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.     

Studies on Antioxidative Substances of Microbial Origin

Aminopeptidase T (AP-T) is a metallo-dependent dimeric enzyme of Thermus aquaticus YT-1, an extremely thermophilic bacterium. We cloned the AP-T gene from T. aquaticus YT-1 into Escherichia coli using a synthetic oligonucleotide as a hybridization probe. The nucleotide sequence of the AP-T gene was found to encode 408 amino acid residues with GTG as a start codon. The molecular weight was calculated to be 44,820. The AP-T was overproduced in E. coli (about 5% of total soluble protein) when the start codon of the gene was changed from GTG to ATG, and the gene was downstream from the tac promoter. The AP-T expressed in E. coli was heat stable and easily purified by heat treatment (80°C, 30 min). The N-terminal amino acid sequence of AP-T showed similarity with that of aminopeptidase II from Bacillus stearothermophilus.     

Cloning and nucleotide sequence of the gene coding for a subunit of the respiratory NADH dehydrogenase from Bacillus stearothermophilus

The putative gene coding for a subunit of the respiratory NADH dehydrogenase from Bacillus stearothermophilus was cloned in Escherichia coli and the nucleotide sequence was determined. A large open reading frame (ORF1) was recognized, which was composed of 879 bp corresponding to 293 amino acids and a molecular weight of 33,600. Possible promoter and Shine-Dalgarno sequences were found upstream from the initiation codon. The deduced amino acid sequence of the gene was homologous to the NADH dehydrogenase of Paramecium aurelia.     

Colorimetric Determination of α-Amino Nitrogen in Urine and Plasma with Ninhydrin Reaction

Bacillus stearothermophilus TH 6–2 has two kinds of purine nucleoside phosphorylases (Pu-NPase I and Pu-NPase II). The Pu-NPase I is a functional homolog of eukaryotic purine nucleoside phosphorylases that can catalyze the phosphorolysis of inosine and guanosine, but not adenosine, the primary substrate of Pu-NPase II. The Pu-NPase I gene of TH 6–2 has been cloned, sequenced, and expressed in E. coli. The gene corresponded to an open reading frame of 822 nucleotides that translates into a putative 274-amino acid protein with a molecular weight of 29,637. The deduced amino terminus sequence completely coincided with that found for the purified enzyme. The cloned gene was overexpressed in E. coli by using the trc promoter to produce an active enzyme in large quantities. The amino acid sequence of Pu-NPase I shared 50% similarity with those of human and mouse purine nucleoside phosphorylases.     

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

The structural gene for a thermostable α-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 α-amylase (20.9 U/mg of dry cells) than did the wild-type strain of B. stearothermophilus. Some properties of the α-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 α-amylase, with a molecular weight of 53,000, retained about 60% of its activity even after treatment at 80°C for 60 min.     

Molecular characterization and sequence of phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis

The gene encoding monophosphatidylinositol inositol phosphohydrolase (PI-specific phospholipase C, PI-PLC) of Bacillus thuringiensis was cloned in Staphylococcus carnosus TM300. The complete coding region comprises 987 base pairs corresponding to a precursor protein of 329 amino acids (molecular weight, 38095). The NH₂-terminal sequence of the purified enzyme from Escherichia coli indicated that the mature PI-PLC consists of 299 amino acid residues with a molecular weight of 34586. Polyacrylamide gel electrophoresis revealed the same molecular weight for the purified enzyme isolated from the DNA-donor strain of B. thuringiensis and from the E. coli clone. By computer analysis, the secondary structure was predicted. The enzyme from the E. coli recombinant shows no activity on other phospholipids and sphingo-myelin. The cleaving specifity of PI-PLC was examined by thin layer chromatography.     

Overproduction and single-step purification of Bacillus stearothermophilus peroxidase in Escherichia coli

Summary The cloned peroxidase gene from Bacillus stearothermophilus was highly expressed in Escherichia coli. Using the high copy number plasmid which is temperature-sensitive and its own strong promoter, this thermostable peroxidase was produced at 28% of the total cell proteins when the cells were grown at 42°C. The enzyme could be easily purified from E. coli by heat treatment and single-column Sephadex G-200 chromatography. From a 200 ml culture, 30 mg of purified enzyme was obtained. The peroxidase produced by E. coli showed a thermostability, haem type and content identical with those of the peroxidase produced by B. stearothermophilus.Offprint requests to: H. Okada     

Molecular Cloning of the Genes for Pyruvate Kinase of Two Bacilli,Bacillus psychrophilus and Bacillus licheniformis,and Comparison of the Properties of the Enzymes Produced in Escherichia coli

The genes for the pyruvate kinases of a psychrophile, Bacillus psychrophilus, and a mesophile, Bacillus licheniformis, have been cloned in Escherichia coli, and all their nucleotides were sequenced. The two bacterial enzymes each had an extra C-terminal sequence consisting of about 110 amino acid residues, which has been found in the B. stearothermophilus enzyme. Both enzymes were overexpressed in E. coli and the properties of the purified enzymes were compared to those of the B. stearothermophilus enzyme. Both enzymes were less stable than the B. stearothermophilus one. The B. psychrophilus enzyme was more stable than the B. licheniformis one. Similarly to the B. licheniformis and B. stearothermophilus pyruvate kinases, the B. psychrophilus enzyme was activated by AMP or ribose 5-phosphate, and inhibited by A TP or fructose 1,6-bisphosphate. Thus, these enzymes were very similar in the sigmoidal saturation curve for phosphoenolpyruvate and allosteric effectors, but their optimum temperatures and thermostabilities were very different.     

FKBP-type peptidyl-prolyl cis–trans isomerase from a sulfur-dependent hyperthermophilic archaeon,Thermococcus sp. KS-1

A gene encoding an FK506 binding protein (FKBP)-type peptidyl-prolyl cis–trans isomerase (PPIase) was cloned from a hyperthermophilic archaeon, Thermococcus sp. KS-1, and sequenced. This gene encoded an FKBP with 159 amino-acid residues with a molecular mass of 17.6 kDa. Two insertion sequences with 13 and 44 amino acids were found in the regions corresponding to the bulge and flap regions of human FKBP-12, respectively. Comparison with other archaeal FKBP sequences obtained from reported genome sequences revealed that the insertion sequences in the bulge and flap regions were common to archaeal FKBPs. It was also revealed that archaeal FKBPs are classified into two groups: one is approx. 17 kDa and the other 27 kDa. This Thermococcus FKBP (TcFK) belonged to the smaller archaeal FKBP. In this TcFK, 9 out of 15 amino acid residues forming the FK506 binding pocket of human FKBP12 were found. This gene was expressed in Escherichia coli and the recombinant protein was purified. The purified protein showed PPIase activity and its activity was inhibited by FK506 with an IC₅₀ of 7 μM. This enzyme showed high kinetic stability with a half-life of 40 min at 100°C. Catalytic efficiency of this recombinant PPIase was 1.2-times higher with the substrate N-succinyl-A-L-P-F-p-nitroanilide than with N-succinyl-A-A-P-F-p-nitroanilide.     

Cloning and sequence analysis of the heat-stable acrylamidase from a newly isolated thermophilic bacterium, Geobacillus thermoglucosidasius AUT-01

A thermophilic bacterium capable of degrading acrylamide, AUT-01, was isolated from soil collected from a hot spring area in Montana, USA. The thermophilic strain grew with 0.2 % glucose as the sole carbon source and 1.4 mM acrylamide as the sole nitrogen source. The isolate AUT-01 was identified as Geobacillus thermoglucosidasius based on 16S rDNA sequence. An enzyme from the strain capable of transforming acrylamide to acrylic acid was purified by a series of chromatographic columns. The molecular weight of the enzyme was estimated to be 38 kDa by SDS-PAGE. The enzyme activity had pH and temperature optima of 6.2 and 70 ºC, respectively. The influence of different metals and amino acids on the ability of the purified protein to transform acrylamide to acrylic acid was evaluated. The gene from G. thermoglucosidasius encoding the acrylamidase was cloned, sequenced, and compared to aliphatic amidases from other bacterial strains. The G. thermoglucosidasius gene, amiE, encoded a 38 kDa, monomeric, heat-stable amidase that catalysed the cleavage of carbon–nitrogen bonds in acrylamide. Comparison of the amino acid sequence to other bacterial amidases revealed 99 and 82 % similarity to the amino acid sequences of Bacillus stearothermophilus and Pseudomonas aeruginosa, respectively.     

A new aryl acylamidase from Rhodococcus sp. strain Oct1 acting on ω-lactams: Its characterization and gene expression in Escherichia coli

Rhodococcus sp. strain Oct1 utilizing ω-octalactam as a sole source of carbon and nitrogen was isolated from soil. ω-Octalactam hydrolyzing enzyme was purified to homogeneity. The purified enzyme has a molecular weight of approximately 48,100 by SDS polyacrylamide gel electrophoresis and 99,100 by gel filtration, indicating that the enzyme consists of 2 subunits. The purified enzyme catalyzed the hydrolysis of ω-octalactam to form 8-aminooctanoic acid at a rate of 3.95 U/mg. The purified enzyme also acted on ω-heptalactam, ω-laurolactam, nitroacetoanilide substitutions, and various aliphatic amides. The most suitable substrate was o-nitroacetanilide for the enzyme (11.6 U/mg). The enzyme belongs to aryl acylamidase. The gene for the enzyme was cloned and the deduced amino acid sequence showed similarity to ω-laurolactam hydrolase from Rhodococcus sp. U224 (51%) and putative aryl acylamidase from Nocardia farcinica IFM 10152 (98%), and N-terminal amino acid sequence (28 residues) of aryl acylamidase from Nocardia globerula IFO 13510 (92%). Aryl acylamidases and 6-aminohexanoate-cyclic-dimer hydrolases are in the same phylogenic lineage. These enzymes were mostly active toward non-natural amides. From phylogenic analysis, these enzymes were classified into amidase signature family. The enzyme was produced in a soluble form as a fusion protein (extension of 13 amino acids at C-terminal) in Escherichia coli.     

Cloning and nucleotide sequence of Bacillus stearothermophilus pyruvate carboxylase

A gene for prokaryotic pyruvate carboxylase (PC) was cloned from Bacillus stearothermophilus. It has an open reading frame of 3441 base pairs which can code for a protein of 128 353 Da. Not only the molecular size and domain organization but also the deduced amino acid sequence of B. stearothermophilus PC are similar to those of eukaryotic PCs.     

Gene cloning,purification, and characterization of the highly thermostable leucine dehydrogenase of Bacillus sp.

The leucine dehydrogenase (l-leucine: NAD⁻ oxidoreductase, deaminating, EC 1.4.1.9) gene from Bacillus sp. DSM730 was cloned into Escherichia coli C600 with a vector plasmid, pBR322. The E. coli cells carrying a recombinant plasmid, pKULD1 (9.5 kb), produced a highly thermostable leucine dehydrogenase. The enzyme from E. coli cells carrying pKULD103, a deletion plasmid (6.5 kb) of pKULD1, was purified to homogeneity from the crude extract of clone cells by only one ion-change chromatography application with a yield of 73%. The leucine dehydrogenase of Bacillus sp. DSM730 is very similar in enzymological properties to those of other bacteria, except for molecular weight and stability. It has a molecular weight of about 280,000 and consists of six subunits identical in molecular weight (47,000). The enzyme is not inactivated by heat treatment at 80°C for 10 min, and incubation in the pII range of 5.4 to 10.3 at 55°C for 10 min. The Bacillus sp. DSM730 leucine dehydrogenase is the most thermostable of the leucine dehydrogenases so far purified, and is very useful for structure and stability studies, as well as being applicable to l-leucine production.     

Cloning and functional analysis of molecular chaperone genes from Bacillus stearothermophilus SIC1

Genes homologous to groES and groEL, which are recognized as molecular chaperone genes, from Bacillus stearothermophilus SIC1 were cloned and sequenced. By addition of GroES, GroEL and ATP in vitro, remaning activity of the alcohol dehydrogenase from Saccharomyces cerevisiae after heat treatment at 50°C for 6 min was improved from 55% to 90%. Furthermore, even though inclusion bodies were formed when a single chain Fv(sFv) was expressed in E. coli cells, during in vivo coexpression with molecular chaperone, a significant amount of the antibody protein could be recovered from the soluble fraction.     

Cloning, expression and characterization of xylose isomerase, XylA, from Caldanaerobacter subterraneus subsp. yonseiensis

The xylA gene, coding for xylose isomerase, from the extreme thermophile, Caldanaerobacter subterraneus subsp. yonseiensis was cloned, sequenced, and expressed in Escherichia coli. The nucleotide sequence of the xylA gene encoded a polypeptide of 438 residues with a calculated molecular weight of 50,170 Da. The purified XylA showed high sequence homology (92% identity) with that of Thermoanaerobacter thermohydrosulfuricus. The recombinant enzyme expressed in Escherichia coli was purified by heat treatment and gel chromatography. The purified enzyme was thermostable with optimal activity at 95°C. The enzyme required divalent cations including Zn²⁺ for its maximal activity and thermostability.     

Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (k_cat/K_m) measured at 15°C for the peptidyl substrates, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM⁻¹ s⁻¹, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicus FK506-binding protein). The MTFK gene (462 bp) was cloned from an M. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.     

Cloning, Characterization, and Expression of a New cry1Ab Gene from DOR Bt-1, an Indigenous Isolate of Bacillus thuringiensis

A new cry1Ab gene was cloned from the promising local isolate, DOR Bt-1, a Bacillus thuringiensis strain isolated from castor semilooper (Achaea janata L.) cadavers from castor bean (Ricinus communis L.) field. The nucleotide sequence of the cloned cry1Ab gene indicated that the open reading frame consisted of 3,465 bases encoding a protein of 1,155 amino acid residues with an estimated molecular weight of 130 kDa. Homology comparisons revealed that the deduced amino acid sequence of cry1Ab had a variation of seven amino acid residues compared to those of the known Cry1Ab proteins in the NCBI database and this gene has been designated as cry1Ab26 by the B. thuringiensis δ-endotoxin Nomenclature Committee. cry1Ab26 was cloned into pET 29a(+) vector and expressed in E. coli strain BL21 (DE3) under the control of T7 promoter with IPTG induction. ELISA, SDS-PAGE, and Western blot analysis confirmed the expression of 130-kDa protein. Insect bioassays with neonate larvae of Helicoverpa armigera showed that the partially purified Cry1Ab26 caused 97 % mortality within 5 days of feeding.     

Molecular cloning,sequence analysis,and characterization of a new cell wall hydrolase,CwIL, of Bacillus licheniformis

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38 994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-l-alanine amidase, which has a M_r value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.     

Thermostable NADP+-Dependent Medium-Chain Alcohol Dehydrogenase from Acinetobacter sp. Strain M-1: Purification and Characterization and Gene Expression in Escherichia coli

NADPH-dependent alkylaldehyde reducing enzyme, which was greatly induced by n-hexadecane, from Acinetobacter sp. strain M-1 was purified and characterized. The purified enzyme had molecular masses of 40 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kDa as determined by gel filtration chromatography. The enzyme, which was shown to be highly thermostable, was most active toward n-heptanal and could use n-alkylaldehydes ranging from C₂ to C₁₄ and several substituted benzaldehydes, including the industrially important compounds cinnamyl aldehyde and anisaldehyde, as substrates. The alrA gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the alrA gene exhibited homology to the amino acid sequences of zinc-containing alcohol dehydrogenases from various sources. The gene could be highly expressed in Escherichia coli, and the product was purified to homogeneity by simpler procedures from the recombinant than from the original host. Our results show that this enzyme can be used for industrial bioconversion of useful alcohols and aldehydes.