Characterization of a thermostable L-arabinose (D-galactose) isomerase from the hyperthermophilic eubacterium Thermotoga maritima

The araA gene encoding L-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni(2+) affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90 degrees C and pH 7.5 under the assay conditions used. Its apparent K(m) values for L-arabinose and D-galactose were 31 and 60 mM, respectively; the apparent V(max) values (at 90 degrees C) were 41.3 U/mg (L-arabinose) and 8.9 U/mg (D-galactose), and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 74.8 mM(-1).min(-1) (L-arabinose) and 8.5 mM(-1).min(-1) (D-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn(2+) and/or Co(2+) than in the absence of these ions. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield of 56% for 6 h at 80 degrees C.     

Purification and partial characterization of an intracellular aminopeptidase from Streptococcus salivarius subsp. thermophilus strain ACA-DC 114.

An intracellular aminopeptidase from Streptococcus salivarius subsp. thermophilus strain ACA-DC 114, isolated from traditional Greek yoghurt, was purified by chromatography on DEAE-cellulose and Sephadex G-100. The enzyme had a molecular weight of 89,000. It was active over a pH range 4.5-9.5 and had optimum activity on L-lysyl-4-nitroanilide at pH 6.5 and 35 degrees C with Km = 1.80 mmol/l; above 55 degrees C the enzyme activity declined rapidly. The aminopeptidase was capable of degrading substrates by hydrolysis of the N-terminal amino acid; it had very low endopeptidase and no carboxypeptidase activity. The enzyme was strongly inactivated by EDTA. Serine and sulphydryl group reagents had no effect on enzyme activity.     

极端嗜热菌Thermus thermophilus HB8中天冬氨酸转氨酶在大肠杆菌中的表达、纯化及酶学性质研究

为获得具有热稳定性的天冬氨酸转氨酶,从极端嗜热细菌Thermus thermophilus HB8中克隆得到天冬氨酸转氨酶基因aspC,并在大肠杆菌BL21(DE3)和Rosetta(DE3)中进行表达,发现在Rosetta(DE3)中具有较高的表达量。重组酶的最适反应pH是7.0,37℃下在pH8～10的缓冲液中保温1h酶活几乎不改变。重组酶反应的最适温度为75℃,酶活稳定的温度范围为25～55℃。重组酶在65℃时半衰期为3.5h,75℃时为2.5h。重组酶的KmKG为7.559mmol/L,VmaxKG为0.086mmol/(L·min),KmAsp为2.031mmol/L,VmaxAsp为0·024mmol/(L·min)。Ca2 、Fe3 、Mn2 等金属离子对酶活性有微弱抑制作用。     

Heterologous expression, purification, and characterization of recombinant rat cysteine dioxygenase

Cysteine dioxygenase (CDO, EC 1.13.11.20) catalyzes the oxidation of cysteine to cysteine sulfinic acid, which is the first major step in cysteine catabolism in mammalian tissues. Rat liver CDO was cloned and expressed in Escherichia coli as a 26.8-kDa N-terminal fusion protein bearing a polyhistidine tag. Purification by immobilized metal affinity chromatography yielded homogeneous protein, which was catalytically active even in the absence of the secondary protein-A, which has been reported to be essential for activity in partially purified native preparations. As compared with those existing purification protocols for native CDO, the milder conditions used in the isolation of the recombinant CDO allowed a more controlled study of the properties and activity of CDO, clarifying conflicting findings in the literature. Apo-protein was inactive in catalysis and was only activated by iron. Metal analysis of purified recombinant protein indicated that only 10% of the protein contained iron and that the iron was loosely bound to the protein. Kinetic studies showed that the recombinant enzyme displayed a K(m) value of 2.5 +/- 0.4 mm at pH 7.5 and 37 degrees C. The enzyme was shown to be specific for l-cysteine oxidation, whereas homocysteine inhibited CDO activity.     

Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana.

The nitrilase AtNIT1 from Arabidopsis thaliana was overexpressed in Escherichia coli with an N-terminal His6 tag and purified by zinc chelate affinity chromatography in a single step almost to homogeneity in a 68% yield with a specific activity of 34.1 U.mg-1. The native enzyme (approximately 450 kDa) consists of 11-13 subunits (38 kDa). The temperature optimum was determined to be 35 degrees C and a pH optimum of 9 was found. Thus, recombinant AtNIT1 resembles in its properties the native enzyme and the nitrilase from Brassica napus. The stability of AtNIT1 could be significantly improved by the addition of dithiothreitol and EDTA. The substrate range of AtNIT1 differs considerably from those of bacterial nitrilases. Aliphatic nitriles are the most effective substrates, showing increasing rates of hydrolysis with increasing size of the residues, as demonstrated in the series butyronitrile, octanenitrile, phenylpropionitrile. In comparison with 3-indolylacetonitrile, the rate of hydrolysis of 3-phenylpropionitrile is increased by a factor of 330, and the Km value is reduced by a factor of 23. With the exception of fluoro, substituents in the alpha position to the nitrile function completely inhibit the hydrolysis.     

Human recombinant prolidase from eukaryotic and prokaryotic sources. Expression, purification, characterization and long-term stability studies

Prolidase is a Mn(2+)-dependent dipeptidase that cleaves imidodipeptides containing C-terminal proline or hydroxyproline. In humans, a lack of prolidase activity causes prolidase deficiency, a rare autosomal recessive disease, characterized by a wide range of clinical outcomes, including severe skin lesions, mental retardation, and infections of the respiratory tract. In this study, recombinant prolidase was produced as a fusion protein with an N-terminal histidine tag in eukaryotic and prokaryotic hosts and purified in a single step using immobilized metal affinity chromatography. The enzyme was characterized in terms of activity against different substrates, in the presence of various bivalent ions, in the presence of the strong inhibitor Cbz-Pro, and at different temperatures and pHs. The recombinant enzyme with and without a tag showed properties mainly indistinguishable from those of the native prolidase from fibroblast lysate. The protein yield was higher from the prokaryotic source, and a detailed long-term stability study of this enzyme at 37 degrees C was therefore undertaken. For this analysis, an 'on-column' digestion of the N-terminal His tag by Factor Xa was performed. A positive effect of Mn(2+) and GSH in the incubation mixture and high stability of the untagged enzyme are reported. Poly(ethylene glycol) and glycerol had a stabilizing effect, the latter being the more effective. In addition, no significant degradation was detected after up to 6 days of incubation with cellular lysate. Generation of the prolidase in Escherichia coli, because of its high yield, stability, and similarity to native prolidase, appears to be the best approach for future structural studies and enzyme replacement therapy.     

Functional expression of the keratinolytic serine protease gene sfp2 from Streptomyces fradiae var. k11 in Pichia pastoris

We report the initial characterization and expression of sfp2, a gene encoding a keratinolytic serine protease from Streptomyces fradiae var. k11. Recombinant SFP2 was expressed in and secreted from the yeast Pichia pastoris with a final yield of 78 mg/L (136.2 U/mL caseinolytic activity) after 25 h of induction. The recombinant enzyme was purified using by ammonium sulfate precipitation and gel filtration chromatography to electrophoretic homogeneity, which was appropriately glycosylated and had a molecular mass of 26.0 kDa. The purified recombinant SFP2 was characterized. The optimal pHs and temperatures of SFP2 for proteolysis of casein and keratin azure were pH 10.0, 60 degrees C, and pH 9.0, 55 degrees C, respectively. SFP2 activity was stable from pH 3.0 to pH 11.0. The enzyme activity was inhibited by Co(2+) and Cr(3+) and enhanced by Ni(2+) and Cu(2+). The K(m) of 0.45 mmol/L and V(max) of 19.84 mmol/min mg were calculated using N-succinyl-Ala-Ala-Pro-Phe-pNA as a substrate. We tested the activity of SFP2 with soluble and insoluble substrates; SFP2 was more specific for keratinous substrates compared with proteinase K and other commercial proteases.     

重组大肠杆菌热稳定性过氧化氢酶的纯化及性质研究

将产热稳定性过氧化氢酶的重组大肠杆菌培养后菌体破碎得到的粗酶液经热处理、硫酸铵分级沉淀、DEAE\|Sephadex A\|50离子交换层析、HiPrep16/10 Phenyl疏水作用层析、Superdex200 HR 10/30凝胶层析提纯后得到电泳纯的酶,比酶活达到15629U/mg。此酶的最适温度为70℃,最适pH70,在60℃保温60min酶活力基本不变,在pH3～8的范围内比较稳定。此酶的Km和Vmax分别为775mmol/L和278mmol\5min\+\{-1\}·mg-1。1mmol/L的Zn2+、Ba2+、Mn2+可使该酶完全失活,KCN、NaN\-3、Na\-2S\-2O\-4、巯基乙醇对酶活力有抑制作用,50mmol/L的EDTA不影响酶活性。     

Purification and characterization of a feruloyl esterase from the intestinal bacterium Lactobacillus acidophilus

Dietary ferulic acid (FA), a significant antioxidant substance, is currently the subject of extensive research. FA in cereals exists mainly as feruloylated sugar ester. To release FA from food matrices, it is necessary to cleave ester cross-linking by feruloyl esterase (FAE) (hydroxycinnamoyl esterase; EC 3.1.1.73). In the present study, the FAE from a human typical intestinal bacterium, Lactobacillus acidophilus, was isolated, purified, and characterized for the first time. The enzyme was purified in successive steps including hydrophobic interaction chromatography and anion-exchange chromatography. The purified FAE appeared as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 36 kDa. It has optimum pH and temperature characteristics (5.6 and 37 degrees C, respectively). The metal ions Cu(2+) and Fe(3+) (at a concentration of 5 mmol liter(-1)) inhibited FAE activity by 97.25 and 94.80%, respectively. Under optimum pH and temperature with 5-O-feruloyl-L-arabinofuranose (FAA) as a substrate, the enzyme exhibited a K(m) of 0.0953 mmol liter(-1) and a V(max) of 86.27 mmol liter(-1) min(-1) mg(-1) of protein. Furthermore, the N-terminal amino acid sequence of the purified FAE was found to be A R V E K P R K V I L V G D G A V G S T. The FAE released FA from O-(5-O-feruloyl-alpha-L-arabinofuranosyl)-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) and FAA obtained from refined corn bran. Moreover, it released two times more FA from FAXX in the presence of added xylanase.     

Heterologous expression of the Saccharomyces cerevisiae PGU1 gene in Schizosaccharomyces pombe yields an enzyme with more desirable properties for the food industry

The Saccharomyces cerevisiae PGU1 gene was successfully expressed in Schizosaccharomyces pombe. The optimum pH and temperature for the recombinant enzyme were 5 and 40 degrees C, respectively, these being around 0.5 U higher and 5 degrees C lower than those shown by the native enzyme. The K(m) value was about fourfold higher than that of the S. cerevisiae enzyme. The recombinant endopolygalacturonase was more efficient in reducing the viscosity of polygalacturonic acid and was also more stable at different pHs and temperatures than the native enzyme.     

Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp.

An extracellular lipase was isolated from the cell-free broth of Bacillus sp. GK 8. The enzyme was purified to 53-fold with a specific activity of 75.7 U mg(-1) of protein and a yield of 31% activity. The apparent molecular mass of the monomeric protein was 108 kDa as estimated by molecular sieving and 112 kDa by SDS-PAGE. The proteolysis of the native molecule yields a low molecular weight component of 11.5 kDa that still retains the active site. It was stable at the pH range of 7.0-10.0 with optimum pH 8.0. The enzyme was stable at 50 degrees C for 1 h with a half life of 2 h, 40 min, and 18 min at 60, 65, and 70 degrees C, respectively. With p-nitrophenyl laurate as substrate the enzyme exhibited a K(m) and V(max) of 3.63 mM and 0.26 microM/min/ml, respectively. Activity was stimulated by Mg(2+) (10 mM), Ba(2+) (10 mM), and SDS (0.1 mM), but inhibited by EDTA (10 mM), phenylmethane sulfonyl fluoride (100 mM), diethylphenylcarbonate (10 mM), and eserine (10 mM). It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C(4) and C(18:1). Thermostability of the proteolytic fragment at 60 degrees C was observed to be 37% of the native protein. The native enzyme was completely stable in ethylene glycol and glycerol (30% v/v each) for 60 min at 65 degrees C.     

Physicochemical properties of a novel alpha-L-arabinofuranosidase from Rhizomucor pusillus HHT-1

The zygomycete fungus Rhizomucor pusillus HHT-1, cultured on L(+)arabinose as a sole carbon source, produced extracellular alpha-L-arabinofuranosidase. The enzyme was purified by (NH4)2SO4 fractionation, gel filtration, and ion exchange chromatography. The molecular mass of this monomeric enzyme was 88 kDa. The native enzyme had a pI of 4.2 and displayed a pH optimum and stability of 4.0 and 7.0-10.0, respectively. The temperature optimum was 65 degrees C, and it was stable up to 70 degrees C. The Km and Vmax for p-nitrophenyl alpha-L-arabinofuranoside were 0.59 mM and 387 micromol x min(-1) x mg(-1) protein, respectively. Activity was not stimulated by metal cofactors. The N-terminal amino acid sequence did not show any similarity to other arabinofuranosidases. Higher hydrolytic activity was recorded with pnitrophenyl alpha-L-arabinofuranoside, arabinotriose, and sugar beet arabinan; lower hydrolytic activity was recorded with oat-spelt xylan and arabinogalactan, indicating specificity for the low molecular mass L(+)-arabinose containing oligosaccharides with furanoside configuration.     

Identification and characterization of a novel ferric reductase from the hyperthermophilic Archaeon Archaeoglobus fulgidus

Archaeoglobus fulgidus, a hyperthermophilic sulfate-reducing Archaeon, contains high Fe(3+)-EDTA reductase activity in its soluble protein fraction. The corresponding enzyme, which constitutes about 0.75% of the soluble protein, was purified 175-fold to homogeneity. Based on SDS-polyacrylamide gel electrophoresis, the ferric reductase consists of a single subunit with a M(r) of 18,000. The M(r) of the native enzyme was determined by size exclusion chromatography to be 40,000 suggesting that the native ferric reductase is a homodimer. The enzyme uses both NADH and NADPH as electron donors to reduce Fe(3+)-EDTA. Other Fe(3+) complexes and dichlorophenolindophenol serve as alternative electron acceptors, but uncomplexed Fe(3+) is not utilized. The purified enzyme strictly requires FMN or FAD as a catalytic intermediate for Fe(3+) reduction. Ferric reductase also reduces FMN and FAD, but not riboflavin, with NAD(P)H which classifies the enzyme as a NAD(P)H:flavin oxidoreductase. The enzyme exhibits a temperature optimum of 88 degrees C. When incubated at 85 degrees C, the enzyme activity half-life was 2 h. N-terminal sequence analysis of the purified ferric reductase resulted in the identification of the hypothetical gene, AF0830, of the A. fulgidus genomic sequence. The A. fulgidus ferric reductase shares amino acid sequence similarity with a family of NAD(P)H:FMN oxidoreductases but not with any ferric reductases suggesting that the A. fulgidus ferric reductase is a novel enzyme.     

Purification and properties of peroxidase from Pinus pinaster needles

Peroxidase (Ec 1.11.1.7) was purified from needles of Pinus pinaster to apparent homogeneity by DE-52 cellulose chromatography with a final recovery of enzyme activity of about 85%. The purified enzyme (A402/A275 = 1.05) had a specific activity of about 948 U/mg of protein and ran as a single protein band both on SDS-PAGE and native PAGE with Mr of 37,000 and 151,000, respectively. Both native PAGE and isoelectric focusing gels of the purified enzyme were stained for activity which coincided with the protein band. The pI of the purified enzyme was found to be 3.2 by isoelectric focusing on an ultrathin polyacrylamide gel. The enzyme has an optimum pH of activity of 5.0 and temperature optimum of 30 degrees C. Stability studies of the enzyme as a function of pH and temperature suggest that it is most stable at pH 5.0 and 0-40 degrees C, respectively.     

Enzymatic properties of two beta-glucosidases from Ceriporiopsis subvermispora produced in biopulping conditions

AIMS: Ceriporiopsis subvermispora produces endoglucanase and beta-glucosidase when cultivated on cellulose or wood, but biodegradation of cellulose during biopulping by C. subvermispora is low even after long periods. To resolve this discrepancy, we grew C. subvermispora on Pinus taeda wood chips and purified the major beta-glucosidases it produced. Kinetic parameters were determined to clear if this fungus produces enzymes capable of yielding assimilable glucose from wood. METHODS AND RESULTS: Ceriporiopsis subvermispora was grown on P. taeda wood chips under solid-state fermentation. After 30 days, the crude extract obtained from enzyme extraction with sodium acetate buffer 50 mmol l(-1), pH 5.4, was filtrated in membranes with a molecular mass exclusion limit of 100 kDa. Enzyme purification was carried out using successively Sephacryl S-300 gel filtration. The retained fraction attained 76% of beta-glucosidase activity with 3.7-fold purification. Two beta-glucosidases were detected with molecular mass of 110 and 53 kDa. We have performed a characterization of the enzymatic properties of the beta-glucosidase of 110 kDa. The optimum pH and temperature were 3.5 and 60 degrees C, respectively. The K(m) and V(max) values were respectively 3.29 mmol l(-1) and 0.113 micromol min(-1) for the hydrolysis of p-nitrophenyl-beta-glucopyranoside (pNPG) and 2.63 mmol l(-1) and 0.103 micromol min(-1), towards cellobiose. beta-Glucosidase activity was strongly increased by Mn(2+) and Fe(3+), while Cu(2+) severely inhibited it. CONCLUSIONS: Ceriporiopsis subvermispora produces small amounts of beta-glucosidase when grown on wood. The gel filtration and polyacrylamide gel electrophoresis data revealed the existence of two beta-glucosidases with 110 and 53 kDa. The 110 kDa beta-glucosidase from C. subvermispora can be efficiently purified in a single step by gel filtration chromatography. The enzyme has an acid pH optimum with similar activity on pNPG and cellobiose and is thus typical beta-glucosidase. SIGNIFICANCE AND IMPACT OF THE STUDY: Ceriporiopsis subvermispora produces beta-glucosidase with limited action during wood decay making able its use for the production of biomechanical and biochemical pulps. The results presented in this paper show the importance of studying the behaviour of beta-glucosidases during biopulping.     

短双歧杆菌α-D-半乳糖苷酶基因aga1在大肠杆菌中的高效表达

短双歧杆菌（Bifidobacterium breve 203）α_D_半乳糖苷酶基因(aga1)被克隆到大肠杆菌温度诱导表达质粒pBV220中,构建重组质粒pBVaga1,转入大肠杆菌进行温度诱导表达,得到的重组酶Aga1在大肠杆菌DH5α、DH10B和BL21中的比活分别为28.08、19.44和13.85U/mg, 均高于短双歧杆菌α_D_半乳糖苷酶的比活1.76U/mg。重组质粒pBVaga1在E. coli BL21中稳定性较好。重组酶Aga1蛋白亚基分子量约67kD,最适反应温度为45℃,酶在40℃以下稳定,60℃仅剩余约5%的酶活性,70℃时酶全部失活;最适反应pH为4.0～4.4,酶在pH 3.6～6.0范围内稳定;酶对p_硝基苯酚_α_半乳糖苷的Km＝1.43mmol/L,Vmax＝35.71μmol/(L·min),对蜜二糖的Km＝261mmol/L,Vmax＝63.69μmol/(L·min);酶在蜜二糖、棉子糖水解体系中不显示转糖基活性。结果说明Aga1与已经报道的一种短双歧杆菌的α_D_半乳糖苷酶不同,是新发现的一种短双歧杆菌的α_D_半乳糖苷酶。     

Characterization of a novel zinc-containing, lysine-specific aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus

Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of L-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 +/- 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100 degrees C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100 degrees C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.     

Characterization of a thermostable extracellular beta-galactosidase from a thermophilic fungus Rhizomucor sp

An extracellular beta-galactosidase from a thermophilic fungus Rhizomucor sp. has been purified to homogeneity by successive DEAE cellulose chromatography followed by gel filtration on Sephacryl S-300. The native molecular mass of the enzyme is 250,000 and it is composed of two identical subunits with molecular mass of 120,000. It is an acidic protein with a pI of 4.2. Purified beta-galactosidase is a glycoprotein and contains 8% neutral sugar. The optimum pH and temperature for enzyme activity are 4.5 and 60 degrees C, respectively. The enzyme is stable at 60 degrees C for 4 h, and has a t(1/2) of 150 min(-1) at 70 degrees C which is one of the highest reported for fungal beta-galactosidases. Substrate specificity studies indicated that the enzyme is specific for beta-linked galactose residues with a preference for p-nitrophenyl-beta-D-galactopyranoside (pNPG). The Km and Vmax values for the synthetic substrates pNPG and o-nitrophenyl-beta-D-galactopyranoside (oNPG) were 0.66 mM and 1.32 mM; and 22.4 mmol min(-1) mg(-1) and 4.45 mmol min(-1) mg(-1), respectively, while that for the natural substrate, lactose, was 50.0 mM and 12 mmol min(-1) mg(-1). The end product galactose and the substrate analogue isopropyl thiogalactopyranoside (ITPG) inhibited the enzyme with Ki of 2.6 mM and 12.0 mM, respectively. The energy of activation for the enzyme using pNPG and oNPG were 27.04 kCal and 9.04 kCal, respectively. The active site characterization studies using group-specific reagents revealed that a tryptophan and lysine residue play an important role in the catalytic activity of the enzyme.     

The oxidized pyrimidine ribonucleotide, 5-hydroxy-CTP,is hydrolyzed efficiently by the Escherichia coli recombinant Orf135 protein

The Escherichia coli orf135 gene encodes a 15.4kDa protein with homology to the MutT family of nucleotide hydrolases. The orf135 gene was cloned within a glutathione S-transferase (GST) fusion protein expression vector, which was used to overproduce the GST-Orf135 fusion protein in E. coli. The fusion protein thus obtained was purified by affinity column chromatography and gel filtration chromatography from the crude extract. The recombinant Orf135 protein was obtained by removing the GST tag from the purified fusion protein. Various oxidized nucleotides were tested as substrates for the recombinant Orf135 protein. As a result, we found a novel 5-hydroxy-CTPase activity of Orf135, but the hydrolyzing activities for the other nucleotides, including 5-hydroxy-dCTP, were very low. The activation constant (K(a)) of Mg(2+) for the 5-hydroxy-CTPase activity was 1.2 mM, and the pH optimum was 8.5. The catalytic efficiency (k(cat)/K(m)) for this activity was 630 s(-1) mM(-1) at 30 degrees C, which was 30-fold higher than that for the CTPase activity. This result indicates that 5-hydroxy-CTP is the best substrate of Orf135 among the nucleotides tested.     

Purification and characterization of a novel mannitol dehydrogenase from Lactobacillus intermedius

Mannitol 2-dehydrogenase (MDH) catalyzes the pyridine nucleotide dependent reduction of fructose to mannitol. Lactobacillus intermedius (NRRL B-3693), a heterofermentative lactic acid bacterium (LAB), was found to be an excellent producer of mannitol. The MDH from this bacterium was purified from the cell extract to homogeneity by DEAE Bio-Gel column chromatography, gel filtration on Bio-Gel A-0.5m gel, octyl-Sepharose hydrophobic interaction chromatography, and Bio-Gel Hydroxyapatite HTP column chromatography. The purified enzyme (specific activity, 331 U/mg protein) was a heterotetrameric protein with a native molecular weight (MW) of about 170 000 and subunit MWs of 43 000 and 34 500. The isoelectric point of the enzyme was at pH 4.7. Both subunits had the same N-terminal amino acid sequence. The optimum temperature for the reductive action of the purified MDH was at 35 degrees C with 44% activity at 50 degrees C and only 15% activity at 60 degrees C. The enzyme was optimally active at pH 5.5 with 50% activity at pH 6.5 and only 35% activity at pH 5.0 for reduction of fructose. The optimum pH for the oxidation of mannitol to fructose was 7.0. The purified enzyme was quite stable at pH 4.5-8.0 and temperature up to 35 degrees C. The K(m) and V(max) values of the enzyme for the reduction of fructose to mannitol were 20 mM and 396 micromol/min/mg protein, respectively. It did not have any reductive activity on glucose, xylose, and arabinose. The activity of the enzyme on fructose was 4.27 times greater with NADPH than NADH as cofactor. This is the first highly NADPH-dependent MDH (EC 1.1.1.138) from a LAB. Comparative properties of the enzyme with other microbial MDHs are presented.