Production of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine by entrapped ACV-synthetase fromStreptomyces clavuligerus

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV)-synthetase fromStreptomyces clavuligerus was studied under conditions that enabled the reuse of the enzyme. Coupling of ACV-synthetase to DEAE-Trisacryl and aminopropyl-glass resulted in an immobilized enzyme product of little or no catalytic activity. However, an enzyme reactor was designed by physical confinement of partially-purified ACV-synthetase in an ultrafiltration cell. This system was stimulated by phosphoenolpyruvate at lower concentrations of ATP, an effect not observed with purified enzyme. Up to 30% conversion of the limiting substrate, cysteine, to ACV occurred under semi-continuous conditions. Reaction products were investigated as potential inhibitors: AMP was the most inhibitory, but only when used at concentrations in excess of those produced in reaction mixtures. Under a nitrogen atmosphere, both product and enzyme stabilities were greatly improved and the enzyme retained 45–46% of its initial activity after five uses at room temperature during a 24-h period. Extrapolations based on these data suggest that 1.3 g partially purified enzyme (0.13 U g^–1) would be capable of producing 411 mg of ACV in a 1-L reaction mixture in this period.     

Production kinetics and stability properties of ϖ(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase fromStreptomyces clavuligerus

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV)-synthetase is a key enzyme that channels primary metabolites to a tripeptide common to cephalosporin and cephamycin biosynthesis inStreptomyces clavuligerus. Time-course studies indicated that theS. clavuligerus ACV-synthetase was stable during the cephamycin C fermentation: the enzyme was produced early in the growth phase and its activity remained high up to 96 h of growth. The detection of crude ACV-synthetase activity in older cultures was best achieved with an assay medium supplemented with 5 mM phosphoenolpyruvate, at lower ATP concentrations. During storage at 4°C, a progressive decrease in the stability of crude ACV-synthetase was observed with increasing culture age. Although a proteinolytic activity with a pH optimum at 8.2 was detected in crude cell-free extracts, no significant variation was observed in its activity with increasing culture age to account for the instability of ACV-synthetase in vitro. Addition of proteinase inhibitors did not improve the stability of the enzyme. However, a stabilization cocktail containing dithiothreitol. MgCl₂, the three substrate amino acids, and glycerol increased the stability of the enzyme isolated from cultures grown for 30–40 h, which was shortly after the appearance of antibiotics in the culture fluid. This stabilized enzyme retained half of its initial activity after 6 days at 4°C.     

Efficient chemoenzymatic synthesis of (S)-α-amino-4-fluorobenzeneacetic acid using immobilized penicillin amidase

An efficient chemoenzymatic route was developed for synthesis of (S)-α-amino-4-fluorobenzeneacetic acid, a valuable chiral intermediate of Aprepitant, using immobilized penicillin amidase catalyzed kinetic resolution of racemic N-phenylacetyl-4-fluorophenylglycine. The optimum temperature, pH and agitation rate of the reaction were determined to be 40 °C, 9.5 and 300 rpm, respectively. Kinetic resolution of 80 g L⁻¹ N-phenylacetyl-4-fluorophenylglycine by immobilized amidase 20 g L⁻¹ resulted in 49.9% conversion and >99.9% e.e. within 3 h. The unreacted N-phenylacetyl-4-fluorophenylglycine can be easily racemized and then recycled as substrate. The production of (S)-α-amino-4-fluorobenzeneacetic acid was further amplified in 1 L reaction system, affording excellent conversion (49.9%) and enantioselectivity (99.9%). This chemoenzymatic approach was demonstrated to be promising for industrial production of (S)-α-amino-4-fluorobenzeneacetic acid.     

Hydrolysis of di-substituted hydantoins,by an enzyme preparation from lentil (Lens esculenta) seeds,for the synthesis of α,α-dialkylated amino acids with linear and cyclic substituents

Hydrolysis of 5,5-di-substituted hydantoins with linear and cyclic side chains, to produce the corresponding ,-dialkylated amino acids and 1-aminocycloalkane-1-carboxylic acids, using an enzyme preparation from lentil (Lens esculenta) seeds is reported for the first time. This method could be developed on a large scale and should be of interest to the chemical and pharmaceutical industries.R. Rai and V. Taneja are with the Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi-221 005, India; R. Balaji Rao is with the Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi-221 005, India.     

Synthesis of poly(ε-caprolactone) by an immobilized lipase coated with ionic liquids in a solvent-free condition

Polycaprolactone (PCL) was synthesized by ring-opening polymerization of ε-caprolactone through two different enzymatic processes. The lipase from Candida antarctica B, immobilized on macroporous acrylic acid beads, was employed either untreated or coated with small amounts of ionic liquids (ILs). Monocationic ionic liquids, [C _n MIm][NTf₂] (n = 2, 6, 12), as well as a dicationic ionic liquid, ([C₄(C₆Im)₂][NTf₂]₂), were used to coat the immobilized lipase and also as the reaction medium. In both methods, the polarity, anion of the ILs concentration and viscosity strongly influenced the reaction. Coating the immobilized enzyme with ILs improved catalytic activity and less ILs was required to produce PCL with a higher molecular weight and reaction yield. At 60 °C and ILs/Novozyme-435 coating ratio of 3:1 (w/w) for 48 h, the highest M _w and reaction yield of PCL were 35,600 g/mol and 62 % in the case of [C₁₂MIm][NTf₂], while the M _w and reaction yield of PCL was 20,300 g/mol and 54 % with [C₁₂MIm][NTf₂] and catalyzed by untreated lipase.     

Synthesis of the α,ω-dicarboxylic acid precursor of biotin by the canonical fatty acid biosynthetic pathway

Biotin synthesis requires the C7 α,ω-dicarboxylic acid, pimelic acid. Although pimelic acid was known to be primarily synthesized by a head to tail incorporation of acetate units, the synthetic mechanism was unknown. It has recently been demonstrated that in most bacteria the biotin pimelate moiety is synthesized by a modified fatty acid synthetic pathway in which the biotin synthetic intermediates are O-methyl esters disguised to resemble the canonical intermediates of the fatty acid synthetic pathway. Upon completion of the pimelate moiety, the methyl ester is cleaved. A very restricted set of bacteria have a different pathway in which the pimelate moiety is formed by cleavage of fatty acid synthetic intermediates by BioI, a member of the cytochrome P450 family.     

A Facile Method of Preparing l-α-Methyl DOPA (l-3-(3,4-Dihydroxyphenyl)-2-methylalanine)

To develop a practical method for production of l-α-methyl DOPA, the optical resolution of its precursor, dl-3-(3,4-methylenedioxyphenyl)-2-methylalanine, was studied. The monohydrochloride of dl-3-(3,4-methylenedioxyphenyl)-2-methylalanine was resolved by a preferential crystallization procedure. Optically pure l-α-methyl DOPA was obtained in good yield. Industrial production of l-α-methyl DOPA by the present simple method is considered to be very promising.     

Inhibition of activity of the ethylene-forming enzyme by α(p-chlorophenoxy)isobutyric acid

(p-Chlorophenoxy)isobutyric acid (PCIB) inhibited indole-3-acetic acid (IAA)-induced ethylene production in etiolated mung bean hypocotyl sections. The endogenous level of 1-aminocyclopropane-1-carboxylic acid (ACC) was not significantly affected by PCIB, indicating that PCIB exerted its effect primarily by inhibiting the activity of the ethylene-forming enzyme (EFE). This conclusion was supported by the observations that PCIB inhibited the conversion of exogenously applied ACC to ethylene. The inhibitory effect of PCIB was already evident with 0.05 mM PCIB, and it increased with time after application of the inhibitor. PCIB also significantly inhibited ethylene production in apple fruit tissues, but it only slightly reduced the level of endogenous ACC. Similar to mung bean, EFE activity in apple tissue was significantly inhibited by PCIB. The possibility that PCIB also inhibits auxin-induced ACC synthase activity is discussed.     

Liquefaction of dulse (Palmaria palmata (L.) Kuntze) by a commercial enzyme preparation and a purified endo ,β-1,4-D-xylanase

Liquefaction of dry and freshPalmaria palmata by food grade enzyme preparations and a purified endo--1,4-D-xylanase was studied.The endo--1,4-D-xylanase (EC 3.2.1.8) was purified to homogeneity from a commercial food grade enzyme prepared fromAspergillus niger. It has a molecular weight of 22 500, a pI of 3.5, is inactive toward corn arabinoxylan,p-nitrophenyl--D-xylose, carboxymethyl cellulose but shows a weak activity toward microcrystalline cellulose. It hydrolyzes oat and dulse xylan equally well in seawater and deionized water essentially into xylose and xylobiose. It is stable between pH 5.5 to 9.0 and 0 to 30 °C and its activity is optimal at pH 4.5–5.5 and 40–60 °C. It has a K_m of 2.2 and 2.8 mg ml^-1 and V_max of 3600 and 3900 nkat mg^-1 of protein on oat and dulse xylan, respectively.Acetate buffer, deionized water and seawater alone extracted 62.6 to 64.5 % of the dry weight of dry dulse, but the use of commercial food grade enzyme preparations or the purified xylanase improved liquefaction to 81.2–87.1 %. Xylose and galactose were the only sugars present in the soluble extracts. Deionized and seawater extracted 58.8–52.7 and 39.1–42.2% of the dry weight of the fresh algae collected in fall and summer, respectively. Only galactose was found in the seawater extract, while some xylose with galactose were measured in the deionized water extract of the fresh autumn algal sample. Purified and crude xylanase improved liquefaction of fresh algae to 79.8–81.4 and 71.9–77.9% of the fresh dry weight (fall and summer, respectively) in deionized and seawater, respectively, and increased the xylose content of the soluble fractions. Polysaccharides in the soluble residues were composed of 1,3/1,4-linked xylose, 1-linked galactose (floridoside) and 1,4-linked glucose (cellulose) and contained essentially 1,4-linked xylose and 1,4-linked glucose in insoluble fractions obtained after enzymatic treatment.The use of xylanase-containing food grade enzyme preparations improves liquefaction ofPalmaria palmata, particularly from fresh alga. This study indicates that processing such as drying may modify markedly the solubility ofP. palmata cell wall polysaccharides, which would imply the existence of some organization and/or other components in the fresh cell wall that lower xylan solubility in seawater.     

Synthesis of Guaiacol-α-D-glucoside and Curcumin-bis-α-D-glucoside by an Amyloglucosidase from Rhizopus

Guaiacol (2-methoxyphenol) and curcumin [1E,6E-1,7-di(4-hydroxy-3-methoxy-phenyl)-1,6-heptadiene-3,5-dione] were converted into their corresponding glucosides using glucose and an amyloglucosidase from Rhizopus. Guaiacol-α-D-glucoside yields ranged from 3 to 52% with the highest at pH 7.0. Curcumin-bis-α-D-glucoside yields ranged from 3 to 48% with the highest at pH 4.0 with 50% (w/w D-glucose) of enzyme. The phenolic hydroxyl group of guaiacol and both phenolic hydroxyl groups of curcumin were glucosylated at the C1 carbon of α-D-glucose indicating that the enzymatic reaction is stereospecific. Both guaiacol-α-D-glucoside and curcumin-bis-α-D-glucosides had antioxidant activities.     

The incorporation of cytidine 5′-monophosphate and other ribonucleotides by an enzyme preparation from rat-liver microsomes

1. An enzyme preparation from rat-liver microsomes incorporated all four ribonucleotides from the corresponding triphosphates into ribosomal RNA. The reaction was Mn(2+)-dependent, but UMP incorporation also occurred in the presence of Mg(2+). 2. The incorporation of any one ribonucleotide was inhibited by the presence of the other three ribonucleoside triphosphates and by denatured DNA. 3. The product of the reaction consisted of short chains of homopolymer attached to the primer ribosomal RNA. 4. ;Soluble' RNA, synthetic polyribonucleotides, and oligoribonucleotides were also effective primers for CMP incorporation. 5. When phosphodiesterase-treated ;soluble' RNA was the primer, CMP was incorporated into positions usually occupied by the normal terminal trinucleotide sequence of intact ;soluble' RNA, but the enzyme did not synthesize a specific terminal sequence consisting of a defined number of CMP residues.     

The use of biosensors in bioorganic synthesis: Peptide synthesis by immobilized α-chymotrypsin assessed with an enzyme thermistor

Summary Biosensor analysis of peptide synthesis in organic solvents using immobilized -chymotrypsin is described. It is shown that with the enzyme thermistor sensor used, a direct correlation exists between the negative T-values registered and the amount of peptides formed (essentially various N-acetyldipeptide amides) allowing concentrations of 0.1 mM peptide and lower to be determined directly in the reaction medium.     

An Alternative Synthesis of (2R,6R)-(–)-2,6,10-Trimethylundecyl Bromide,the Key Intermediate in the Synthesis of Natural α-Tocopherol

Crystalline aromatic l-amino acid decarboxylase from Micrococcus percitreus is inactive in the absence of pyridoxal phosphate (PLP). The inactive form of the enzyme shows absorption at 340 nm and contains one mol of PLP per mol of enzyme. Binding of PLP to the inactive form is accompanied by a pronounced increase in absorbance at 415 nm. The amount of PLP that binds to this holoenzyme is 2 mol per mol of enzyme. The inactive half-resolved form, i. e. semiapoenzyme, is obtained again by dialysis of the holoenzyme against phosphate buffer. When the semiapoenzyme is dialyzed against phosphate buffer containing 3,4-dihydroxyphenyl-l-alanine, it loses the absorption at 340 nm with the loss of PLP. This apoenzyme regains the activity and absorption at 340 nm and 415 nm on association with PLP.     

Synthesis of 1-(4-Keto-2, 3-O-isopropylidene-α-L-rhamnopyranosyl) uracil

Abstract

Synthesis of 1-(2, 3, 4-tri-0-acetyl-α-L-rhamnopyranosyl) uracil (3), 1-(α-L-rhamnopyranosyl) uracil (4), 1-(2, 3-0-isopropylidene-α-L-rhamnosyl) uracil (5), and 1-(2, 3-0-isopropylidene-4-keto-α-L-rhamnopyranosyl) uracil (6) are reported. Oxidation of (5) to (6) was effected using pyridinium chlorochromate in presence of molecular sieves.     

Synthesis, characterization and properties of uridine 5′-(α-d-apio-d-furanosyl pyrophosphate)

1. A method was developed for synthesizing UDP-apiose [uridine 5'-(alpha-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5'-(alpha-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5'-(alpha-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [(3)H]UDP-[U-(14)C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the (3)H/(14)C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100 degrees C for 15min; (b) degraded at pH8.0 and 100 degrees C for 3min; (c) used as a substrate in the enzymic synthesis of [(14)C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [(3)H]UDP-[U-(14)C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-(14)C]apiose and phosphate formed on alkaline degradation of UDP-[U-(14)C]apiose was alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-(14)C]apiose and phosphate formed on acid hydrolysis of alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-(14)C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-(14)C]apiose to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80 degrees C, at pH8.0 and 25 degrees C and at pH8.0 and 4 degrees C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-(14)C]-apiose to d-[U-(14)C]apiose and UDP at pH3.0 and 40 degrees C was 4.67min. After 20 days at pH6.2-6.6 and 4 degrees C, 17% of the starting UDP-[U-(14)C]apiose was degraded to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-(14)C]apiose and UDP. After 120 days at pH6.4 and -20 degrees C 2% of the starting UDP-[U-(14)C]apiose was degraded and 4% was hydrolysed.     

Kinetic behaviour of α-galactosidase produced by Absidia griseola: a comparison between free and immobilized forms of the enzyme

In this research the characteristics of free (partially purified) and immobilized (mould pellets of Absidia griseola) -galactosidase have been investigated. Inhibition studies of the enzyme showed that p-nitrophenol and sucrose do not have any inhibitory effect on the enzyme, but that galactose is a competitive inhibitor. In the immobilized form, inhibition was lower than in the free enzyme and the level of inhibition decreased as the temperature increased. The activity and stability of free and immobilized enzyme were investigated with respect to temperature, and the results showed that the optimal temperature range of the free enzyme was 45–50 °C, while the immobilized enzyme had an optimum at 55–60 °C. The optimum pH for the free enzyme was 6.0 and the value was decreased to 5.0 by immobilizing. The experimental effectiveness factors were found to be represented as a single function of the modified Thiele modulus, including parameters such as pellet size, enzyme concentration in the pellets and substrate concentration. Both experimental and theoretical data concerning effectiveness factors are nearly the same.     

Synthesis,characterization, and biological activity of Nα-(N-fluoresceinthiocarbamoyl)-(Asp1, Ile5)-angiotensin II

A fluorescent analog of angiotensin II was synthesized by reacting fluorescein 5′-isothiocyanate with (Asp¹, Ile⁵)-angiotensin II. N^α-(N-Fluoresceinthiocarbamoyl)-(Asp¹, Ile⁵)-angiotensin II was purified by chromatography on DEAE-cellulose and Sephadex G-25. Analysis of the analog by thin-layer chromatography, thin-layer electrophoresis, and reversed-phase high-performance liquid chromatography indicated that the analog was free of angiotensin II and fluorescein 5′-isothiocyanate. N-Terminal sequence analysis demonstrated that fluorescein 5′-isothiocyanate reacted with the N-terminal aspartic acid residue of angiotensin II. N^α-(N-Fluoresceinthiocarbamoyl)-(Asp¹, Ile⁵)-angiotensin II has an absorption maximum at 492 nm, and the value of the molar extinction coefficient, ?, is 7.7 × 10⁴m^?1 cm^?1. The fluorescence emission maximum occurs at 520 nm. Infusion of the analog (0.69 μg/min/kg body wt) directly into the renal artery of an anesthetized rat reduced the blood flow by 12 to 27% within 2 min. Infusion of angiotensin II (0.48 μg/min/kg body wt) reduced renal arterial blood flow by 35 to 53% within 2 min. Saralasin, a partial agonist and antagonist of angiotensin II, inhibited the biologic effect of the fluorescent analog and angiotensin II by 75 and 70%, respectively. The purity, spectral properties, and in vivo biologic activity of N^α-(N-fluoresceinthiocarbamoyl)-(Asp¹, Ile⁵)-angiotensin II indicate that this analog should facilitate characterization of angiotensin II receptors.     

Practical Synthesis of the Disaccharide Epitope,D-Galactopyranosyl-α-1,3-D- galactopyranose,by using 1,2;5,6-Di-O-cyclohexylidene-α-D-galactofuranose as the Glycosyl Acceptor

D-Galactosyl-α-1,3-D-galactopyranose (1) was chemically prepared in a good yield by coupling phenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-galactopyranoside (5) or 2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl bromide (8) with 1,2:5,6-di-O-cyclohexylidene-α-D-galactofuranose (3) with subsequent de-O-benzylation and de-O-cyclohexylidenation of the resulting protected α-1,3-disaccharide.     

Synthesis and Antileishmanial Activity of 3-(α-Azolylbenzyl)indoles

The goal of the present study was to evaluate several azolyl-substituted indoles as new antileishmanial agents. Ten 3- (α -azolylbenzyl)indoles have been synthesized using Friedel-Crafts acylation as a key-step. All the target compounds were found to display high levels of activity when tested against Leishmania mexicana promastigotes in vitro. The most active compounds, showing an IC 50 <1 μM, were 5-bromo-1-ethyl-3-[(2,4-dichlorophenyl)(1 H -imidazol-1-yl)methyl]-1 H -indole 15 and its triazole analogue 17. Four representative compounds 15, 17, 22 and, 23 were also tested against intracellular amastigotes of L. mexicana using ketoconazole and meglumine antimoniate as reference compounds, the results of which are discussed.