Pore diffusion model for a two-substrate enzymatic reaction: Application to galactose oxidase immobilized on porous glass particles

An analysis of the pore diffusion model involving a two-substrate enzymatic reaction is presented. The resulting equations have been applied to the case of galactose oxidase catalyzed oxidation of galactose when the enzyme is immobilized on porous glass particles. The physical constants of the system were obtained by theoretical predictions and the enzyme concentration in the porous medium was derived from the experimental results. The calculations were performed with the assumption that the kinetic parameters of the enzyme remain unchanged upon immobilization. The theoretically calculated effectiveness factors were compared with the experimental effectiveness factors determined from the batch kinetic experiments and were found to be in agreement. The results are presented as effectiveness factor plots graphed as functions of bulk galactose and oxygen concentrations. The model was extended in order to study the effect of external mass transfer coefficients and pore enzyme concentrations on the effectiveness factors.     

Radioimmunoassay for pig angiotensin I converting enzyme: a comparison of immunologic with enzymatic activity.

Angiotensin I converting enzyme in body fluids and extracts of various pig tissues was measured by radioimmunoassay and by enzymic assay. Based on the ratios of enzymic to immunologic activity, the extracts could be separated into two groups. One group, with ratios around 4 U/mg, included urine and extracts from the adrenal, choroid plexus, epididymis, gall bladder, heart, liver, retina, spleen, and testis. The other group, with ratios around 12 U/mg, contained serum and extracts from lung and kidney. Explanations are offered for why one group had a lower enzymic to immunologic ratio than the other.     

Leukotriene A: stereochemistry and enzymatic conversion to leukotriene B

Leukotriene A was assigned the structure 5(S)-$\text{trans}$-5,6-oxido-7,9-$\text{trans}\text{-11,14-}\text{cis}$-eicosatetraenoic acid by the enzymatic conversion of a synthetic product of known stereochemistry into the naturally occurring isomer of 5(S),12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid in human polymorphonuclear leukocytes.     

New assays for the enzymatic conversion of cholesterol to pregnenolone

Cholesterol side chain cleavage is determined by means of separation of (26-¹⁴C)-cholesterol and its radioactively labeled side chain (1-¹⁴C)-isocaproic acid. Alumina minicolumn assay (AMCA): adsorption of cholesterol from an aqueous phase by aluminium oxide, while isocaproic acid can percolate through the column. In modification of a previously described technique (1), cholesterol is quantitatively eluted by ethanol. Filter assay (FA): retention of cholesterol by a membrane filter (pore size ≦ 0.1 um) while isocaproic acid can pass the filter. Two-phase scintillation assay (TPSA): pH-dependent partition of isocaproic acid between an organic scintillation mixture and an aqueous phase. The TPSA can be applied for all enzymatic reactions in which the polarity of the radioactive residue which is split off depends on pH values or when the total charge of a polar molecule is changed to an apolar state by cleaving one non-radioactive group (e.g. steroid sulfates) and vice versa.The criteria of reliability of the test systems are described Bovine adrenal mitochondria were incubated and the side chain cleavage of (26-¹⁴C)-cholesterol was studied by the new test systems and compared to the conversion rates of (¹⁴C)-cholesterol to its metabolites as determined by thin layer chromatography. A good agreement of all tests was found.     

ETH 6022: an artificial enzyme? A comparison between enzymatic and chemical recognition for sensing ethanol

Routinely the quantification of ethanol in clinical chemistry and food chemistry is performed by enzymatic methods in addition to chemical methods. Liver and yeast alcohol:NAD⁺ oxidoreductase (ADH, EC. 1.1.1.1) and alcohol oxidase AOD (EC 1.1.3.13) are mainly applied for selective evaluations by different kinds of bioassay. Nevertheless, drawbacks of ethanol sensing make the evaluation of ethanol concentrations in alcoholic beverages by an optical sensor, in a concentration range between 0·7% and 40%, most attractive. The chemical recognition process is based on a lipophilic amide of trifluoroacetylaniline (ETH 6022) as a ligand for ethanol implanted in a solvent polymeric membrane. The interaction leads to a hypsochromic shift of the absorption band in the ultraviolet region (305 nm). The chemical reaction in the apolar environment of the optode membrane is reversible. The principle of this recognition process is compared to the enzymatic reaction of ADH. In both cases a highly electrophilic centre participates in the interaction between the hydroxy group of the alcohol and the ligand. The selectivity behaviour of the artificial recognition process may be tailored by the substituents of the aromatic trifluoroacetyl compound. The response characteristics of the optochemical sensors are compared.     

Performance evaluation of a silk protein-based matrix for the enzymatic conversion of tyrosine to L-DOPA

L-DOPA (3,4-dihydroxyphenyl-L-alanine), one of the most important intermediates in the melanin biosynthesis pathway, is used for the treatment of Parkinson's disease. With a view of developing a cheaper and more effective method for the bioconversion of tyrosine to L-DOPA, the potential and performance of a novel fibrous matrix prepared from Bombyx mori silk protein fibroin were evaluated for the immobilization of tyrosinase. Cross-linkage between fibroin and tyrosinase using glutaraldehyde was evident from Fourier transform infra red spectroscopy. Maximum product formation occurred when 1000 U enzyme was immobilized on 20 mg fibroin. The optimum conditions for maximal L-DOPA production using immobilized tyrosinase were 40 degrees C and pH 5.5, conditions that caused a 50% loss of free enzyme activity. Immobilized tyrosinase also showed to have a higher degree of stability during storage and it retained 80% of its original activity after repeated reuses. The efficiency of this immobilized tyrosinase system to produce L-DOPA was high, as evident from a high effectiveness factor, between 0.7 and 0.8, thereby making this method feasible for the large-scale production of L-DOPA.     

Spectral and enzymatic properties of human recombinant myeloperoxidase: comparison with the mature enzyme.

Human recombinant myeloperoxidase (recMPO), purified from an engineered Chinese hamster ovary (CHO) cell line, has been characterized and compared to the mature enzyme isolated from polymorphonuclear leukocytes. Both molecules appear essentially similar in physicochemical enzymatic terms according to the following observations. 1. The unprocessed recombinant protein displays the characteristic light absorption spectra of ferric mature MPO and exhibits its typical spectral changes in the presence of dithionite or hydrogen peroxide. 2. The addition of 14C-labeled 5-aminolevulinic acid, a heme precursor, to the culture medium of recombinant CHO cells yields labeled recMPO, indicating the presence of a heme-like structure in the molecule. 3. Like mature MPO, recMPO has a peroxidatic activity and catalyzes the oxidation of chloride ions in the presence of hydrogen peroxide, producing hypochlorous acid as measured by the monochlorodimedon assay. For both enzymes, the chlorinating activity optimally occurs around pH 5.0 at about 100 microM of hydrogen peroxide and is strongly inhibited by methimazole. 4. Diethylpyrocarbonate significantly reduces the enzymatic activity of both molecules, suggesting that histidine residues may be of prime importance in the active site of the enzymes. 5. According to infrared spectroscopy data, both enzymes present a very similar secondary structure organization. In conclusion, the data suggest that the processing of the precursor enzyme (recMPO) into the mature form occurs without major structural and functional consequences.     

Leukotriene A4: preparation and enzymatic conversion in a cell-free system to leukotriene B4

Treatment of leukotriene A4 (LTA4) methyl ester with sodium hydroxide in aqueous methanol at 4 degrees C afforded LTA4, the presence of which was inferred from the UV spectrum of the compound, its rate of reaction with water, and the identity of the hydration products obtained. The half-life of LTA4 in water (pH 7.4, room temperature) was increased from 14 to 500 s by 1 mg/ml of bovine serum albumin. This stabilized (chiral) LTA4 was converted to LTB4 by an epoxide hydrolase activity in the 100,000 x g supernatant fraction from sonified rat basophilic leukemia cells. Neither the ester of LTA4 nor the biologically incorrect enantiomer of LTA4 was metabolized to LTB4 under these conditions.     

Assay for the enzymatic conversion of indoleacetic acid to 3-methylindole in a ruminal Lactobacillus species

An assay to measure the rate of enzymatic formation of 3-methylindole (3MI) from indoleacetic acid (IAA) in Lactobacillus sp. strain 11201 was developed. The reaction mixture contained 50 micrograms of microbial protein per ml (range, 25 to 100 mg/ml), essential low-molecular-weight reaction ingredients, and radiolabeled IAA as substrate (range, 0 to 2 mM IAA). The reaction was anaerobic for 25 min at 39 degrees C. The apparent Michaelis-Menten constants were: Km, 0.14 mM IAA; and Vmax, 64 nmol 3MI.mg-1.min-1. The inhibitors avidin, aminopterin, and EDTA had no effect on the 3MI-forming enzyme. Dithionite stimulated the 3MI-forming enzyme. The product of the reaction, 3MI, acted as a noncompetitive inhibitor of the enzyme. Enzyme activity was associated with the cell wall fraction after sonication; treatment with the French press; or treatment with detergents, proteolytic enzymes, and EDTA.     

Carnitine biosynthesis in Neurospora crassa: enzymatic conversion of lysine to epsilon-N-trimethyllysine.

The enzymatic conversion of L-lysine, epsilon-N-trimethyl-L-lysine the first series of reactions in the biosynthesis of carnitine in Neurospora crassa, proceeds via sequential methylation of free L-lysine, epsilon-N-methyl-L-lysine, and epsilon -N-dimethyl-L-lysine. The latter two compounds have been shown to be intermediates in the biosynthesis of carnitine by radioisotope dilution and incorporation experiments in growing cultures of N. crassa 33933 (lys-) and 38706 (met-). Methionine but not choline, has been recognized as an effective methyl donor in vivo. Inclusion of choline in the growth medium of strain 33933 does, however, enhance incorporation of the methyl groups of L-[methyl-3H]methionine into carnitine in an apparent "sparing" effect on methionine synthesis. Studies in cell-free extracts of the lysine auxotroph strain 33933 of N. crassa have established that lysine and epsilon-N-methyl and epsilon-N-dimethyllysine are enzymatically methylated, with S-adenosyl-L-methionine as the methyl group donor. The enzyme system appears to have no essential cofactors. Lysine does not induce synthesis of the enzyme system in the wild-type strain 262, whereas both carnitine and epsilon-N-trimethyllysine repress its synthesis in strain 33933.     

Assay for the enzymatic conversion of indoleacetic acid to 3-methylindole in a ruminal Lactobacillus species.

An assay to measure the rate of enzymatic formation of 3-methylindole (3MI) from indoleacetic acid (IAA) in Lactobacillus sp. strain 11201 was developed. The reaction mixture contained 50 micrograms of microbial protein per ml (range, 25 to 100 mg/ml), essential low-molecular-weight reaction ingredients, and radiolabeled IAA as substrate (range, 0 to 2 mM IAA). The reaction was anaerobic for 25 min at 39 degrees C. The apparent Michaelis-Menten constants were: Km, 0.14 mM IAA; and Vmax, 64 nmol 3MI.mg-1.min-1. The inhibitors avidin, aminopterin, and EDTA had no effect on the 3MI-forming enzyme. Dithionite stimulated the 3MI-forming enzyme. The product of the reaction, 3MI, acted as a noncompetitive inhibitor of the enzyme. Enzyme activity was associated with the cell wall fraction after sonication; treatment with the French press; or treatment with detergents, proteolytic enzymes, and EDTA.     

Biocatalytic preparation of a chiral synthon for a vasopeptidase inhibitor: enzymatic conversion of N(2)-

[4S-(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01) is a key chiral intermediate for the synthesis of Omapatrilat (BMS-186716), a new vasopeptidease inhibitor under development. By using a selective enrichment culture technique we have isolated a strain of Sphingomonas paucimobilis SC 16113, which contains a novel L-lysine epsilon-aminotransferase. This enzyme catalyzed the oxidation of the epsilon-amino group of lysine in the dipeptide dimer N(2)-[N[phenyl-methoxy)-carbonyl] L-homocysteinyl] L-lysine)1,1-disulphide (BMS-201391-01) to produce BMS-199541-01. The aminotransferase reaction required alpha-ketoglutarate as the amino acceptor. Glutamate formed during this reaction was recycled back to alpha-ketoglutarate by glutamate oxidase from Streptomyces noursei SC 6007. Fermentation processes were developed for growth of S. paucimobilis SC 16113 and S. noursei SC 6007 for the production of L-lysine epsilon-amino transferase and glutamate oxidase, respectively. L-lysine epsilon-aminotransferase was purified to homogeneity and N-terminal and internal peptides sequences of the purified protein were determined. The mol wt of L-lysine epsilon-aminotransferase is 81 000 Da and subunit size is 40 000 Da. L-lysine epsilon-aminotransferase gene (lat gene) from S. paucimobilis SC 16113 was cloned and overexpressed in Escherichia coli. Glutamate oxidase was purified to homogeneity from S. noursei SC 6003. The mol wt of glutamate oxidase is 125 000 Da and subunit size is 60 000 Da. The glutamate oxiadase gene from S. noursei SC 6003 was cloned and expressed in Streptomyces lividans. The biotransformation process was developed for the conversion of BMS-201391-01 to BMS-199541-01 by using L-lysine epsilon-aminotransferase expressed in E. coli. In the biotransformation process, for conversion of BMS-201391-01 (CBZ protecting group) to BMS-199541-01, a reaction yield of 65-70 M% was obtained depending upon reaction conditions used in the process. Phenylacetyl or phenoxyacetyl protected analogues of BMS-201391-01 also served as substrates for L-lysine epsilon-aminotransferase giving reaction yields of 70 M% for the corresponding BMS-199541-01 analogs. Two other dipeptides N-[N[(phenylmethoxy)carbonyl]-L-methionyl]-L-lysine (BMS-203528) and N,2-[S-acetyl-N-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine (BMS-204556) were also substrates for L-lysine epsilon-aminotransferase. N-alpha-protected (CBZ or BOC)-L-lysine were also oxidized by L-lysine epsilon-aminotransferase.     

Demonstration of a cyclic pyrophosphate intermediate in the enzymatic conversion of neryl pyrophosphate to borneol

A soluble enzyme preparation obtained from young sage (Salvia officinalis) leaves catalyzes the conversion of neryl pyrophosphate to (+)-borneol and the oxidation of (+)-borneol to (+)-camphor. Attempts to purify the borneol synthetase activity by gel permeation column chromatography resulted in the apparent loss of catalytic capability; however, subsequent recombination of column fractions demonstrated that two separable enzymatic activities were required for the conversion of neryl pyrophosphate to borneol. Several lines of evidence indicated that a water-soluble, dialyzable intermediate was involved in this transformation. The intermediate was isolated and subsequently identified as bornyl pyrophosphate by direct chromatographic analysis and by the preparation of derivatives and chromatographic analysis of both the hydrogenolysis (LiAlH₄) and enzymatic hydrolysis products of bornyl pyrophosphate. The results presented indicate that borneol is derived by cyclization of neryl pyrophosphate to bornyl pyrophosphate, followed by hydrolysis. This is the first demonstration of a cyclic pyrophosphorylated intermediate in the biosynthesis of bicyclic monoterpenes.     

Specificity of enzymatic in vitro glycosylation by PNGase F: a comparison of enzymatic and non-enzymatic glycosylation

Enzymatic in vitro glycosylation is possible using a reverse reaction of peptide-N-glycosidase F (PNGase F), and non-enzymatic in vitro glycosylation occurs when the sugar residue is one or two units long. To identify the differences between enzymatic and non-enzymatic glycosylation, glycosylation sites were analyzed by the acid hydrolysis of glycopeptides followed by MALDI-TOF mass spectrometric analysis. Pentapeptide (Arg-Lys-Asp-Val-Tyr) and octapeptide (Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr) were used in this study, and the sequence of the octapeptide was appropriately chosen to investigate the specificity of enzymatic glycosylation by considering the characteristics of PNGase F and non-enzymatic glycosylation. N,N′-Diacetylchitobiose was aminated prior to the glycosylation reaction at an amination extent of 60%. The glycosylation site was very specific to the aspartate residue in the enzymatic reaction, while non-enzymatic glycosylation occurred at arginine or lysine residues. PNGases F can be effectively used for the glycosylation of the non-glycosylated recombinant proteins produced in prokaryotic cells.     

Extending the limits to enzymatic catalysis: diffusion of ribonuclease A in one dimension

Bovine pancreatic ribonuclease A (RNase A) is a distributive endoribonuclease that catalyzes the cleavage of the P-O5' bond of RNA on the 3' side of pyrimidine residues. Here, RNase A is shown to cleave the P-O5' bond of a pyrimidine ribonucleotide faster when the substrate is embedded within a longer tract of poly(adenylic acid) [poly(A)] or poly(deoxyadenylic acid) [poly(dA)]. These data indicate that a ribonuclease can diffuse in one dimension along a single-stranded nucleic acid. This facilitated diffusion is mediated by Coulombic interactions, as the extent is diminished by the addition of NaCl. RNase A is more effective at cleaving a pyrimidine ribonucleotide embedded within a poly(dA) tract than within a poly(deoxycytidylic acid) [poly(dC)] tract. T45G RNase A, which catalyzes the processive cleavage of poly(A) but the distributive cleavage of poly(cytidylic acid) [poly(C)], has the same preference. Apparently, processive catalysis by the T45G enzyme arises from the expanded substrate specificity of the variant superimposed upon an intrinsic ability to diffuse along poly(A). Homologous ribonucleases with cytotoxic activity may rely on facilitated diffusion along poly(A) tails for efficient degradation of the essential information encoded by cellular mRNA.     

RNA is required for enzymatic conversion of glutamate to delta-aminolevulinate by extracts of Chlorella vulgaris

Formation of delta-aminolevulinic acid (ALA) from glutamete catalyzed by a soluble extract from the unicellular green alga, Chlorella vulgaris, was abolished after incubation of the cell extract with bovine pancreatic ribonuclease A (RNase). Cell extract was prepared for the ALA formation assay by high-speed centrifugation and gel-filtration through Sephadex G-25 to remove insoluble and endogenous low-molecular-weight components. RNA hydrolysis products did not affect ALA formation, and RNase did not affect the ability of ATP and NADPH to serve as reaction substrates, indicating that the effect of RNase cannot be attributed to degradation of reaction substrates or transformation of a substrate or cofactor into an inhibitor. The effect of RNase was blocked by prior addition of placental RNase inhibitor (RNasin) to the cell extract, but RNasin did not reverse the effect of prior incubation of the cell extract with RNase, indicating that RNase does not act by degrading a component generated during the ALA-forming reaction, but instead degrades an essential component already present in active cell extract at the time the ALA-forming reaction is initiated. After inactivation of the cell extract by incubation with RNase, followed by administration of RNasin to block further RNase action, ALA-forming activity could be restored to a higher level than originally present by addition of a C. vulgaris tRNA-containing fraction isolated from an active ALA-forming preparation by phenol extraction and DEAE-cellulose chromatography. Baker's yeast tRNA, wheat germ tRNA, Escherichia coli tRNA, and E. coli tRNAglu type II were unable to reconstitute ALA-forming activity in RNase-treated cell extract, even though the cell extract was capable of catalyzing the charging of some of these RNAs with glutamate.     

Acid-mechanical,alkaline and enzymatic treatment of agricultural wastes to obtain substrate for microbiological conversion

The present paper deals with almost wasteless technologies of pretreatment and obtaining of sugars and biomass from straw and potato tops. Several variants ensure a 1 t of straw per hour productivity using original auger reactors 290 and 150 mm in diameter. The authors have studied the submerged and solid state cultivation of various microorganisms applying the obtained substrates.