Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Penicillin acylase of E. coli NCIM 2400 has been purified to homogeneity using a combination of hydrophobic interaction chromatography and DEAE-cellulose treatment. A variety of substituted matrices were synthesized using D- or DL-phenylglycine, norleucine, ampicillin, or amoxycillin as ligands, all of which retained penicillin acylase at high concentrations of ammonium sulfate or sodium sulfate. The enzyme could be eluted nonbiospecifically by buffer of lower ionic strength with over 95% recovery of the activity. Ammonium chloride, ammonium nitrate, sodium chloride, sodium nitrate, and potassium chloride were ineffective in either adsorption or elution of the enzyme on these columns. Further purification of this partially pure enzyme with DEAE-cellulose at pH 7.0-7.2 yielded an enzyme preparation of very high purity according to electrophoretic and ultracentrifugal analyses, its specific activity being as high as 37 U/mg protein. The purified enzyme has a molecular weight of 67,000 a sedimentation coefficient of 4.0S, and resolves into two forms upon isoelectric focusing. Overall recoveries ranged between 75 and 85%. Ease of operation, high recoveries, high purity of the enzyme and prolonged reuse of the conjugates make the process economically feasible and possibly of great commercial importance.
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PMID:Novel approaches to the purification of penicillin acylase. 639 70

Acid catheptic activity was measured in crude extracts of muscle, liver, heart, spleen and gonads from the fishes Mujil auratus, Sparus aurata and Lightonatus mormyrus. The spleen was the organ which showed the highest activity. A comparative study of the seven most commonly used extraction methods was made. Some were modified to account for the characteristics of the fish organs and the activity extracted from them. The Siebert method resulted as the best extraction method only if 1 mM EDTA was present in the medium. The activity from Mujil auratus muscle was strongly inhibited by iodoacetate, N-ethylmaleimide, p-hydroxy mercuribenzoate, and diazo-acetyl-DL-norleucine methyl ester. The results indicated the presence of a carboxyl-proteinase and a thiol-proteinase. According to inhibition studies, the levels of proteinase and amidase activities shown by different organs of Mujil auratus were re-examined. The spleen extract showed the maximum activity for both cathepsins, but muscle extract accounted for more than 95% of total catheptic activity.
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PMID:Acid proteinase activity in fish--I. Comparative study of extraction of cathepsins B and D from Mujil auratus. 674 25

Aminoacylase (EC 3.5.1.14) from Aspergillus oryzae was purified from a commercially available crude material by heat treatment, precipitation by polyethyleneimine and ammoniumsulfate, gel chromatography and preparative disc-gel-electrophoresis. The purified product was homogenous as judged by polyacrylamide gel electrophoresis. SDS-gel electrophoresis, polyacrylamide-gel-gradient electrohoresis, gel chromatography and amino acid analysis demonstrated the enzyme to be composed of two subunits with Mr of 36 600. The kinetic properties of the enzyme were studied with chloracetyl derivatives of alanine, phenylalanine, methionine, leucine, norleucine and tryptophan. The pH optimum of the acylase activity with chloroacetyl-alanine as substrate is at pH 8.5. Acyl derivatives of hydrophobic amino acids are preferred substrates. The enzyme has no dipeptidase activity. Aminoacylase is not inhibited by SH-blocking agents and no SH-groups could be detected with Ellman's reagent in the native and denatured enzyme. The enzyme activity is insensitive to phenylmethylsulfonyl fluoride and N-alpha-p-tosyl-L-lysine chloromethyl ketone. The microbial acylase is zince metallo enzyme. Mental chelating agents are strong inhibitors; it is further inhibited by Cd2+, Mn2+ and activated by Co2+. The properties of pig kidney and Aspergillus acylase are compared.
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PMID:Aminoacylase from Aspergillus oryzae. Comparison with the pig kidney enzyme. 677 95

Biocatalytic processes were used to prepare chiral intermediates for pharmaceuticals. These include the following processes. Enzymatic synthesis of [4S-(4a,7a,10ab)]1-octahydro-5-oxo-4-[[(phenylmethoxy) carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01), a key chiral intermediate for synthesis of a new vasopeptidase inhibitor. Enzymatic oxidation of the epsilon-amino group of lysine in dipeptide dimer N2-[N[[(phenylmethoxy)carbonyl] L-homocysteinyl] L-lysine)1,1-disulfide (BMS-201391-01) to produce BMS-199541-01 using a novel L-lysine epsilon-aminotransferase from S. paucimobilis SC16113 was demonstrated. This enzyme was overexpressed in E. coli, and a process was developed using recombinant enzyme. The aminotransferase reaction required alpha-ketoglutarate as the amine acceptor. Glutamate formed during this reaction was recycled back to alpha-ketoglutarate by glutamate oxidase from S. noursei SC6007. Synthesis and enzymatic conversion of 2-keto-6-hydroxyhexanoic acid 5 to L-6-hydroxy norleucine 4 was demonstrated by reductive amination using beef liver glutamate dehydrogenase. To avoid the lengthy chemical synthesis of ketoacid 5, a second route was developed to prepare the ketoacid by treatment of racemic 6-hydroxy norleucine (readily available from hydrolysis of 5-(4-hydroxybutyl) hydantoin, 6) with D-amino acid oxidase from porcine kidney or T. variabilis followed by reductive amination to convert the mixture to L-6-hydroxynorleucine in 98% yield and 99% enantiomeric excess. Enzymatic synthesis of (S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (allysine ethylene acetal, 7), one of three building blocks used for synthesis of a vasopeptidase inhibitor, was demonstrated using phenylalanine dehydrogenase from T. intermedius. The reaction requires ammonia and NADH. NAD produced during the reaction was recycled to NADH by oxidation of formate to CO2 using formate dehydrogenase. Efficient synthesis of chiral intermediates required for total chemical synthesis of a beta 3 receptor agonist was demonstrated. These include: (a) microbial reduction of 4-benzyloxy-3-methanesulfonylamino-2'-bromoacetophenone 9 to corresponding (R)-alcohol 10 by S. paucimobilis SC16113, (b) enzymatic resolution of racemic alpha-methyl phenylalanine amide 11 and alpha-methyl-4-hydroxyphenylalanine amide 13 by amidase from M. neoaurum ATCC 25795 to prepare corresponding (S)-amino acids 12 and 14, and (c) asymmetric hydrolysis of methyl-(4-methoxyphenyl)-propanedioic acid ethyl diester 15 to corresponding (S)-monoester 16 by pig liver esterase. (S)[1-(acetoxyl)-4-(3-phenyl)butyl]phosphonic acid diethyl ester 21, a key chiral intermediate required for total chemical synthesis of BMS-188494 (an anticholesterol drug) was prepared by stereoselective acetylation of racemic [1-(hydroxy)-4-(3-phenyl)butyl]phosphonic acid diethyl ester 22 using G. candidum lipase. Lipase-catalyzed stereoselective acetylation of racemic 7-[N,N'-bis-(benzyloxy-carbonyl)N-(guanidinoheptanoyl)]-alpha-hydroxy-glycine 24 to corresponding S-(-)-acetate 25 was demonstrated. S-(-)-acetate 25 is a key intermediate for total chemical synthesis of (-)-15-deoxyspergualin 23, an immunosuppressive agent and antitumor antibiotic. Stereoselective microbial reduction of (1S)[3-chloro-2-oxo-1-(phenyl-methyl)propyl] carbamic acid, 1,1-dimethyl-ethyl ester 26 to corresponding chiral alcohol 27a (a key chiral intermediate for HIV protease inhibitors) was also demonstrated. Stereospecific enzymatic hydrolysis of racemic epoxide RS-1-[2',3'-dihydro benzo[b]furan-4'-yl]-1,2-oxirane 29 the corresponding R-diol 30 and unreacted chiral S-epoxide 28 was demonstrated using R. glutinis and A. niger. Dynamic resolution of racemic diol RS-1-[2',3'-dihydrobenzo[b]furan-4'-yl]-ethane-1,2-diol 32 to corresponding S-diol S-1-[2',3'-dihydrobenzo[b]furan-4'-yl]-ethane-1,2-diol 31 was demonstrated using C. boidinii and P. methanolica. Chiral (S)-epoxide 28 and (S)-diol 31 are key intermediates for a new prospective circadian modulator drug. Enzymatic resolution of racemic 2-pentanol and 2-heptanol by lipase B from Candida antarctica was demonstrated. S-(+)-2-pentanol is a key chiral intermediate required for synthesis of anti-Alzheimer's drugs.
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PMID:Microbial/enzymatic synthesis of chiral drug intermediates. 1287 94