Identification and characterization of a novel inulin binding module (IBM) from the CFTase of Bacillus macerans CFC1

A novel inulin-binding module (IBM), which was identified from the N-terminal region of the cycloinulinooligosaccharide fructanotransferase (CFTase) in Bacillus macerans CFC1, was characterized using the discrete entity of IBM produced by the recombinant Escherichia coli strains. Deletion analyses located the inulin binding activity in the N-terminal region between 241 and 389 amino acid residues, which was removed from the mature enzyme by processing when secreted from the B. macerans CFC1 cells. IBM bound specifically to polyfructans such as inulin and levan but it did not interact with any of the glycan polymers tested in this study including cellulose, xylan, and starch. Binding studies on the IBM revealed that the equilibrium dissociation constant K(d) and the maximum amount of protein bound [(PC)(max)] were 4.7 microM and 22 microM g(-1), respectively. Together, these results indicate that the IBM of CFTase has a relatively high and specific affinity for inulin. Adsorption of the IBM to inulin was highest at pH 7.0 and lowered slowly with decreasing pH down to 3.0. At pH 7.0, the binding activity was enhanced about twofold by the presence of 1 M MgCl(2). Chemical modification experiments with the aromatic amino acid-specific modifiers implied that tryptophan and tyrosine residues in the IBM are likely to participate in the interaction with the inulin molecules.     

Characterization of cyclofructans from inulin by Saccharomyces cerevisiae Strain displaying cell-surface cycloinulooligosaccharide fructanotransferase

The cycloinulooligosaccharide fructanotransferase (CFTase) gene (cft) from Paenibacillus macerans (GenBank access code AF222787) was expressed on the cell surface of Saccharomyces cerevisiae by fusing with Aga2p linked to the membrane-anchored protein Aga1p. The surface display of CFTase was confirmed by immunofluorescence microscopy and enzymatic assay. The optimized reaction conditions of surface-displayed CFTase were as follows; pH, 8.0; temperature, 50 degrees C; enzyme amount, 30 milliunit; substrate concentration, 5%; inulin source, Jerusalem artichoke. As a result of the reaction with inulin, cycloinulohexaose was produced as a major product along with cycloinuloheptaose and cycloinulooctaose as minor products.     

类芽孢杆菌环果寡糖糖基转移酶的分离纯化和酶学性质

采用硫铵沉淀、疏水层析和DEAE离子交换层析对产自类芽孢杆菌Paenibacillus sp.Lfos16的环果寡糖糖基转移酶(Cycloinulooligosaccharide Fructanotransferase,CFTase)进行分离纯化,得到电泳纯的CFTase,最终纯化倍数为13. 9,比活力为33. 3 U/mg。纯化的CFTase经SDSPAGE和Native-PAGE电泳分析得出其相对分子量大小约为120 000,且是双亚基蛋白。探究了CFTase的酶学性质,最适反应条件为:40℃～45℃,p H 7. 0;温度稳定范围为30℃～45℃,p H稳定范围为5. 0～9. 0。并对CFTase降解菊糖的催化机制进行了分析,初步确定为外切加环化作用形成环果寡糖。     

Role of glutamate dehydrogenase in ammonia assimilation in nitrogen-fixing Bacillus macerans.

Pathways of ammonia assimilation into glutamic acid in Bacillus macerans were investigated by measurements of the specific activities of glutamate dehydrogenase (GDH), glutamine synthetase, and glutamate synthase. In ammonia-rich medium, GDH was the predominant pathway of ammonia assimilation. In nitrogen-fixing cells in which the intracellular NH4+ concentration was 1.4 +/- 0.5 mM, the activity of GDH with a Km of 2.2 mM for NH4+ was found to be severalfold higher than that of glutamate synthase. The result suggests that GDH plays a significant role in the assimilation of NH4+ in N2-fixing B. macerans.     

环果寡糖果糖基转移酶研究进展

环果寡糖果糖基转移酶(CFT酶)是一种多功能型酶,能催化三种转糖基反应(环化、歧化和耦合)以及水解反应,而其中能将菊糖水解为环果寡糖的环化反应是其特征性反应。环果寡糖的独特结构使其在医药、化工等领域具有潜在应用价值。简要论述了环果寡糖的结构和应用,重点论述了环果寡糖果糖基转移酶的酶学性质、基因克隆和表达,以及功能和催化机理等方面的最新研究进展。     

Purification and some properties of cycloinulo-oligosaccharide fructanotransferase from Bacillus circulans OKUMZ 31B

Cycloinulo-oligosaccharide fructanotransferase was purified from the cultured medium of Bacillus circulans OKUMZ 31B, to electrophoretic homogeneity, by anion-exchange column chromatography on DEAE-Toyopearl 650M, hydrophobic column chromatography on Butyl-Toyopearl 650M, gel-filtration column chromatography on Sephacryl S-2000HR and anion-exchenge column chromatography on SuperQ-Toyopearl 650M. The enzyme has a molecular weight of 132 000 and a pI of 4.1. The enzyme was most active at pH 7.5 and 40°C, and was stable at pH 6.0–9.0 and below 40°C. The enzyme catalyses the conversion of inulin into cycloinulohexaose and cycloinuloheptaose in the ratio of ca. 4:1, and a small amount of cycloinulo-octaose. The enzyme has an isoform which may be a proteolyticaly modified species of the CFTase because of its reduced molecular weight, 126 000.     

Partial purification and some properties of homoserine O-acetyltransferase of a methionine auxotroph of Saccharomyces cerevisiae.

A wild-type strain and six methionine auxotrophs of Saccharomyces cerevisiae were cultured in a synthetic medium supplemented with 0.1 mM L-cysteine or L-methionine and analyzed for the synthesis of homoserine O-acetyltransferase (EC 2.3.1.31). Among them, four mutant strains exhibited enzyme activity in cell extracts. Methionine added to the synthetic medium at concentrations higher than 0.1 mM repressed enzyme synthesis in two of these strains. The enzyme was partially purified (3,500-fold) from an extract of a mutant strain through ammonium sulfate fractionation and chromatography on columns of DEAE-cellulose, Phenyl-Sepharose C1-4B, and Sephadex G-150. The enzyme exhibited optimal pH at 7.5 for activity and at 7.8 for stability. The reaction product was ascertained to be O-acetyl-L-homoserine by confirming that it produced L-homocysteine in an O-acetyl-L-homoserine sulfhydrylase reaction. The Km for L-homoserine was 1.0 mM, and for acetyl coenzyme A it was 0.027 mM. The molecular weight of the enzyme was estimated to be approximately 104,000 by Sephadex G-150 column chromatography and 101,000 by sucrose density gradient centrifugation. The isoelectric point was at pH 4.0. Of the hydroxy amino acids examined, the enzyme showed reactivity only to L-homoserine. Succinyl coenzyme A was not an acyl donor. In the absence of L-homoserine, acetyl coenzyme A was deacylated by the enzyme, with a Km of 0.012 mM. S-Adenosylmethionine and S-adenosylhomocysteine slightly inhibited the enzyme, but methionine had no effect.     

The probiotic Lactobacillus johnsonii NCC 533 produces high-molecular-mass inulin from sucrose by using an inulosucrase enzyme

Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. The probiotic bacterium Lactobacillus johnsonii strain NCC 533 possesses a single fructansucrase gene (open reading frame AAS08734) annotated as a putative levansucrase precursor. However, (13)C nuclear magnetic resonance (NMR) analysis of the fructan product synthesized in situ revealed that this is of the inulin type. The ftf gene of L. johnsonii was cloned and expressed to elucidate its exact identity. The purified L. johnsonii protein was characterized as an inulosucrase enzyme, producing inulin from sucrose, as identified by (13)C NMR analysis. Thin-layer chromatographic analysis of the reaction products showed that InuJ synthesized, besides the inulin polymer, a broad range of fructose oligosaccharides. Maximum InuJ enzyme activity was observed in a pH range of 4.5 to 7.0, decreasing sharply at pH 7.5. InuJ exhibited the highest enzyme activity at 55 degrees C, with a drastic decrease at 60 degrees C. Calcium ions were found to have an important effect on enzyme activity and stability. Kinetic analysis showed that the transfructosylation reaction of the InuJ enzyme does not obey Michaelis-Menten kinetics. The non-Michaelian behavior of InuJ may be attributed to the oligosaccharides that were initially formed in the reaction and which may act as better acceptors than the growing polymer chain. This is only the second example of the isolation and characterization of an inulosucrase enzyme and its inulin (oligosaccharide) product from a Lactobacillus strain. Furthermore, this is the first Lactobacillus strain shown to produce inulin polymer in situ.     

Purification and characterization of the Bacillus subtilis levanase produced in Escherichia coli.

The enzyme levanase encoded by the sacC gene from Bacillus subtilis was overexpressed in Escherichia coli with the strong, inducible tac promoter. The enzyme was purified from crude E. coli cell lysates by salting out with ammonium sulfate and chromatography on DEAE-Sepharose CL-6B, S-Sepharose, and MonoQ-Sepharose. The purified protein had an apparent molecular mass of 75,000 Da in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which is in agreement with that expected from the nucleotide sequence. Levanase was active on levan, inulin, and sucrose with Km values of 1.2 microM, 6.8 mM, and 65 mM, respectively. The pH optimum of the enzyme acting on inulin was 5.5, and the temperature optimum was 55 degrees C. Levanase was rapidly inactivated at 60 degrees C, but activity could be retained for longer times by adding fructose or glycerol. The enzyme activity was completely inactivated by Ag+ and Hg2+ ions, indicating that a sulfhydryl group is involved. A ratio of sucrase to inulinase activity of 1.2 was found for the purified enzyme with substrate concentrations of 50 mg/ml. The mechanism of enzyme action was investigated. No liberation of fructo-oligomers from inulin and levan could be observed by thin-layer chromatography and size exclusion chromatography-low-angle laser light scattering-interferometric differential refractive index techniques. This indicates that levanase is an exoenzyme acting by the single-chain mode.     

A synthesis of acylphosphonic acids and of 1-aminoalkylphosphonic acids: the action of pyruvate dehydrogenase and lactate dehydrogenase on acetylphosphonic acid.

Acylphosphonic acids, R-CO-PO(OH)2, have been synthesized by the steps [formula: see text] of which the last is new and provides a mild method for de-esterifying acylphosphonic acids. Their reductive amination gives a simple way of making 1-aminoalkylphosphonic acids. Acetylphosphonic acid inhibited NAD+ reduction by pyruvate with the pyruvate dehydrogenases from Escherichia coli and Bacillus stearothermophilus. The inhibition was competitive with pyruvate, with Ki of 6 microM for the E. coli enzyme (pyruvate Km 0.5 mM) and one of 0.4 mM of the B. stearothermophilus enzyme (pyruvate Km 0.1 mM). Acetylphosphonate and its monomethyl ester are substates for pig heart lactate dehydrogenase, with Km values of 15 mM and 10 mM respectively (pyruvate Km 0.05 mM) and specificity constants one thousandth that for pyruvate.     

Purification and properties of a heat-stable exoinulinase isoform from Aspergillus fumigatus

An inducible extracellular exoinulinase (isoform II) was purified from the extracellular extract of Aspergillus fumigatus by ammonium sulphate precipitation, followed by successive chromatographies on DEAE-Sephacel, Octyl-Sepharose (HIC), Sephacryl S-200, affinity chromatography on ConA-CL Agarose and Sephacryl S-100 columns. The enzyme was purified 75-folds with 3.2% activity yield from the starting culture broth. The purified isoform II was a monomeric 62 kDa protein with a pI value of 4.5. The enzyme showed maximum activity at pH 6.0 and was stable over a pH range of 4.0-7.0, whereas the optimum temperature for enzyme activity was 60 degrees C. The inulinase isoform II showed exo-inulinolytic activity and retained 72% and 44% residual activity after 12 h at 60 degrees C and 70 degrees C, respectively. The inulin hydrolysis activity was completely abolished with 5 mM Hg2+ and Fe2+, whereas K+ and Cu2+ enhanced the inulinase activity. As compared to sucrose, stachyose and raffinose the purified enzyme had a lower Km (1.25 mM) and higher catalytic center activity (Kcat = 3.47 x 10(4) min(-1)) for inulin. As compared to exoinulinase isoform I of A. fumigatus, purified earlier, the isoform II is more thermostable and is a potential candidate for commercial production of fructose from inulin.     

Isolation and molecular kinetic properties of the angiotensin-converting enzyme from the human heart

An angiotensin-converting enzyme was isolated from human heart using N[-1(S)-carboxy-5-aminopentyl]glycyl-glycine as an affinity adsorbent. The isolation procedure resulted in an enzyme purified 1650-fold. The enzyme specific activity was 38.0 u./mg protein, Mr = 150 kD. The pH optimum for the angiotensin-converting enzyme towards Hip-His-Leu lies at 7.8, Km = 1.2 mM. The enzyme was inhibited by the substrate (Ks' = 14 mM). The enzyme effectively catalyzed the hydrolysis of angiotensin I (Km = 10 microM; kcat = 250 s-1). NaCl, CaCl2 as well as Na2SO4 in the absence of Cl- activated the enzyme, whereas CH3COONa and NaNO3 did not influence the enzyme activity. It was found that the bradykinin-potentiating factor inhibited the cardiac angiotensin-converting enzyme with IC50 = 4.0 X 10(-8) M.     

Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast

Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, [hereafter called (S)- and (R)-enzymes] have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH. The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme. Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.     

Temperature dependence of kinetic parameters for hyperthermophilic glutamate dehydrogenase from Aeropyrum pernix K1

The temperature dependence of the steady-state kinetic parameters for a glutamate dehydrogenase from Aeropyrum pernix K1 was investigated. The enzyme showed a biphasic kinetic characteristic for L-glutamate and a monophasic one for NADP at 50-90 degrees C. At low concentrations of L-glutamate the Km decreased from 2.02 to 0.56 mM and the catalytic efficiency (Vmax/Km) markedly increased (4-150 micromol x mg(-1) x mM(-1)) along with the increase of temperature from 50 to 90 degrees C. At high concentrations of the substrate the Km was fairly high and approximately constant (around 225 mM), and the catalytic efficiency was low and its temperature-dependent change was small. The Km (0.039 mM) for NADP did not change with the increase of temperature. In the reductive amination, the Kms for 2-oxoglutarate (1.81 and 9.37 mM at low and high levels of ammonia, respectively) were independent on temperature, but the Kms for ammonia and NADPH rose from 86 to 185 mM and 0.050 to 0.175 mM, respectively.     

Method for Enrichment of Rhodopseudomonas sphaeroides from Fresh Water

An inulinase was highly purified from the culture broth of Penicillium purpurogenum by chromatographies on DEAE-Sepharose CL-6B, Toyopearl HW-65, and Bio-Gel P-100. The enzyme was homogeneous by disc electrophoretic analysis. The molecular weight was 6.4 × 10⁴ by SDS-disc electrophoresis and gel filtration on Bio-Gel P-150. The isoelectric point was pH 3.6 by isoelectric focusing. The enzyme hydrolyzed inulin rapidly, but did not affect sucrose. By paper chromatography analysis, the major products from inulin were tri-, tetra-, penta-, and hexa-saccharides. The substrate specificity of the enzyme on hydrolyses of fructo-oligosaccharides[1^F(1-β-d-fructofuranosyl)n sucrose (n = 1 to 6 and n (average of polymerization degree) = 8)] were examined. The Km values and relative maximum velocities for the hydrolyses of inulin and fructo-oligosaccharides (GF_n, n = 2 to 7 and n = 9) were as follows: inulin, (DP = 35) 0.21 mM and 100; GF₉, 0.24 mM and 86.5; GF₇, 0.33 mM and 132; GF₆, 0.85 mM and 71.2; GF₅, 3.8 mM and 25.4; GF₄, 2.8 mM and 28.8; GF₃, (nystose) 16 mM and 0.8; GF₂ (1-kestose), 8.4 mM and 0.2. The molecular activities for the hydrolyses of fructo-oligosaccharides (GF_n, n = 2 to 6) were increased depending on the degree of polymerization of fructosyl residues, and were nearly constant if the polymerization degree was over seven. These results strongly suggested that the endo-type inulinase from Penicillium purpurogenum had a subsite structure consisting of at least seven subsites.     

Recombinant thermostable cycloinulo-oligosaccharide fructanotransferase produced by Saccharomyces cerevisiae.

A truncated fragment of the cycloinulo-oligosaccharide fructanotransferase (CFTase) gene of Bacillus circulans MCI-2554 was fused to the prepro secretion sequence of the alpha-factor and expressed in Saccharomyces cerevisiae under the control of the 5' upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL). Efficiently secreted recombinant CFTase protein (yeast CFTase) was purified. Yeast CFTase consisted of three protein molecules, each of which had CFTase activity (yeast CFTase 1 [116 kDa], yeast CFTase 2 [117 kDa], and yeast CFTase 3 [116 kDa]). Yeast CFTase 2 was the major product of the expression system employed and was shown to be N glycosylated by endoglycosidase H treatment. Yeast CFTase 1 was N glycosylated but had a short truncation at its N terminus, while yeast CFTase 3 did not contain an N-glycosylated carbohydrate chain(s). Yeast CFTase 2 showed an optimum pH, an optimum temperature, and a pH stability similar to those of CFTase purified from B. circulans but exhibited a significant increase in thermostability. Production of yeast CFTase by the strain which had two copies of the CFTase gene integrated into its chromosomes reached 391 U per liter of culture at 120 h, which corresponded to 8.40 mg of protein per liter, by shake-flask cultivation.     

Characteristics of an inulinase produced by Bacillus subtilis 430A, a strain isolated from the rhizosphere of Vernonia herbacea (Vell Rusby).

Bacillus subtilis 430A, isolated from the Vernonia herbacea (Vell Rusby) rhizosphere, produced an exocellular inulinase that fits the requirements for the production of syrups on an industrial scale. The partially purified enzyme, obtained by acetone precipitation, displayed a higher specificity for inulin (Km, 8 mM) than for sucrose (56 mM) and a total invertase/total inulase ratio of 0.62. In addition, it is stable at an optimal temperature of 45 to 50 degrees C for at least 7 h and is inhibited by the end product, fructose, at 14 mM.     

Characteristics of an inulinase produced by Bacillus subtilis 430A, a strain isolated from the rhizosphere of Vernonia herbacea (Vell Rusby).

Bacillus subtilis 430A, isolated from the Vernonia herbacea (Vell Rusby) rhizosphere, produced an exocellular inulinase that fits the requirements for the production of syrups on an industrial scale. The partially purified enzyme, obtained by acetone precipitation, displayed a higher specificity for inulin (Km, 8 mM) than for sucrose (56 mM) and a total invertase/total inulase ratio of 0.62. In addition, it is stable at an optimal temperature of 45 to 50 degrees C for at least 7 h and is inhibited by the end product, fructose, at 14 mM.     

Properties of purified L-ornithine decarboxylase (EC 4.1.1.17) from Tetrahymena thermophila

Ornithine decarboxylase (ornithine carboxy lyase; EC 4.1.1.17) (ODC) from Tetrahymena thermophila was purified 6,300 fold employing fractionated ammonium sulfate precipitation, gel permeation chromatography on Sephadex G-150, ion exchange chromatography on DEAE-Sepharose CL-6B, and preparative isoelectric focussing. The product obtained in 24% yield was a preparation of the specific activity of 10,200 nmol CO2.h-1.mg-1. The purified enzyme was rather stable at 37 degrees C (14% loss of activity within 1 h). The molecular and catalytic properties of this enzyme were investigated. The isoelectric point was 5.7 and the molecular weight (MW) was estimated to be 68,000 under nondenaturing conditions. The pH optimum was between 6.0 and 7.0, the Km for the substrate L-ornithine was 0.11 mM, and the Km for the cofactor pyridoxal 5-phosphate was 0.12 microM; the product of ODC catalysis, putrescine, was a poor inhibitor with an estimated Ki of about 10 mM. The enzyme was inhibited competitively by D-ornithine with a Ki of 1.6 mM and by alpha-difluoromethylornithine with a Ki of 0.15 mM. The latter one, an enzyme activated irreversible inhibitor of mammalian ODC, inactivated the enzyme from T. thermophila at high concentrations with a half life time of 14 min. Other basic amino acids, e.g. L-lysine, L-arginine, and L-histidine, were neither substrates nor inhibitors of the enzyme, as were the diamines 1,3-diaminopropanol and cadaverine, the polyamines spermidine and spermine and the cosubstrate analogues pyridoxal and pyridoxamine-5-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of particulate malate dehydrogenase and phosphoenolpyruvate carboxykinase from Leishmania mexicana mexicana

The particulate activities of Leishmania mexicana mexicana amastigote malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) have been purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5'AMP-Sepharose 4B, and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. Phosphoenolpyruvate carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SDS-gel electrophoresis were 39 800 and 33 300 for malate dehydrogenase and 63 100 and 65 100 for phosphoenolpyruvate carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km(NADH) of 41 microM and a Km(oxaloacetic acid) of 39 microM. For malate oxidation there was a Km(malate) of 3.6 mM and a Km(NAD) of 0.79 mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than 0.83 mM and malate was found to be a product inhibitor at high concentrations. However, there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors, suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts; nevertheless, their apparent importance and unique subcellular organization in the parasite make them potential targets for chemotherapeutic attack.