Overexpression and Purification of Asparagine Synthetase from Escherichia coli

Overexpression of the asnA gene from Escherichia coli K-12 coding for asparagine synthetase (EC 6.3.1.1) was achieved with a plasmid, pUNAd37, a derivative of pUCI8, in E. coli. The plasmid was constructed by optimizing a DNA sequence between the promoter and the ribosome binding region. The enzyme, comprising ca. 15%, of the total soluble protein in the E. coli cell, was readily purified to apparent homogeneity by DEAE-Cellulofine and Blue-Cellulofine column chromatographies. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein agreed with the predicted values based on the DNA sequence of the gene. Furthermore the native molecular weight measured by gel filtration confirmed that asparagine synthetase exists as a dimer of identical subunits.     

Purification and characterization of NADP+-specific glutamate dehydrogenase fromEscherichia coli

The NADP⁺-specific glutamate dehydrogenase fromEscherichia coli has been purified to electrophoretic homogeneity. The enzyme was purified 40-fold and has a specific activity of 23. Glutamate dehydrogenase fromE. coli is a hexameric enzyme with a native molecular weight of 275 KDa composed of monomers each with a molecular weight of 44.5 KDa. In nondenaturing isoelectric focusing gels, the purified enzyme is resolved into six catalytically active species, each with a molecular weight of 275 KDa and with isoelectric points ranging between pH 5.3 and 5.7. The K_m values for substrates and coenzymes have been determined, and the effect of several divalent ions on catalytic activity has been investigated.     

Phospholipase D fromStreptomyces antibioticus: Cloning,sequencing, expression,and relationship to other phospholipases

The extracellular phospholipase D (PLD) gene fromStreptomyces antibioticus was cloned, sequenced, and expressed inEscherichia coli. Analysis of DNA sequence data revealed a putative ribosome-binding site and an open reading frame encoding a 556-amino-acid protein that included amino acid sequences obtained from the purified enzyme. The protein was expressed in an insoluble form inE. coli, but reacted with antibody against PLD. After solubilization of the protein with guanidine-HCI and 2-mercaptoethanol, subsequent dialysis restored the PLD activity. Comparison of the nucleotide sequence data with the N-terminal protein sequence indicates that this secreted protein is synthesized as a larger precursor with a 47-amino-acid N-terminal extension to the mature enzyme of 509 amino acids. The amino acid sequence of the S.antibioticus PLD was extensively compared with other PLDs and phospholipase C (PLC). The deduced amino acid sequence of the cloned PLD was highly homologous to PLDs from S. acidomyceticus andStreptomyces sp., and contained a conserved region with S.chromofuscus PLD. From comparisons of the structural similarity and properties of the various PLDs, a classification of PLDs into two subgroups has been proposed and the highly conserved region designated tentatively region X_PLD, which may be important in the catalytic function, has been identified. The homology comparison between our PLD and phosphatidylinositol-specific phospholipase C (PI-PLC) is also discussed.     

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 expression of the gene encoding <Emphasis Type="BoldItalic">Streptomyces coelicolor</Emphasis> A3(2) α-galactosidase belonging to family 36

The α-galactosidase gene of Streptomyces coelicolor A3(2) was cloned, expressed in Escherichia coli and characterized. It consisted of 1497 nucleotides encoding a protein of 499 amino acids with a predicted molecular weight of 57,385. The observed homology between the deduced amino acid sequences of the enzyme and α-galactosidase from Thermus thermophilus was over 40%. The α-galactosidase gene was assigned to family 36 of the glycosyl hydrolases. The enzyme purified from recombinant E. coli showed optimal activity at 40 °C and pH 7. The enzyme hydrolyzed p-nitrophenyl-α-D-galactopyroside, raffinose, stachyose but not melibiose and galactomanno-oligosaccharides, indicating that this enzyme recognizes not only the galactose moiety but also other substrates.     

l-Stereoselective amino acid amidase with broad substrate specificity from Brevundimonas diminuta: characterization of a new member of the leucine aminopeptidase family

Brevundimonas diminuta TPU 5720 produces an amidase acting l-stereoselectively on phenylalaninamide. The enzyme (LaaA_Bd) was purified to electrophoretic homogeneity by ammonium sulfate fractionation and four steps of column chromatography. The final preparation gave a single band on SDS-PAGE with a molecular weight of ≈53,000. The native molecular weight of the enzyme was about 288,000 based on gel filtration chromatography, suggesting that the enzyme is active as a homohexamer. It had maximal activity at 50°C and pH 7.5. LaaA_Bd lost its activity almost completely on dialysis against potassium phosphate buffer (pH 7.0), and the amidase activity was largely restored by the addition of Co²⁺ ions. The enzyme was, however, inactivated in the presence of ethylenediaminetetraacetic acid even in the presence of Co²⁺, suggesting that LaaA_Bd is a Co²⁺-dependent enzyme. LaaA_Bd had hydrolyzing activity toward a broad range of l-amino acid amides including l-phenylalaninamide, l-glutaminamide, l-leucinamide, l-methioninamide, l-argininamide, and l-2-aminobutyric acid amide. Using information on the N-terminal amino acid sequence of the enzyme, the gene encoding LaaA_Bd was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 4,446 bp of the cloned DNA revealed the presence of seven open-reading frames (ORFs), one of which (laaA _Bd ) encodes the amidase. LaaA_Bd is composed of 491 amino acid residues (calculated molecular weight 51,127), and the deduced amino acid sequence exhibits significant similarity to that of ORFs encoding hypothetical cytosol aminopeptidases found in the genomes of Caulobacter crescentus, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Mesorhizobium loti, and Agrobacterium tumefaciens, and leucine aminopeptidases, PepA, from Rickettsia prowazekii, Pseudomonas putida ATCC 12633, and Escherichia coli K-12. The laaA _Bd gene modified in the nucleotide sequence upstream from its start codon was overexpressed in an E. coli transformant. The activity of the recombinant LaaA_Bd in cell-free extracts of the E. coli transformant was 25.9 units mg⁻¹ with l-phenylalaninamide as substrate, which was 50 times higher than that of B. diminuta TPU 5720.     

Overexpression in Escherichia coli and functional reconstitution of anchovy trypsinogen from the bacterial inclusion body

We have synthesized and optimized a high-yielding Escherichia coli expression system to produce trypsinogen from anchovy Engraulis japonicus and have developed conditions for its successful refolding. Recombinant anchovy trypsinogen precipitated in E. coli Rosetta (DE3) placI strain as inclusion bodies was denatured by 6 M guanidine-HCl followed by refolding with drop wise addition to a large excess of a folding buffer containing 0.5 M non-detergent sulfobetaine (NDSB-251) and a redox potential of oxidized and reduced glutathiones. The folded trypsinogen was autocatalytically activated to its mature form, trypsin, and purified with a MonoQ ion-exchange column. NH₂-terminal amino acid sequencings revealed that E. coli efficiently processed NH₂-terminal methionine residue from the expressed trypsinogen and that trypsinogen was activated at the correct site to generate active trypsin. The recombinant enzyme showed kinetic properties comparable to those of the native enzyme and demonstrated a typical cleavage preference for arginine over lysine residue against a protein substrate. The optimized expression and folding procedures yielded 12 mg of purified, active trypsin from 1 L of bacterial culture or 45 g wet weight cells, which is quite enough for various analytical and semipreparative purposes.     

Cloning and heterologous expression of a novel arylmalonate decarboxylase gene from Alcaligenes bronchisepticus KU 1201

We have cloned and sequenced a DNA fragment that encodes the arylmalonate decarboxylase (AMDase) gene from Alcaligenes bronchisepticus KU 1201. The AMDase gene consists of an open reading frame of 720 nucleotides, which specifies a 240-amino-acid protein of relative molecular mass (M_r) 24734. The M_r deduced from the AMDase gene is in good agreement with that of the AMDase isolated from A. bronchisepticus. No TATA or TTGA sequence was observed within the cloned DNA fragment, but the fragment was expressed in Escherichia coli by the lac promoter of pUC19. The enzyme produced in E. coli has the same M_r and the same enzyme activity as the purified from A. bronchisepticus. Comparison of the DNA sequence and the deduced amino acid sequence of AMDase with available DNA and amino acid sequence data bases revealed that there are no significant sequence homologies.Correspondence to: Hiromichi Ohta     

Methionine-repressible homoserine dehydrogenase of serratia marcescens: Purification and properties

Summary Serratia marcescens Sa-3 possesses two homoserine dehydrogenases and neither has any aspartokinase activity unlike the case ofEs-cherichia coli enzymes. The two enzymes have been separated. One of them is active with either NAD^– or NADP⁺ and has been purified about 180-fold to homogeneity. This enzyme is completely repressed by the presence of 1mm methionine or homoserine in the growth medium, but its activity is unaffected by any amino acid of the aspartate family either singly or together. In many of its properties (such as pH optimum, K_m for substrate and cofactors), it resembles its counterpart inE. coli K₁₂. Potassium ions stabilize the enzyme but are not essential for activity. Its molecular weight is around 155,000 as determined by gel filtration and approximately 76,000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme has two subunits (polypeptide chains) in the molecule: 8m urea has no effect on enzyme activity. This enzyme represents approximately 30% of the total homoserine dehydrogenase activity ofS. marcescens unlike inSalmonella typhimurium andE. coli K₁₂ where it is a minor or a negligible component.     

Cloning and Nucleotide Sequencing of l-Lactate Dehydrogenase Gene from Streptococcus thermophilus M-192

The gene encoding l-lactate dehydrogenase (LDH) was cloned from an industrial dairy strain of Streptococcus thermophilus M-192 using a synthetic oligonucleotide probe based on the N-terminal amino acid sequence of the purified enzyme, and its nucleotide sequence was determined. The enzyme was deduced to have 328 amino acid residues with a molecular weight of 35,428 and found to have high sequence similarity to LDHs from other lactic acid bacteria (89.0% to Streptococcus mutans, 76.3% to Lactococcus lactis subsp. lactis, 67% to Lactobacillus casei, and 60% to Lactobacillus plantarum). The gene contained a promoter-like sequence similar to the Escherichia coli promoter consensus, and expression of the S. thermophilus LDH gene was observed in E. coli cells.     

E. coli galactose-1-phosphate uridyl transferase: N-terminal and C-terminal sequences

Summary A modified procedure for the purification of E. coli galactose-1-phosphate uridyl transferase (E.C. 2.7.6.12) was developed which reproducibly gives pure enzyme. The purified enzyme was shown to be a dimeric protein with a subunit molecular weight of 41,000 and its amino acid composition and content of free sulfhydryl groups were determined. The N-terminal and C-terminal amino acid sequences were found to be NH₂-thr-gln-phe-asn-pro-val-asp and -ser(val leu)-ala-COOH respectively. This N-terminal sequence allowed the identification of the start of the transferase gene in the DNA sequence determined by GRINDLEY. Furthermore it appears to define a nine base intercistronic region between the epimerase and transferase genes.Abbreviations Cyclic AMP Cyclic adenosine 2¹5¹ monophosphate - DPN Diphosphopyridine nucleotide - UDP Uridine diphosphate - EDTA Ethylene diamine tetra acetic acid - SDS sodium dodecyl sulfate - NEM N-ethylmaleimide     

Analysis of a cDNA encoding arginine decarboxylase from oat reveals similarity to the Escherichia coli arginine decarboxylase and evidence of protein processing

Summary Arginine decarboxylase is the first enzyme in one of the two pathways of putrescine synthesis in plants. We purified arginine decarboxylase from oat leaves, obtained N-terminal amino acid sequence, and then used this information to isolate a cDNA encoding oat arginine decarboxylase. Comparison of the derived amino acid sequence with that of the arginine decarboxylase gene from Escherichia coli reveals several regions of sequence similarity which may play a role in enzyme function. The open reading frame (ORF) in the oat cDNA encodes a 66 kDa protein, but the arginine decarboxylase polypeptide that we purified has an apparent molecular weight of 24 kDa and is encoded in the carboxyl-terminal region of the ORF. A portion of the cDNA encoding this region was expressed in E. coli, and a polyclonal antibody was developed against the expressed polypeptide. The antibody detects 34 kDa and 24 kDa polypeptides on Western blots of oat leaf samples. Maturation of arginine decarboxylase in oats appears to include processing of a precursor protein.     

Purification and characterization of isoamyl acetate-hydrolyzing esterase encoded by the IAH1 gene of Saccharomyces cerevisiae from a recombinant Escherichia coli

The IAH1 gene of Saccharomyces cerevisiae encodes an esterase that preferentially acts on isoamyl acetate; however, the enzyme has not yet been completely purified from the yeast S. cerevisiae. We constructed the IAH1 gene expression system in Escherichia coli, and purified the IAH1 gene product (Iah1p). The amount of Iah1p produced by recombinant E. coli was more than 40% of total cellular proteins. The molecular size of Iah1p was 28 kDa by SDS-polyacrylamide gel electrophoresis. Judging from the molecular weight estimation by gel filtration of purified Iah1p, the enzyme was thought to be a homodimer. The K _m values for isoamyl acetate and isobutyl acetate were 40.3 mM and 15.3 mM, respectively. The enzyme activity was inhibited by Hg²⁺, p-chloromercuribenzoate, and diisopropylfluorophosphate. Received: 23 May 1999 / Received revision: 27 October 1999 / Accepted: 5 November 1999     

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.     

Nucleotide sequence of the gene encoding novel delta-endotoxin from Bacillus thuringiensis serovar japonensis strain Buibui specific to scarabaeid beetles

A new isolate of Bacillus thuringiensis serovar japonensis strain Buibui, which was specific to scarab beetles (M. Ohba et al., Lett. Appl. Microbiol. 14:54, 1992), was shown to have a 130-kDa insecticidal crystal protein (ICP) (H. Hori et al., J. Appl. Bacteriol. 76:307, 1994). CalI restriction enzyme fragments of total cell DNA of the isolate were cloned into E. coli (Sato et al., Curr. Microbiol. 28:15, 1994). Whole 3480-bp nucleotide sequence of the gene encoding 130-kDa ICP was determined, and the molecular weight of the ICP was estimated to be 130,424. The strongly conserved five blocks that occur in almost all ICP genes of B. thuringiensis were detected in the ORF with the same order and almost the same intervals as elsewhere. The amino acid sequence homologies of the whole ICP or N-terminus half portion to that of the CryIIIA, B, C, D, and CryV were about 35%.     

Molecular cloning and characterization of the gene encoding a fibrinolytic enzyme from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Strain A1

A fibrinolytic metalloprotease gene from Bacillus subtilis has been cloned in Escheridria coliXL1-Blue and the bacterial expressed enzyme was purified. The nucleotide sequence of the cloned fibrinolytic enzyme gene revealed a single open reading frame of 1023 bp coding for 341 amino acids (M _r 37708.21 Da). N-terminal amino acid sequencing of the fibrinolytic enzyme excreted from E. coli host cells revealed that the mature fibrinolytic enzyme consists of 288 amino acids (M _r 31391.1 Da). The deduced amino acid sequence showed significant homology with Erwina carotovora neutral metalloprotease and Serratia marcescens minor metalloprotease by 65 and 58% amino acid sequence identity, respectively. The protein showed significant alignments with the conserved domain of catalytic activity and the -helix domain in Bacillus anthracisthermolysis metalloprotease. The biochemical properties of the purified enzyme suggested that the enzyme is a fibrinolytic metalloprotease, which has optimal activity at pH 7.0 and 50 °C.     

Thermostable xylanase from Streptomyces thermocyaneoviolaceus for optimal production of xylooligosaccharides

A thermo stable xylanase was purified from Streptomyces thermocyaneoviolaceus M049 for the production of xylooligosaccharides from xylan. The enzyme showed thermostability by maintaining 65% of remaining enzyme activity after 1 h heat treatment at 70°C. The molecular weight of the purified protein was 35 kDa in SDS-PAGE, and the optimal pH and temperature for the enzymatic activity were pH 5.0 and 60°C, respectively. N-terminal amino acid sequences of the purified xylanase, DTITSNQTGTHNGYF, were similar to StxII from S. Thermoviolaceus and XlnB from S. lividans. Using those two genes, stxll and xlnB as probe DNA, a gene encoding xylanase, xynB, was cloned from genomic library of S. thermocyaneoviolaceus M049. The open reading frame of the xynB was composed of 1008 nucleotide sequences. Compared to N-terminal sequences from purified enzyme, it was proposed that the XynB contained a 40 amino acid long signal peptide to the N-terminus. For easy production and purification, a XynB overproduction strain was constructed using pET21a(+) and strain E. coli BLR(DE3). Consequently, the recombinant enzyme was tested for the production of xylooligosaccharides through TLC and HPLC analyses.     

Functional studies of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunits A and B expressed in Escherichia coli: formation of highly active A4 and B4 homotetramers and evidence that aggregation of the B4 complex is mediated by the B subunit carboxy terminus

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (phosphorylating, E.C. 1.2.1.13) (GAPDH) of higher plants exists as an A₂B₂ heterotetramer that catalyses the reductive step of the Calvin cycle. In dark chloroplasts the enzyme exhibits a molecular mass of 600 kDa, whereas in illuminated chloroplasts the molecular mass is altered in favor of the more active 150 kDa form. We have expressed in Escherichia coli proteins corresponding to the mature A and B subunits of spinach chloroplast GAPDH (GapA and GapB, respectively) in addition to a derivative of the B subunit lacking the GapB-specific C-terminal extension (CTE). One mg of each of the three proteins so expressed was purified to electrophoretic homogeneity with conventional methods. Spinach GapA purified from E. coli is shown to be a highly active homotetramer (50–70 U/mg) which does not associate under aggregating conditions in vitro to high-molecular-mass (HMM) forms of ca. 600 kDa. Since B₄ forms of the enzyme have not been described from any source, we were surprised to find that spinach GapB purified from E. coli was active (15–35 U/mg). Spinach GapB lacking the CTE purified from E. coli is more highly active (130 U/mg) than GapB with the CTE. Under aggregating conditions, GapB lacking the CTE is a tetramer that does not associate to HMM forms whereas GapB with the CTE occurs exclusively as an aggregated HMM form. The data indicate that intertetramer association of chloroplast GAPDH in vitro occurs through GapB-mediated protein-protein interaction.Abbreviations GAPDH glyceraldehyde-3-phosphate dehydrogenase - CTE carboxy-terminal extension - HMM high molecular mass - ATP adenosine triphosphate - 3PGA 3-phosphoglycerate - 1,3bisPGA 1,3-bisphosphoglycerate - HMM high-molecular mass     

Cloning of a superoxide dismutase gene from Listeria ivanovii by functional complementation in Escherichia coli and characterization of the gene product.

Summary A gene encoding superoxide dismutase (EC 1.15.1.1., SOD) was isolated from a plasmid library of chromosomal DNA from Listeria ivanovii by functional complementation of an SOD-negative Escherichia coli host. The nucleotide sequence of the cloned gene was determined and contained an open reading frame which codes for a protein of 202 amino acid residues (calculated molecular weight 22 755 Da including the amino-terminal methionine residue). Comparison of the deduced amino acid sequence of L. ivanovii SOD with previously reported SOD amino acid sequences revealed considerable homologies with Fe- and Mn-dependent SODs. Enzymatic analyses using cell lysates and the purified recombinant enzyme indicated that this SOD is manganese-dependent. The recombinant SOD accounted for up to 30% of the total soluble protein in recombinant E. coli and protected sodA sodB mutants against the toxic effects of paraquat. Subunits of the recombinant Listeria SOD and of both E. coli SODS formed enzymatically active hybrids in vivo.Some of our preliminary observations have been published as a conference report of SOD V (Jerusalem, 1989) in Free Rad Res Commun (1991) 12–13:371     

Reversal Effect of Potassium Ion on Microbial Growth Inhibition by Combinations of NaCl,Ethanol and Perillaldehyde

The glutamate racemase (EC 5.1.1.3) gene of a lactic acid bacterium, Pediococcus pentosaceus, was cloned into Escherichia coli C600 with a vector plasmid, pBR322. The requirement of l-glutamate for the growth of E. coli in the minimum medium containing d-glutamate and the formation of a red pigment in a coupled enzyme reaction mixture were used to select clones expressing glutamate racemase activity. Glutamate racemase overproduced as 0.3— 2.0% of the total soluble proteins in a clone carrying the plasmid pICR221, 10.3 kb of DNA, was purified from cell extracts about 130-fold to homogeneity. The purified enzyme has a molecular weight of about 40,000 and is a single polypeptide chain. Glutamate is the sole substrate for the enzyme. Unlike many other amino acid racemases, glutamate racemase is devoid of cofactors: there is no evidence for pyridoxal 5’-phosphate or FAD in the ultraviolet spectrum of the purified enzyme, and the enzyme is not inactivated by carbonyl reagents such as hydroxylamine and sodium borohydride.