A Model System for Convenient Fluorescent Labeling of Sugar Chain in Taka-amylase A

A convenient detection of sugar chains in Taka-amylase A (TAA) was done by using 40 μg of enzyme, where a decrease in the UV absorption of NaIO₄ during the periodate oxidation reaction was monitored. The periodate-oxidized sugar chain was labeled with a fluorescent reagent, N-1-ethylenediaminonaphthalene (EDAN), by incubation at pH 9.5 and 30°C for 1 h. The excess EDAN was removed by either quenching with o-phthaladehyde or Bio-Gel P-2 gel adsorption. Among the peptide fragments prepared from the EDAN-labeled TAA, a fluorescent peptide corresponding to the sugar chain was distinguished by the ODS column. These results suggest that periodate oxidation and subsequent fluorescent labeling were useful for the sensitive analysis of various glycoprotein samples.     

Pluripotentialities of a quenched fluorescent peptide substrate library: enzymatic detection, characterization, and isoenzymes differentiation

Protease inhibitors represent a major class of drugs, even though a large number of proteases remain unexplored. Consequently, a great interest lies in the identification of highly sensitive substrates useful for both the characterization and the validation of these enzyme targets and for the design of inhibitors as potential therapeutic agents through high-throughput screening (HTS). With this aim, a synthetic substrate library, in which the highly fluorescent (L)-pyrenylalanine residue (Pya) is efficiently quenched by its proximity with the p-nitro-(L)-phenylalanine (Nop) moiety, was designed. The cleavage between Pya and Nop leads to a highly fluorescent metabolite providing the required sensitivity. This library, characterized by a water-soluble primary sequence Ac-SGK-Pya-(X)_n-Nop-GGK-NH₂, X being a mixture of 10 natural amino acids (A, I, L, K, F, W, E, Q, T, P) and n varying from 0 to 3, was validated using enzymes belonging to the four main types of hydrolases: serine-, metallo-, cystein-, and aspartyl-proteases. The selectivity of substrates belonging to this library was evidenced by characterizing specific substrates for the isoenzymes NEP-1 and NEP-2. This library easily synthesized is of great interest for the identification and development of selective and specific substrates for still uncharacterized endoproteases.     

Influence of rheopolyglucin and dextran dialdehyde on physicochemical properties and thermostability of human hemoglobin

A study was made of the influence of rheopolyglucin and dextran dialdehyde derived therefrom on the structural characteristics and thermostability of human hemoglobin. The effects of solution pH, incubation time and temperature, and the degree of dextran oxidation on the conjugation between human hemoglobin and dextran dialdehyde were assessed. Formation of the hemoglobin-dextran dialdehyde complex resulted in shielding of the protein chromophore groups by the polysaccharide and transition of a part of hemoprotein molecules from a low-spin (HbO₂) to a high-spin (Hb and MetHb) state. It was found that the temperature of denaturation transition for the native protein and hemoglobin in the presence of rheopolyglucin was 60°C versus 80°C for the hemoprotein-dextran dialdehyde conjugate. Presumably, the latter is determined by the enhancement of hydrophobic interactions within the protein globule caused by dextran dialdehyde and the ability of the surface-bound carbohydrate components to prevent the association of hemoglobin molecules.     

Phospholipase A2 from Trypanosoma brucei gambiense and Trypanosoma brucei brucei: Inhibition by Organotins

Activity and kinetics of phospholipase A₂ (PLA₂) from Trypanosoma brucei gambiense (Wellcome strain) and Trypanosoma brucei brucei (GUTat 3.1) were examined using two different fluorescent substrates. The activity in the supernatants of sonicated parasites was Ca²⁺-independent, strongly stimulated by Triton X-100 with optimum activity at 37°C and pH 6.5–8.5. To encourage a possible interaction between the parasite enzyme and organotin compounds, fatty acid derivatives of dibutyltin dichloride were synthesized and evaluated as potential inhibitors of PLA₂. The enzyme from the two-trypanosome species differ with respect to kinetic parameters and are noncompetitively inhibited by the organotin compounds. The Michaelis constant (K_M) for PLA₂ from T. b. brucei is 63.87 and 30.90 μM while for T. b. gambiense it is 119.64 and 32.90 μM for the substrates l,2-bis-(1-pyrenebutanoyl-sn-glycero-3-phosphocholine (PBGPC) and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dode-canoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBDC₁₂-HPC), respectively.     

Isolation and Characterization of a Prolidase from Streptococcus cremoris H61

A prolidase with a molecular weight of 43,000 was purified to homogeneity from a cell-free extract of Streptococcus cremoris H61. The optimum pH of the enzyme was in the range of 6.5 to 7.5. The hydrolyzing activity was specific for dipeptides of the X-Pro type. Kinetic constants for 4 dipeptides (Leu-Pro, Phe-Pro, Val-Pro and Ala-Pro) were estimated. Km values were not very different for these substrates, but V_max values were quite different (Leu-Pro > Phe-Pro, Val-Pro > Ala-Pro). The enzyme was activated by cobalt ion and inactivated by metal-chelating agents or with 2-mercaptoethanol.     

一种几丁质裂解性多糖单加氧酶的活性评价及稳定性研究

【目的】裂解性多糖单加氧酶(LPMO)是一类铜离子依赖型的单加氧酶,能够通过氧化的方式断裂糖苷键,进而显著提高多糖的降解效率,受到广泛的关注。但是LPMO单加氧酶的性质使其容易被自身氧化而失活,且底物的聚合性质和释放产物的多样性使得对LPMO催化过程活性的评估变得十分困难。【方法】本研究以2,6-二甲氧基苯酚(2,6-DMP)和H2O2为底物,建立了测定几丁质裂解性多糖单加氧酶(BtLPMO10A)活性的评价体系,并研究该酶在降解几丁质底物过程中的稳定性。【结果】研究发现,在测定BtLPMO10A活性的过程中,较高的酶浓度,过氧化氢浓度和2,6-DMP浓度均使得反应过程脱离了线性范围,而抗坏血酸的加入能够提高灵敏度,但是对活性测定过程有较大影响。BtLPMO10A对2,6-DMP和H2O2的Km分别为0.53mmol/L和5.31 mmol/L,亲和性高于纤维素裂解活性的NcLPMO9C。BtLPMO10A在还原剂抗坏血酸存在的条件下容易失活,但底物几丁质的加入能够一定程度上稳定LPMO的活性,但是其在降解几丁质过程中活性依然会下降。【结论】本研究以2,6-二甲氧基苯酚为底物检测BtL...     

A study of the substrate specificity of aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2

A systematic study was made of the ability of aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2 to hydrolyse different peptide substrates. The enzyme showed a marked preference for substrates containing arginine as the N-terminal residue but, to a lesser extent, was also capable of cleaving other residues such as lysine and leucine. There was a tendency for the activity to increase with the hydrophobicity index of the C-terminal residue of dipeptide substrates. It was also observed that the enzyme tended to have higher affinities but lower V _max values for tripeptides with hydrophobic C-terminal residues. The values determined for K _m and V _max increased with chain length for oligopeptides of the general formula Lys-Phe-(Gly) _n , the optimum, as determined from V _max/K _m, being when n = 4. Typical K _m values for the most effective substrates were in the range 0.2–0.6 mM.     

Mutation affecting the charging reaction of alanyl-tRNA synthetase from Escherichia coli K 10

Summary A temperature-sensitive mutant of Escherichia coli was identified as having an altered alanyl-tRNA synthetase. Specific activity of wild type and mutant cell-free extracts showed no difference in the hydroxamate assay; the charging activity, however, was more than 10 fold lower for mutant extract protein. Wild type alanyl-tRNA synthetase has been purified 344 fold, the mutant enzyme was enriched 45 fold. With these preparations the following results were obtained:Sedimentation analysis in sucrose gradients indicates a molecular weight of the mutant enzyme of half the size of the wild type enzyme. Analytical gel filtration yields an approximate size for the native enzyme of 165000 and for the mutant enzyme material of 95,000. The mutant alanyl-tRNA synthetase differs from the wild type enzyme by a 10 fold increase in the k _mfor tRNA; no true difference in the k _m-values for the other substrates was detected. Temperature studies indicate an unusual low temperature-optimum for the charging reaction of both enzymes, whereas hydroxamate fromation capacity increases linearly up to almost 50°C. High temperature treatment of the native enzyme selectively affects the aminoacylation reaction but not the activation step; no effect of such treatment of the mutant enzyme was detected. It is proposed that the mutation causes the enzyme to dissociate and that the resulting subunits possess and altered tRNA binding site.     

Cathepsins B, H and L in Peritoneal Macrophages and Hepatopancreas of Carp Cyprinus carpio

Cathepsin B was purified from the crude extract of carp hepatopancreas by a modified method, and the specific activity increased about 3,400-fold with a 17% recovery. The purified enzyme gave a single protein band on Native-PAGE, whereas two bands with molecular weights of 30,000 (single chain) and 26,000 (heavy chain) migrated on SDS-PAGE. The enzyme potently hydrolyzed Z-Arg-Arg-MCA and Z-Phe-Arg-MCA but lacked the ability to hydrolyze most of the other MCA substrates. The kinetic constants of the enzyme with two Z-blocked substrates revealed that V_max and K_cat values of Z-Phe-Arg-MCA were much higher than Z-Arg-Arg-MCA, while their K_m values were approximate. The optimum hydrolysis pH and temperature of the enzyme for these two substrates were determined to be pH 6.0 and 45°C, respectively. A variety of protease inhibitors such as E-64, DTNB, antipain, leupeptin, chymostatin, TLCK and TPCK and metal compounds such as CuCl₂, CdCl₂, HgCl₂, and Zn(CH₃COO)₂ were able to significantly inactivate the enzyme. In contrast, cysteine and 2-mercaptoethanol activated the enzyme, with cysteine being more effective for activation than 2-mercaptoethanol over the tested concentrations.     

A cel44C-man26A gene of endophytic Paenibacillus polymyxa GS01 has multi-glycosyl hydrolases in two catalytic domains

A bacterial strain Paenibacillus polymyxa GS01 was isolated from the interior of the roots of Korean cultivars of ginseng (Panax ginseng C. A. Meyer). The cel44C-man26A gene was cloned from this endophytic strain. This 4,056-bp gene encodes for a 1,352-aa protein which, based on BLAST search homologies, contains a glycosyl hydrolase family 44 (GH44) catalytic domain, a fibronectin domain type 3, a glycosyl hydrolase family 26 (GH26) catalytic domain, and a cellulose-binding module type 3. The multifunctional enzyme domain GH44 possesses cellulase, xylanase, and lichenase activities, while the enzyme domain GH26 possesses mannanase activity. The Cel44C enzyme expressed in and purified from Escherichia coli has an optimum pH of 7.0 for cellulase and lichenase activities, but is at an optimum pH of 5.0 for xylanase and mannanase activities. The optimum temperature for enzymatic activity was 50°C for all substrates. No detectable enzymatic activity was detected for the Cel44C-Man26A mutants E91A and E222A. These results suggest that the amino acid residues Glu₉₁ and Glu₂₂₂ may play an important role in the glycosyl hydrolases activity of Cel44C-Man26A.     

Further Properties of the New Type of Exopolygalacturonate Lyase from Streptomyces nitrosporeus

The new type of exopolygalacturonate lyase from Streptomyces nitrosporeus had a molecular weight between 32,400 to 39,000 in three different determination methods, a S⁰₂₀, _w value of 3.4 S and an isoelectric point of pH 4.05. The purified enzyme contained a relatively large amount of aspartic acid, glycine and about 13% carbohydrate. The enzyme had higher affinity on 10~30% esterified polygalacturonate than on de-esterified polygalacturonate, but the degradation limit of the substrates by the enzyme was diminished by an increase in methyl ester groups of the substrates.     

Stabilization of Escherichia coli penicillin G acylase against thermal inactivation by cross-linking with dextran dialdehyde polymers

The thermostabilization of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11105 by cross-linking with dextran dialdehyde molecules, at a molecular mass of 11 500, 37 700 and 71 000 Da, was studied. The thermal inactivation mechanisms of the native and modified PGA were both considered to obey first-order inactivation kinetics during prolonged heat treatment, forming fully active but temperature-sensitive transient states. The highest enhancement to the thermostability of PGA was obtained using dextran-71000-dialdehyde modification, as a␣nearly ninefold increase at temperatures above 50 °C. The modification of PGA by dextran-11500-dialdehyde resulted in a considerable reduction of the V _m and K _m parameters of the enzyme. However, other dextran dialdehyde derivatives used for modification did not cause a meaningful change in either V _m and K _m. Modification by dextran dialdehyde derivatives did not result in significant change to either the optimal temperature or the activation energy of PGA. All modified PGA preparations showed lower inactivation rate constants but higher half-lives for inactivation than those of the native PGA at all temperatures studied. As indicated by the half-life times and k _i values, dextran 71000-dialdehyde was found to be more effective at cross-linking in the thermo-stabilization of PGA than any other agent studied in this work. Received: 3 December 1996 / Received revision: 17 March 1997 / Accepted: 22 March 1997     

Identification of tandemly repeated type VI cellulose-binding domains in an endoglucanase from the aerobic soil bacterium Cellvibrio mixtus

Cellulose-binding domains (CBD) play a pivotal role during plant cell wall hydrolysis by cellulases and xylanases from aerobic soil bacteria. Recently we␣have reported the molecular characterisation of a single-domain endoglucanase from Cellvibrio mixtus, suggesting that some cellulases produced by this aerobic bacterium preferentially hydrolyse soluble cellulosic substrates. Here we describe the complete nucleotide sequence of a second cellulase gene, celB, from the soil bacterium C. mixtus. It revealed an open reading frame of 1863 bp that encoded a polypeptide, defined as cellulase B (CelB), with a predicted M _r of 66 039. CelB contained a glycosyl hydrolase family 5 catalytic domain at its N terminus followed by two repeated domains, which exhibited sequence identity with type VI CBD previously found in xylanases. Full-length CelB bound to cellulose while catalytically active truncated cellulase derivatives were unable to bind the polysaccharide, confirming that CelB is a modular enzyme and that the type VI CBD homologues were functional. Analysis of the biochemical properties of CelB revealed that the enzyme hydrolyses a range of cellulosic substrates, although it was unable to depolymerise Avicel. We propose that type VI CBD, usually found in xylanases, provide an additional mechanism by which cellulases can accumulate on the surface of the plant cell wall, although they do not potentiate cellulase activity directly. These results demonstrate that C. mixtus, in common with other aerobic bacteria, is able to produce cellulases that are directed to the hydrolysis of cellulose in its natural environment, the plant cell wall. Received: 6 October 1997 / Received revision: 22 December 1997 / Accepted: 2 January 1998     

Fluorogenic cephalosporin substrates for β-lactamase TEM-1

Cephalosporin was used to synthesize soluble and precipitating fluorogenic β-lactam substrates that demonstrated differential catalytic hydrolysis by three different subtypes of β-lactamase: TEM-1 (class A), p99 (class C), and a Bacillus cereus enzyme sold by Genzyme (class B). The most successful soluble substrate contained difluorofluorescein (Oregon Green 488) ligated to two cephalosporin moieties that, therefore, required two turnovers to produce the fluorescent Oregon Green 488 leaving group. The bis-cephalosporin modification was required so that the final reaction product was the Oregon Green 488 carboxylic acid rather than a less bright phenolic adduct of the dye. Hydrolysis in pH 5.5 Mes and pH 7.2 phosphate-buffered saline (PBS) buffers was similar, but in pH 8.0 Tris the hydrolysis rate nearly doubled. Activity of the β-lactamases on the various substrates was shown to depend highly on the linker between the cephalosporin and the fluorophore, with an allyl linker promoting faster turnover than a phenol ether linker. Measured K_m values for dichlorofluorescein and difluorofluorescein cephalosporin substrates were approximately the same as K_m values for penicillin G and ampicillin found in the literature (∼30–40 μM).     

Heterologous expression and biochemical characterization of novel pyranose 2-oxidases from the ascomycetes <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> and <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

A gene encoding a pyranose 2-oxidase (POx; pyranose/oxygen 2-oxidoreductase; glucose 2-oxidase; EC 1.1.3.10) was identified in the genome of the ascomycete Aspergillus nidulans. Attempts to isolate POx directly from A. nidulans cultures or to homologously overexpress the native POx (under control of the constitutive gpdA promoter) in A. nidulans were unsuccessful. cDNA encoding POx was synthesized from mRNA and expressed in Escherichia coli, and the enzyme was subsequently purified and characterized. A putative pyranose 2-oxidase-encoding gene was also identified in the genome of Aspergillus oryzae. The coding sequence was synthetically produced and was also expressed in E. coli. Both purified enzymes were shown to be flavoproteins consisting of subunits of 65 kDa. The A. nidulans enzyme was biochemically similar to POx reported in literature. From all substrates, the highest catalytic efficiency was found with D-glucose. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones and 2,6-dichloroindophenol. As judged by the catalytic efficiencies (k _cat/k _m), some of these quinone electron acceptors are better substrates for pyranose oxidase than oxygen. The enzyme from A. oryzae was physically similar but showed lower kinetic constants compared to the enzyme from A. nidulans. Distinct differences in the stability of the two enzymes may be attributed to a deletion and an insertion in the sequence, respectively.     

A Branched-chain Amino Acid Aminotransferase from the Rumen Ciliate Genus Entodinium

A branched-chain amino acid aminotransferase was extracted from rumen ciliates of the genus Entodinium and was partially purified by Sephadex G-200, DEAE-cellulose and DEAE-Sephadex A-50 column chromatography. The purified enzyme was active only with leucine, isoleucine and valine, and required pyridoxal phosphate as cofactor. The amino acids competed with each other as substrates. The enzyme had optimal activity at pH 6.0 in phosphate buffer. The K_m values for the substrates and cofactor are as follows: 1.66 for leucine; 0.90 for isoleucine; 0.79 for valine; 0.29 mM for α-ketoglutarate: and 0.1 μM for pyridoxal phosphate. Enzyme activity was inhibited by p-chloromercuribenzoate and HgCl₂. Gel filtration indicated the enzyme to have a molecular weight of 34,000.     

Sialic acid and biomass production by Streptococcus agalactiae under different growth conditions

A fermentation process to increase type capsular polysaccharide production by different serotypes of Streptococcus agalactiae (group B Streptococcus) was established. As sialic acid is an integral component of the polysaccharide, its synthesis was used to monitor polysaccharide, its synthesis was used to monitor polysaccharide production. Culture conditions, examined both on laboratory and pilot-plant scales, allowed optimal bacterial growth and high polysaccharide production in a medium composed of ultrafiltered Todd Hewitt broth supplemented with 2% (w/v) glucose and 1.5% (w/v) Na₂HPO₄, at a constant pH of 7.2. Studies using different gas atmospheres (air, CO₂ or their mixture) showed that air greatly enhanced polysaccharide production. Correspondence to: C. von Hunolstein     

Kinetic study of a membrane enzyme reactor

A flat-membrane dialyzer was used as enzyme reactor by introducing enzyme solution into one of the membrane-separated chambers. The apparent Michaelis constant K_m(app) of urease was always larger (ten times at [urease] = 1 mg/ml) than that of free enzyme because the permeation of substrate through the membrane was rate determining. K_m(app) for urease decreased from 125 to 20mM with increasing flow rate of the substrate solution because of the turbulent flow near the membrane. In the case of glucose oxidase or creatine kinase, the reaction rate was limited by the permeation of less permeable substrates such as oxygen or ATP. Therefore, K_m(app) of more permeable substrates such as glucose or creatine became smaller than that of free enzyme. The reaction amount calculated from the permeation data agreed well with experimental results. By designing spacers for the reactor to give turbulence to the solution, the effectiveness of the reactor was improved fivefold.     

An investigation on acarbose inhibition and the number of active sites in an amylopullulanase (L14-APU) from an Iranian Bacillus sp.

An amylopullulanase (L₁₄-APU) from an Iranian thermophilic bacterium was purified and the effect of acarbose, as a general inhibitor of α-amylases, on pullulan and starch hydrolysis catalyzed by L₁₄-APU was investigated. The inhibition is a competitive type whereas inhibition constants for pullulan and starch are 99 μM and 72 μM, respectively. Investigation of the reaction rate in a system contains competitive substrates and the inhibition type of acarbose in presence of different substrates suggests that L₁₄-APU possesses only one active site for two activities. The analysis of metal ions and other reagents effects has shown that Ca²⁺, Mg²⁺, Mn²⁺ and Co²⁺ enhanced both activities of the enzyme while N-bromosuccinimide treatment leads to the complete inactivation of the enzyme. The enzyme activity increased in the presence of low concentration of SDS as a surfactant.     

Purification and properties of mitochondrial and peroxisomal 3-hydroxyacyl-CoA dehydrogenase from rat liver

There are two 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) in rat liver, one in mitochondria (type I enzyme), and another in peroxisomes (type II enzyme). In a series of the studies on the properties and the physiological roles of fatty acid oxidation systems in both organelles, the two enzymes were purified and compared for their properties. The final preparations obtained were judged to be homogeneous based on the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation velocity analysis. Type I enzyme was composed of two identical subunits of molecular weight of 32,000, whereas type II enzyme was a monomeric enzyme having a molecular weight of 70,000–77,000. These subunit structures were confirmed by the results of fluorescence studies. Both enzymes were different in amino acid compositions, especially in the contents of tryptophan and half-cystine. Antibodies against them formed single precipitin lines for the corresponding enzymes, but not for the others when subjected to an Ouchterlony double-diffusion test. The K_m values of type II enzyme for various substrates were lower than those of type I enzyme except those for acetoacetyl-CoA. As for 3-hydroxyacyl-CoA substrates, both enzymes had lower K_m's for longer-chain substrates. The V for the substrates of C₄C₁₀ were similar for each enzyme, though the value of type II enzyme for C₁₀ substrate was rather lower. The results of fluorescence studies suggested that their dissociation constants for NADH were lower and those for NAD⁺ were higher at lower pH. Both enzymes were specific to l-form of 3-hydroxyacyl-CoA substrate. The optimal pH of the forward reaction of type I and type II enzymes was 9.6 and 9.8, and of the reverse reaction, 4.5 and 6.2, respectively. From these results they were concluded to be completely different enzymes.