Production of galacto-oligosaccharide from lactose by Sterigmatomyces elviae CBS8119.

Our stock cultures were screened for microorganisms that can produce galacto-oligosaccharide (Gal-OS) from lactose. Of the 574 strains of bacteria and yeasts tested, Sterigmatomyces elviae CBS8119, Rhodotorula minuta IFO879, and Sirobasidium magnum CBS6803 were found to be efficient producers of Gal-OS from lactose and S. elviae CBS8119 was selected as a representative, high-level producing strain. With toluene-treated resting S. elviae CBS8119 cells, 135 mg of Gal-OS per ml was produced from 360-mg/ml lactose. During this reaction, the by-product glucose was found to inhibit Gal-OS production. Therefore, in order to remove the glucose from the reaction mixture, a culture method in which cell growth followed the enzymatic reaction was devised, which increased the yield of Gal-OS considerably because of the consumption of glucose for cell growth. Under such conditions, 232 mg of Gal-OS per ml was produced from 360-mg/ml lactose after incubation at 30 degrees for 60 h. The structure of the major product was identified as O-beta-D-galactopyranosyl-(1-->4)-O-beta-D-galactopyranosyl-(1-->4)-D- glucopyranose (4'-galactosyl-lactose) by 13C nuclear magnetic resonance spectroscopy.     

Production of Galacto-Oligosaccharide from Lactose by Sterigmatomyces elviae CBS8119

Volume 61, no. 11, p. 4022, column 2, line 10: "K(inf2)HSO(inf4)" and "KH(inf2)SO(inf4)" should read "K(inf2)HPO(inf4)" and "KH(inf2)PO(inf4)," respectively. Line 13: "K(inf2)HSO(inf4)" should read "K(inf2)HPO(inf4)." Line 14: "KH(inf2)SO(inf4)" and "0.2 g of CaCO(inf3)" should read "KH(inf2)PO(inf4)" and "1 g of CaCO(inf3)," respectively. [This corrects the article on p. 4022 in vol. 61.].     

Purification and Properties of a Novel Thermostable Galacto-Oligosaccharide-Producing (beta)-Galactosidase from Sterigmatomyces elviae CBS8119

Volume 61, no. 11, p. 4028, column 1, line 37: "p-nitrophenyl-(beta)-d-galactopyranoside" should read "p-nitrophenyl-(beta)-d-glucopyranoside." Page 4028, column 1, line 38: "p-nitrophenyl-(beta)-l-arabinopyranoside" should read "p-nitrophenyl-(alpha)-l-arabinopyranoside." Page 4028, column 2, Table 2, last line: "PNP-(beta)-l-arabinopyranoside" should read "PNP-(alpha)-l-arabinopyranoside." Page 4028, column 2, Table 2, footnote a, line 4: "PNP-(alpha)-l-arabinopyranoside" should read "PNP-(beta)-l-arabinopyranoside." [This corrects the article on p. 4026 in vol. 61.].     

Galacto-oligosaccharide production using a recycling cell culture of Sterigmatomyces elviae CBS8119

Addition of small amounts of Fe²⁺, Zn²⁺, Cu²⁺ and thiamine-HCl to the culture medium was required for promoting the galacto-oligosaccharide (Gal-OS)-producing activity of Sterigmatomyces elviae CBS8119, when the concentration of yeast extract in the medium was lowered to 0·1 g l⁻¹. Galacto-oligosaccharide production using a recycling cell culture was performed in a medium containing 360 mg ml⁻¹ of lactose supplemented with optimal concentrations of Fe²⁺ (1·5 mg l⁻¹ of FeSO₄.7H₂O), Zn²⁺ (15 mg l⁻¹ of ZnSO₄.7H₂O), Cu²⁺ (0·5 mg l⁻¹ of CuSO₄.5H₂O) and thiamine-HCl (1 mg l⁻¹ ) . Galacto-oligosaccharide production was maintained at high levels during six cycles of production, with the amount of Gal-OS produced in each cycle being more than 216 mg ml⁻¹ (weight yield of more than 60%).     

Purification and characterization of a novel thermostable lipase from Pseudomonas cepacia.

A thermostable lipase from Pseudomonas cepacia has been purified to homogeneity as judged by SDS-PAGE and isoelectric focusing. The purification included treatment of the culture supernatant with acrinol, hydrophobic interaction chromatography, and gel filtration. The enzyme was a monomeric protein with M(r) of 36,500 and pI of 5.1. The optimal pH at 50 degrees C and optimal temperature at pH 6.5 were 5.5-6.5 and 55-60 degrees C, respectively, when olive oil was used as the substrate. Simple triglycerides of short and middle chain fatty acids (C < or = 12) were the preferred substrates over those of long chain fatty acids. The enzyme cleaved all the ester bonds of triolein, with some preference for the 1,3-ester bonds. The enzyme retained all its activity even after incubation at 75 degrees C (pH 6.5) for 30 min. Further, the activity was not impaired during 21 h storage at pH 6.5 in 40% water-miscible solvents including methanol, ethanol, acetone, acetonitrile, dimethylformamide, dimethylsulfoxide, and dioxane. The addition of dimethylsulfoxide or acetone to the assay mixture in the range of 0-35% stimulated the enzyme, whereas benzene or n-hexane had an inhibitory effect. These properties together with the N-terminal amino acid sequence confirmed that the enzyme differs from the known Pseudomonas sp. lipases.     

Purification and characterization of a highly thermostable novel pullulanase from Clostridium thermohydrosulfuricum.

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.     

Purification and characterization of a novel thermostable lipase from Bacillus sp

A thermostable lipase from Bacillus sp. has been purified to homogeneity as judged by disc-PAGE, SDS-PAGE, and isoelectric focusing. The purification included ammonium sulfate fractionation, treatment with acrinol, and sequential column chromatographies on DEAE-Sephadex A-50, Toyopearl HW-55F, and Butyl Toyopearl 650M. The purified enzyme was found to be a monomeric protein with Mr of 22,000, and pI of 5.1. The optimal pH at 30 degrees C, and optimal temperature at pH 5.6 were 5.5-7.2, and 60 degrees C, respectively, when olive oil was used as the substrate. The substrate specificity towards simple triglycerides was broad and 1- and 3-positioned ester bonds were hydrolyzed in preference to a 2-positioned ester bond. The addition of acetone to the assay mixture in the range of 0-60% (v/v) stimulated the enzyme remarkably, whereas n-hexane had an inhibitory effect.     

Purification and properties of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520.

A chitinase was purified from the culture filtrate of Streptomyces thermoviolaceus OPC-520. The enzyme showed a high optimum temperature (70 to 80 degrees C), a high optimum pH level (8.0 to 10.0), and heat stability. This enzyme showed high sequence homology with chitinases from Serratia marcescens QMB1466 and Bacillus circulans WL-12.     

Purification and characterization of a thermostable beta-galactosidase from kidney beans (Phaseolus vulgaris L.) cv. PDR14

Using five different steps, beta-Galactosidase has been purified from kidney beans to apparent electrophoretic homogeneity with approximately 90-fold purification with a specific activity of 281 units mg-1 protein. A single band was observed in native PAGE. Activity staining of the native gel with 5-bromo 4-chloro 3-indoxyl beta-D-galactopyranoside (X-Gal) at pH 4.0 also produced a single band. Analytical gel filtration in Superdex G-75 revealed the molecular mass of the native protein to be approximately 75 kD. 10% SDS-PAGE under reducing conditions showed two subunits of molecular masses, 45 and 30 kD, respectively. Hence, beta-galactosidase from kidney beans is a heterodimer. A typical protein profile with lambda max at 280 nm was observed and A280/A260 ratio was 1.52. The N-terminal sequence of the 45 kD band showed 86% sequence homology with an Arabidopsis thaliana and 85% with Lycopersicon esculentum putative beta-galactosidase sequences. The Electrospray Mass Spectrometric analysis of this band also revealed a peptide fragment that had 90% sequence homology with an Arabidopsis thaliana putative beta-galactosidase sequence. The N-terminal sequencing of the 30 kD band as well as mass spectrometric analysis both by MALDI-TOF and ES MS revealed certain sequences that matched with phytohemagglutinin of kidney beans. The optimum pH of the enzyme was 4.0 and it hydrolysed o- and p-nitrophenyl beta-D galactopyranoside with a Km value of 0.63 mmol/L and 0.74 mmol/L, respectively. The energy of activation calculated from the Arrhenius equation was 14.8 kcal/mol enzyme site. The enzyme was found to be comparatively thermostable showing maximum activity at 67 degrees C. Thermal denaturation of the enzyme at 65 degrees C obeys single exponential decay with first order-rate constant 0.105 min-1. Galactose, a hydrolytic product of this enzyme was a competitive inhibitor with a Ki of 2.7 mmol/L.     

Thermostable beta-galactosidase from the archaebacterium Sulfolobus solfataricus. Purification and properties

A thermophilic and thermostable beta-galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Sulfolobus solfataricus, by a procedure including ion-exchange and affinity chromatography. The homogeneous enzyme had a specific activity of 116.4 units/mg at 75 degrees C with o-nitrophenyl beta-galactopyranoside as substrate. Molecular mass studies demonstrated that the S. solfataricus beta-galactosidase was a tetramer of 240 +/- 8 kDa composed of similar or identical subunits. Comparison of the amino acid composition of beta-galactosidase from S. solfataricus with that from Escherichia coli revealed a lower cysteine content and a lower Arg/Lys ratio in the thermophilic enzyme. A rabbit serum, raised against the homogeneous enzyme did not cross-react with beta-galactosidase from E. coli. The enzyme, characterized for its reaction requirements and kinetic properties, showed a thermostability and thermophilicity notably greater than those reported for beta-galactosidases from other mesophilic and thermophilic sources.     

Purification and properties of beta-galactosidase from chicken seminal plasma

1. beta-Galactosidase (EC 3.2.1.23) from chicken seminal plasma was purified approx. 111-fold to homogeneity. 2. pH optimum of the enzyme ranged from 3.6 to 4.0 and its Km was 0.65 mM with p-nitrophenyl-beta-D-galactoside as substrate. 3. The enzyme was unstable at its optimal activity pH and was activated by Cl- ions. 4. The enzyme had pI value of 4.0. 5. The active enzyme had Mr approx. 100,000 by Sephacryl S-300 chromatography. SDS electrophoresis in the presence of beta-mercaptoethanol showed four bands corresponding to Mr of approx. 90,000, 75,000, 65,000 and 13,000.     

Characterization of a thermostable recombinant beta-galactosidase from Thermotoga maritima

AIMS: Characterization of a thermostable recombinant beta-galactosidase from Thermotoga maritima for the hydrolysis of lactose and the production of galacto-oligosaccharides. METHODS AND RESULTS: A putative beta-galactosidase gene of Thermotoga maritima was expressed in Escherichia coli as a carboxyl terminal His-tagged recombinant enzyme. The gene encoded a 1100-amino acid protein with a calculated molecular weight of 129,501. The expressed enzyme was purified by heat treatment, His-tag affinity chromatography, and gel filtration. The optimum temperatures for beta-galactosidase activity were 85 and 80 degrees C with oNPG and lactose, respectively. The optimum pH value was 6.5 for both oNPG and lactose. In thermostability experiments, the enzyme followed first-order kinetics of thermal inactivation and its half-life times at 80 and 90 degrees C were 16 h and 16 min, respectively. Mn2+ was the most effective divalent cation for beta-galactosidase activity on both oNPG and lactose. The Km and Vmax values of the thermostable enzyme for oNPG at 80 degrees C were 0.33 mm and 79.6 micromol oNP min(-1) mg(-1). For lactose, the Km and Vmax values were dependent on substrate concentrations; 1.6 and 63.3 at lower concentrations up to 10 mm of lactose and 27.8 mm and 139 micromol glucose min(-1) mg(-1) at higher concentrations, respectively. The enzyme displayed non-Michaelis-Menten reaction kinetics with substrate activation, which was explained by simultaneous reactions of hydrolysis and transgalactosylation. CONCLUSIONS: The results suggest that the thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galacto-oligosaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge of hydrolysis and transgalactosylation performed by beta-galactosidase of hyperthermophilic bacteria.     

Purification and some properties of thermostable invertase from wine

Invertase from a white table wine made from Semillon grapes was purified to homogeneity on polyacrylamide gel electrophoresis. The enzymatic and physicochemical properties of the enzyme were compared with those of invertase purified from Semillon grape juice. The invertases from the two sources showed similar properties, suggesting that the wine invertase originated from the juice and was stable during the vinification and aging processes.     

Purification and some properties of a thermostable DNA polymerase from a Thermotoga species

A stable DNA polymerase (EC 2.7.7.7) has been purified from the extremely thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1 by a five-step purification procedure. First, the crude extract was treated with polyethylenimine to precipitate nucleic acids. The endonuclease activity coprecipitated. DEAE-Sepharose, CM-Sephrarose, and hydroxylapatite column chromatography were used to purify the preparation. As a final step on a small scale, preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used. The purified DNA polymerase exhibited a molecular weight of 85,000, as determined by both SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography. Its pH optimum was in the range pH 7.5-8. When assayed over the temperature range 30-80 degrees C, the maximum activity in a 30-min assay was at 80 degrees C. The enzyme was moderately thermostable and exhibited half-lives of 3 min at 95 degrees C and 60 min at 50 degrees C in the absence of substrate. Several additives such as Triton X-100 enhanced thermostability. During storage at 4 degrees C and -70 degrees C, the stability of the enzyme was improved by the addition of gelatin.     

疏绵状嗜热丝孢菌热稳定几丁质酶的纯化及其性质研究

采用硫酸铵沉淀、DEAE SepharoseFastFlow阴离子层析、Phenyl Sepharose疏水层析等步骤获得了凝胶电泳均一的疏绵状嗜热丝孢菌 (Thermomyceslanuginosus)几丁质酶。经SDS PAGE和凝胶过滤层析测得纯酶蛋白的分子量在 4 8～ 4 9 .8kD之间。该酶反应的最适温度和最适pH分别为 5 5℃和 4 5 ,在pH4 5条件下 ,该酶在 5 0℃以下稳定 ;6 5℃的半衰期为 2 5min ;70℃保温 2 0min后 ,仍保留 2 4 %的酶活性。其N 端氨基酸序列为AQGYLSVQYFVNWAI。金属离子对几丁质酶的活性影响较大 ,Ca2 、Na 、K 、Ba2 对酶有激活作用 ;Ag 、Fe2 、Cu2 、Hg2 对酶有显著的抑制作用 ;以胶体几丁质为底物的Km 和Vmax值分别为 9 .5 6mg mL和 2 2 . 12 μmol min。抗菌活性显示 ,该酶对供试病原菌有不同程度的抑制作用。     

Purification and some properties of a thermostable acid esterase from Acidiphilium sp. AIU 409

Extracellular and cell-bound esterases produced by Acidiphilium sp. AIU 409 were homogeneously purified from culture broth and cells, respectively, and some properties were investigated. Both esterases more rapidly hydrolyzed p-nitrophenyl acyl esters containing long-chain fatty acids from C 8:0 to C 18:0 than those containing short-chain fatty acids from C 2:0 to C 6:0. The Km values for p-nitrophenyl long-chain fatty acid esters from C 8:0 to C 18:0 were approximately 1.3-1.5 mM. The enzymes were stable at 50 degrees C for 2 days between pH 3.0 and 6.5, and optimum pH and temperature were 5.0 and 70 degrees C, respectively. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride and SDS. The molecular mass of both enzymes was estimated to be approximately 64 kDa by SDS-PAGE. The 23 amino acid sequence from the NH(2)-terminus was also the same in both enzymes. These results suggest that extracellular esterase might be composed of the same components as cell-bound esterase.     

Purification and properties of Streptococcus lactis beta-galactosidase

beta-Galactosidase of Streptococcus lactis 7962 was partially purified, and its properties were studied. Enzyme from only this strain of numerous lactic streptococci tested was stable in cell exudates prepared by various means. Cell-free extracts of the 7962 strain were prepared by sonic treatment of washed cells previously grown in presence of lactose to fully induce enzyme synthesis. Protamine sulfate precipitation of the nucleic acids and ammonium sulfate precipitation of protein were used for partial purification of the enzyme. The resulting enzyme, when resuspended in cold (5 C) phosphate buffer, was extremely labile. However, ammonium sulfate in high concentrations (0.85 m) stabilized and stimulated beta-galactosidase activity. Sephadex G-200 gel filtration was used to achieve further purification and to monitor homogeneity of the enzyme. Separation of the beta-galactosidase in buffer at 5 C yielded an enzyme elution pattern showing two peaks of activity. However, addition of the enzyme solution in 0.85 m ammonium sulfate to the column equilibrated with the same salt concentration yielded only one peak of enzyme activity. The data suggested that the native enzyme was dissociating into active subunits which were stabilized in the presence of the ammonium sulfate.     

Purification and properties of 4-hydroxybenzoate 1-hydroxylase (decarboxylating), a novel flavin adenine dinucleotide-dependent monooxygenase from Candida parapsilosis CBS604.

A novel flavoprotein monooxygenase, 4-hydroxybenzoate 1-hydroxylase (decarboxylating), from Candida parapsilosis CBS604 was purified to apparent homogeneity. The enzyme is induced when the yeast is grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, or 3,4-dihydroxybenzoate as the sole carbon source. The purified monooxygenase is a monomer of about 50 kDa containing flavin adenine dinucleotide as weakly bound cofactor. 4-Hydroxybenzoate 1-hydroxylase from C. parapsilosis catalyzes the oxidative decarboxylation of a wide range of 4-hydroxybenzoate derivatives with the stoichiometric consumption of NAD(P)H and oxygen. Optimal catalysis is reached at pH 8, with NADH being the preferred electron donor. By using (18)O2, it was confirmed that the oxygen atom inserted into the product 1,4-dihydroxybenzene is derived from molecular oxygen. 19F nuclear magnetic resonance spectroscopy revealed that the enzyme catalyzes the conversion of fluorinated 4-hydroxybenzoates to the corresponding hydroquinones. The activity of the enzyme is strongly inhibited by 3,5-dichloro-4-hydroxybenzoate, 4-hydroxy-3,5-dinitrobenzoate, and 4-hydroxyisophthalate, which are competitors with the aromatic substrate. The same type of inhibition is exhibited by chloride ions. Molecular orbital calculations show that upon deprotonation of the 4-hydroxy group, nucleophilic reactivity is located in all substrates at the C-1 position. This, and the fact that the enzyme is highly active with tetrafluoro-4-hydroxybenzoate and 4-hydroxy-3-nitrobenzoate, suggests that the phenolate forms of the substrates play an important role in catalysis. Based on the substrate specificity, a mechanism is proposed for the flavin-mediated oxidative decarboxylation of 4-hydroxybenzoate.