Cloning, Expression, and Characterization of a DNA Ligase from a Hyperthermophilic Archaeon Thermococcus Sp.

Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. NA1, revealed an ORF of 1689 bases encoding 562 amino acids that showed a high similarity to DNA ligases from other hyperthermophilic archaea. The ligase, which was designated TNA1_lig (Thermococcus sp. NA1 ligase), was cloned and expressed in Escherichia coli. The recombinant TNA1_lig was purified by metal affinity chromatography. The optimum ligase activity of the recombinant TNA1_lig occurred at 80 °C and pH 7.5. The enzyme was activated by MgCl₂ and ZnCl₂ but was inhibited by MnCl₂ and NiCl₂. Additionally, the enzyme was activated by either ATP or NAD⁺. Revisions requested 27 October 2005; Revisions received 14 December 2005     

Cloning, expression, and characterization of aminopeptidase P from the hyperthermophilic archaeon Thermococcus sp. strain NA1

Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. strain NA1, revealed the presence of a 1,068-bp open reading frame encoding a protein consisting of 356 amino acids with a calculated molecular mass of 39,714 Da (GenBank accession no. DQ144132). Sequence analysis showed that it was similar to the putative aminopeptidase P (APP) of Thermococcus kodakaraensis KOD1. Amino acid residues important for catalytic activity and the metal binding ligands conserved in bacterial, nematode, insect, and mammalian APPs were also conserved in the Thermococcus sp. strain NA1 APP. The archaeal APP, designated TNA1_APP (Thermococcus sp. strain NA1 APP), was cloned and expressed in Escherichia coli. The recombinant enzyme hydrolyzed the amino-terminal Xaa-Pro bond of Lys(Nepsilon-Abz)-Pro-Pro-pNA and the dipeptide Met-Pro (Km, 0.96 mM), revealing its functional identity. Further enzyme characterization showed the enzyme to be a Co2+-, Mn2+-, or Zn2+-dependent metallopeptidase. Optimal APP activity with Met-Pro as the substrate occurred at pH 5 and a temperature of 100 degrees C. The APP was thermostable, with a half-life of >100 min at 80 degrees C. This study represents the first characterization of a hyperthermophilic archaeon APP.     

Purification and properties of an intracellular leucine aminopeptidase from the fungus,Penicillium citrinum strain IFO 6352

An intracellular leucine aminopeptidase (LAP) fromPenicillium citrinum (IFO 6352) was purified to homogeneity using three successive purification steps. The enzyme has a native molecular mass of 63 kDa using HPLC gel filtration analysis and a molecular mass of 65 kDa when using SDS-polyacrylamide gel electrophoresis. This monomeric aminopeptidase showed maximum enzyme activity at pH 8.5. An optimum temperature was 45–50°C whenl-Leu-p-nitroanilide (pNA) was the substrate, and enzyme activity drastically decreased above 60°C. The Michaelis-Menten constants forl-Leu-pNA andl-Met-pNA were 2.7 mM and 1.8 mM, respectively. When the enzyme reacted with biosynthetic methionyl human growth hormone, it showed high specificity for N-terminal methionine residue and recognized a stop sequence (Xaa-Pro). The aminopeptidase was inactivated by EDTA or 1,10-phenanthroline, indicating that it is a metallo-exoprotease. Enzyme activity was restored to 90% of maximal activity by addition of Co²⁺ ions. The activity of EDTA-treated enzyme was restored by addition of Zn²⁺, but reconstitution with Ca²⁺, Mg²⁺ or Mn²⁺ restored some enzyme activity. It is likely that Co²⁺ ions play an important role in the catalysis or stability of thePenicillium citrinum aminopeptidase, as zinc plays a similar function in other leucine aminopeptidases.     

Characterization of Hyperthermostable Fructose-1,6-Bisphosphatase from <Emphasis Type="Italic">Thermococcus onnurineus</Emphasis> NA1

To understand the physiological functions of thermostable fructose-1,6-bisphosphatase (TNA1-Fbp) from Thermococcus onnurineus NA1, its recombinant enzyme was overexpressed in Escherichia coli, purified, and the enzymatic properties were characterized. The enzyme showed maximal activity for fructose-1,6-bisphosphate at 95°C and pH 8.0 with a half-life (t _1/2) of about 8 h. TNA1-Fbp had broad substrate specificities for fructose-1,6-bisphosphate and its analogues including fructose-1-phosphate, glucose-1-phosphate, and phosphoenolpyruvate. In addition, its enzyme activity was increased five-fold by addition of 1 mM Mg²⁺, while Li⁺ did not enhance enzymatic activity. TNA1-Fbp activity was inhibited by ATP, ADP, and phosphoenolpyruvate, but AMP up to 100 mM did not have any effect. TNA1-Fbp is currently defined as a class V fructose-1,6-bisphosphatase (FBPase) because it is very similar to FBPase of Thermococcus kodakaraensis KOD1 based on sequence homology. However, this enzyme shows a different range of substrate specificities. These results suggest that TNA1-Fbp can establish new criterion for class V FBPases.     

Characterization of a dUTPase from the Hyperthermophilic Archaeon <Emphasis Type="Italic">Thermococcus onnurineus</Emphasis> NA1 and Its Application in Polymerase Chain Reaction Amplification

Genomic analysis of the hyperthermophilic archaeon Thermococcus onnurineus NA1 (TNA1) revealed the presence of a 471-bp open reading frame with 93% similarity to the dUTPase from Pyrococcus furiosus. The dUTPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dUTP at about a 10-fold higher rate than dCTP. The protein behaved as a dimer in gel filtration chromatography, even though it contains five motifs that are conserved in all homotrimeric dUTPases. The dUTPase showed optimum activity at 80°C and pH 8.0, and it was highly thermostable with a half-life (t _1/2) of 170 min at 95°C. The enzymatic activity of the dUTPase was largely unaffected by variations in MgCl₂, KCl, (NH₄)₂SO₄, and Triton X-100 concentrations, although it was reduced by bovine serum albumin. Addition of the dUTPase to polymerase chain reactions (PCRs) run with TNA1 DNA polymerase significantly increased product yield, overcoming the inhibitory effect of dUTP. Further, addition of the dUTPase allowed PCR amplification of targets up to 15 kb in length using TNA1 DNA polymerase. This enzyme also improved the PCR efficiency of other archaeal family B type DNA polymerases, including Pfu and KOD.     

Overexpression and characterization of a carboxypeptidase from the hyperthermophilic archaeon Thermococcus sp. NA1

Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. NA1, revealed the presence of an 1,497 bp open reading frame, encoding a protein of 499 amino acids. The deduced amino acid sequence was similar to thermostable carboxypeptidase 1 from Pyrococcus furiosus, a member of peptidase family M32. Five motifs, including the HEXXH motif with two histidines coordinated with the active site metal, were conserved. The carboxypeptidase gene was cloned and overexpressed in Escherichia coli. Molecular masses assessed by SDS-PAGE and gel filtration were 61 kDa and 125 kDa respectively, which points to a dimeric structure for the recombinant enzyme, designated TNA1_CP. The enzyme showed optimum activity toward Z-Ala-Arg at pH 6.5 and 70-80 degrees C (k(cat)/K(m)=8.3 mM(-1) s(-1)). In comparison with that of P. furiosus CP (k(cat)/K(m)=667 mM(-1) s(-1)), TNA1_CP exhibited 80-fold lower catalytic efficiency. The enzyme showed broad substrate specificity with a preference for basic, aliphatic, and aromatic C-terminal amino acids. This broad specificity was confirmed by C-terminal ladder sequencing of porcine N-acetyl-renin substrate by TNA1_CP.     

Cloning, purification, and characterization of a thermostable alpha-L-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari

The gene, AbfAC26Sari, encoding an α-l-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari, was isolated, cloned, sequenced, and characterizated. On the basis of amino acid sequence similarities, this 57-kDa enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. Characterization of the purified recombinant α-l-arabinofuranosidase produced in Escherichia coli BL21 revealed that it is active at a broad pH range (pH 4.5 to 9.0) and at a broad temperature range (45–85°C) and it has an optimum pH of 5.5 and an optimum temperature of 65°C. Kinetic experiment at 65°C with p-nitrophenyl α-l-arabinofuranoside as a substrate gave a V _max and K _m values of 1,019 U/mg and 0.139 mM, respectively. The enzyme had no apparent requirement of metal ions for activity, and its activity was strongly inhibited by 1 mM Cu²⁺ and Hg²⁺. The recombinant arabinofuranosidase released l-arabinose from arabinan, arabinoxylan, oat spelt xylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. Endoarabinanase activity was not detected. These findings suggest that AbfAC26Sari is an exo-acting enzyme.     

Purification and properties of thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60

Thermostable N-acylamino acid recemase from Amycolatopsis sp. TS-1-60, a rare actinomycete strain selected for its ability to grow on agar plates incubated at 40° C, was purified to homogeneity and characterized. The relative molecular mass (M _r) of the native enzyme and the subunit was estimated to be 300 000 and 40 000 on gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis respectively. The isoelectric point (pI) of the enzyme was 4.2. The optimum temperature and pH were 50° C and 7.5 respectively. The enzyme was stable at 55° C for 30 min. The enzyme catalyzed the racemization of optically active N-acylamino acids such as N-acetyl-l-or d-methionine, N-acetyl-l-valine, N-acetyl-l-tyrosine and N-chloroacetyl-l-valine. In addition, the enzyme also catalyzed the recemization of the dipeptide l-alanyl-l-methionine. By contrast, the optically active amino acids, N-alkyl-amino acids and methyl and athyl ester derivatives of N-acetyl-d- and l-methionine were not racemized. The apparent K _m values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 18.5 mM and 11.3 mM respectively. The enzyme activity was markedly enhanced by the addition of divalent metal ions such as Co²⁺, Mn²⁺ and Fe²⁺ and was inhibited by addition of EDTA and P-chloromercuribenzoic acid. The similarity between the NH₂-terminal amino acid sequence of the enzyme and that of Streptomyces atratus Y-53 [Tokuyama et al. (1994) Appl Microbiol Biotechnol 40:835–840] was above 80%.     

Purification and characterization of an alkaline cellulase from a newly isolated alkalophilic Bacillus sp. HSH-810

An alkaline cellulase from Bacillus sp. HSH-810 was purified 8.7-fold with a 30% yield and a specific activity of 71 U mg^–1 protein. It was optimally active at pH 10 and 50 °C and was stable from pH 6 to 10 with more than 60% activity remaining after heating at 60 °C for 60 min. The molecular mass of cellulase was 80 kDa. It was inhibited by 50% by Fe³⁺ (1 mM) and Mn²⁺ (0.1 mM) but was relatively insensitive to Hg²⁺ and Pb²⁺ at 1 mM.Revisions requested: 8 October 2004/1 December 2004; Revisions received 29 November 2004/5 January 2005     

Gene cloning,expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18

A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence LipX was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET28a(+)/lipX gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipX_His with a 6× histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0 with p-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipX_His is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn²⁺, Co²⁺, Mn²⁺, Fe³⁺ and EDTA. Most non-ionic detergents, such as DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Additionally, the highest lipolytic rate of the recombinant LipX_His lipase was achieved when p-nitrophenyl myristate was used as a substrate, among all the p-nitrophenyl esters tested.     

Expression, purification, and characterization of alanine racemase from Pseudomonas putida YZ-26

Alanine racemase catalyzes the interconversion of d- and l-alanine and plays an important role in supplying d-alanine, a component of peptidoglycan biosynthesis, to most bacteria. Alanine racemase exists mostly in prokaryotes and is generally absent in higher eukaryotes; this makes it an attractive target for the design of new antibacterial drugs. Here, we present the cloning and characterization of a new gene-encoding alanine racemase from Pseudomonas putida YZ-26. An open reading frame (ORF) of 1,230 bp, encoding a protein of 410 amino acids with a calculated molecular weight of 44,217.3 Da, was cloned into modified vector pET32M to form the recombinant plasmid pET–alr. After introduction into E.coli BL21, the strain pET-alr/E.coli BL21 expressed His₆-tagged alanine racemase. The recombinant alanine racemase was efficiently purified to homogeneity using Ni²⁺–NTA and a gel filtration column, with 82.5% activity recovery. The amino acid sequence deduced from the alanine racemase gene revealed identity similarities of 97.0, 93, 23, and 22.0% with from P. putida F1, P. putida200, P. aeruginosa, and Salmonella typhimurium, respectively. The recombinant alanine racemase is a monomeric protein with a molecular mass of 43 kDa. The enzyme exhibited activity with l-alanine and l-isoleucine, and showed higher specificity for the former compared with the latter. The enzyme was stable from pH 7.0–11.0; its optimum pH was at 9.0. The optimum temperature for the enzyme was 37°C, and its activity was rapidly lost at temperatures above 40°C. Divalent metals, including Sr²⁺, Mn²⁺, Co²⁺, and Ni²⁺ obviously enhanced enzymatic activity, while the Cu²⁺ ion showed inhibitory effects.     

Thermococcus peptonophilus sp. nov., a fast-growing,extremely thermophilic archaebacterium isolated from deep-sea hydrothermal vents

Two extremely thermophilic archaebacteria, strains OG-1 and SM-2, were isolated from newly discovered deep-sea hydrothermal vent areas in the western Pacific ocean. These strains were cocci, obligately anaerobic Archaea about 0.7–2 μm in diameter. Optimum growth conditions for OG-1 and SM-2 were at 85–90°C (range 60–100°C), pH 6 (range pH 4–8), a NaCl concentration of 3% (range 1–5%), and a nutrient concentration (tryptone plus yeast extract) of 0.2% (range 0.005–5%). Elemental sulfur stimulated the growth rate fourfold. Ammonium slightly stimulated growth. Both tryptone and yeast extract allowed growth as sole carbon sources; these isolates were not able to utilize or grow exclusively on sucrose, glucose, maltose, succinate, pyruvate, propionate, acetate, or free amino acids. OG-1 showed the fastest growth rate within the genus Thermococcus. Growth was inhibited by rifampicin. The DNA G+C content was 52 mol%. Sequencing of their 16S rDNA gene fragment indicated that these isolates belonged to the genus Thermococcus. OG-1 and SM-2 were different than the described Thermococcus species. We propose that OG-1 belongs to a new species: Thermococcus peptonophilus. Received: 8 March 1995 / Accepted: 24 May 1995     

Overexpression, one-step purification, and biochemical characterization of a recombinant gamma-glutamyltranspeptidase from Bacillus licheniformis

A truncated gene from Bacillus lichenifromis ATCC 27811 encoding a recombinant γ-glutamyltranspeptidase (BLrGGT) was cloned into pQE-30 to generate pQE-BLGGT, and the overexpressed enzyme was purified from the crude extract of IPTG-induced E. coli M15 (pQE-BLGGT) to homogeneity by nickel-chelate chromatography. This protocol yielded over 25 mg of purified BLrGGT per liter of growth culture under optimum conditions. The molecular masses of the subunits of the purified enzyme were determined to be 41 and 22 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature for the recombinant enzyme were 6–8 and 40 °C, respectively. The chloride salt of metal ions Mg²⁺, K⁺, and Na⁺ can activate BLrGGT, whereas that of Pb²⁺ dramatically inhibited it. The substrate specificity study showed that l-γ-glutamyl-p-nitroanilide (l-γ-Glu-p-NA) is a preference for the enzyme. Steady-state kinetic study revealed that BLrGGT has a k _cat of 105 s⁻¹ and a K _m of 21 μM when using l-γ-Glu-p-NA as the substrate. With this overexpression and purification system, BLrGGT can now be obtained in quantities necessary for structural characterization and synthesis of commercially important γ-glutamyl compounds.     

Production and properties of asparaginase from a new Erwinia sp

Asparaginase production by a mesophilic strain ofErwinia sp. was examined; the maximum of activity was found at 40°C and pH 8.5. Among the various carbon sources, mannitol was shown to be the best for production of activity. Inorganic nitrogen sources were better than the organic ones. The enzyme activity was not inhibited by 10 mmol/L metal ions (Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Co²⁺, Ni²⁺, Zn²⁺); the activity was strongly inhibited by addition of EDTA.l-Arginine,dl-alanine,l-asparagine andl-glutamine stimulated thel-asparaginase production by 3.9, 1.7, 4.3 and 4.0 fold, respectively. The combination ofl-arginine,l-asparagine andl-glutamine synergistically stimulated the asparaginase up to 5.8 fold.     

Purification and Characterization of an Esterase from <Emphasis Type="Italic">Acinetobacter lwoffii</Emphasis> I6C-1

EstA was purified from the supernatant by A. lwoffii 16C-1. Its molecular mass was determined to be 45 kDa, and the optimal activity occurred when the pH level was 8.0 at a temperature of 37°C. The activation energies for the hydrolysis of p-nitrophenyl butyrate was determined to be 11.25 kcal/mol in the temperature range of 10–37°C. The enzyme was unstable at temperatures higher than 50°C. The Michaelis constant (K _m ) and V _max for p-nitrophenyl butyrate were 11 μM and 131.6 μM min⁻¹ mg of protein-1, respectively. The enzyme was strongly inhibited by Hg²⁻, Ca²⁺, Mg²⁺, Fe²⁺, Cu²⁺, Zn²⁺, Mn²⁺, Co²⁺, ethylemediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), and diisopropyl fluorophosphate (DFP). Received: 20 August 2001 / Accepted: 20 September 2001     

Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete <Emphasis Type="Italic">Pseudonocardia thermophila</Emphasis>

A constitutively expressed thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila was purified to homogeneity by applying hydrophobic interaction, anion exchange and gel filtration chromatography, giving a yield of 26% and a specific activity of 19.5 units mg^–1. The purified enzyme has an estimated molecular mass of 108 kDa and an isoelectric point of 4.2. The amidase is active at a broad pH range (pH 4–9) and temperature range (40–80°C) and has a half-life of 1.2 h at 70°C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co²⁺, Hg²⁺, Cu²⁺, Ni²⁺, and thiol reagents. The amidase has a broad substrate spectrum, including aliphatic, aromatic and amino acid amides. The presence of a double bond or a methyl group near the carboxamide group of aliphatic and amino acid amides enhances the enzymatic activity. Among aromatic amides with substitutions at the o-, m-, or p-position, the p-substituted amides are the preferred substrates. The highest acyl transferase activity was detected with hexanoamide, isobutyramide and propionamide. The K_m values for propionamide, methacrylamide, benzamide and 2-phenylpropionamide are 7.4, 9.2, 4.9 and 0.9 mM, respectively. The amidase is highly S-stereoselective for 2-phenylpropionamide; and the racemic amide was converted to the corresponding S-acid with an enantiomeric excess of >95% at 50% conversion of the substrate. In contrast, the d,l-tryptophanamide and d,l-methioninamide were converted to the corresponding d,l-acids at the same rate. This thermostable enzyme represents the first reported amidase from a thermophilic actinomycete.     

Characterization of thermostable deblocking aminopeptidases of archaeon Thermococcus onnurineus NA1 by proteomic and biochemical approaches

Thermococcus onnurineus NA1 is a hyperthermophilic archaeon that grows optimally at >80°C. The deblocking aminopeptidase (DAP) (TNA1-DAP1) encoded in Ton_1032 of T. onnurineus NA1 is considered a major DAP. However, four genes encoding putative DAP have been identified from a genomic analysis of T. onnurineus NA1. A proteomic analysis revealed that all four DAPs were differentially induced in YPS culture medium and, particularly, two DAPs (TNA1-DAP1 and TNA1-DAP2) were dominantly expressed in T. onnurineus NA1. The biochemical properties and enzyme activity of DAPs induced in an E. coli expression system suggested that the two major DAPs play complementary roles in T. onnurineus NA1.     

Adhesion forces in the self-recognition of oligosaccharide epitopes of the proteoglycan aggregation factor of the marine sponge <Emphasis Type="Italic">Microciona prolifera</Emphasis>

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular-mass aggregation factor, termed MAF. Earlier investigations revealed that the cell aggregation activity of MAF depends on two functional domains: (i) a Ca²⁺-independent cell-binding domain and (ii) a Ca²⁺-dependent proteoglycan self-interaction domain. Structural analysis of involved carbohydrate fragments of the proteoglycan in the self-association established a sulfated disaccharide β-d-GlcpNAc3S-(1→3)-α-l-Fucp and a pyruvated trisaccharide β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp. Recent UV, SPR, and TEM studies, using BSA conjugates and gold nanoparticles of the synthetic sulfated disaccharide, clearly demonstrated self-recognition on the disaccharide level in the presence of Ca²⁺-ions. To determine binding forces of the carbohydrate–carbohydrate interactions for both synthetic MAF oligosaccharides, atomic force microscopy (AFM) studies were carried out. It turned out that, in the presence of Ca²⁺-ions, the force required to separate the tip and sample coated with a self-assembling monolayer of thiol-spacer-containing β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- was found to be quantized in integer multiples of 30 ± 6 pN. No binding was observed between the two monolayers in the absence of Ca²⁺-ions. Cd²⁺-ions could partially induce the self-interaction. In contrast, similar AFM experiments with thiol-spacer-containing β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- did not show a binding in the presence of Ca²⁺-ions. Also TEM experiments of gold nanoparticles coated with the pyruvated trisaccharide could not make visible aggregation in the presence of Ca²⁺-ions. It is suggested that the self-interaction between the sulfated disaccharide fragments is stronger than that between the pyruvated trisaccharide.     

Induction of an Increase in Nerve Growth Factor Content in the Cell-conditioned Medium of Mouse L-M Cells by Basic Peptides

Thermostable trehalose synthase, which catalyzes the conversion of maltose into trehalose by intramolecular transglucosylation, was purified from a cell-free extract of the thermophilic bacterium Thermus aquaticus ATCC 33923 to an electrophoretically homogeneity by successive column chromatographies. The purified enzyme had a molecular weight of 105,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.6 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The optimum pH and temperature were pH 6.5 and 65°C, respectively. The enzyme was stable from pH 5.5 to 9.5 and up to 80°C for 60min. The trehalose synthase from Thermus aquaticus is more thermoactive and thermostable than that from Pimelobacter sp. R48. The yield of trehalose from maltose by the enzyme was independent of the substrate concentration, and tended to increase at lower temperatures. The maximum yield of trehalose from maltose by the enzyme reached 80–82% at 30–40°C. The activity was inhibited by Cu²⁺ , Hg²⁺, Zn²⁺, and Tris.     

Purification and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-tagatose

l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI activity was stimulated by Mn²⁺, Fe³⁺, Fe²⁺, Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Hg²⁺, Pb²⁺. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K _m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production corresponds to a 39% equilibrium.