Proteins in the nervous system — structure and function : Progress in clinical and biological research; volume 79

We have purified a unique enzyme, α-amino-?-caprolactam racemase 945-fold from an extract of Achromobacter obae by Octyl—Sepharose CL-4B and Thiopropyl—Sepharose 6B and some other chromatographies. The purified enzyme was found homogeneous by sodium dodecyl sulfate—polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a monomeric structure with M_r ～ 50 000 and a sedimentation coefficient (s_20,w) of 4.28 S. The enzyme contains pyridoxal 5'-phosphate as a coenzyme. The pH optimum for the enzyme activity is ～9.0. D- and L-α-amino-?-caprolactams are the only substrates. The K_m values for the D- and L-isomers are, 8 and 6 mM, respectively.     

High catalytic activity of alanine racemase from psychrophilic Bacillus psychrosaccharolyticus at high temperatures in the presence of pyridoxal 5'-phosphate

We examined the effect of the pyridoxal 5'-phosphate (PLP) cofactor on the activity and stability of the psychrophilic alanine racemase, having a high catalytic activity at low temperature, from Bacillus psychrosaccharolyticus at high temperatures. The decrease in the enzyme activity at incubation temperatures over 40 degrees C was consistent with the decrease in the amount of bound PLP. Unfolding of the enzyme at temperatures above 40 degrees C was suppressed in the presence of PLP. In the presence of 0.125 mM PLP, the specific activity of the psychrophilic enzyme was higher than that of a thermophilic alanine racemase, having a high catalytic activity at high temperature, from Bacillus stearothermophilus even at 60 degrees C.     

Purification and characterization of alanine racemase from hepatopancreas of black-tiger prawn, Penaeus monodon

Alanine racemase has been purified to homogeneity from the hepatopancreas of the black tiger prawn, Panaeus mondon. The enzyme depends on pyridoxal 5′-phosphate and consists of two subunits with an identical molecular weight of 41,000. V_max and K_m values for -alanine are 460 μmol/min/mg and 50 mM, and those for -alanine are 94 μmol/min/mg and 24 mM, respectively. The enzyme is highly specific toward alanine. Among other amino acids examined, only serine served as a substrate: -serine was racemized at a rate of approximately 0.5% of that of -alanine. The prawn enzyme is immunochemically distinguishable from the enzymes of Bacillus stearothermophilus and Schizosaccharomyces pombe, which resemble each other. The prawn enzyme is activated and stabilized by the presence of monovalent anions including chloride. This is consistent with the previous hypothesis (e.g. E. Fujita, E. Okuma, H. Abe, Comp. Biochem. Physiol. 116A (1997) 83–87) that -alanine serves as an osmoregulator in marine and euryhaline animals.     

Stereochemistry of the hydrogen abstraction from pyridoxamine phosphate catalyzed by alanine racemase of Bacillus stearothermophilus

Alanine racemase of Bacillus stearothermophilus catalyzes transamination as a side reaction. Stereospecificity for the hydrogen abstraction from C-4′ of pyridoxamine 5′-phosphate occurring in the latter half transamination was examined. Both apo-wild-type and apo-fragmentary alanine racemases abstracted approximately 20 and 80% of tritium from the stereospecifically-labeled (4′S)- and (4′R)-[4′-³H]PMP, respectively, in the presence of pyruvate. Alanine racemase catalyzes the abstraction of both 4′S- and 4′R-hydrogen like amino acid racemase with broad substrate specificity. However, R-isomer preference is a characteristic property of alanine racemase.     

Purification and characterization of a novel enzyme, L-threo-3-hydroxyaspartate dehydratase, from Pseudomonas sp. T62

L-threo-3-Hydroxyaspartate dehydratase (L-threo-3-hydroxyaspartate hydro-lyase), which exhibited specificity for L-threo-3-hydroxyaspartate (K(m)=0.74 mM, V(max)=37.5 micromol min(-1) (mg protein)(-1)) but not for D-threo or D, L-erythro-3-hydroxyaspartate, was purified from a cell-free extract of Pseudomonas sp. T62. The activity of the enzyme was inhibited by hydroxylamine and EDTA, which suggests that pyridoxal 5'-phosphate and divalent cations participate in the enzyme reaction. The NH(2)-terminal amino acid sequence showed significant similarity to the Saccharomyces cerevisiae YKL218c gene product, a hypothetical threonine dehydratase. However, the purified enzyme showed no threonine dehydratase activity.     

假单胞菌L-半胱氨酸合成酶的纯化和性质研究

假单胞菌TS-1138的细胞浆液通过硫铵沉淀、SephadexG-75凝胶过滤、DEAE-Cellulose52离子交换、SephadexG 100凝胶过滤等分离纯化手段分别将从L-ATC合成L 半胱氨酸的两个酶——L-ATC水解酶和L-SCC水解酶纯化了 83.9和 90.3倍。SDS-PAGE鉴定均为单一条带 ,两种酶的相对分子质量分别为 37.5和 42.8kD ;酶反应的最适温度均为 35℃ ,最适pH分别为 7.0和 8.0 ;酶的米氏常数分别为 0.67mmol L和 0.15mmol L ,     

Enzymic properties of β-mannanase from Polyporus versicolor

Substrate specificities and the kinetic parameters, K_m and V_max, of the four multiple enzyme forms of extracellular β-mannanase activity purified from Polyporus versicolor were determined. Although K_m values were significantly greater than those encountered in other β-mannanase systems V_max values were equivalent or much greater, rendering the physiological efficiencies of the β-mannanase comparable to those of other β-mannanases. All enzymes preferred glucomannan as substrate, were highly refractory at low concentrations to n-octylglucopyranoside, sodium deoxylcholate, and sodium dodecylsulfate, and were largely insensitive to methanol, ethanol, acetonitrile, and dimethylsulfoxide.     

Purification and properties of the β-fructofuranosidase from Kluyveromyces fragilis

The β-fructofuranosidase from Kluyveromyces fragilis was purified to one band on electrophoresis by 3 different methods. Two of the preparations were found to be impure by isoelectric focusing. This demonstrates the need for more than one criteria of homogeneity when purifying this enzyme. The enzyme was found to be a glycoprotein, stable at 50°C, with a pH optimum of 4.5. The cations Hg²⁺, Ag⁺, Cu²⁺ and Cd²⁺ exhibited a marked inhibition of the enzyme. Competitive inhibition was observed with the fructose analog 2,5-anhydro-D-mannitol suggesting that the enzyme is inhibited by the furanose form of fructose.     

Kinetic characterization of recombinant human cystathionine β-synthase purified from E. coli

Cystathionine β-synthase (CBS) catalyzes the pyridoxal-5′-phosphate-dependent condensation of l-serine and l-homocysteine to form l-cystathionine in the first step of the transsulfuration pathway. Although effective expression systems for recombinant human CBS (hCBS) have been developed, they require multiple chromatographic steps as well as proteolytic cleavage to remove the fusion partner. Therefore, a series of five expression constructs, each incorporating a 6-His tag, were developed to enable the efficient purification of hCBS via immobilized metal ion affinity chromatography. Two of the constructs express hCBS in fusion with a protein partner, while the others bear only the affinity tag. The addition of an amino-terminal, 6-His tag, in the absence of a protein fusion partner and in the absence or presence of a protease-cleavable linker, was found to be sufficient for the purification of soluble hCBS and resulted in enzyme with 86–91% heme saturation and with activity similar to that reported for other hCBS expression constructs. The continuous assay for l-Cth production, employing cystathionine β-lyase and l-lactate dehydrogenase as coupling enzymes, was employed here for the first time to determine the steady-state kinetic parameters of hCBS, via global analysis, and revealed previously unreported substrate inhibition by l-Hcys (K_i^l-Hcys = 2.1 ± 0.2 mM). The kinetic parameters for the hCBS-catalyzed hydrolysis of l-Cth to l-Ser and l-Hcys were also determined and the k_cat/K_m^l-Cth of this reaction is only 2-fold lower than the k_cat/K_m^l-SER of the physiological, condensation reaction.     

变色栓菌锰过氧化物酶同工酶的纯化及其性质研究

变色栓菌(Trametes versicolor)胞外产酶培养液经硫酸铵沉淀、DEAE-cellulose DE52离子交换柱层析后,获得两个活性组分D1和D2,其中活性组分D2经Phenyl SepharoseTM6Fast Flow疏水层析后,所得样品MnP1经SDS-PAGE检测已达到电泳纯。活性组分D1经Phenyl SepharoseTM6Fast Flow疏水层析、Sephacryl S-200HR凝胶过滤层析后,所得样品MnP2经SDS-PAGE检测已达到电泳纯。两种同工酶MnP1及MnP2,各自的比活力为579.09、425.00U/mg;纯化倍数为17.51、12.85;活力回收率为6.17%、2.47%。由SDS-PAGE法测得MnP1及MnP2的表观分子量分别为46.3kD、43.0kD。两种同工酶催化DMP(2,6-二甲氧基酚)氧化反应的最适pH值及最适反应温度有所不同,最适pH值分别为pH5.8、pH6.2,最适反应温度分别为60℃、65℃。在45℃以下,pH4.0~7.0之间,MnP1及MnP2的稳定性好。DMP为最佳酶促反应底物,以DMP为底物的Km分别为13.43μmol/L、12.45μmol/L。在无Mn2 存在的条件下,酶促反应几乎不发生。EDTA在较高浓度时抑制酶的活性,DTT在所试浓度下都完全抑制酶的活性。     

副干酪乳杆菌β-葡糖苷酶的表达、纯化及酶学性质研究 *

为了实现糖苷类物质的高效转化,将来源于副干酪乳杆菌(Lactobacillus paracasei)TK1501 β-葡糖苷酶基因连接于表达载体pET28a(+)上,在E. coli BL21中表达,重组酶经镍离子亲和层析分离得到纯酶,其分子质量和比酶活分别为86.63kDa和675.56U/mg。最适作用温度和pH分别为30℃和6.5。 Mg ²⁺和Ca ²⁺对β-葡糖苷酶酶活抑制作用最小,Cu ²⁺几乎使其丧失催化活性。其底物特异性较宽泛,对大豆异黄酮、栀子苷、水杨苷、七叶苷、虎杖苷、熊果苷均有降解作用。以β-pNPG为底物时,该酶的K_m和V_max分别为1.44mmol/L和58.32mmol/(L·s),催化系数k_cat为3 982/s。结果与分析表明,来源于副干酪乳杆菌TK1501 β-葡糖苷酶对水解大豆异黄酮和合成糖苷将会发挥重要作用。     

Purification and substrate characterization of α-ketobutyrate decarboxylase from Pseudomonas putida

α-Ketobutyrate decarboxylase encoded in the -methionine catabolism operon of Pseudomonas putida is homologous with the E1 component of pyruvate dehydrogenase complex from gram-negative bacteria. The enzyme was purified to homogeneity from the cell extract of an Escherichia coli transformant. The purified enzyme was homodimeric with a subunit of M_r 93,000 on SDS-PAGE. The enzyme activity was activated by the addition of both thiamine pyrophosphate (TPP) and a divalent cation, such as Mg²⁺, Mn²⁺, and Co²⁺. The enzyme showed high activity for α-ketobutyrate and α-keto-n-valerate rather than pyruvate, but the α-keto acids with increasing length of the side chain as well as branching, such as α-keto-n-caproate and α-keto-3-methylvalerate, were not used by the enzyme. The K_m values for α-ketobutyrate and pyruvate were 0.016 and 0.147 mM, respectively, and the k_cat/K_m value (10.69 s⁻¹ mM⁻¹) for α-ketobutyrate was 29-fold greater than that for pyruvate. Thus, α-ketobutyrate decarboxylase is distinguished from the pyruvate dehydrogenase E1 component with respect to the substrate specificity, although their structural and enzymological properties were similar. These results suggest that the unique substrate specificity of α-ketobutyrate decarboxylase is due to a slight difference in the highly conserved active sites of both enzymes.     

Production, purification and properties of γ-glutamyltranspeptidase from a newly isolated Bacillus subtilis NX-2

Production, purification and properties of γ-glutamyltranspeptidase from a newly isolated Bacillus subtilis NX-2 was investigated. At the optimum conditions for enzyme formation, a high level, 3.2 U/ml of γ-GTP was obtained. The extracellular γ-GTP from this strain was purified 111.15-fold to homogeneity from the culture supernatant by acetone precipitation, hydrophobic interaction chromatography and ion exchange chromatography. The purified enzyme was a heterodimer consisting of one large subunit (43 kDa) and one small subunit (32 kDa), and exhibited high activity at 40–60 °C, pH 8.0. It preferred basic amino acids as γ-glutamyl acceptor in transpeptidation, and the stereochemistry of the γ-glutamyl acceptor had no influence on the enzyme activity, which was different from other γ-GTPs reported. Furthermore, it was proved that γ-GTP of this strain could catalyze the transfer of l-glutamine to glycylglycine to synthesize Gln–Gly–Gly, which was promising for the synthesis of valuable γ-glutamyl peptides.     

Purification and enzymatic properties of α-galactosidase from Penicillium janthinellum

α-Galactosidase (E.C.3.2.1.22) from Penicillium janthinellum was purified by precipitation and fractionation with ammonium sulphate, cold acetone or ethanol, calcium phosphate gel, and column chromatographies on Sephadex G-100 and G-200. The enzyme was purified about 110.39-fold when Sephadex G-100 was used. α-Galactosidase exhibited the optimum pH and temperature at 4.5 and 60°C, respectively. The optimum enzyme stability was obtained at pH 3.5 for 24 h (at room temperature). The enzyme was found to be thermostable below 65°C up to 40 minutes and was gradually inactivated by increasing the temperature above this degree. The MICHAELIS constant was 0.55 mM for p-nitrophenyl-α-D-galactoside. The α-galactosidase activity was strongly inhibited by Hg⁺⁺ and slightly activated by Mn⁺⁺. The results show the possibility of producing a thermostable enzyme from a low-priced agricultural product, for instance, lupine.     

Biochemical characterization of a novel lysine racemase from Proteus mirabilis BCRC10725

A lysine racemase gene (lyr) that consisted of an open reading frame of 1224-bp and encoded a protein with a calculated molecular mass of 45 kDa was cloned from the Proteus mirabilis BCRC10725 and expressed in Escherichia coli BL21(DE3). The purified His₆-tagged Lyr was most active towards lysine, exhibiting a specific activity of 2828 ± 97 U/mg. This enzyme also racemized arginine with a specific activity of 568 ± 28 U/mg but not other amino acids. The optimal conditions for Lyr activity to l-lysine were pH 8.0–9.0 and 50 °C. The racemization activity of Lyr was completely inhibited by 5 mM hydroxylamine and was partially restored by the addition of pyridoxal 5′-phosphate. The S394 residue of Lyr was subjected to site-directed mutagenesis. The arginine racemization activities of the S394Y, S394N, S394C and S394T variant proteins were increased by 1.5–1.8 fold compared to the wild-type Lyr, indicating that the S394 residue played a crucial role in determining the preference of Lyr to lysine and arginine.     

极端嗜盐古生菌(Natrinema sp.)R6-5胞外嗜盐蛋白酶的纯化和性质研究

采用bacitracin-Sepharose 4B亲和层析的方法得到凝胶电泳均一的来自极端嗜盐古生菌(Natrinema sp.)R6-5的胞外嗜盐蛋白酶。经SDS-PAGE分析该酶亚基分子量为62kDa。PMSF对它的活性完全抑制,表明它是一种丝氨酸蛋白酶,该酶反应的最适NaCl浓度为3mol/L,最适温度为45℃,最适pH值为8.0。在高盐条件下能维持高活性并十分稳定,具有重要的潜在应用价值。     

Substrate spectrum of mandelate racemase: Part 1: Variation of the α-hydroxy acid moiety

Enzymatic racemization of mandelic acid derivatives modified at the α-hydroxy acid moiety was achieved using mandelate racemase [EC 5.1.2.2]. Whereas α-amino acid derivatives, such as phenyl glycine and mandelic acid hydrazide were not accepted, the mandelic acid amide was racemized at an acceptable rate. The latter was significantly enhanced by an electron-withdrawing substituent in the phenyl moiety. Based on the catalytic mechanism of the enzyme, the relative activities of non-natural substrates could be explained by steric and electronic reasons.     

Purification and characterization of two minor endo-β-1,4-xylanases of Schizophyllum commune

Two minor extracellular endo-β-1,4-xylanases (XynB and XynC, EC 3.2.1.8) were purified from the culture filtrate of Schizophyllum commune grown on cellulose. The molecular mass of enzymes was estimated to be 30.5 kDa for XynB and 30 kDa for XynC according to SDS-PAGE. Both enzymes were acidic, with pI value 2.8 for XynB and 3.6 for XynC. The highest activities were achieved at 50 °C and pH 5.5 and enzymes were stable up to 40 °C in the pH range 5–7. A comparison of hydrolysis products of glucuronoxylan, rhodymenan and acetylxylan showed different mode of action of all three xylanases of S. commune. Known XynA generated products typical for family 11 of glycoside hydrolase – aldopentaouronic acid from glucuronoxylan and isomeric xylotetraose from rhodymenan. XynB released fragments by one xylopyranosyl unit shorter – aldotetraouronic acid MeGlcA1-2Xylβ1-4Xylβ1-4Xyl from glucuronoxylan and isomeric xylotriose from rhodymenan, products usually generated by xylanases from glycoside hydrolase family 10. XynC liberated aldotetraouronic acid Xylβ-1,4-(MeGlcA-1,2-)Xylβ-1,4-Xyl with glucuronoyl unit attached to the middle xylopyranosyl unit from glucuronoxylan and isomeric xylotetraose from rhodymenan. XynC was also able to release xylose from the reducing end of aldotetraouronic acid MeGlcA1-2Xylβ1-4Xylβ1-4Xyl.     

Purification and characterization of a thermostable intracellular β-xylosidase from the thermophilic fungus Sporotrichum thermophile

An intracellular β-xylosidase from the thermophilic fungus Sporotricum thermophile strain ATCC 34628 was purified to homogeneity by Q-Sepharose and Mono-Q column chromatographies. The protein properties correspond to molecular mass and pI values of 45 kDa and 4.2, respectively. The enzyme is optimally active at pH 7.0 and 50 °C. The purified β-xylosidase is fully stable at pH 6.0–8.0 and temperatures up to 50 °C and retained over 58% of its activity after 1 h at 60 °C. The enzyme hydrolyzes β-1,4-linked xylo-oligosaccharides with chain lengths from 2 to 6, releasing xylose from the non-reducing end, but is inactive against xylan substrates. The apparent K_m and V_max values from p-nitrophenyl β-d-xylopyranoside are 1.1 mM and 114 μmol p-nitrophenol min⁻¹ mg⁻¹, respectively. Alcohols inactivate the enzyme, ethanol at 10% (v/v) yields a 30% decrease of its activity. The enzyme is irreversibly inhibited by 2,3-epoxypropyl β-d-xylobioside while alkyl epoxides derived from d-xylose were not inhibitors of the enzyme. The enzyme catalyses the condensation reaction using high donor concentration, up to 60% (w/v) xylose.