Probing the essential catalytic residues and substrate affinity in the thermoactive Bacillus stearothermophilus US100 L-arabinose isomerase by site-directed mutagenesis

The L-arabinose isomerase (L-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of l-arabinose isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 L-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 L-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the L-fucose isomerase and the L-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 L-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 L-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for L-fucose and decreased the affinity for L-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.     

Cloning and characterization of acetohydroxyacid synthase from Bacillus stearothermophilus

Five genes from the ilv-leu operon from Bacillus stearothermophilus have been sequenced. Acetohydroxyacid synthase (AHAS) and its subunits were separately cloned, purified, and characterized. This thermophilic enzyme resembles AHAS III of Escherichia coli, and regulatory subunits of AHAS III complement the catalytic subunit of the AHAS of B. stearothermophilus, suggesting that AHAS III is functionally and evolutionally related to the single AHAS of gram-positive bacteria.     

Overproduction, purification and characterization of GroES and GroEL from thermophilic Bacillus stearothermophilus

Abstract Biofilms containing diverse microflora were developed on bitumen-painted steel and glass tiles suspended in a chemostat model of a water distribution system. Escherichia coli , taken from a naturally occurring biofilm, was transformed with a plasmid containing the anaerobically induced nirB promoter fused to the lacZ reporter gene. The resulting transformant, PRB1, was introduced into the chemostat. After 7 and 13 days, an E. coli strain with an anaerobically induced Lac⁺ phenotype was present in the biofilm. Development of an episcopic differential interference contrast technique combined with UV fluorescence microscopy enabled the simultaneous visualization of E. coli in the biofilm using a fluorescent probe to detect expression of the gusA reporter gene and a lacZ fluorescent probe to monitor anaerobic expression of β-galactosidase from pnirB .     

The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus.

Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34. The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000. Kinetic parameters for the enzyme were determined with a variety of substrates. The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine. The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose. The glucokinase was maximally active at pH 9.0.     

Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis

The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg-1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65℃ and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65℃.     

Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis

The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg-1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65℃ and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65 ℃ .     

Primary structure of triosephosphate isomerase from Bacillus stearothermophilus

1. Triosephosphate isomerase from Bacillus stearothermophilus is a dimeric enzyme comprising two chemically identical polypeptide chains. 2. The nearly complete amino acid sequence of the subunit polypeptide chain has been established from sequences of tryptic, chymotryptic and lysine-blocked tyrptic fragments of S-[2-14C]carboxymethylated enzyme. Overlaps not established by experimental data have been provisionally established from considerations of sequence homology with previously established sequences for the rabbit, chicken and coelacanth enzymes. The nearly complete sequence of the 249 residues is as follows. (See Text). 3. Comparison of the thermophile and chicken muscle enzymes shows that 40% of the residues are in identical sequence. 4. Correlation of the sequence of the thermophile enzyme with the three-dimensional structure of the muscle enzyme shows that residues in the catalytic site and in the subunit interface are strongly conserved. Possible correlations between sequence changes and thermal stabilisation of the dimeric structure are also noted.     

Cloning, overexpression, purification and crystallisation of ribosomal protein L9 from Bacillus stearothermophilus.

The cloning, sequencing and overexpression of the gene coding for Bacillus stearothermophilus ribosomal protein L9 is described. The sequence corresponds directly to that presented for the protein itself by classical methods, differing at only a few amino acid positions. The purification and crystallisation of the corresponding L9 protein is presented. The crystals are isomorphous to those described for L9 obtained by conventional methods.     

Partial purification and characterization of type I DNA topoisomerase from Bacillus stearothermophilus

Type I DNA topoisomerase was partially purified from Bacillus stearothermophilus by ammonium sulfate precipitation and column chromatographies on phosphocellulose, DEAE-cellulose and heparin-agarose. On heparin-agarose chromatography, topoisomerase I activity was separated into three fractions (designated Fractions A, B, and C). Each fraction was further subjected to gel filtration on Sephacryl S-200. From electrophoretic analysis on polyacrylamide gel, Fraction A was found to contain two enzyme species having molecular weights of 110,000 and 100,000, and Fraction B one enzyme species with a molecular weight of 80,000. The molecular weight of the enzyme in Fraction C was estimated to be around 150,000 by gel filtration. The enzymes in Fractions A and B exhibited little activity in the presence of Mg2+, while the activity was increased remarkably by NaCl with Mg2+. No activity was observed in the presence of NaCl alone. The enzyme in Fraction C required only Mg2+ for full activity. With Fraction A, the topoisomerase I-induced cleavage sites on tetracycline-resistant plasmid pNS1 (2.55 megadaltons) were mapped. Fraction A cleaved the DNA at ten specific sites. These sites were compared to those of the Haemophilus gallinarum enzyme, which have already been mapped (Shishido et al. (1983) Biochem. Biophys. Acta 740, 108). The results showed that there is a remarkably coincidence between the cleavage sites induced by the B. stearothermophilus and H. gallinarum enzymes.     

Cloning, expression, and biochemical characterization of a thermostable lipase from Geobacillus stearothermophilus JC

A thermophilic lipase gene of Geobacillus stearothermophilus JC was cloned and expressed in a pET 28-a (+) expression vector. The biochemical properties of the recombinant enzyme and its enantioselective hydrolysis of (RS)-1-phenylethyl acetate were studied. Removal of the signal peptide greatly increased the enzyme’s expression level by 4.3 times. The purified JC lipase had an optimum temperature of 55°C and optimum pH of 9. Furthermore, comparisons with other enzymes suggest that a few amino acid alterations may significantly change the thermostability of this enzyme. The hydrolysis of (RS)-1-phenylethyl acetate with the crude recombinant JC lipase at 25°C produce (R)-1-phenylethanol in 97.7% e.e. and 46.1% yield after 24 h, corresponding to an E value of 237.     

Thermostable dipeptidase from Bacillus stearothermophilus: its purification, characterization, and comparison with aminoacylase

Dipeptidase (dipeptide hydrolase [EC 3.4.13.11]) has been purified to homogeneity and crystallized from the cell extract of Bacillus stearothermophilus IFO 12983. The enzyme has a molecular weight of about 86,000, and is composed of two subunits identical in molecular weight (43,000). The enzyme contains 2 g atoms of zinc per mol of protein. A variety of dipeptides consisting of glycine or only L-amino acids serve as substrates of the enzyme; Km and Vmax values for L-valyl-L-alanine are 0.5 mM and 68.0 units/mg protein, respectively. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturants such as urea and guanidine hydrochloride. The enzyme also catalyzes hydrolysis of several N-acylamino acids with Vmax values 3-30% of those for the hydrolysis of dipeptides. The thermostable dipeptidase shares various properties with bacterial aminoacylase [EC 3.5.1.14]: their subunit molecular weight, metal content and requirement, amino acid composition, and amino acid sequence in the N-terminal region are very similar.     

Towards efficient enzymatic conversion of d-galactose to d-tagatose: purification and characterization of l-arabinose isomerase from Lactobacillus brevis

Bioprocess and Biosystems Engineering - l-arabinose isomerase (l-AI) (EC 5. 3. 1. 4. l-AI) that mediates the isomerization of d-galactose to d-tagatose was isolated from Lactobacillus brevis (MF...     

Cloning and Expression of Thermostable alpha-Amylase Gene from Bacillus stearothermophilus in Bacillus stearothermophilus and Bacillus subtilis

The structural gene for a thermostable alpha-amylase from Bacillus stearothermophilus was cloned in plasmids pTB90 and pTB53. It was expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about fivefold more alpha-amylase (20.9 U/mg of dry cells) than did the wild-type strain of B. stearothermophilus. Some properties of the alpha-amylases that were purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant differences were observed among the enzyme properties despite the difference in host cells. It was found that the alpha-amylase, with a molecular weight of 53,000, retained about 60% of its activity even after treatment at 80 degrees C for 60 min.     

Cloning and characterization of the gene for a methanol-utilising alcohol dehydrogenase from Bacillus stearothermophilus

The cloning and characterization of the alcohol dehydrogenase (ADH) gene (adh) from Bacillus stearothermophilus strain DSM2334, an obligate aerobe, are described. The clone directed the synthesis of ADH as judged on Western blots, activity gels and tetrazolium plates. It specified an enzyme that oxidised methanol as well as ethanol. The enzyme was found to be encoded by a single gene in B. stearothermophilus which did not cross-hybridize to adh clones from Escherichia coli, yeast or maize. The cloned gene was expressed in E. coli but activity was not detected in Bacillus subtilis, despite stable maintenance of the recombinant plasmid in this host. The gene is catabolite-repressed in DSM2334.     

Overexpression,purification, and characterization of the recombinant leucine aminopeptidase II of Bacillus stearothermophilus

For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32 to generate pQE-LAPII. The His(6)-tagged enzyme was overexpressed in IPTG-induced E. coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 425 U/mg protein with a final yield of 76%. The subunit molecular mass of the purified protein was estimated to be 44.5 kDa by SDS-PAGE. The temperature and pH optima for the purified protein were 60 degrees C and 8.0, respectively. Under optimal condition, the purified enzyme showed a marked preference for Leu- p-nitroanilide, followed by Arg- and Lys-derivatives. The His(6)-tagged enzyme was stimulated by Co(2+) ions, but was strongly inhibited by Cu(2+) and Hg(2+) and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co(2+) ions, indicating that it is a cobalt-dependent exopeptidase. Taking the biochemical characteristics together, we found that the recombinant LAP II exhibits no important differences from those properties described for the native enzyme.     

Gene cloning,purification and characterization of thermostable alanine dehydrogenase of Bacillus stearothermophilus

The alanine dehydrogenase (l-alanine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.1) gene of Bacillus stearothermophilus IFO12550 was cloned and expressed in Escherichia coli C600 with a recombinant plasmid, pICD301, which was constructed from pBR322 and the alanine dehydrogenase gene derived from B. stearothermophilus. The enzyme overproduced in the clone was purified about 30 fold to homogeneity by heat treatment and two subsequent steps with a yield of 46%. The enzyme of E. coli-pICD301 was immunochemically identical with that of B. stearothermophilus. The enzyme has a molecular weight of about 240,000 and consists of six subunits identical in molecular weight (40,000). The enzyme is not inactivated by heat treatment: at pH 7.2 and 75°C for 30 min; at 55°C and various pHs between 6.0 and 11.5 for 10 min. The enzymological properties are very similar to those of the mesophilic B. sphaericus enzyme (Ohshima, T. and Soda, K., Eur. J. Biochem., 100, 29–39, 1979) except for thermostability.     

Thermostable alanine racemase from Bacillus stearothermophilus: molecular cloning of the gene, enzyme purification, and characterization

The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, was cloned and expressed in Escherichia coli C600 with vector plasmid pICR301, which was constructed from pBR322 and the L-alanine dehydrogenase gene derived from B. stearothermophilus. A coupled assay method with L-alanine dehydrogenase and tetrazolium salts was used to detect visually the alanine racemase activity in the clones. Alanine racemase overproduced in a clone carrying the plasmid pICR4, 12 kilobases of DNA, was purified from cell extracts about 340-fold to homogeneity by five steps including heat treatment. The overproduced enzyme was confirmed to originate from B. stearothermophilus by an immunochemical cross-reaction with the enzyme of B. stearothermophilus. The purified enzyme has a molecular weight of about 78 000 and consists of two identical subunits of Mr of 39 000. At the optimum temperature (50 degrees C), the enzyme has a specific activity of 1800 units/mg (Vmax, D- to L-alanine). Resolution and reconstitution experiments together with the absorption spectrum of the enzyme clearly indicate that alanine racemase of B. stearothermophilus is a pyridoxal 5'-phosphate enzyme.     

Stable ammonia-specific NAD synthetase from Bacillus stearothermophilus: purification,characterization, gene cloning,and applications

Bacillus stearothermophilus H-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5). The enzyme specifically used NH3 as an amide donor for the synthesis of NAD as it formed AMP and pyrophosphate from deamide-NAD and ATP. None of the l-amino acids tested, such as l-asparagine or l-glutamine, or other amino compounds such as urea, uric acid, or creatinine was used instead of NH3. Mg2+ was needed for the activity, and the maximum enzyme activity was obtained with 3 mM MgCl2. The molecular mass of the native enzyme was 50 kDa by gel filtration, and SDS-PAGE showed a single protein band at the molecular mass of 25 kDa. The optimum pH and temperature for the activity were from 9.0 to 10.0 and 60 degrees C, respectively. The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60 degrees C. The Km for NH3, ATP, and deamide-NAD were 0.91, 0.052, and 0.028 mM, respectively. The gene encoding the enzyme consisted of an open reading frame of 738 bp and encoded a protein of 246 amino acid residues. The deduced amino acid sequence of the gene had about 32% homology to those of Escherichia coli and Bacillus subtilis NAD synthetases. We caused the NAD synthetase gene to be expressed in E. coli at a high level; the enzyme activity (per liter of medium) produced by the recombinant E. coli was 180-fold that of B. stearothermophilus H-804. The specific assay of ammonia and ATP (up to 25 microM) with this stable NAD synthetase was possible.