel A-1.5m. These preparations gave a single protein staining band (Mr = 115,000) on sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is homogeneous. The native Mr of nitrate reductase was found to be 230,000, with a minor form of Mr = 420,000 also occurring. These results indicate that the native nitrate reductase is a homodimer of Mr = 115,000 subunits. Acidic amino acids predominate over basic amino acids, as shown both by the amino acid composition of the enzyme and an isoelectric point for nitrate reductase of 5.7. The homogeneous nitrate reductase had a UV/visible spectrum typical of a b-type cytochrome. The enzyme was found to contain one each of flavin (as FAD), heme iron, molybdenum, and Mo-pterin/Mr = 115,000 subunit. A model is proposed for squash nitrate reductase in which two Mr = 115,000 subunits are joined to made the native enzyme. Each subunit contains 1 eq of FAD, cytochrome b, and molybdenum/Mo-pterin.     

Atropine metabolism by a Pseudimanas sp.: Diauxic growth and oxidation of the products from esterase-mediated cleavage

Abstract Pseudomonas AT3 was isolated by elective culture with atropine as sole carbon source. Growth on atropine involved cleavage of the ester bond and was diauxic, with tropic acid being used in the first phase of growth followed by growth on the accumulated tropine moiety. During growth in N-limited medium, tropic acid was still used preferentially, nitrogen was provided from the tropine and novel keto compounds accumulated in the media. An inducible atropine esterase was synthesized during growth on atropine and, to a lesser extent, during growth on tropic acid and tropine. Organisms harvested from either phase of diauxic growth were able to oxidise tropic acid atropine. The ability of Pseudomonas AT3 to oxidase tropine was evident only in the second phase of growth and the greater extent of atropine oxidation in this phase reflected the ability to oxidase both cyclic components.     

Bile acid: CoASH ligases from guinea pig and porcine liver microsomes. Purification and characterization

A procedure for the purification of the enzyme bile acid:CoA ligase from guinea pig liver microsomes was developed. Activity toward chenodeoxycholate, cholate, deoxycholate, and lithocholate co-purified suggesting that a single enzyme form catalyzes the activation of all four bile acids. Activity toward lithocholate could not be accurately assayed during the earlier stages of purification due to a protein which interfered with the assay. The purified ligase had a specific activity that was 333-fold enriched relative to the microsomal cell fraction. The purification procedure successfully removed several enzymes that could potentially interfere with assay procedures for ligase activity, i.e. ATPase, AMPase, inorganic pyrophosphatase, and bile acid-CoA thiolase. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified ligase gave a single band of approximately 63,000 Mr. A molecular size of 116,000 +/- 4,000 daltons was obtained by radiation inactivation analysis of the ligase in its native microsomal environment, suggesting that the functional unit of the ligase is a dimer. The purified enzyme was extensively delipidated by adsorption to alumina. The delipidated enzyme was extremely unstable but could be partially stabilized by the addition of phospholipid vesicles or detergent. However, such additions did not enhance enzymatic activity. Kinetic analysis revealed that chenodeoxycholate, cholate, deoxycholate, and lithocholate were all relatively good substrates for the purified enzyme. The trihydroxy bile acid cholate was the least efficient substrate due to its relatively low affinity for the enzyme. Bile acid:CoA ligase could also be solubilized from porcine liver microsomes and purified 180-fold by a modification of the above procedure. The final preparation contains three polypeptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three peptides range in size from 50,000 to 59,000, somewhat smaller than the guinea pig enzyme. The functional size of the porcine enzyme in its native microsomal environment was determined by the technique of radiation inactivation analysis to be 108,000 +/- 5,000 daltons. Thus, the functional form of the porcine enzyme also appears to be a dimer.     

Properties of a cGMP-dependent monomeric protein kinase from bovine aorta

A form of cGMP-dependent protein kinase (cGK) that was different from previously described cGK was purified from bovine aorta smooth muscle. The partial amino-terminal sequencing of this enzyme indicated that it was derived by endogenous proteolysis of the type I beta isozyme of cGK. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this form migrated as a smaller protein (Mr = 70,000) than the parent cGK (Mr = 80,000), and since the calculated nondenatured Mr was approximately 89,000 compared to Mr = 170,000 for the dimeric native enzyme, it represented a monomeric form of cGK. The monomer bound approximately 2 mol of [3H]cGMP per mol of monomer, although it had only one rapid component in [3H]cGMP dissociation assays as compared to one rapid and one slow component for the native cGK. The specific catalytic activity of the kinase was similar to that of the native enzyme, suggesting that the catalytic domain was essentially intact. The monomeric cGK incorporated significant 32P when incubated with Mg2+ and [gamma-32P]ATP in the presence of cGMP, although the phosphorylation proceeded at a slower rate than that obtained with native cGK. In contrast to previous reports of monomeric forms of cGK, this monomer was highly cGMP-dependent, although it had a slightly higher Ka (0.8 microM) for cGMP than that of the native enzyme (0.4 microM) and a low Hill coefficient of 1.0 (1.6 for the native enzyme). The cGMP dependence of the monomer did not decrease with dilution, implying that the cGMP dependence was not due to monomer-monomer interactions in the assay. The results indicated that the catalytic domain, cGMP binding domain(s), and inhibitory domain of cGK interact primarily within the same subunit rather than between subunits of the dimer as previously hypothesized for dimeric cGK.     

Purification and characterization of a novel cholesterol esterase from Pseudomonas aeruginosa,with its application to cleaning lipid-stained contact lenses

With the aim of developing a new cholesterol esterase for eliminating lipids on used contact lenses, microorganisms were screened for the enzyme activity. A Pseudomonas aeruginosa isolated from soil was found to produce a desirable enzyme. The enzyme had an isoelectric point of 3.2, and molecular mass of 58 kDa. The optimal temperature was around 53 degrees C at pH 7.0, and the optimal pH was from 5.5 to 9.5. The enzyme was stable between pH 5 and 10 for 19 h at 25 degrees C, and retained its activity up to 53 degrees C on 30 min of incubation at pH 7.0. The rates of hydrolysis of cholesteryl esters of different fatty acids were in the following order: linoleate > oleate > stearate > palmitate > caprylate > myristate > laurate, caprate > caproate > butyrate, acetate. Addition of (tauro)cholate to a final concentration of 100 mM markedly promoted the hydrolysis of triglycerides of short-, medium-, and long-chain fatty acids. When used with taurocholate, the enzyme acted as an effective cleaner for contact lenses stained with lipids consisting of cholesteryl oleate, tripalmitin, and stearyl stearate.     

一种具有立体选择性新酯酶的菌种筛选和基因克隆

从土样分离到一株产生具有立体选择性酯水解酶的恶臭假单孢菌(Pseudomonas putida NH33)。构建P.putida NH33的基因组文库,并在E.coli中进行酯酶活性筛选,得到一个含有4.7kb插入片段的阳性克隆。对这个克隆的DNA片段进行序列分析,表明它含有一个1142个碱基的开放阅读框,为编码381个氨基酸的酯酶。推测的酯酶氨基酸序列与其它丝氨酸酯水解酶具有共同的保守基序GXSXG。把该蛋白在E.coli BL21(DE3)中进行表达,并用金属亲和层析纯化至单一条带。利用纯化的酶水解2-芳基丙酸乙酯制备2-芳基丙酸的S型异构体,产物的光学纯度ee_p>99%,说明此酶可8用于手性药物的合成。该酯酶是一个新酶,其基因序列已递交GenBank,登记号为AY896293。     

Liver microsomes contain two distinct NADPH-Monooxygenases with NH2-terminal segments homologous to the flavin containing NADPH-monooxygenase of Pseudomonas fluorescens

Two NADPH-reductase preparations (FAD-containing monooxygenases) were isolated from rabbit liver microsomes, referred to as from 1 and from 2. Purification was achieved by means of anion-exchange, cation-exchange and hydroxylapatite chromatography in the presence of cholate and Nonidet P-40. Affinity chromatography on 2', 5'-ADP Sepharose was used to increase the purity and to concentrate the enzyme. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, form 1 exhibited a single band at Mr 58,500 and form 2 at Mr 58,000. The NH2- terminus of form 1 is blocked, whereas the NH2-terminus of form 2 is homologous to the NADPH-phydroxybenzoate hydrolase from Pseudomonas fluorescens. The latter and the form 2 enzyme share 11 identical residues in the NH2-terminal segment of 15 residues. Both forms were subjected to tryptic cleavages and peptide mapping. Sequence analysis of the peptides obtained indicated that forms 1 and 2 are similar but not identical proteins. A tryptic peptide, homologous to residues 3 to 32 of form 2 enzyme was isolated from the form 1 protein. This segment has 24 residues that are identical to the form 2 and contains the consensus sequence Gly-X-Gly-X-X-Gly, found in most FAD binding proteins. These results indicate that the NADPH-monooxygenase system consists of at least two distinct proteins representing different gene products.     

A peptidase in human platelets that deamidates tachykinins. Probable identity with the lysosomal "protective protein".

We discovered an enzyme in human platelets that deamidates substance P and other tachykinins. Because an amidated carboxyl terminus is important for biological activity, we purified and characterized this deamidase. The enzyme, released from human platelets by thrombin, was purified to homogeneity by ammonium sulfate precipitation, followed by chromatography on an octyl-Sepharose column and chromatofocusing on PBE 94. The purified enzyme exhibits esterase, peptidase, and deamidase activities. The peptidase activity (with furylacryloyl-Phe-Phe) is optimal at pH 5.0 while the esterase (benzoyl-tyrosine ethyl ester) and deamidase (D-Ala2-Leu5-enkephalinamide) activities are optimal at pH 7.0. With biologically important peptides, the enzyme acts both as a deamidase (substance P, neurokinin A, and eledoisin) and a carboxy-peptidase (with bradykinin, angiotensin I, substance P-free acid, oxytocin-free acid) at neutrality, although the carboxypeptidase action is faster at pH 5.5. Enkephalins, released upon deamidation of enkephalinamides, were not cleaved. Gly9-NH2 of oxytocin was released without deamidation. Peptides with a penultimate Arg residue were not hydrolyzed. Some properties of the deamidase are similar to those reported for cathepsin A. The deamidase is inhibited by diisopropylfluorophosphate, inhibitors of chymotrypsin-type enzymes, and mercury compounds while other inhibitors of catheptic enzymes, trypsin-like enzymes, and metalloproteases were ineffective. In gel filtration, the native enzyme has an Mr = 94,000 while in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr = 52,000 indicating it exists as a dimer. After reduction, deamidase dissociates into two chains of Mr = 33,000 and 21,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]diisopropylfluorophosphate labeled the active site serine in the Mr = 33,000 chain. The first 25 amino acids of both chains were sequenced. They are identical with the sequences of the two chains of lysosomal "protective protein" which, in turn, has sequence similarity to the KEX1 gene product and carboxypeptidase Y of yeast. This protective protein complexes with beta-galactosidase and neuraminidase in lysosomes and is vitally important in maintaining their activity and stability. A defect in this protein is the cause of galactosialidosis, a severe genetic disorder. The ability of physiological stimuli (e.g. thrombin or collagen) to release the deamidase from platelets indicates that it may also be involved in the local metabolism of bioactive peptides.     

Microbial metabolism of quinoline and related compounds. VIII. Xanthine dehydrogenase from a quinoline utilizing Pseudomonas putida strain.

The xanthine dehydrogenase from Pseudomonas putida 86 was purified 68-fold to homogeneity with 47% recovery. SDS-polyacrylamide gel electrophoresis of the enzyme revealed two protein bands corresponding to an Mr of 87,000 and 52,000. The Mr of the native enzyme was calculated to 550,000 by gel chromatography. The enzyme contained 4 atoms of molybdenum, 16 atoms of iron, 16 atoms of acidlabile sulphur and 4 molecules of FAD. Due to the composition of the cofactors the xanthine dehydrogenase belongs to the class of molybdo-iron/sulphur-flavoproteins. Form A, an oxidation product of the molybdenum cofactor, was identified. Methanol and cyanide were effective inhibitors.     

利用乳酸乳球菌AcmA表面展示b-1, 3-1, 4-葡聚糖酶

采用PCR扩增乳酸乳球菌(Lactococcus lactis)MB191菌株的全长肽聚糖水解酶基因acmA, 通过C-末端融合构建了与绿色荧光基因gfp的融合基因acmA-gfp, 再连接于表达载体pMG36k上后得到可组成型表达AcmA-GFP融合蛋白的重组质粒pMB137, 然后将该质粒电转化导入到乳酸乳球菌AS1.2829中获得重组菌MB137。经SDS-PAGE检测, 重组菌MB137可表达预期的分子量约74 kD的蛋白质。Western blotting、细胞分级分离组分的荧光活性测定和特异GFP二抗标记的流式细胞仪检测证实GFP被成功锚定在重组菌细胞表面, 被锚定蛋白约占总表达融合蛋白的35%。进一步通过从枯草芽胞杆菌BF7658基因组中扩增去信号肽序列的b-1, 3-1, 4葡聚糖酶基因gls, 来取代pMB137中的gfp, 得到携带融合基因acmA-gls的重组质粒pMB138, 经导入到乳酸乳球菌AS1.2829后得到重组菌MB138, 其全细胞b-1, 3-1, 4-葡聚糖水解酶的活性约为12 U/mL菌液, 明显高于对照菌株。