Cold-active serine alkaline protease from the psychrotrophic bacterium Shewanella strain ac10: gene cloning and enzyme purification and characterization

The gene encoding serine alkaline protease (SapSh) of the psychrotrophic bacterium Shewanella strain Ac10 was cloned in Escherichia coli. The amino acid sequence deduced from the 2,442-bp nucleotide sequence revealed that the protein was 814 amino acids long and had an estimated molecular weight of 85,113. SapSh exhibited sequence similarities with members of the subtilisin family of proteases, and there was a high level of conservation in the regions around a putative catalytic triad consisting of Asp-30, His-65, and Ser-369. The amino acid sequence contained the following regions which were assigned on the basis of homology to previously described sequences: a signal peptide (26 residues), a propeptide (117 residues), and an extension up to the C terminus (about 250 residues). Another feature of SapSh is the fact that the space between His-65 and Ser-369 is approximately 150 residues longer than the corresponding spaces in other proteases belonging to the subtilisin family. SapSh was purified to homogeneity from the culture supernatant of E. coli recombinant cells by affinity chromatography with a bacitracin-Sepharose column. The recombinant SapSh (rSapSh) was found to have a molecular weight of about 44,000 and to be highly active in the alkaline region (optimum pH, around 9.0) when azocasein and synthetic peptides were used as substrates. rSapSh was characterized by its high levels of activity at low temperatures; it was five times more active than subtilisin Carlsberg at temperatures ranging from 5 to 15 degreesC. The activation energy for hydrolysis of azocasein by rSapSh was much lower than the activation energy for hydrolysis of azocasein by the subtilisin. However, rSapSh was far less stable than the subtilisin.     

Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan

A strong fibrinolytic enzyme (nattokinase) was purified from the vegetable cheese natto. Nattokinase was extracted from natto with saline and isolated by sequential use of hydrophobic chromatography on Butyl-Toyopearl, ion-exchange chromatography on CM-Toyopearl, and gel-filtration on Sephadex G-50. The isolated protein gave a single sharp band on SDS-PAGE either before or after reduction. The sequence, as determined by automated Edman degradation of the uncleaved molecule and its enzymatically derived peptide, consisted of a total 275 amino acid residues (M.W = 27,728) and exhibited a high homology with the subtilisins. The purified nattokinase digested not only fibrin but also several synthetic substrates. Among the synthetic substrates, the most sensitive substrate was Suc-Ala-Ala-Pro-Phe-pNA for subtilisin. PMSF inhibited both the fibrinolytic activity and the amidolytic activity. The results indicate that nattokinase is a subtilisin-like serine protease.     

Cloning and high-level expression of alpha-galactosidase cDNA from Penicillium purpurogenum

The cDNA coding for Penicillium purpurogenum alpha-galactosidase (alphaGal) was cloned and sequenced. The deduced amino acid sequence of the alpha-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic alphaGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was to Trichoderma reesei AGLI. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P. purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides.     

Primary structure of Streptomyces griseus metalloendopeptidase II

Streptomyces griseus metalloendopeptidase II (SGMPII) is a unique protease, since it shows anomalous susceptibility to the proteinaceous "serine protease inhibitors" produced by Streptomyces, such as Streptomyces subtilisin inhibitor (SSI) and its homologous proteins. In this study, we analyzed the amino acid sequence of SGMPII by analyzing various peptide fragments produced enzymatically. The sequence of SGMPII, which is composed of 334 amino acids, showed no extensive similarity to SSI-insensitive metalloproteases produced by other species of Streptomyces, except for the amino acid residues essential for catalysis and zinc binding. However, SGMPII is 35-41% similar to thermolysin and its related metalloproteases, which are not inhibited by SSI, and the residues presumed to be critical for catalysis and zinc-binding are well conserved in SGMPII. Glu137 in a "His-Glu-Xaa-His" motif of SGMPII was identified as the residue modified by CICH2 CO-DL-(N-OH)Leu-Ala-Gly-NH2, an active-site-directed irreversible inhibitor of thermolysin-like metalloproteases. Based on the sequence comparison of SGMPII and other bacterial metalloproteases, we discuss the structural basis for the differences in substrate specificity and stability between SGMPII and other thermolysin-like proteases. A possible SSI-binding locus of SGMPII is also proposed.     

L-allo-threonine aldolase from Aeromonas jandaei DK-39: gene cloning, nucleotide sequencing, and identification of the pyridoxal 5'-phosphate-binding lysine residue by site-directed mutagenesis

We have isolated the gene encoding L-allo-threonine aldolase (L-allo-TA) from Aeromonas jandaei DK-39, a pyridoxal 5'-phosphate (PLP)-dependent enzyme that stereospecifically catalyzes the interconversion of L-allo-threonine and glycine. The gene contains an open reading frame consisting of 1,014 nucleotides corresponding to 338 amino acid residues. The protein molecular weight was estimated to be 36,294, which is in good agreement with the subunit molecular weight of the enzyme determined by polyacrylamide gel electrophoresis. The enzyme was overexpressed in recombinant Escherichia coli cells and purified to homogeneity by one hydrophobic column chromatography step. The predicted amino acid sequence showed no significant similarity to those of the currently known PLP-dependent enzymes but displayed 40 and 41% identity with those of the hypothetical GLY1 protein of Saccharomyces cerevisiae and the GLY1-like protein of Caenorhabditis elegans, respectively. Accordingly, L-allo-TA might represent a new type of PLP-dependent enzyme. To determine the PLP-binding site of the enzyme, all of the three conserved lysine residues of L-allo-TA were replaced by alanine by site-directed mutagenesis. The purified mutant enzymes, K51A and K224A, showed properties similar to those of the wild type, while the mutant enzyme K199A was catalytically inactive, with corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys199 of L-allo-TA probably functions as an essential catalytic residue forming an internal Schiff base with PLP of the enzyme to catalyze the reversible aldol reaction.     

Isocitrate dehydrogenase from Thermus aquaticus YT1: purification of the enzyme and cloning, sequencing, and expression of the gene

Isocitrate dehydrogenase from an extremely thermophilic bacterium, Thermus aquaticus YT1, was purified to homogeneity, and the gene was cloned by using a degenerate oligonucleotide probe based on the N-terminal sequence. The gene consisted of a single open reading frame of 1,278 bp preceded by a Shine-Dalgarno ribosome binding site, and a terminator-like sequence was detected downstream of the open reading frame. The G+C content of the coding region was 65%, and that of the third nucleotide of the codons was 93%. The amino acid sequence of the enzyme showed a relatively low level of similarity to the counterpart from T. thermophilus (35% identity) but showed higher levels of similarity (54 to 69% identity) to the other bacterial counterparts so far reported, including those from Escherichia coli, Bacillus subtilis, Vibrio sp., and Anabaena sp. The cloned gene was highly expressed in E. coli and easily purified to homogeneity by heat treatment (70 degrees C, 30 min) and DEAE column chromatography to yield approximately 10 mg of protein from 1 g of wet cells. The recombinant enzyme showed high thermostability and almost the same heat denaturation profile as the intact enzyme purified from the thermophile cells, implying that the recombinant protein has the same structure as the intact one.     

Effects of hypertonia on voltage-gated ion currents in freshly isolated hippocampal neurons, and on synaptic currents in neurons in hippocampal slices

A NAD-dependent mannitol dehydrogenase (MtlD) was purified to homogeneity from P. fluorescens DSM50106 and the N-terminal amino acid sequence was determined. An oligonucleotide deduced from this peptide sequence was used as a probe to isolate the mannitol dehydrogenase gene (mtlD) from a genomic library of P. fluorescens. Nucleotide sequence analysis of a 1.8 kb NruI fragment containing the entire mtlD gene revealed an open reading frame of 1482 bp encoding a protein with a calculated molecular weight of 54.49 kDa. The enzyme shared a high similarity with a mannitol dehydrogenase from Rhodobacter sphaeroides and a putative mannitol dehydrogenase of Saccharomyces cerevisae with an overall identity in amino acid sequence of 44% and 42%, respectively, whereas the similarity to mannitol-1-phosphate dehydrogenases of Escherichia coli or Enterococcus faecalis was only about 23% of identical amino acids. By construction of inducible expression plasmids the specific activity of the mannitol dehydrogenase synthesized in E. coli was increased from 0.02 U (mg protein)(-1) to 10 U (mg protein)(-1). After fusion of six histidine codons to the 3' end of mtlD gene and expression in E. coli active mannitol dehydrogenase could be purified in a two-step procedure by affinity chromatography using a Ni2+ matrix column. The purified enzyme exhibited a specific activity of 46 U (mg protein)(-1) and was shown to be a polyol dehydrogenase with a broad substrate spectrum oxidizing efficiently mannitol, sorbitol and arabitol.     

Dobutamine-induced hypoperfusion without transient wall motion abnormalities: less severe ischemia or less severe stress?

We describe the first cDNA sequence encoding a juvenile hormone-specific epoxide hydrolase from an insect. A full-length cDNA clone revealed a 462-amino-acid open reading frame encoding an amino acid sequence with 44% identity and 64% similarity to human microsomal epoxide hydrolase. All residues in the catalytic triad (residues Asp227-His428-Asp350 in the M. sexta protein) were present, as was the conserved Trp154 corresponding to the oxyanion hole. The surprising similarity of insect juvenile hormone epoxide hydrolase to vertebrate microsomal epoxide hydrolases, coupled with the ancient lineage of the epoxide hydrolases and haloalkane dehalogenases, suggests that this catabolic enzyme evolved from an original ubiquitous detoxication function to a more recent role in hormonal regulation.     

Enhancing regional perinatal care: a clinical traineeship for perinatal nurses in a predominantly rural area

The gene nprM encoding the calcium-dependent extracellular proteinase from Bacillus megaterium ATCC 14581 was cloned in the vector pBR322 and expressed in Escherichia coli HB101. The DNA sequence of the cloned 3.7 kb fragment revealed only one open reading frame consisting of 1686 bp with a coding capacity of 562 amino acid residues. A predicted Shine-Dalgarno (SD) sequence was observed 9 bp upstream from the presumptive translation start site (ATG). A possible promoter sequence (TAGACG for the -35 region and TATAAT for the -10 region) was found about 69 bp upstream of the ATG start site. The deduced amino acid sequence exhibited a 24 amino acid residue signal peptide and an additional polypeptide 'pro' sequence of 221 amino acids preceding the putative mature protein of 317 amino acid residues. Amino acid sequence comparison revealed 84.5% homology between the mature protein and that of a thermolabile neutral protease from B. cereus. It also shares 73% homology with the thermostable neutral proteases of B. thermoproteolyticus and B. stearothermophilus. The zinc-binding sites and the catalytic residues are completely conserved in all four proteases. NprM has a temperature optimum of 58 degrees C, a pH optimum of between 6.4 and 7.2, and is stimulated by calcium ions and inhibited by EDTA. These results indicate that the enzyme is a neutral (metallo-) protease.     

The HPr(Ser) kinase of Streptococcus salivarius: purification, properties, and cloning of the hprK gene

In gram-positive bacteria, HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system, is phosphorylated on a serine residue at position 46 by an ATP-dependent protein kinase. The HPr(Ser) kinase of Streptococcus salivarius ATCC 25975 was purified, and the encoding gene (hprK) was cloned by using a nucleotide probe designed from the N-terminal amino acid sequence. The predicted amino acid sequence of the S. salivarius enzyme showed 45% identity with the Bacillus subtilis enzyme, the conserved residues being located mainly in the C-terminal half of the protein. The predicted hprK gene product has a molecular mass of 34,440 Da and a pI of 5.6. These values agree well with those found experimentally by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, molecular sieve chromatography in the presence of guanidine hydrochloride, and chromatofocusing using the purified protein. The native protein migrates on a Superdex 200 HR column as a 330,000-Da protein, suggesting that the HPr(Ser) kinase is a decamer. The enzyme requires Mg2+ for activity and functions optimally at pH 7.5. Unlike the enzyme from other gram-positive bacteria, the HPr(Ser) kinase from S. salivarius is not stimulated by FDP or other glycolytic intermediates. The enzyme is inhibited by inorganic phosphate, and its Kms for HPr and ATP are 31 microM and 1 mM, respectively.     

Protease evolution in Streptomyces griseus. Discovery of a novel dimeric enzymes

This report describes the cloning and sequencing of a novel protease gene derived from Streptomyces griseus. Also described is the heterologous expression of the gene in Bacillus subtilis and characterization of the gene product. The sprD gene encodes a prepro mature protease of 392 amino acids tentatively named S. griseus protease D (SGPD). A significant component of the enzyme preregion was found to be homologous with the mitochondrial import signal of hsp60. The sprD gene was subcloned into an Escherichia coli/B. subtilis shuttle vector system such that the pro mature portion of SGPD was fused in frame with the promoter, ribosome binding site, and signal sequences of subtilisin. The gene fusion was subsequently expressed in B. subtilis DB104, and active protease was purified. SGPD has a high degree of sequence homology to previously described S. griseus proteases A, B, C, and E and the alpha-lytic protease of Lysobacter enzymogenes, but unlike all previously characterized members of the chymotrypsin superfamily, the recombinant SGPD forms a stable alpha 2 dimer. The amino acid sequence of the protein in the region of the specificity pocket is similar to that of S. griseus proteases A, B, and C. The purified enzyme was found to have a primary specificity for large aliphatic or aromatic amino acids. Nucleotide sequence data were used to construct a phylogenetic tree using a method of maximum parsimony which reflects the relationships and potentially the lineage of the chymotrypsin-like proteases of S. griseus.     

Disproportionating enzyme (4-alpha-glucanotransferase; EC 2.4.1.25) of potato. Purification, molecular cloning, and potential role in starch metabolism

Disproportionating enzyme (D-enzyme, 4-alpha-glucanotransferase; EC 2.4.1.25) has been purified to homogeneity from potato tubers and its activity characterized. The enzyme catalyzes the transfer of maltooligosaccharides from one 1,4-alpha-D-glucan molecule to another, or to glucose. Maltooligosaccharides are effective donor molecules, but short chain amylose and amylopectin may also function as donors. Enzyme activity is not affected by inorganic phosphate, 3-phosphoglycerate, or hexose phosphates. A cDNA clone encoding the enzyme was isolated using oligonucleotide probes derived from partial peptide sequences of the purified enzyme. The identity of the cDNA clone was confirmed by expression in Escherichia coli resulting in D-enzyme activity. The amino acid sequence deduced from the cDNA shows significant homology with a 4-alpha-glucanotransferase from Streptococcus. The deduced sequence indicates the presence of an amino-terminal plastid transit peptide of 52 amino acid residues and a mature polypeptide of 524 residues. D-enzyme mRNA is present in leaves, stems, roots, and stolons but is most abundant in developing and mature tubers. The amount of mRNA in leaves increases in response to light and to sucrose added to the medium. These results are discussed in terms of the function of D-enzyme in potato starch metabolism.     

Purification and biochemical analysis of WprA, a 52-kDa serine protease secreted by B. subtilis as an active complex with its 23-kDa propeptide

The Gram-positive bacterium Bacillus subtilis produces numerous proteases that are secreted to the extracellular milieu, and as strains are generated which lack the more prominent proteases, minor ones become detectable. We have isolated a 52-kDa secreted protease from the protease-deficient strain WB600. It is encoded by the wprA gene which encompasses a signal sequence, a 46-kDa propeptide further processed to 23 kDa, and the 52-kDa mature protease. The 52-kDa and 23-kDa polypeptides were previously detected in cell-wall preparations of a wild-type strain. We have co-purified these proteins from culture supernatant, and confirmed the same N-termini and molecular weights as the membrane-bound species. The WprA protease domain has 28.5% identity to subtilisin A, and like other subtilisins, it displays a broad substrate specificity. WprA and subtilisin A have similar pH profiles, showing optimal activity near pH 7.5 for substrates with Met, Gln, or Lys residues at P1. Using a substrate with Asp at P1, another peak of activity was observed for WprA at pH 5 and at pH 6 for subtilisin A. The pH dependence of some bacterial proteases in their interaction with substrates and inhibitors may be biologically relevant.     

Purification and characterization of NADH-dependent glutamate synthase from the silkworm fat body (Bombyx mori)

NADH-dependent glutamate synthase (Nadh-Gogat; EC 1.41.14) was purified 766-fold from the fat body of 5th instar larvae of the silkworm with a final specific activity of 13.8 units/mg protein by a produce including ammonium sulfate fraction, Q-Sepharose HP ion exchange column chromatography, Blue Sepharose FF affinity column chromatography and Superdex 200 HR gel filtration. The purified enzyme yielded a single band corresponding to a molecular mass of 195kDa by SDS-polyacrylamide gel electrophoresis. Molecular mass of the native enzyme was estimated to be 190 kDa by Superdex 200HR gel filtration, suggesting that the enzyme is composed of a monomeric polypeptide. The enzyme showed an absorption spectrum with maximum values at 272, 375, and 435 nm, suggesting the presence of a flavin prosthetic group in the enzyme. The N-terminal amino acid sequence showed a high similarity to those of other GOGATs from plants, yeast and bacteria, but no similarity to other known proteins was detected. The enzyme exhibited a strong specificity to the electron donor and substrates; NADH as electron donor, 2-oxoglutarate as amino acceptor and glutamine as amino donor were essential for the catalytic activity. The optimum pH was around 7.5, at which Km values for 2-oxoglutarate, glutamine and NADH were 17, 220 and 5.7 micro M, respectively. Azaserine, 6-diazo-5-oxonorleucine and p-chloromercuribenzoic acid were strong inhibitors of the activity. These result show that NADH-GOGAT in the silkworm fat body strongly resembles other eukaryotic NADH-GOGATs, suggesting that it plays a significant role in ammonia assimilation in the same manner as the other enzymes.     

A novel protease in the pupal yellow body of Sarcophaga peregrina (flesh fly). Its purification and cDNA cloning

We purified a novel serine protease with a molecular mass of 26 kDa from Sarcophaga pupae. This protease appeared almost exclusively in the yellow body, an organ that develops temporarily in the pupae of dipteran insects and expands to form the adult midgut by engulfing the larval midgut. cDNA analysis revealed that this protease consists of 239 amino acid residues and has significant structural similarity with bovine trypsin (about 40% sequence identity). The 26-kDa protease gene was transiently activated in 1-day-old pupae. The protease was found to cross-react immunologically with antibody against sarcotoxin IA, an antibacterial protein produced by this insect. It is suggested that this protease participates in the decomposition of the larval midgut in the yellow body during metamorphosis.     

Discriminable stimuli produced by hallucinogens

Bison pancreatic ribonuclease was isolated by affinity chromatography. Thermolysin and tryptic digestion of denaturated protein, and subtilisin digestion of native protein yielded peptides, which were purified and submitted to amino acid analysis. These peptides, together with partial sequence data obtained by Stewart & Stevenson (16) overlap the entire amino acid sequence of bison ribonuclease. No differences with bovine ribonuclease were found, although there may be differences in state of amidation of some residues.     

Mortality prediction in head trauma patients: performance of Glasgow Coma Score and general severity systems

A glycerol-ester hydrolase was purified to homogeneity from porcine intestinal mucosa using a partial delipidation method and an eight-step purification procedure. The isolation scheme used gave a 483-fold purification, resulting in a pure enzyme with a specific activity on tributyrin of 290 micromol x min(-1) x mg(-1). The molecular mass of the enzyme was estimated at 240 kDa, based on the results of size-exclusion chromatography, and at 60 kDa, as determined by SDS/PAGE analysis. The isoelectric focusing data obtained indicated that only one isoform with a pI of 5.1 was present. Complete identity was found to exist between the N-terminal sequence of the first 25 amino acid residues and that of a porcine liver carboxylesterase. A full-length cDNA coding for the enzyme was isolated from pig small intestine. We observed that the corresponding protein originally named intestinal glycerol-ester hydrolase definitely belongs to the carboxylesterase family. The deduced amino acid sequence consisted of 565 residues and showed 97% identity with that of porcine liver carboxylesterase and more than 50% identity with those of other carboxylesterases from different mammalian species.     

Isolation and amino acid sequence of a protein-synthesis inhibitor from the seeds of rye (Secale cereale)

A protein-synthesis inhibitor, designated RPSI, was isolated from the seeds of rye (Secale cereale) using gel filtration and S-Sepharose column chromatography. RPSI is a basic protein with an isoelectric point of over 10, and the concentration of protein required for 50% inhibition of protein synthesis (IC50) of purified RPSI was about ten-fold the concentration of ricin A-chain. The complete amino acid sequence of RPSI was discovered by analyzing the peptides and fragments obtained from the proteolytic digests and by the cyanogen bromide- and hydroxylamine-cleavages of RPSI. RPSI consists of 280 amino acid residues and has a molecular weight of 30,171. RPSI has only 21% sequence identity with that of ricin A-chain, but all five amino acid residues involved in the active site of ricin A-chain are conserved in RPSI.