Enzymatic synthesis of p-nitrophenyl 35-O-β-N-acetylglucosaminyl|-α-maltopentaoside by lysozyme; a novel substrate for human amylase assay

Transglycosylation from di-N-acetylchitobiose to the 3-position at the nonreducing end glucosyl group of p-nitrophenyl α-maltopentaoside was regioselectively induced through the use of hen egg-white lysozome. The enzyme formed p-nitrophenyl 3⁵-O-β-N-acetylglucosaminyl-α-maltopentaoside (5% of the enzyme-catalyzed net decreased of p-nitrophenyl α-maltopentaoside) from di-N-acetylchitobiose as a donor and p-nitrophenyl α-maltopentaoside as an acceptor. The rate of the transglycosylation depended on the concentration of substrate, the temperature and the pH. The hydrolytic actions of human pancreatic and salivary α-amylase on this derivative were examined. The maltopentaoside derivative was shown to be useful as a substrate for α-amylase assay through a coupled reaction involving α-D-glucosidase and glucoamylase.     

The assay of the bromelains using N alpha-CBZ-L-lysine p-nitrophenyl ester and N-CBZ-glycine p-nitrophenyl ester as substrates

Two new esterolytic assays of the pineapple stem bromelains are described. They use as substrates the p-nitrophenyl esters of N^α-CBZ-l-lysine (CLN) and N-CBZ-glycine (CGN). The activity is monitored by the direct spectrophotometric measurement of the enzyme-catalyzed hydrolysis of these esters at 340 nm. The bromelains are rapidly activated with 1 mm l-cysteine at pH 4.6 for the CLN assay and pH 6.1 for the CGN assay. EDTA has no measurable effect. The sensitivities of the assays approach 10 μg/ml in a reaction time of 3 min.     

Enantiomer-selective solvolysis catalysed by a histidine-containing cyclic dipeptide carrying chiral side chain

Tripeptides with cyclic dipeptide backbones, cyclo[l-Glu(l-Leu-O Bzl)-t-His] and cyclo[l-Glu(l-Leu-OH)-Ir His, and the corresponding tripeptides with linear backbones, Me₃COCO-l-Glu(l-Leu-OBzl)-l-His-OMe and Me₃COCO-l-Glu(l-Leu-OH)-l-His-OMe, were synthesized and used as catalysts for the hydrolysis of carboxylic acid active esters of various types. The experimental results are summarized as follows. (I) In the hydrolysis of a neutral and hydrophobic substate, p-nitrophenyl laurate, in 20% dioxane/H₂O mixture of pH 7.8, a hydrophobic and flexible peptide, Me₃COCO-l-Glu(l-Leu-OH)-l-His-OMe, was more reactive than imidazole. On the other hand, cyclo[l-Glu(l-Leu-OBzl)-l-His] and cyclo[l-Leu-OH)-l-His], which have rigid backbone chain and fixed sidechain conformation, were not particularly reactive. (2) in the solcolysis of a positively charged substrate, p-nitrophenyl glycinate hydrochloride, in 42% i-PrOH/H₂O mixture at pH 6.95, a positively charged substrate, p-nitrophenyl glycinate hydrochloride, in 42% i-PrOH/H₂O mixture at pH 6.95, a negatively charged and flexible peptide, Me₃COCO-l-Glu(l-Leu-OH)-l-His-OMe, was more reactive than imidazole. However, cyclo [l-Glu(l-Leu-OH)-l-His] was not particularly reactive in the same reaction. In the hydrolysis of p-nitrophenyl glycinate hydrochloride in aqueous solution at pH 7.8 a hydrophobic and rigid peptide, cyclo[(l-Glu(l-Leu-OBzl)-l-His], was more reactive than imidazole. However, in the hydrolysis of p-nitrophenyl CO-AMINODODECANOATE hydrochloride, which has a positive charge and a rective site separated by a long hydrophobic chain, peptide catalysts did not show efficient catalysis. (3) In the hydrolysis of a positively charged, hydrophobic and chiral substrate, p-nitrophenyl leucinate hydrochloride, in aqueous solution at pH 6.95, the d-enantiomer was hydrolysed more quickly that the t-enantiomer with cyclo[l-Glu(l-Leu-OBzl)l-His] or cyclo[t-Glu(l-Leu-OH)-l-His] as catalyst. On the other hand, the tripeptides with linear backbone did not effect an enantiomer-selective catalysis. The solvolytic reaction catalysed by the tripeptides with cyclic dipeptide backbone in 42% i-PrOH/water mixture was also enantiomer-selective.     

The p-nitrophenyl phosphatase activity of muscle carbonic anhydrase

Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO₂ hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a K_m of 2.8 mm. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate (K_i = 1.22 × 10^?3M), arsenate (K_i = 1.17 × 10^?3M), and molybdate (K_i = 1.34 × 10^?7M), with these inhibitors having no effect on the CO₂ hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO₂ hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO₂ hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites.     

Organophosphorus compounds as active site-directed inhibitors of elastase

Four ethyl $\text{p}$-nitrophenyl alkylphosphonates were studied for the inhibition of elastase. A pH-dependence study using the assay substrate BOC-Ala-ONp or the phosphonate inhibitors showed the participation of an ionizing group with an apparent pK_a of 6.9 and a decrease of reaction or inhibition at higher pH. Out of the four compounds investigated ethyl $\text{p}$-nitrophenyl pentylphosphonate was found to be the best inhibitor of elastase as judged from the value of $\text{k}{}_2\text{K}{}_{\mathrm{<mtext>I</mtext>}}$. This value, which is the measure of inhibitory capacity, is the highest reported so far for the inhibition of elastase.     

Human liver acid phosphatases: Purification and properties of a low-molecular-weight isoenzyme

A low-molecular-weight human liver acid phosphatase was purified 2580-fold to homogenity by a procedure involving ammonium sulfate fractionation, acid treatment, and SP-Sephadex ion-exchange chromatography with ion-affinity elution. The purified enzyme contains a single polypeptide chain and has a molecular weight of 14,400 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of this enzyme (E) is reported. A pH dependence study using p-nitrophenyl phosphate as a substrate (S) revealed the effect of substrate ionization (pK_a 5.2) and the participation of a group in the ES complex having a pK_a value of 7.8. The enzyme is readily inactivated by sulfhydryl reagents such as heavy metal ions. Alkylation of the enzyme with iodoacetic acid and iodoacetamide causes complete inactivation of the enzyme and this inactivation is prevented by the presence of phosphate ion. The enzyme is also inactivated by treatment with diethyl pyrocarbonate; protection against this reagent is afforded by phosphate ion. The substrate specificity of this enzyme is unusual for an acid phosphatase. Of the many alkyl and aryl phosphomonoesters tested, the only possibly physiological substrate hydrolyzed by this enzyme was flavin mononucleotide, which exhibits a V which is 3-fold larger at pH 5.0 and 6-fold larger at pH 7.0 than that for p-nitrophenyl phosphate. However, the enzyme also catalyzes the hydrolysis of acetyl phosphate at pH 5.0 with a velocity eight times larger than that reported for an acyl phosphatase from human erythrocytes.     

Specific spectrophotometric assays for cathepsin B1.

Cathepsin B₁ from bovine spleen was partially purified by acetone fractionation and by chromatography on Sephadex G-150 and DEAE Sephadex A-50. The enzyme was shown to catalyze the hydrolysis of p-nitrophenyl benzyloxycarbonylglycinate and p-nitrophenyl α-N-benzyloxycarbonyl-l-lysinate. Under the assay conditions, cathepsin B₁ is the major enzyme present in bovine spleen homogenates hydrolyzing these substrates. The kinetic parameters for the hydrolysis of p-nitrophenyl benzyloxycarbonylglycinate and p-nitrophenyl α-N-benzyloxycarbonyl-l-lysinate were measured and compared with those obtained for other cathepsin B₁ substrates. These results form the basis of an improved spectrophotometric assay for this enzyme in which the liberation of p-nitrophenol from either the N-benzyloxycarbonyl glycine or lysine p-nitrophenyl ester is monitored continuously at 326 nm.     

Enzymatic properties,crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans

A high-isoelectric-point (pI), alkaline endo-1,4-β-glucanase (Egl-257) of Bacillus circulans KSM-N257 was purified to homogeneity and crystallized. The purified enzyme hydrolyzed carboxymethyl cellulose (CMC) with optima of pH 8.5 and 55 °C. The molecular mass was 43 kDa, and the pI was pH 9.3. The structural gene contained a single open reading frame of 1221 bp, corresponding to 407 amino acids (aa), including a 30-aa signal peptide (377 aa and 41,680 Da for the mature enzyme). Egl-257 hydrolyzed lichenan and showed 76.3% aa identity to a lichenase from B. circulans WL-12 belonging to glycosyl hydrolase family 8 but did not hydrolyze laminarin, curdran, and xylan at all. This indicates that Egl-257 is a true endo-1,4-β-glucanase. However, this enzyme was not active on p-nitrophenyl β-d-cellotrioside and p-nitrophenyl β-d-cellotetraoside. It was crystallized by the hanging-drop vapor-diffusion method with phosphate plus CdCl₂ as precipitant. Pyramid-like crystals were formed, and they diffracted X-rays beyond 2.2 Å resolution. It belongs to the space group P2₁2₁2₁ with unit cell parameters of a=62.5 Å, b=71.7 Å, and c=88.6 Å.     

Tetrazolium Reduction-Malachite Green Method for Assessing the Viability of Filamentous Bacteria in Activated Sludge

A method was developed to assess the activity of filamentous bacteria in activated sludge. It involves the incubation of activated sludge with 2(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride followed by staining with malachite green. Both cells and 2(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride-formazan crystals can be observed in prepared specimens by using bright-field microscopy. This procedure allowed us to distinguish between inactive and actively metabolizing filaments after chlorine application to control the bulking of activated sludge.     

Leukocidin, Tetraethylammonium Ions, and the Membrane Acyl Phosphatases in Relation to the Leukocyte Potassium Pump

The response of the leukocyte to leukocidin and its relevance to excitable and secreting tissues are described. New data are presented on the leukocyte membrane phosphatases and the action of tetraethylammonium ion (TEA) on the leukocyte. The leukocyte surface membrane lacks a cation-sensitive ATPase but possesses a potassium- and ouabain-sensitive p-nitrophenyl phosphatase. The p-nitrophenyl phosphatase shows peak activity at three pH values and the pH dependence and potassium sensitivity depend on the state of the membranes. In the presence of magnesium, potassium can stimulate over the range pH 6 to 8. The relation of the leukocyte p-nitrophenyl phosphatase to electrolyte control in the leukocyte and to the properties of cation-sensitive phosphatases in other cells suggests that the leukocyte enzyme is a component of an electrogenic potassium pump. Leukocidin stimulates the leukocyte p-nitrophenyl phosphatase under all the conditions studied. The effect is specific and occurs under conditions that induce cytotoxic effects in the cell. It is concluded that the potassium pump is the site of action of leukocidin. TEA prevents the effects of leukocidin by inhibiting the action of leukocidin and not the responses of the cell to injury. TEA does not inhibit the p-nitrophenyl phosphatase nor prevent its stimulation by leukocidin. The enhancement of leukocidin by diisopropylphosphofluoridate (DFP) is briefly described. It is concluded that TEA acts in the opposite way to DFP and blocks the ion pathway activated by leukocidin in the leukocyte potassium pump.     

A spectrophotometric transesterification-based assay for lipases in organic solvent

A new method to evaluate lipase activities in nonaqueous conditions using vinyl ester absorbance at ultraviolet (UV) wavelengths is described. The model reaction is the transesterification between vinyl stearate and pentanol in hexane at 30 °C or in decane at 50 °C. The conversion of vinyl stearate into pentyl stearate is monitored through decreasing UV absorbance at 200 nm. Six commercial lipases were tested with this method, and results were compared with gas chromatography (GC) quantification and a classical spectrophotometric method using p-nitrophenyl palmitate. Results from the new spectrophotometric assay are similar both to results from GC quantification (R² = 0.999) and to results from p-nitrophenyl palmitate (R² = 0.989). The proposed method is able to evaluate both high activity from immobilized lipases such as immobilized Candida antarctica B lipase (3060 ± 350 U g⁻¹) and low activity from crude enzymatic extracts such as Carica papaya dried latex (0.1 ± 0.04 U g⁻¹). The method has also been used to measure kinetic parameters of C. antarctica B lipase for vinyl stearate and the correlation between its synthesis activity and its concentration. The method has also proved to be effective in studying the acyl selectivity of a lipase by comparing its activities with increasing chain lengths of vinyl esters.     

A Cold-Adapted Lipase of an Alaskan Psychrotroph, Pseudomonas sp. Strain B11-1: Gene Cloning and Enzyme Purification and Characterization

A psychrotrophic bacterium producing a cold-adapted lipase upon growth at low temperatures was isolated from Alaskan soil and identified as a Pseudomonas strain. The lipase gene (lipP) was cloned from the strain and sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (924 bp) corresponded to a protein of 308 amino acid residues with a molecular weight of 33,714. LipP also has consensus motifs conserved in other cold-adapted lipases, i.e., Lipase 2 from Antarctic Moraxella TA144 (G. Feller, M. Thiry, J. L. Arpigny, and C. Gerday, DNA Cell Biol. 10:381–388, 1991) and the mammalian hormone-sensitive lipase (D. Langin, H. Laurell, L. S. Holst, P. Belfrage, and C. Holm, Proc. Natl. Acad. Sci. USA 90:4897–4901, 1993): a pentapeptide, GDSAG, containing the putative active-site serine and an HG dipeptide. LipP was purified from an extract of recombinant Escherichia coli C600 cells harboring a plasmid coding for the lipP gene. The enzyme showed a 1,3-positional specificity toward triolein. p-Nitrophenyl esters of fatty acids with short to medium chains (C₄ and C₆) served as good substrates. The enzyme was stable between pH 6 and 9, and the optimal pH for the enzymatic hydrolysis of tributyrin was around 8. The activation energies for the hydrolysis of p-nitrophenyl butyrate and p-nitrophenyl laurate were determined to be 11.2 and 7.7 kcal/mol, respectively, in the temperature range 5 to 35°C. The enzyme was unstable at temperatures higher than 45°C. The K_m of the enzyme for p-nitrophenyl butyrate increased with increases in the assay temperature. The enzyme was strongly inhibited by Zn²⁺, Cu²⁺, Fe³⁺, and Hg²⁺ but was not affected by phenylmethylsulfonyl fluoride and bis-nitrophenyl phosphate. Various water-miscible organic solvents, such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30% (vol/vol) activated the enzyme.     

Hydroxo-complexes of Hg2+ chelates as models of hydrolytic metalloenzymes

Hydroxo-complexes of chelates of Hg²⁺ were found to catalyze hydrolysis of active esters and amides. Thus, 10⁻²M of the hydroxo-complex of Hg²⁺ pentamethyl diethylenetriamine (Hg-PDETA) at pH 7 enhanced the rate of hydrolysis of p-nitrophenyl esters and of cinnamoyl imidazole by factors of 400 and 1900, respectively. In the latter case the rates of reaction were linear with catalyst concentration. The hydroxo-complexes of Hg²⁺ phenanthroline (Hg-Phen) exhibited kinetic specificity toward p-nitrophenyl carbalkoxyglycinates. With these specific substrates acceleration factors of 1000 and more were obtained at 5 × 10⁻³M Hg-Phen, pH 8. The dependence of rates upon catalyst concentration was found to be curvilinear. This latter behavior was attributed to attack of one molecule of Hg-Phen, in the form of a hydroxo-complex, on a ternary complex Hg-Phen-substrate. The general features of metal-ion-catalyzed hydrolytic reactions are discussed and compared with the mode of action of hydrolytic metalloenzymes such as carboxypeptidase A and carbonic anhydrase.     

Coconut α-d-galactosidase isoenzymes: isolation,purification and characterization

α-d-Galactosidases (α-d-galactoside galactohydrolase, EC 3.2.1.22) from normal coconut endosperm were isolated and partially purified by a combination of ammonium sulfate fractionation, SP-Sephadex C50–120 ion-exchange chromatography and Sephadex G-200 and G-100 gel filtration. Two molecular forms of the enzyme, designated as A and B, were eluted after SP-Sephadex C50–120 ion-exchange chromatography. α-d-Galactosidase A, which is the major isoenzyme, was partially purified 43-fold on Sephadex G-200 and has a MW of about 23 000 whereas α-d-galactosidase B was partially purified 23-fold on Sephadex G-100 and has a similar MW of about 26 600. Both isoenzymes exhibited optimum activity at pH 7.5. The apparent K_m and V_max of α-d-galactosidase A were obtained at 3.46 × 10^?4M and 1.38 × 10^?3 M p-nitrophenyl α-<d-galactoside, respectively. A distinct substrate inhibition was noted. The enzyme was inhibited strongly by d-galactose and to a lesser extent by myo-inositol, d-glucose-6-phosphate, l-arabinose, melibiose and iodoacetic acid. Similarly, makapuno α-d-galactosidase was localized in the 40–70 % (NH₄)₂SO₄ cut but its optimum activity at pH 7.5 was considerably lower as compared to the normal. Its K_m was obtained at 6.75 × 10^?4 M p-nitrophenyl α-d-galactoside while the V_max was noted at 5.28 × 10^?3 M p-nitrophenyl α-d-galactoside. Based on the above kinetic data, the possible cause(s) of the deficiency of α-d-galactosidase activity in makapuno is discussed.     

Characterization of a ribonuclease from Anacystis nidulans infected with cyanophage as-1

In Anacystis nidulans the ribonuclease (RNase) activity is very low but is greatly increased upon phage-infection. A RNase was isolated and purified over 300-fold from A. nidulans cells infected by cyanophage AS-1. The enzyme did not attack single- or double-stranded DNA, was inactive on p-nitrophenyl phosphate or bis-p-nitrophenyl phosphate as substrates, and had neither 3′- nor 5′-nucleotidase activity. The approximate MW of the enzyme was 12000. Maximal enzyme activity was at pH 7.5. No absolute requirement for metal ions was observed, but Fe³⁺ stimulated and Co²⁺ and Ni²⁺ inhibited enzyme activity. The enzyme is an endonuclease which, upon exhaustive hydrolysis, produces mainly oligonucleotides (average chain-length: 3) with 3′-P termini. Analysis of the base composition of these oligonucleotides and determination of their 3′-terminal nucleosides, together with the investigation of the rate of hydrolysis of synthetic polyribonucleotides, have shown that the enzyme has a relative specificity for uridylic acid.     

Zinc enzyme modelling with zinc complexes of polar pyrazolylborate ligands

The Zn-OH₂ and Zn-OH complexes of the new tris(pyrazolyl)borate ligands with pyridyl and carboxamido substituents were investigated for their reactivity towards hydrolyzeable substrates. Tp^4−Py,MeZn-OH inserted CO₂ and CS₂ in methanol forming the Zn-OCOOMe and Zn-SCSOMe products. In non-aqueous media, both types of complexes with both types of substituents on the Tp ligands effected stoichiometric cleavage of tris(p-nitrophenyl)phosphate and p-nitrophenyl acetate. In solutions containing water and the MOPS buffer, up to eight p-nitrophenyl groups per equivalent of zinc complex could be cleaved from the esters, and the resulting bis(p-nitrophenyl)phosphate was also degraded to mono(p-nitrophenyl)phosphate. This is the first time that pyrazolylborate-zinc complexes have shown catalytic activity in hydrolytic reactions.     

Alpha-chymotrypsin: effects of bicyclic substrate geometry and hydrophobicity on reactivity

The α-chymotrypsin-catalyzed hydrolysis rates of p-nitrophenyl cyclopentane-carboxylate (I), p-nitrophenyl indan-2-carboxylate (II), and p-nitrophenyl spiro-[4.4]nonane-2-carboxylate (III) were measured at pH 8.1 in 20% methanol. After correction for variations in reactivity owing to stereoelectronic effects inherent to the substrates, the deacylation rate constants (k_c)_n of I and II are not significantly different. In $\text{(}\text{k}{}_{\mathrm{c}}\text{K}{}_{\mathrm{m}}\text{)}{}_{\mathrm{n}}$ II is 50 times more reactive than I, which demonstrates that the aromatic ring of the former substrate contributes significantly to its reactivity. The nearly equal reactivities of II and III indicate that the enzyme is rather insensitive to the geometry of the nonester-bearing ring of these compounds.     

A specific alkaline phosphatase from Saccharomyces cerevisiae with protein phosphatase activity

In this paper, specific PHO13 alkaline phosphatase from Saccharomyces cerevisiae was demonstrated to possess phosphoprotein phosphatase activity on the phosphoseryl proteins histone II-A and casein. The enzyme is a monomeric protein with molecular mass of 60 kDa and hydrolyzes p-nitrophenyl phosphate with maximal activity at pH 8.2 with strong dependence on Mg²⁺ ions and an apparent K_m of 3.6×10⁻⁵ M. No other substrates tested except phosphorylated histone II-A and casein were hydrolyzed at any significant rate. These data suggest that the physiological role of the p-nitrophenyl phosphate-specific phosphatase may involve participation in reversible protein phosphorylation.     

An examination of the rate assay to determine the active site normality of papain

Data are presented which demonstrate that the α-N-benzoyl-l-argine ethyl ester rate assay procedure, based on a burst titration with N-benzyloxy-carbonyl-l-tyrosine p-nitrophenyl ester as previously desribed (1), is an accurate and reliable method for determining the normality of papain in solution.     

Cloning, expression and characterization of a novel esterase from Bacillus pumilus

The gene encoding esterase (CE1) from Bacillus pumilus ARA with a calculated molecular weight of 28.4 kDa was cloned, sequenced and efficiently expressed in Escherichia coli. The open reading frame of 747 nucleotides encoded a protein, which was classified as a carboxylesterase with an identity of 87 % to esterase from Bacillus subtilis 168. Recombinant CE1 was purified in a single step to electrophoretic homogeneity by IMAC (Ni²⁺). The enzyme displayed maximum activity toward p-nitrophenyl (pNP) acetate at 37–40 °C and pH?6.5–7.0. It was stable in the pH range from 6.5 to 8.0, and at temperature from 25 to 40 °C. Among four p-nitrophenyl esters tested, the best substrate was pNP acetate with K _m and k _cat values of 0.33 mM and 4.07 s^?1, respectively. Amounts of 2 mM Ca²⁺ and Co²⁺ significantly increased the esterase activity to 190 and 121 %, respectively. These results suggest that CE1 has very attractive applications of increasing feed digestibility in animal nutrition in this moderate temperature range.