Genetic analysis and characterization of poly(aspartic acid) hydrolase-1 from Sphingomonas sp. KT-1

Sphingomonas sp. KT-1 hydrolyzes poly(aspartic acid) (PAA) containing alpha- and beta-amide units and has at least two different types of PAA hydrolases. The PAA hydrolase-1 hydrolyzes selectively beta-beta amide units in PAA. Molecular cloning of PAA hydrolase-1 from Sphingomonas sp. KT-1 has been carried out to characterize its gene products. Genetic analysis shows that the deduced amino acid sequence of PAA hydrolase-1 has a similarity with those of the catalytic domain of poly(3-hydroxybutyric acid) (PHB) depolymerases from Alcaligenes faecalis AE122 and Pseudomonas lemoignei. Site-specific mutation analysis indicates that (176)Ser is a part of a strictly conserved pentapeptide sequence (Gly-Xaa-Ser-Xaa-Gly), which is the lipase box, and plays as an active residue.     

Poly(aspartate) hydrolases: biochemical properties and applications

Thermally synthesized poly(aspartate) (tPAA) shows potential for use in a wide variety of products and applications as a biodegradable replacement for non-biodegradable polycarboxylates, such as poly(acrylate). The tPAA molecule has unnatural structures, and the relationship between its biodegradability and structures has been investigated. Two tPAA-degrading bacteria, Sphingomonas sp. KT-1 and Pedobacter sp. KP-2, were isolated from river water; from them, two PAA-hydrolyzing enzymes, PAA hydrolases-1 and -2, were purified and biologically and genetically characterized. Interestingly, not only are PAA hydrolases-1 from those two strains novel in terms of structural genes and substrate specificities (they specifically cleave the amide bond between β-aspartate units in tPAA), they also probably play a central role in tPAA biodegradation by both strains. In green polymer chemistry, one active area of research is the use of purified enzymes for the enzyme-catalyzed synthesis of polypeptides by taking advantage of their substrate specificities. Recently, β-peptides have attracted academic and industrial interest as functional materials as they possess both functions of α-peptides and excellent metabolic stability. As one of the attractive applications of PAA hydrolases, we report here the enzyme-catalyzed synthesis of poly(α-ethyl β-aspartate), which is composed of only β-linkages and belongs to β-peptides, using the unique substrate specificity of the enzyme from Pedobacter sp. KP-2.     

Poly(Aspartic Acid) Degradation by a Sphingomonas sp. Isolated from Freshwater

A poly(aspartic acid) degrading bacterium (strain KT-1 [JCM10459]) was isolated from river water and identified as a member of the genus Sphingomonas. The isolate degraded only poly(aspartic acid)s of low molecular masses (<5 kDa), while the cell extract hydrolyzed high-molecular-mass poly(aspartic acid)s of 5 to 150 kDa to yield aspartic acid monomer.     

COMPARATIVE HYDROLYSIS OF GELATIN BY PEPSIN,TRYPSIN, ACID,AND ALKALI

A comparison has been made of the relative velocity of hydrolysis of the various peptid linkings of the gelatin molecule when hydrolyzed by acid, alkali, pepsin or trypsin. It has been found that: 1. Those linkages which are most rapidly split by pepsin or trypsin are among the more resistant to acid hydrolysis. 2. Those linkages which are hydrolyzed by pepsin are also hydrolyzed by trypsin. 3. Trypsin hydrolyzes linkages which are not attacked by pepsin. 4. Of the linkages which are hydrolyzed by both enzymes, those which are most rapidly hydrolyzed by pepsin are only slowly attacked by trypsin. 5. Those linkages which are attacked by trypsin or pepsin are among the ones first (most rapidly) hydrolyzed by alkali. In general it may be said that the course of the early stages of hydrolysis of gelatin is similar with alkali, trypsin, or pepsin and quite different with acid.     

Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan.

The action of neopullulanase from Bacillus stearothermophilus on many oligosaccharides was tested. The enzyme hydrolyzed not only alpha-(1----4)-glucosidic linkages but also specific alpha-(1----6)-glucosidic linkages of several branched oligosaccharides. When pullulan was used as a substrate, panose, maltose, and glucose, in that order, were produced as final products at a final molar ratio of 3:1:1. According to these results, we proposed a model for the pattern of action of neopullulanase on pullulan as follows. In the first step, the enzyme hydrolyzes only alpha-(1----4)-glucosidic linkages on the nonreducing side of alpha-(1----6) linkages of pullulan and produces panose and several intermediate products composed of some panose units. In the second step, taking 6(2)-O-alpha-(6(3)-O-alpha-glucosyl-maltotriosyl)-maltose as an example of one of the intermediate products, the enzyme hydrolyzes either alpha-(1----4) (the same position as that described above) or alpha-(1----6) linkages and produces panose or 6(3)-O-alpha-glucosyl-maltotriose plus maltose, respectively. In the third step, the alpha-(1----4) linkage of 6(3)-O-alpha-glucosyl-maltotriose is hydrolyzed by the enzyme, and glucose and another panose are produced. To confirm the model of the pattern of action, we extracted intermediate products produced from pullulan by neopullulanase and analyzed the structures by glucoamylase, pullulanase, and neopullulanase analyses. The experimental results supported the above-mentioned model of the pattern of action of neopullulanase on pullulan.     

Lysophospholipase-catalyzed hydrolysis of lysophospholipids in Mycoplasma gallisepticum membranes.

Mycoplasma gallisepticum strains have a membrane-bound lysophospholipase which hydrolyzes lysophospholipid generated in these membranes by treatment with an external phospholipase. This paper studies the hydrolysis of the membranous lysophospholipids by an enzyme residing in the same membrane (intramembrane utilization) or in adjacent membranes (intermembrane utilization). To study intermembrane hydrolysis, the phospholipids of M. gallisepticum were labeled with [3H]oleic acid. Membranes were prepared, heated at 65 degrees C, and subsequently treated with pancreatic phospholipase A2. This resulted in membranes whose enzyme was heat inactivated, but which contained lysophospholipid. When these membranes were mixed with M. gallisepticum cells or membranes, the lysophospholipid was hydrolyzed by the membranous lysophospholipase. To study intramembrane hydrolysis, [3H]oleyl-labeled membranes of M. gallisepticum were treated with pancreatic phospholipase A2 at pH 5.0. At this pH, lysophospholipid was generated but not hydrolyzed. Adjustment of the pH to 7.4 resulted in hydrolysis of the lysophospholipid by the membranous lysophospholipase. These procedures permitted measuring the initial rates of intramembrane and intermembrane hydrolysis of the lysophospholipid, showing that the time course and dependence on endogenous substrate concentration were different in the intramembrane and intermembrane modes of utilization. They also permitted calculation of the molar concentration of the lysophospholipid in the membrane and its rate of hydrolysis, expressed as moles per minute per cell or per square centimeter of cell surface.     

Enzymatic Production of l-Alanine by Pseudomonas dacunhae

To establish an advantageous method for the production of l-alanine, a procedure was studied for converting l-aspartic acid to l-alanine by microbial l-aspartic beta-decarboxylase. A number of organisms were screened to test their ability to form and accumulate alanine from aspartic acid. Pseudomonas dacunhae was selected as the most advantageous organism. With this organism, enzyme activity as high as 3,910 muliters of CO(2) per hr per ml of medium could be produced by shaking the culture at 30 C in the medium containing ammonium fumarate, sodium fumarate, corn steep liquor, peptone, and inorganic salts. For the enzymatic conversion of l-aspartic acid to l-alanine, the culture broth was employed as the enzyme source. A large amount of l-aspartic acid (as much as 40% of the broth) was converted stoichiometrically to alanine in 72 hr at 37 C. Furthermore, appropriate addition of a surface-active agent to the reaction mixture was found to be highly effective in shortening the time required for the conversion. Accumulated l-alanine was readily isolated in pure form by ordinary procedures with ion-exchange resins. Yields of isolated l-alanine of over 90% from l-aspartic acid were easily attainable.     

Preparative separation of alpha- and beta-naphthols catalyzed by immobilized sulfatase

It has been found that sulfatase from Helix pomatia hydrolyzes beta-naphthyl sulfate much faster than alpha-naphthyl sulfate; e.g., at pH 7.8, while the former is readily hydrolyzed, the latter undergoes no appreciable hydrolysis. Kinetic investigations of both enzymatic and acid hydrolysis of naphthyl sulfates and their analogs indicate that in the enzymatic reaction the difference in reactivities is due to steric hindrances exerted in alpha-naphthyl sulfate by the benzene ring adjacent to the one bearing the sulfate group. (In the beta-ester this ring is remote from the site of hydrolysis.) The enzyme was immobilized and employed for the preparative resolution of alpha- and beta-naphthols: a mixture of the isomers was first sulfated with chlorosulfonic acid and then incubated with sulfatase covalently attached to alumina. The beta-naphthol produced was extracted with benzene, followed by acid hydrolysis of alpha-naphthyl sulfate in the remaining aqueous solution and extraction of the alpha-naphthol formed. Helix pomatia sulfatase also expresses a marked regiospecificity in the hydrolysis of ortho and para substituted phenyl sulfates. Therefore, the enzyme can be used for the preparative separation of naphthols as well as a variety of isomeric phenols.     

Chemotaxonomic and phylogenetic analyses of Sphingomonas strains isolated from ears of plants in the family Gramineae and a proposal of Sphingomonas roseoflava sp. nov

Chemotaxonomic and phylogenetic characteristics of Sphingomonas strains isolated from plants of the family Gramineae were investigated. All strains contained the monosaccharide (glucuronic acid) type of glycosphingolipid (GSL-1). Most were found also to contain the oligosaccharide-type glycosphingolipids. Fatty acid and sphingosine profiles of the isolates were identical, although the ratio of the contents varies among the isolates. They all contained ubiquinone Q-10, and the G1C contents were from 66 to 68%. Phylogenetic analysis using 16S rRNA gene base sequences revealed that all the isolates were placed in the phylogenetic group of Sphingomonas paucimobilis in the alpha-4 subclass of Proteobacteria. By DNA-DNA hybridization experiments, the plant isolates were divided into five genotypic groups (groups 1 to 5). The strains of group 5 showed common physiological characteristics and formed pink-yellow colored colonies. Based on these results, Sphingomonas roseoflava sp. nov. was proposed for that homology group.     

Sucrose 6-alpha-D-glucosyltransferase from Streptococcus sobrinus: characterization of a glucosyl-enzyme complex

A covalent glucosyl-enzyme was isolated from a quenched reaction of Streptococcus sobrinus sucrose 6-alpha-D-glucosyltransferase and radiolabeled sucrose. No complex was observed with heat-inactivated enzyme or when sucrose was replaced with radiolabeled maltose or glucose. The complex was stable at pH 2 in 1% sodium dodecyl sulfate, 6.0 M urea, and 4.0 M guanidine hydrochloride, but became increasingly labile with increased pH (32-min half-life at pH 7.0). D-Glucose was the exclusive radiolabeled compound identified when all radioactivity was released under mild alkaline conditions. Glucosyl-enzyme hydrolysis rates were linearly dependent on hydroxide ion concentration, giving a second-order rate constant of 2.15 x 10(5) M-1 min-1. When compared to the base lability of known glycosyl amino acid derivatives, the pH dependency of the glucosyl-enzyme most closely paralleled a glucosyl linkage to a carboxyl group. A novel application of a carbohydrate high-performance liquid chromatography column in aqueous solution was used to identify the anomeric form of D-glucose released on (i) alkaline hydrolysis of denatured glucosyl-enzyme and (ii) native enzyme hydrolysis of sucrose. The beta-anomer was identified in the former case and the alpha-anomer in the latter. The results with the denatured glucosyl-enzyme are consistent with a beta-glucosyl ester linkage to an aspartic or glutamic acid that hydrolyzes at the ester carbon with retention of anomeric configuration; for native glucosyltransferase catalysis, the data are consistent with a beta-glucosyl covalent intermediate as well, where deglucosylation occurs by attack at the acetal carbon with anomeric inversion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzymic properties of thermopsin

The specificity of thermopsin, a thermostable acid protease from Sulfolobus acidocaldarius, was studied using oxidized insulin B chain as substrate followed by peptide isolation and identification. The following bonds were hydrolyzed: Leu-Val, Leu-Tyr, Phe-Phe, Phe-Tyr, and Tyr-Thr. Thus, the specificity of thermopsin is similar to that of pepsin, that is, it prefers large hydrophobic residues at both sides of the scissile bond. We confirmed this by the use of a synthetic substrate, Lys-Pro-Ala-Glu-Phe-p-nitro-phenylalanyl-Ala-Leu, which was cleaved by thermopsin between Phe and p-nitro-phenylalanyl. Using this substrate, enzyme inhibition and kinetic properties of thermopsin have been studied. Thermopsin optimally hydrolyzes this substrate at 75 degrees C and pH 2 with Km and kcat values under these conditions of 5.3 x 10(-5) M and 14.3 s-1, respectively. Pepstatin competitively inhibits thermopsin with a Ki of 2 x 10(-7) M. Other known aspartic protease inhibitors, diazoacetylnorleucine ethyl ester and 1,2-epoxy-3-(p-nitrophenoxy)propane inhibited thermopsin only slowly and with nonspecific reactions. Although thermopsin contains a single cysteine, iodoacetic acid and p-chloromercuric benzoate had no effect on activity. Mercuric chloride inhibited the enzyme, and the inhibition was reversible by mercaptoethanol. However, the enzyme was not labeled by [14C]iodoacetic acid either before or after sodium dodecyl sulfate denaturation. Thus, the thiol group is likely blocked, and the inhibition effect of mercuric ion is unrelated to the thiol group. These observations suggest that thermopsin has a different active site than the aspartic protease family but may have a similar transition state structure. The temperature dependence of Km and kcat was studied for thermopsin hydrolysis of the synthetic substrate between 26-78 degrees C. Both parameters increased with temperature, and the rise of kcat value was particularly sharp above 65 degrees C. Hydrolysis activity measured at high substrate concentration has a maximum at 76 degrees C, which is near the physiological temperature for the optimal growth of this organism. Thus, thermopsin appears to function best at high temperature and high substrate concentration. It may be utilized by the organism to response to the presence of high substrate concentration in the medium. Thermopsin is also competitively inhibited by urea, acetamide, and phenylalaninamide with Ki values of 0.5, 0.4, and 0.01 M, respectively.     

Depolymerization and de-N-acetylation of chitin oligomers in hydrochloric acid

The monosaccharide 2-amino-2-deoxy-D-glucose (glucosamine, GlcN) has recently drawn much attention in relation to its use to treat or prevent osteoarthritis in humans. Glucosamine is prepared from chitin, a process that is performed in concentrated acid, such as hydrochloric acid. This process involves two acid-catalyzed processes, that is, the hydrolysis of the glycosidic linkages (depolymerization) and of the N-acetyl linkages (de-N-acetylation). The depolymerization reaction has previously been found to be much faster compared to the deacetylation, with the consequence that the chitin chain will first be hydrolyzed to the monomer 2-acetamido-2-deoxy-D-glucose (N-acetylglucosamine, GlcNAc) which is subsequently deacetylated. We have found that the chitin disaccharide GlcNAc(1-->4)GlcNAc could be completely hydrolyzed to the monosaccharide GlcNAc with negligible concomitant de-N-acetylation, and the chitin disaccharide and monosaccharide were further used to study the depolymerization reaction and the de-N-acetylation reaction, respectively. The reactions were performed in hydrochloric acid as a function of acid concentration (3-12 M) and temperature (20-35 degrees C), and 1H-NMR spectroscopy was used to monitor the reaction rates. The 1H NMR spectrum of GlcNAc in concentrated (12 M) and deuterated hydrochloric acid at 25 degrees C was assigned. The glucofuranosyl oxazolinium (3) ion was found to exist in equilibrium with the alpha- and beta-anomers of the pyranose form of GlcNAc, where 3 was present in half the total molar concentrations of the two anomeric forms of GlcNAc. At lower acid concentration (3-6 M), only trace concentrations of 3 could be detected. The rate of de-N-acetylation of GlcNAc was determined as a function of hydrochloric acid concentration, showing a maximum at 6 M and decreasing by a factor of 2 upon decreasing or increasing the acid concentration to 3 or 12 M. The activation energy for hydrolysis of the N-acetyl linkage of GlcNAc was determined to be 102 +/- 7, 116 +/- 8, and 110 +/- 8 kJ mol(-1) at 3, 6, and 12 M hydrochloric acid concentration, respectively. The results are in accordance with the proposed SN2 reaction mechanism of the acid-catalyzed hydrolysis of the N-acetyl linkage where the rate-limiting step is the addition of water to the carbonium ion. The 1H NMR spectrum of the dimer GlcNAc-GlcNAc in concentrated (12 M) and deuterated hydrochloric acid at 25 degrees C was assigned. The rate of the acid-catalyzed cleavage of the glycosidic linkage of the dimer was determined as a function of hydrochloric acid concentration, showing a 6-fold increase from 3 to 6 M HCl concentration and a further 6-fold increase from 6 to 12 M HCl concentration, in contrast to the much smaller effect of acid concentration on the deacetylation reaction. Activation energy for hydrolysis of the glycosidic linkage of GlcNAc-GlcNAc was determined to be 110 +/- 6, 111 +/- 6, and 112 +/- 4 kJ mol(-1) at 3, 6 and 12 M hydrochloric acid concentration, respectively, that is, very similar to the activation energies determined for the deacetylation reaction. The results are in accordance with the proposed SN1 reaction mechanism of the acid-catalyzed hydrolysis of the glycosidic linkage, where the rate-limiting step is the formation of the carbonium ion.     

Oligo(methionyl) proteins. Enzymatic hydrolysis of the model isopeptides N epsilon-oligo(L-methionyl)-L-lysine

A number of model isopeptides containing oligo(methionine) chains varying in length (2-5 residues) covalently linked to the epsilon-amino group of lysine were synthesized by solid-phase procedures. Hydrolysis of these peptides by pepsin, chymotrypsin, cathepsin C (dipeptidyl peptidase IV) and intestinal aminopeptidase N was investigated using high-performance liquid chromatography to identify and quantify the hydrolysis products. Methionine oligomers grafted onto lysine were cleaved to tripeptides by pepsin. Chymotrypsin preferentially hydrolyzed the methionyl-methionine bond preceding the isopeptide bond. Cathepsin C released dimethionyl units from the covalently attached polymers. Intestinal aminopeptidase caused efficient hydrolysis of both peptides and isopeptide bonds although free methionine decreased the cleavage of the latter bond. Hydrophobic characteristics of oligo(methionine) chains promoted enzyme-catalyzed transpeptidations resulting probably from acyl-transfer-type reactions. Complementary hydrolysis of the isopeptides by these digestive enzymes suggests that covalent attachment of oligo(amino acid)s to food proteins may improve their nutritional value.     

Relative rates of hydrolysis by rat pancreatic lipase of esters of C2-C18 fatty acids with C1-C18 primary n-alcohols

The rate at which rat pancreatic lipase (glycerol-ester hydrolase, EC 3.1.1.3) hydrolyzes the esters of primary n-alcohols containing from 1 to 18 carbon atoms with fatty acids containing from 2 to 18 carbon atoms was determined. The speed of hydrolysis was influenced, apparently independently, by both the acyl and the alkyl chains. With respect to the fatty acid moiety, the esters of dodecanoic acid were usually split at the most rapid rate. Esters of butyric acid were the next most susceptible. In the case of the alcohol moiety, esters of heptyl alcohol were hydrolyzed most rapidly. On the basis of the pattern of the relative rates of hydrolysis, it is proposed that the influence of the alcohol component is a result of its orienting the ester molecule at the oil/water interface. The fatty acid effect is attributed to enzyme-substrate specificity.     

Purification and characterization of Acremonium implicatum alpha-glucosidase having regioselectivity for alpha-1,3-glucosidic linkage

alpha-Glucosidase with a high regioselectivity for alpha-1,3-glucosidic linkages for hydrolysis and transglucosylation was purified from culture broth of Acremonium implicatum. The enzyme was a tetrameric protein (M.W. 440,000), of which the monomer (M.W. 103,000; monomeric structure was expected from cDNA sequence) was composed of two polypeptides (M.W. 51,000 and 60,000) formed possibly by posttranslational proteolysis. Nigerose and maltose were hydrolyzed by the enzyme rapidly, but slowly for kojibiose. The k(0)/K(m) value for nigerose was 2.5-fold higher than that of maltose. Isomaltose was cleaved slightly, and sucrose was not. Maltotriose, maltotetraose, p-nitrophenyl alpha-maltoside and soluble starch were good substrates. The enzyme showed high affinity for maltooligosaccharides and p-nitrophenyl alpha-maltoside. The enzyme had the alpha-1,3- and alpha-1,4-glucosyl transfer activities to synthesize oligosaccharides, but no ability to form alpha-1,2- and alpha-1,6-glucosidic linkages. Ability for the formation of alpha-1,3-glucosidic linkage was two to three times higher than that for alpha-1,4-glucosidic linkage. Eight kinds of transglucosylation products were synthesized from maltose, in which 3(2)-O-alpha-nigerosyl-maltose and 3(2)-O-alpha-maltosyl-maltose were novel saccharides.     

Intracellular erythrocyte platelet-activating factor acetylhydrolase I inactivates aspirin in blood

Aspirin (acetylsalicylic acid) prophylaxis suppresses major adverse cardiovascular events, but its rapid turnover limits inhibition of platelet cyclooxygenase activity and thrombosis. Despite its importance, the identity of the enzyme(s) that hydrolyzes the acetyl residue of circulating aspirin, which must be an existing enzyme, remains unknown. We find that circulating aspirin was extensively hydrolyzed within erythrocytes, and chromatography indicated these cells contained a single hydrolytic activity. Purification by over 1400-fold and sequencing identified the PAFAH1B2 and PAFAH1B3 subunits of type I platelet-activating factor (PAF) acetylhydrolase, a phospholipase A(2) with selectivity for acetyl residues of PAF, as a candidate for aspirin acetylhydrolase. Western blotting showed that catalytic PAFAH1B2 and PAFAH1B3 subunits of the type I enzyme co-migrated with purified erythrocyte aspirin hydrolytic activity. Recombinant PAFAH1B2, but not its family member plasma PAF acetylhydrolase, hydrolyzed aspirin, and PAF competitively inhibited aspirin hydrolysis by purified or recombinant erythrocyte enzymes. Aspirin was hydrolyzed by HEK cells transfected with PAFAH1B2 or PAFAH1B3, and the competitive type I PAF acetylhydrolase inhibitor NaF reduced erythrocyte hydrolysis of aspirin. Exposing aspirin to erythrocytes blocked its ability to inhibit thromboxane A(2) synthesis and platelet aggregation. Not all individuals or populations are equally protected by aspirin prophylaxis, the phenomenon of aspirin resistance, and erythrocyte hydrolysis of aspirin varied 3-fold among individuals, which correlated with PAFAH1B2 and not PAFAH1B3. We conclude that intracellular type I PAF acetylhydrolase is the major aspirin hydrolase of human blood.     

Molecular cloning, nucleotide sequencing, and expression of the Bacillus subtilis (natto) IAM1212 alpha-amylase gene, which encodes an alpha-amylase structurally similar to but enzymatically distinct from that of B. subtilis 2633.

An alpha-amylase gene of Bacillus subtilis (natto) IAM1212 was cloned in a lambda EMBL3 bacteriophage vector, and the nucleotide sequence was determined. An open reading frame encoding the alpha-amylase (AMY1212) consists of 1,431 base pairs and contains 477 amino acid residues, which is the same in size as the alpha-amylase (AMY2633) of B. subtilis 2633, an alpha-amylase-hyperproducing strain, and smaller than that of B. subtilis 168, Marburg strain. The amino acid sequence of AMY1212 is different from that of AMY2633 at five residues. Enzymatic properties of these two alpha-amylases were examined by introducing the cloned genes into an alpha-amylase-deficient strain, B. subtilis M15. It was revealed that products of soluble starch hydrolyzed by AMY1212 are maltose and maltotriose, while those of AMY2633 are glucose and maltose. From the detailed analyses with oligosaccharides as substrates, it was concluded that the difference in hydrolysis products of the two similar alpha-amylases should be ascribed to the different activity hydrolyzing low-molecular-weight substrates, especially maltotriose; AMY1212 slowly hydrolyzes maltotetraose and cannot hydrolyze maltotriose, while AMY2633 efficiently hydrolyzes maltotetraose and maltotriose. Further analyses with chimeric alpha-amylase molecules constructed from the cloned genes revealed that only one amino acid substitution is responsible for the differences in hydrolysis products.     

Immobilization of microbial cells on inner epidermis of onion bulb scale for biosensor application

Inner epidermis of onion bulb scales was used as a natural support for immobilization of microbial cells for biosensor application. A bacterium Sphingomonas sp. that hydrolyzes methyl parathion into a chromophoric product, p-nitrophenol (PNP), has been isolated and identified in our laboratory. PNP can be detected by electrochemical and colorimetric methods. Whole cells of Sphingomonas sp. were immobilized on inner epidermis of onion bulb scale by adsorption followed by cross-linking methods. Cells immobilized onion membrane was directly placed in the wells of microplate and associated with the optical transducer. Methyl parathion is an organophosphorus pesticide that has been widely used in the field of agriculture for insect pest control. This pesticide causes environmental pollution and ecological problem. A detection range 4-80 μM of methyl parathion was estimated from the linear range of calibration plot of enzymatic assay. A single membrane was reused for 52 reactions and was found to be stable for 32 days with 90% of its initial hydrolytic activity. The applicability of the cells immobilized onion membrane was also demonstrated with spiked samples.     

Mass spectrometric technique for the determination of N-phosphonoacetyl-l-aspartic acid in serum

N-Phosphonoacetyl-l-aspartic acid (PALA), a potent inhibitor of aspartic acid transcarbamylase, is now undergoing Phase I clinical trials. Initial experiments revealed that PALA is not metabolized to phosphonoacetic acid (PAA) in humans. Thus PALA may be quantified in serum after in vitro conversion to PAA. Serum is deproteinized with perchloric acid, lipid extracted with methylene chloride, hydrolyzed with 8 N hydrochloric acid at 100° for 3 h, and evaporated to dryness with nitrogen. The residue is silylated, and PAA is quantified by monitoring the

ions of the protonated molecular ions of trimethylsilyl derivatives of PAA and phosphonopropionic acid (internal standard) obtained in chemical ionization with methane. Limit of detection is 0.5 μM (150 ng/ml) PALA using 1 ml serum. PALA was given by continuous infusion to cancer patients at various doses. Maximum levels of PALA (50–500 μM range) were obtained at the end of infusion, followed in most cases by biexponential decay. Persistent residual PALA levels (5 μM for 48 h after infusion) correlated with increased toxicity.     

Synthesis of tetrapeptide Bz-RGDS-NH2 by a combination of chemical and enzymatic methods

The tetrapeptide Bz-Arg-Gly-Asp-Ser-NH(2) (Bz-RGDS-NH(2)) was successfully synthesized by a combination of chemical and enzymatic methods in this study. Firstly, the precursor tripeptide Gly-Asp-Ser-NH(2) (GDS-NH(2)) was synthesized by a novel chemical method in four steps including chloroacetylation of l-aspartic acid, synthesis of chloroacetyl l-aspartic acid anhydride, the synthesis of ClCH(2)COAsp-SerOMe and ammonolysis of ClCH(2)COAsp-SerOMe. Secondly, lipase (PPL) was used to catalyze the formation of Bz-RGDS-NH(2) in aqueous water-miscible organic cosolvent systems using Bz-Arg-OEt as the acyl donor and GDS-NH(2) as the nucleophile. The optimum conditions were Bz-Arg-OEt 50 mM; GDS-NH(2) 400 mM; 10 degrees C, 0.1M phosphate buffer, pH 7.5; 60% DMF or 58% DMSO, PPL: 10 mg ml(-1) with the maximum yields of the tetrapeptide of 73.6% for DMF and 70.4% for DMSO, respectively. The secondary hydrolysis of the tetrapeptide product did not take place due to the absence of amidase activity of lipase.