Kinetic study of sphingomyelin hydrolysis for ceramide production

Kinetic study of sphingomyelin hydrolysis catalyzed by Clostridium perfringens phospholipase C was, at the first time, conducted for ceramide production. Ceramide has the major role in maintaining the water-retaining properties of the epidermis. Hence, it is of great commercial potential in cosmetic and pharmaceutical industries such as in hair and skin care products. The enzymatic hydrolysis of sphingomyelin has been proved to be a feasible method to produce ceramide. The kinetic performance of sphingomyelin hydrolysis in the optimal two-phase (water:organic solvent) reaction system was investigated to elucidate the possible reaction mechanism and also to further improve the hydrolysis performance. Enzyme in solution had less thermal stability than the enzyme powder and the immobilized enzyme. The thermal inactivation of phospholipase C in all the three forms did not follow the first order reaction at 65 °C. The reactions for both the soluble and immobilized enzymes followed Michaelis–Menten kinetics. K_m's for the soluble and immobilized enzymes were 1.07 ± 0.32 and 1.26 ± 0.19 mM, respectively. The value of V_max was markedly decreased by the immobilization without much change in K_m, as if the immobilization functioned as the non-competitive inhibition. Ceramide as product activated the hydrolysis reaction, however, and its addition mainly caused the increase in the affinity of the enzyme–substrate complex.     

Nonionic detergent-induced activation of an esterase from Bacillus megaterium 20-1

An esterase-producing Bacillus megaterium strain (20-1) was isolated from a soil sample collected in South Korea. The cloned gene showed that the esterase 20-1 composed of 310 amino acids corresponding to a molecular mass (M_r) of 34,638. Based on the M_r and the protein sequence, the esterase 20-1 belonged to the H lipase/esterase group. The optimum temperature and pH of the purified His-tagged enzyme were 20–35 °C and 8.0, respectively. The esterase 20-1 showed a ‘nonionic detergent-induced activation’ phenomenon, which was a detergent type- and concentration-dependent process. In comparison with the native enzyme, the Tween 80-treated enzyme had relatively a similar k_cat value of 274 s⁻¹ but a very low K_m value of 0.037 mM for PNPC (C₆), therefore, it showed a 14-fold increase in k_cat/K_m value.     

Functional characterization of a salt- and thermotolerant glutaminase from Lactobacillus rhamnosus

The deamination of glutamine is a crucial step in the production of enzymatically hydrolyzed plant proteins to reach high glutamic acid yields. The required glutaminase activity usually is provided by addition of technical enzymes or by in situ generation from fungi, yeast or bacteria (i.e. Aspergillus oryzae in soy sauce production). We screened food-grade Lactobacilli for potential glutaminase activity and selected the enzyme found in Lactobacillus rhamnosus for further characterization. Glutaminase from L. rhamnosus was induced by growing the microorganism on hydrolyzed wheat gluten, a glutamine-rich protein source. Glutamine deaminating activity (glutaminase, EC 3.5.1.2) was found to be membrane-bound and lost its activity gradually upon solubilization. Functional studies of the glutaminase showed an optimal working pH of 7.0 and maximum activity at 50 °C. High salt-tolerance of the enzyme was observed, i.e. the presence of 5% (w/v) salt increased glutaminase activity almost two-fold and 90% of the initial activity still remained at 15% (w/v) salt. The glutaminase activity showed typical Michaelis–Menten behavior with an affinity constant K_m of 4.8±0.4 mM for glutamine and a V_max of 101±2 U/l.     

Biochemical and catalytic properties of two intracellular β-glucosidases from the fungus Penicillium decumbens active on flavonoid glucosides

In the presence of rutin as sole carbon source, Penicillium decumbens produces two intracellular β-glucosidases named G_I and G_II, with molecular masses of 56,000 and 460,000 Da, respectively. The two proteins have been purified to homogeneity. G_I and G_II composed of two and four equal sub-units, respectively and displayed optimal activity at pH 7.0 and temperature 65–75 °C. Both β-glucosidases were competitively inhibited by glucose and glucono-δ-lactone. G_I and G_II exhibited broad substrate specificity, since they hydrolyzed a range of (1,3)-, (1,4)- and (1,6)-β-glucosides as well as aryl β-glucosides. Determination of k_cat/K_m revealed that G_II hydrolyzed 3–8 times more efficiently the above-mentioned substrates. The ability of G_I and G_II to deglycosylate various flavonoid glycosides was also investigated. Both enzymes were active against flavonoids glycosylated at the 7 position but G_II hydrolyzed them 5 times more efficiently than G_I. Of the flavanols tested, both enzymes were incapable of hydrolyzing quercetrin and kaempferol-3-glucoside. The main difference between G_I and G_II as far as the hydrolysis of flavanols is concerned, was the ability of G_II to hydrolyze the quercetin-3-glucoside.     

The Mechanism of Salt Activation of Thermolysin: Relation with Pressure Activation and Implications of Hydration Change Coupled with Rate Process

Thermolysin (E.C. 3.4.24.4) shows a remarkable increase in catalytic activity at elevated salt concentrations or hydrostatic pressures. Salt effected K_cat, only, whilst the effect of pressure was related to both K_cat, and K_m. The turnover, derived from k_cat/K_m(V), of the hydrolysis of an N-acyldipeptide amide substrate was scarcely affected by addition of salt. These results were interpreted in terms of the stabilization of increased (or exposed) charges at the transition state of the reaction.     

Purification and characterization of a Na/K dependent alginate lyase from turban shell gut Vibrio sp. YKW-34

An alginate lyase with high specific enzyme activity was purified from Vibrio sp. YKW-34, which was newly isolated from turban shell gut. The alginate lyase was purified by in order of ion exchange, hydrophobic and gel filtration chromatographies to homogeneity with a recovery of 7% and a fold of 25. This alginate lyase was composed of a single polypeptide chain with molecular mass of 60 kDa and isoelectric point of 5.5–5.7. The optimal pH and temperature for alginate lyase activity were pH 7.0 and 40 °C, respectively. The alginate lyase was stable over pH 7.0–10.0 and at temperature below 50 °C. The alginate lyase had substrate specificity for both poly-guluronate and poly-mannuronate units. The k_cat/K_m value for alginate (heterotype) was 1.7 × 10⁶ s⁻¹ M⁻¹. The enzyme activity was completely lost by dialysis and restored by addition of Na⁺ or K⁺. The optimal activity exhibited in 0.1 M of Na⁺ or K⁺. This enzyme was resistant to denaturing reagents (SDS and urea), reducing reagents (β-mercaptoethanol and DTT) and chelating reagents (EGTA and EDTA).     

Enhanced thermostability and tolerance of high substrate concentration of an esterase by directed evolution

A newly isolated enantioselective esterase from Pseudomonas fluorescens KCTC 1767, which is currently considered as a biocatalyst for the production of a commercially valuable (S)-ketoprofen, has revealed a low structural and thermal stability. In order to enhance the stability, directed evolution was attempted on this enantioselective esterase by successive steps of an error prone and staggered extension PCR. After the second round of evolution, the best mutant 6–52 with enhanced thermal stability was selected and analyzed. DNA sequence analyses of 6–52 revealed that the three amino acid residues (L120P, I208V, and T249A) were changed and the mutation L120P was presumed as a structurally important residue due to its presence in all positive variants. The purified mutant 6–52, when incubated at 50 and 55 °C for 2 h, remained its activity over 30 and 10%, respectively, whereas there were no detectable activities in wild-type enzyme. The analysis of 6–52 in the presence of 15% ethanol showed 1.8-fold increase in the activity, compared to that of wild-type enzyme. The K_m and V_max values of 6–52 were estimated to be slightly increased, leading to 1.2-fold-higher the catalytic efficacy k_cat/K_m than that of wild-type enzyme. Additionally, the mutant 6–52 was more resistant to high substrate concentrations than that of wild-type enzyme.     

Kinetic characterization of chiral biocatalysis of cycloarenes by the camphor 5-monooxygenase enzyme system

Redox enzyme mediated biocatalysis has the potential to regio- and stereo-specifically oxidize hydrocarbons producing valuable products with minimal by-product formation. In vitro reactions of the camphor (cytochrome P-450) 5-monooxygenase enzyme system with naphthalene-like substrates yield stereospecifically hydroxylated products from nonactivated hydrocarbons. Specifically, the enzyme system catalyzes the essentially stereospecific conversion of the cycloarene, tetralin (1,2,3,4-tetrahydronaphthalene) to (R)-1-tetralol ((R)-(−)-1,2,3,4-tetrahydro-1-naphthol). It is shown that this reaction obeys Michaelis–Menten kinetics and that interactions between the enzyme subunits are not affected by the identity of the substrate. This subunit independence extends to the efficiency of NADH usage by the enzyme system—subunit ratios do not effect efficiency, but substrate identity does. Tetralin is converted at an efficiency of 13±3%, whereas (R)-1-tetralol is converted at 7.8±0.7%. A model of this system based on Michaelis–Menten parameters for one subunit (Pdx: K_M=10.2±2 μM) and both substrates (tetralin: K_M=66±26 μM, ν_max=0.11±0.04 s⁻¹, and (R)-1-tetralol: K_M=2800±1300 μM, ν_max=0.83±0.22 s⁻¹) is presented and used to predict the consumption and production of all substrates, products and cofactors.     

Purification and characterization of an enantioselective carbonyl reductase from Candida viswanathii MTCC 5158

A highly enantioselective carbonyl reductase produced by a new yeast strain Candida viswanathii MTCC 5158, which was isolated using an acetophenone enriched medium, has been purified and characterized. The enzyme has been purified to near homogeneity using ammonium sulfate precipitation, ion exchange and gel filtration chromatography. The molecular properties of the carbonyl reductase suggested the native enzyme to be tetrameric, with an apparent molecular weight of 120 kDa, the monomer being about 29 kDa. Acetyl aryl ketones were found to be the preferred substrates for the enzyme and the best reaction was the enantioselective reduction of acetophenone. The enzyme yielded (S)-alcohol in preference to (R)-alcohol and utilized NADH, but not NADPH as the cofactor. The purified enzyme exhibited maximum enzyme activity at pH 7.0 and 60 °C. The enzyme retained about 80% of its activity after 7 h incubation at 25 °C in sodium phosphate buffer (50 mM, pH 7.0). The addition of reducing agents like dithiothreitol and β-mercaptoethanol enhanced the enzyme activity while organic solvents, detergents and chaotropic agents had deleterious effect on enzyme activity. Metal chelating agents like hydroxyquinoline and o-phenanthroline have significant effect on enzyme activity suggesting that the carbonyl reductase required the presence of a tightly bound metal ion for activity or stability. The maximum reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) for acetophenone and NADH were 59.21 μmol/(min mg) protein and 0.153 mM and 82.64 μmol/(min mg) protein and 0.157 mM at a concentration range of 0.2–2 mM acetophenone (NADH fixed at 0.5 mM) and 0.1–0.5 mM NADH (acetophenone fixed at 2 mM), respectively.     

A substitutive substrate for measurements of beta-ketoacyl reductases in two fatty acid synthase systems

Bacterial β-ketoacyl-ACP reductase (FabG) and the β-ketoacyl reductase domain in mammalian fatty acid synthase (FAS) have the same function and both are rendered as the novel targets for drugs. Herein we developed a convenient method, using an available compound ethyl acetoacetate (EAA) as the substitutive substrate, to measure their activities by monitoring decrease of NADPH absorbance at 340 nm. In addition to the result, ethyl 3-hydroxybutyrate (EHB) was detected by HPLC analysis in the reaction system, indicating that EAA worked effectively as the substrate of FabG and FAS since its β-keto group was reduced. Then, the detailed kinetic characteristics, such as optimal ionic strength, pH value and temperature, and kinetic parameters, for FabG and FAS with this substitutive substrate were determined. The K_m and k_cat values of FabG obtained for EAA were 127 mM and 0.30 s^− 1, while those of this enzyme for NADPH were 10.0 μM and 0.59 s^− 1, respectively. The corresponding K_m and k_cat values of FAS were 126 mM and 4.63 s^− 1 for EAA; 8.7 μM and 4.09 s^− 1 for NADPH. Additionally, the inhibitory kinetics of FabG and FAS, by a known inhibitor EGCG, was also studied.     

Purification and partial characterization of a thermostable laccase from an unidentified basidiomycete

A thermostable laccase was isolated from an unidentified fungal isolate [Enz. Microb. Technol. 33 (2003) 212], and tentatively named UD4. This work indicates that the enzyme has unique properties other than its thermostability. Investigation into the kinetic parameters of the thermostable laccase yielded an unusually high affinity for ABTS as a substrate (low K_m) when compared with available published data for other laccase isozymes. The specificity constant (k_cat/K_m) was found to be considerably higher than laccase from other sources and is comparable to “white” laccase from Pleurotus ostreatus (POXA1). However, POXA1 isozyme exhibits a large turnover number (k_cat) that contributes to its high specificity constant whereas the high specificity constant for UD4 laccase is achieved by having a high substrate affinity. The UD4 thermostable laccase, like most other laccases, is able to utilize guaiacol as a substrate, whereas POXA1 is unable to oxidize guaiacol, indicating a broader substrate range for the thermostable laccase from UD4. The thermostable laccase is inhibited by sodium azide through non-competitive inhibition, and by thioglycolic acid and hydroxylamine through competitive inhibition. The high specificity constant, substrate affinity and broader substrate range of the thermostable laccase from UD4 indicates that it is a highly favourable candidate enzyme for industrial application.     

A novel spectrophotometric method for determination of kinetic constants of aldehyde oxidase using multivariate calibration method

Although phenanthridine has been frequently used as a specific substrate for the assessment of aldehyde oxidase activity, the use of this method is questionable due to a lower limit of detection and its validity for kinetic studies. In the present study, a novel sensitive multivariate calibration method based on partial least squares (PLS) has been developed for the measurement of aldehyde oxidase activity using phenanthridine as a substrate. Phenanthridine and phenanthridinone binary mixtures were prepared in a dynamic linear range of 0.1–30.0 μM and the absorption spectra of the solutions were recorded in the range of 210–280 nm in Sorenson's phosphate buffer (pH 7.0) containing EDTA (0.1 mM). The optimized PLS calibration model was used to calculate the concentration of each chemical in the prediction set. Hepatic rat aldehyde oxidase was partially purified and the initial oxidation rates of different concentrations of phenanthridine were calculated using the PLS method. The values were used for calculating Michaelis–Menten constants from a Lineweaver–Burk double reciprocal plot of initial velocity against the substrate concentration. The limits of detection for phenanthridine and phenanthridinone were found to be 0.04 ± 0.01 and 0.03 ± 0.01 μM (mean ± SD, n = 5), respectively. Using this method, the K_m value for the oxidation of phenanthridine was calculated as 1.72 ± 0.09 μM (mean ± SD, n = 3). Thus, this study describes a novel spectrophotometric method that provides a suitable, sensitive and easily applicable means of measuring the kinetics of phenanthridine oxidation by aldehyde oxidase without the need for expensive instrumentation.     

Hydrolysis of PET and bis-(benzoyloxyethyl) terephthalate with a new polyesterase from Penicillium citrinum

A polyethylene terephthalate (PET) model substrate, bis-(benzoyloxyethyl)terephthalate (3PET), was used to screen for micro-organisms producing enzymes hydrolyzing PET. From this screen, a strain growing on 3PET was isolated and identified as Penicillium citrinum. The polyesterase responsible for 3PET and PET hydrolysis was purified to electrophoretic homogeneity. The polyesterase had a molecular weight of 14.1 kDa, and the K_m and K_cat values on 4-nitrophenyl butyrate were 0.57 mM and 0.21 s^-1, respectively. Highest enzyme activities were obtained when P. citrinum was grown on a medium containing cutin, which was hydrolyzed by the polyesterase. Surface hydrolysis of PET with the enzyme lead to an increase in hydrophilicity based on rising height (+5.1 cm) and drop dissipation measurements (55 s). Both from PET and 3PET bis-(2-hydroxyethyl)terephthalate and mono-(2-hydroxyethyl)terephthalate were released, while only low amounts of terephthalic acid were liberated.     

Optimization of androstenedione production in an organic–aqueous two-liquid phase system

A systematic evaluation of the effect of key operational parameters on the selective cleavage of sitosterol to 4-androstene-3,17-dione (AD) with Mycobacterium sp. NRRL B-3805 in a dioctyl phthalate: aqueous buffer two-liquid phase system was performed. Of the parameters assessed, buffer composition, biomedium pH, temperature, and biomass and substrate concentration were those that mostly affected overall bioconversion rate. The optimum pH was 7.5 with Tris buffer. The highest bioconversion rate was observed at 35 °C, although at 40 °C bioconversion activity was virtually lost. Michaelis–Menten type kinetics adequately described the bioconversion system. Increasing biomass concentration from 10 to 70 g_{wet cell weight} l⁻¹ favored AD final yield, although the specific AD yield slightly decreased.     

A pH-stable laccase from alkali-tolerant γ-proteobacterium JB: Purification, characterization and indigo carmine degradation

γ-Proteobacterium JB, an alkali-tolerant soil isolate, produced laccase constitutively in unbuffered medium. The enzyme was purified to homogeneity by ammonium sulphate precipitation, DEAE-sepharose anion exchange chromatography and preparatory polyacrylamide gel electrophoresis. The purified enzyme was a monomeric polypeptide (MW 120 kDa) and absorbed at 590 nm indicating the presence of Type I Cu²⁺-centre. It worked optimally at 55 °C and showed different pH optima for different substrates. The enzyme was highly stable in the pH range 4–10 even after 60 days at 4 °C. K_m and V_max values for syringaldazine, catechol, pyrogallol, p-phenylenediamine, l-methyl DOPA and guaiacol substrates were determined. Inhibitors, viz. azide, diethyldithiocarbamate, thioglycollate and cysteine-hydrochloride all inhibited laccase non-competitively using guaiacol as substrate at pH 6.5. The enzyme degraded indigo carmine (pH 9, 55 °C) to anthranilic acid via isatin as determined spectrophotometrically and by HPLC analysis. Degradation was enhanced in the presence of syringaldehyde (571%), vanillin (156%) and p-hydroxybenzoic acid (91.6%) but not HOBT.     

Recombinant expression, characterization, and pulp prebleaching property of a Phanerochaete chrysosporium endo-β-1,4-mannanase

A Phanerochaete chrysosporium cDNA predicted to encode endo-1,4-β-d-mannanase, man5D, was cloned and expressed in Aspergillus niger. The coding region of the gene man5D was predicted to contain, in order from the N-terminal: a secretory signal peptide, cellulose-binding domain, linker region, and glycosyl hydrolase family 5 catalytic site. The enzyme was purified from culture filtrate of A. niger transformants that carried the recombinant man5D. Recombinant Man5D had an apparent molecular size of about 65 kDa by SDS-PAGE, and optimal activity at pH 4.0–6.0 and 60 °C. It was stable from pH 4.0 to 8.0 and up to 60 °C. The enzyme showed affinity for Avicel cellulose, suggesting that the predicted cellulose-binding domain is biologically functional. The specific activities of Man5D on mannan, galactomannan, and glucomannan at pH 5 and 60 °C ranged from 160 to 460 μmol/(min mg), with apparent K_m values from 0.54 to 2.3 mg/mL. Product analysis results indicated that Man5D catalyzes endo-cleavage, and appears to have substantial transglycosylase activity. When used to treat softwood kraft pulp, Man5D hydrolyzed mainly glucomannan and exhibited a positive effect as a prebleaching agent. Compared to a commercial prebleaching with xylanase, the prebleaching effect of Man5D was weaker but with reduced loss of fibre yield as determined by the release of solubilized sugars.     

A novel subtilase from common bean leaves

We describe the isolation of a protease from common bean leaves grown in the field. On the basis of its biochemical properties it was classified as serine proteinase belonging to the subtilisin clan. Isoelectric focusing resulted in a single band at pH 4.6, and SDS–PAGE in a single band corresponding to M_r 72 kDa. The proteinase activity is maximal at pH 9.9 and shows high stability in the alkaline region. The relative activities of the proteinase for eight different synthetic substrates were determined. The requirement for Arg in the P1 position appeared obligatory. k_cat/K_m values indicate that, for highest catalytic efficiency, a basic amino acid is also required in the P2 position, presenting a motif typical of the cleavage site for the kexin family of subtilases. The sequence of the 17 N-terminal amino acids of this proteinase shows similarity to those of other plant subtilases, sharing the highest number of identical amino acids with proteinase C1 from soybean seedling cotyledons and a cucumisin-like proteinase from white gourd (Benincasa hispida).     

Thermostable and alkalophilic maltogenic amylase of Bacillus thermoalkalophilus ET2 in monomer-dimer equilibrium

A gene encoding a thermostable and alkalophilic maltogenic amylase (BTMA) was cloned from the thermophilic bacterium Bacillus thermoalkalophilus ET2. BTMA was composed of 588 amino acids with a predicted molecular mass of 68.8 kDa. The enzyme had an optimal temperature and pH of 70°C and 8, respectively, the highest among maltogenic amylases reported so far. The Tm of BTMA at pH 8 was 76.7°C with an enthalpy of 113.6 kJ mol^-1. Both hydrolysis and transglycosylation activities for various carbohydrates were evident. β-Cyclodextrin (β-CD) and soluble starch were hydrolyzed mainly to maltose, and pullulan to panose. Acarbose, a strong amylase inhibitor, was hydrolyzed by BTMA to glucose and acarviosine-glucose. The K _m and k _cat values of BTMA for β-CD hydrolysis were 0.128 mM and 165.8 s^-1 mM, respectively. The overall catalytic efficiency (k _cat/K _m) of the enzyme was highest toward β-CD. BTMA was present in a monomer-dimer equilibrium with a molar ratio of 54:46 in 50 mM glycine-NaOH buffer (pH 8.0). This equilibrium could be affected by KCl and enzyme concentrations. The multi-substrate specificity of the enzyme was modulated by the structural differences between monomeric and dimeric forms. Starch was hydrolyzed more readily when monomeric BTMA was prevalent, while the opposite was observed for β-CD.     

Arg-94 is crucial to the catalysis of 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8

The crucial role of Arg-94 in 3-isopropylmalate (IPM) dehydrogenase from Thermus thermophilus HB8 was elucidated by replacing the residue to lysine with site-directed mutagenesis. The k_cat value of the R94K mutant enzyme for IPM was significantly reduced to 1/170 compared with that of native enzyme, whereas the K_m for IPM was not much changed. It appeared that the major role of Arg-94 in exerting the enzymatic activity is not for the substrate recognition, but for the reaction catalysis, in such a way that Arg-94 facilitates stabilization of the transition-state in the decarboxylation step.     

Isolation of a renin-like enzyme from the leech Theromyzon tessulatum

This article reports the purification of a renin-like enzyme (an aspartyl protease) from head parts of the leech Theromyzon tessulatum. After four steps of purification including gel permeation and anion exchange chromatographies followed by reversed-phase HPLC, this enzyme was purified to homogeneity. The renin-like enzyme (of 32 kDa) hydrolyses at neutral pH and at 37°C, the Leu¹⁰-Leu¹¹ bond of synthetic porcine angiotensinogen tetradecapeptide yielding the angiotensin I and the Leu¹¹-Val¹²-Tyr¹³-Ser¹⁴ peptide as products, with a specific activity of 1.35 pmol AI/min/mg (K_m 22 μM; K_cat 2.7). The hydrolysis of angiotensinogen is inhibitable at 90% by pepstatin A (IC₅₀ = 4.6 μM), consistent with a renin activity. This is the first biochemical evidence of renin-like enzyme in invertebrates.