Glutathione as an essential factor for chaperon-mediated activation of lactonizing lipase (LipL) from Pseudomonas sp. 109

Pseudomonas sp. 109 produces a unique lipase (LipL) which efficiently catalyzes intramolecular transesterification of omega-hydroxyesters to form macrocyclic lactones. The production of the enzymatically active LipL requires a specific molecular chaperon (LimL protein) together with a low-M(r) lipase-activation-factor (LAF) of unknown structure. From 50 g of Pseudomonas cells, 2.15 mg of LAF was purified as a sulfobenzofurazanyl derivative after methanol extraction, derivatization, and C(18) reverse-phase HPLC. One-dimensional and two-dimensional 600 MHz (1)H-NMR and fast atom bombardment mass spectrometry (FAB-MS) revealed that LAF is glutathione. Because several SH compounds (L-cysteine and mercaptoethanol) were similarly effective to native LAF in the activation of LipL, and because only LipL contains two cysteinyl residues forming an intramolecular disulfide bond, it is concluded that the reduction of and reformation of the intramolecular disulfide bond of LipL is essential to liberate free and fully active LipL.     

Dienelactone hydrolase from Pseudomonas sp. strain B13.

Dienelactone hydrolase (EC 3.1.1.45) catalyzes the conversion of cis- or trans-4-carboxymethylenebut-2-en-4-olide (dienelactone) to maleylacetate. An approximately 24-fold purification from extracts of 3-chlorobenzoate-grown Pseudomonas sp. strain B13 yielded a homogeneous preparation of the enzyme. The purified enzyme crystallized readily and proved to be a monomer with a molecular weight of about 30,000. Each dienelactone hydrolase molecule contains two cysteinyl side chains. One of these was readily titrated by stoichiometric amounts of p-chloromercuribenzoate, resulting in inactivation of the enzyme; the inactivation could be reversed by the addition of dithiothreitol. The other cysteinyl side chain appeared to be protected in the native protein against chemical reaction with p-chloromercuribenzoate. The properties of sulfhydryl side chains in dienelactone hydrolase resembled those that have been characterized for bacterial 4-carboxymethylbut-3-en-4-olide (enol-lactone) hydrolases (EC 3.1.1.24), which also are monomers with molecular weights of about 30,000. The amino acid composition of the dienelactone hydrolase resembled the amino acid composition of enol-lactone hydrolase from Pseudomonas putida, and alignment of the NH2-terminal amino acid sequence of the dienelactone hydrolase with the corresponding sequence of an Acinetobacter calcoaceticus enol-lactone hydrolase revealed sequence identity at 8 of the 28 positions. These observations foster the hypothesis that the lactone hydrolases share a common ancestor. The lactone hydrolases differed in one significant property: the kcat of dienelactone hydrolase was 1,800 min-1, an order of magnitude below the kcat observed with enol-lactone hydrolases. The relatively low catalytic activity of dienelactone hydrolase may demand its production at the high levels observed for induced cultures of Pseudomonas sp. strain B13.     

Renaturation, purification and characterization of streptokinase expressed as inclusion body in recombinant E. coli

Recombinant protein purification is facilitated using high expression systems which produce larger quantities of streptokinase protein as inclusion bodies. As the accumulation of active streptokinase is toxic to the host cells, we have optimized the conditions to achieve large amounts of streptokinase in the form of inclusion bodies. The solubility and yield of pure protein are highly dependent on various combinations of chemical additives, ionic and non-ionic detergents and salts, with solubilizing agents followed by refolding of denatured protein into active form. As the extraction of the purified streptokinase from inclusion bodies requires denaturation and a subsequent refolding step, careful balancing steps were needed to develop under different controlled conditions. Here the purified fragments of refolded proteins were screened to select the conditions that yield the active streptokinase having native conformation. The maximum specific activity of the purified streptokinase was achieved by these methods. The refolded recombinant streptokinase was analyzed by RP-HPLC showing a purity of 99%. Size exclusion chromatography profile shows that there are minimal aggregates in the active streptokinase protein and the percentage of renaturation is around 99%.     

Purification and characterization of an amidase from an acrylamide-degrading Rhodococcus sp.

A constitutively expressed aliphatic amidase from a Rhodococcus sp. catalyzing acrylamide deamination was purified to electrophoretic homogeneity. The molecular weight of the native enzyme was estimated to be 360,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified preparation yielded a homogeneous protein band having an apparent molecular weight of about 44,500. The amidase had pH and temperature optima of 8.5 and 40 degrees C, respectively, and its isoelectric point was pH 4.0. The amidase had apparent K(m) values of 1.2, 2.6, 3.0, 2.7, and 5.0 mM for acrylamide, acetamide, butyramide, propionamide, and isobutyramide, respectively. Inductively coupled plasma-atomic emission spectometry analysis indicated that the enzyme contains 8 mol of iron per mol of the native enzyme. No labile sulfide was detected. The amidase activity was enhanced by, but not dependent on Fe(2+), Ba(2+), and Cr(2+). However, the enzyme activity was partially inhibited by Mg(2+) and totally inhibited in the presence of Ni(2+), Hg(2+), Cu(2+), Co(2+), specific iron chelators, and thiol blocking reagents. The NH2-terminal sequence of the first 18 amino acids displayed 88% homology to the aliphatic amidase of Brevibacterium sp. strain R312.     

Dr fimbriae coding region associated hemolytic activity of Escherichia coli

Abstract Escherichia coli LE392 (pAL28) was previously isolated as a positive clone harboring the alginate lyase gene ( aly ) from an alginate-degrading strain, Pseudomonas sp. OS-ALG-9. The plasmid pAL205, one of the constructs obtained after successive subcloning of pAL28, gave the highest expression of aly in E. coli cells. A 8-fold increase in the alginate lyase (Aly) activity in E. coli JM109 (pAL205) was induced with isopropyl-β-d-thiogalactoside, which was 210 times higher than that in E. coli LE392 (pAL28). The highly significant increase in the expression of the Aly enzyme with pAL205 was investigated through the nucleotide sequence around the 5' region of aly as well as the N -terminal sequence of the purified enzyme. It was found that the Aly expressed in E. coli (pAL205) was a fused protein containing 7 residues from the N -terminus of β-galactosidase α-peptide and the mature protein found in the Pseudomonas sp. except for three residues in the N -terminal.     

Expression, single-step purification, and matrix-assisted refolding of a maize cytokinin glucoside-specific beta-glucosidase.

Availability of highly purified native beta-glucosidase Zm-p60.1 in milligram quantities was a basic requirement for analysis of structure-function relationships of the protein. Therefore, Zm-p60.1 was overexpressed to high levels as a fusion protein with a hexahistidine tag, (His)(6)Zm-p60.r, in Escherichia coli, resulting, however, in accumulation of most of the protein in insoluble inclusion bodies. Native (His)(6)Zm-p60.r was then purified either from the bacterial lysate soluble fraction or from inclusion bodies. In the first case, a single-step purification under native conditions based on immobilized metal affinity chromatography (IMAC) was developed. In the second case, a single-step purification protocol under denaturing conditions followed by IMAC-based matrix-assisted refolding was elaborated. The efficiency of the native protein purification from soluble fraction of bacterial homogenate was compared to the feasibility of purification and renaturation of the protein from inclusion bodies. Gain of authentic biological activity and quaternary structure after the refolding process was confirmed by K(m) determination and electrophoretic mobility under native conditions. The yield of properly refolded protein was assessed based on the specific activity of the refolded product.     

Purification and characterization of 2,4,6-trichlorophenol-4-monooxygenase, a dehalogenating enzyme from Azotobacter sp. strain GP1.

The enzyme which catalyzes the dehalogenation of 2,4,6-trichlorophenol (TCP) was purified to apparent homogeneity from an extract of TCP-induced cells of Azotobacter sp. strain GP1. The initial step of TCP degradation in this bacterium is inducible by TCP; no activity was found in succinate-grown cells or in phenol-induced cells. NADH, flavin adenine dinucleotide, and O2 are required as cofactors. As reaction products, 2,6-dichlorohydroquinone and Cl- ions were identified. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of O2 per mol of TCP and the formation of 1 mol of Cl- ions. No evidence for membrane association or for a multicomponent system was obtained. Molecular masses of 240 kDa for the native enzyme and 60 kDa for the subunit were determined, indicating a homotetrameric structure. Cross-linking studies with dimethylsuberimidate were consistent with this finding. TCP was the best substrate for 2,4,6-trichlorophenol-4-monooxygenase (TCP-4-monooxygenase). The majority of other chlorophenols converted by the enzyme bear a chloro substituent in the 4-position. 2,6-Dichlorophenol, also accepted as a substrate, was hydroxylated in the 4-position to 2,6-dichlorohydroquinone in a nondehalogenating reaction. NADH and O2 were consumed by the pure enzyme also in the absence of TCP with simultaneous production of H2O2. The NH2-terminal amino acid sequence of TCP-4-monooxygenase from Azotobacter sp. strain GP1 revealed complete identity with the nucleotide-derived sequence from the analogous enzyme from Pseudomonas pickettii and a high degree of homology with two nondehalogenating monooxygenases. The similarity in enzyme properties and the possible evolutionary relatedness of dehalogenating and nondehalogenating monooxygenases are discussed.     

Purification and characterization of a 1,2-dihydroxynaphthalene dioxygenase from a bacterium that degrades naphthalenesulfonic acids.

1,2-Dihydroxynaphthalene dioxygenase was purified to homogeneity from a bacterium that degrades naphthalenesulfonic acids (strain BN6). The enzyme requires Fe2+ for maximal activity and consists of eight identical subunits with a molecular weight of about 33,000. Analysis of the NH2-terminal amino acid sequence revealed a high degree of homology (22 of 29 amino acids) with the NH2-terminal amino acid sequence of 2,3-dihydroxybiphenyl dioxygenase from strain Pseudomonas paucimobilis Q1. 1,2-Dihydroxynaphthalene dioxygenase from strain BN6 shows a wide substrate specificity and also cleaves 5-, 6-, and 7-hydroxy-1,2-dihydroxynaphthalene, 2,3- and 3,4-dihydroxybiphenyl, catechol, and 3-methyl- and 4-methylcatechol. Similar activities against the hydroxy-1,2-dihydroxynaphthalenes were also found in cell extracts from naphthalene-degrading bacteria.     

Refolding and purification of yeast carboxypeptidase Y expressed as inclusion bodies in Escherichia coli

The genes encoding carboxypeptidase Y (CPY) and CPY propeptide (CPYPR) from Saccharomyces cerevisiae were cloned and expressed in Escherichia coli. Six consecutive histidine residues were fused to the C-terminus of the CPYPR for facilitated purification. High-level expression of CPY and CPYPR-His(6) was achieved but most of the expressed proteins were present in the form of inclusion bodies in the bacterial cytoplasm. The CPY and CPYPR-His(6) produced as inclusion bodies were separated from the cells and solubilized in 6 and 3 M guanidinium chloride, respectively. The denatured CPYPR-His(6) was refolded by dilution 1:30 into the renaturation buffer (50 mM Tris-HCl containing 0.5 M NaCl and 3 mM EDTA, pH 8.0), and the refolded CPYPR-His(6) was further purified to 90% purity by single-step immobilized metal ion affinity chromatography. The denatured CPY was refolded by dilution 1:60 into the renaturation buffer containing CPYPR-His(6) at various concentrations. Increasing the molar ratio of CPYPR-His(6) to CPY resulted in an increase in the CPY refolding yield, indicating that the CPYPR-His(6) plays a chaperone-like role in in vitro folding of CPY. The refolded CPY was purified to 92% purity by single-step p-aminobenzylsuccinic acid affinity chromatography. When refolding was carried out in the presence of 10 molar eq CPYPR-His(6), the specific activity, N-(2-furanacryloyl)-l-phenylalanyl-l-phenylalanine hydrolysis activity per milligram of protein, of purified recombinant CPY was found to be about 63% of that of native S. cerevisiae CPY.     

重组人白细胞介素4的纯化及N端氨基酸序列分析

本文对重组人白细胞介素4高效表达克隆pBV220/hIL-4a的表达产物进行了纯化,升对纯化的人IL-4进行了N端氨基酸序列分析。人IL-4基因表达产物在大肠杆菌中以不溶性包涵体形式存在,经过超声破菌、包涵体抽提、复性浓缩、离子交換和凝胶过滤层析一系列纯化步骤,终产物纯度达98％以上,按蛋白总量计算回收率为14％,比活性达2×10~6单位/mg蛋白。通过测定纯化人IL-4的N端16个氮基酸序列,与由其DNA序列推导的氨基酸序列完全一致。本文为重组人IL-4的批量生产奠定了基础。     

Purification and some properties of carbon monoxide dehydrogenase from Acinetobacter sp. strain JC1 DSM 3803.

A brown carbon monoxide dehydrogenase from CO-autotrophically grown cells of Acinetobacter sp. strain JC1, which is unstable outside the cells, was purified 80-fold in seven steps to better than 95% homogeneity, with a yield of 44% in the presence of the stabilizing agents iodoacetamide (1 mM) and ammonium sulfate (100 mM). The final specific activity was 474 mumol of acceptor reduced per min per mg of protein as determined by an assay based on the CO-dependent reduction of thionin. Methyl viologen, NAD(P), flavin mononucleotide, flavin adenine dinucleotide, and ferricyanide were not reduced by the enzyme, but methylene blue, thionin, and dichlorophenolindophenol were reduced. The molecular weight of the native enzyme was determined to be 380,000. Sodium dodecyl sulfate-gel electrophoresis revealed at least three nonidentical subunits of molecular weights 16,000 (alpha), 34,000 (beta), and 85,000 (gamma). The purified enzyme contained particulate hydrogenase-like activity. Selenium did not stimulate carbon monoxide dehydrogenase activity. The isoelectic point of the native enzyme was found to be 5.8; the Km of CO was 150 microM. The enzyme was rapidly inactivated by methanol. One mole of native enzyme was found to contain 2 mol of each of flavin adenine dinucleotide and molybdenum and 8 mol each of nonheme iron and labile sulfide, which indicated that the enzyme was a molybdenum-containing iron-sulfur flavoprotein. The ratio of densities of each subunit after electrophoresis (alpha:beta:gamma = 1:2:6) and the number of each cofactor in the native enzyme suggest a alpha 2 beta 2 gamma 2 structure of the enzyme. The carbon monoxide dehydrogenase of Acinetobacter sp. strain JC1 was found to have no immunological relationship with enzymes of Pseudomonas carboxydohydrogena and Pseudomonas carboxydovorans.     

Chimeras of hepatic lipase and lipoprotein lipase. Domain localization of enzyme-specific properties.

Chimeric molecules between human lipoprotein lipase (LPL) and rat hepatic lipase (HL) were used to identify structural elements responsible for functional differences. Based on the close sequence homology with pancreatic lipase, both LPL and HL are believed to have a two-domain structure composed of an amino-terminal (NH2-terminal) domain containing the catalytic Ser-His-Asp triad and a smaller carboxyl-terminal (COOH-terminal) domain. Experiments with chimeric lipases containing the HL NH2-terminal domain and the LPL COOH-terminal domain (HL/LPL) or the reverse chimera (LPL/HL) showed that the NH2-terminal domain is responsible for the catalytic efficiency (Vmax/Km) of these enzymes. Furthermore, it was demonstrated that the stimulation of LPL activity by apolipoprotein C-II and the inhibition of activity by 1 M NaCl originate in structural features within the NH2-terminal domain. HL and LPL bind to vascular endothelium, presumably by interaction with cell surface heparan sulfate proteoglycans. However, the two enzymes differ significantly in their heparin affinity. Experiments with the chimeric lipases indicated that heparin binding avidity was primarily associated with the COOH-terminal domain. Specifically, both HL and the LPL/HL chimera were eluted from immobilized heparin by 0.75 M NaCl, whereas 1.1 M NaCl was required to elute LPL and the HL/LPL chimera. Finally, HL is more active than LPL in the hydrolysis of phospholipid substrates. However, the ratio of phospholipase to neutral lipase activity in both chimeric lipases was enhanced by the presence of the heterologous COOH-terminal domain, demonstrating that this domain strongly influences substrate specificity. The NH2-terminal domain thus controls the kinetic parameters of these lipases, whereas the COOH-terminal domain modulates substrate specificity and heparin binding.     

Avian glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing and activity are dependent upon essential cysteine residues.

Avian glutamine phosphoribosylpyrophosphate amidotransferase contains an NH2-terminal propetide-like sequence. NH2-terminal sequence analysis of immunoaffinity purified enzyme from chicken liver indicates that the propeptide is processed and the mature enzyme starts with Cys. Propeptide processing was investigated by site-directed mutagenesis using a system for expression in HeLa cells. Glutamine-dependent activity and processing were abolished by replacement of the conserved cysteine at position 1, whereas NH3-dependent activity was retained. Cys1 is thus inferred to have a role in glutamine-dependent activity and in propeptide processing. Inactive, insoluble enzymes in which the propeptide was not processed were obtained as a result of replacements of cysteines 415 and 488. Cysteine residues at positions 415 and 488 are inferred to be ligands to an Fe-S cluster on the basis of sequence similarity to the enzyme from Bacillus subtilis. Mutation of Cys269 and Cys295 led to loss of enzyme activity and propeptide processing, although solubility was unchanged. The results suggest that incorporation of an Fe-S cluster is needed for native structure, resultant propeptide processing, and glutamine-dependent activity.     

Molecular Cloning and Expression of a Cold-Adapted Lipase Gene from an Antarctic Deep Sea Psychrotrophic Bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. 7323

A psychrotrophic bacterium producing a cold-adapted lipase was isolated from the deep-sea sediment of Prydz Bay, Antarctic and identified as a Pseudomonas strain. Determination of the nucleotide sequence of the gene encoding a lipase from Pseudomonas sp. 7323 (lipA) revealed that LipA is composed of 617 amino acid residues with a calculated molecular weight of 64,466 Da. LipA has a GXSXG motif, which is conserved in lipases/esterases and generally contains the active-site serine. The lipase purified from the Escherichia coli transformant (rLipA) by metal-chelating chromatography exhibited the same electrophoretic mobility as did the wild-type lipase (wLipA) purified from strain 7323, and both enzymes were quite similar in physicochemical properties. The optimal temperature and pH value for the lipases activity were 30 degrees C and 9.0, respectively. They were unstable at temperatures above 25 degrees C and only retained half of their highest activity after incubation at 60 degrees C for 5 min. These results indicated that the enzymes were typical alkaline cold-adapted enzymes. Both enzymes were particularly activated by Ca(2+). Additionally, the enzymes hydrolyzed p-nitrophenyl caprate and tributyrin at the highest velocity among the other p-nitrophenyl esters and triglycerides.     

Identification of an alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase in Rhodococcus sp. strain RHA1 and cloning of the gene.

Gram-positive Rhodococcus sp. strain RHA1 possesses strong polychlorinated biphenyl-degrading capabilities. An RHA1 bphC gene mutant, strain RDC1, had been previously constructed (E. Masai, A. Yamada, J. M. Healy, T. Hatta, K. Kimbara, M. Fukuda, and K. Yano, Appl. Environ. Microbiol. 61:2079-2085, 1995). An alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD), designated EtbC, was identified in RDC1 cells grown on ethylbenzene. EtbC contained the broadest substrate specificity of any meta cleavage dioxygenase identified in a Rhodococcus strain to date, including RHA1 BphC. EtbC was purified to near homogeneity from RDC1 cells grown on ethylbenzene, and a 58-amino-acid NH2-terminal sequence was determined. The NH2-terminal amino acid sequence was used for the identification of the etbC gene from an RDC1 chromosomal DNA 2,3-DHBD expression library. The etbC gene was successfully cloned, and we report here the determination of its nucleotide sequence. The substrate specificity patterns of cell extract and native nondenaturing polyacrylamide gel electrophoresis analysis identified the coexpression of two 2,3-DHBDs (BphC and EtbC) in RHA1 cells grown on either biphenyl or ethylbenzene. The possible implication of coexpressed BphC extradiol dioxygenases in the strong polychlorinated-biphenyl degradation activity of RHA1 was suggested.     

Purification and characterization of a novel enzyme, L-threo-3-hydroxyaspartate dehydratase, from Pseudomonas sp. T62

L-threo-3-Hydroxyaspartate dehydratase (L-threo-3-hydroxyaspartate hydro-lyase), which exhibited specificity for L-threo-3-hydroxyaspartate (K(m)=0.74 mM, V(max)=37.5 micromol min(-1) (mg protein)(-1)) but not for D-threo or D, L-erythro-3-hydroxyaspartate, was purified from a cell-free extract of Pseudomonas sp. T62. The activity of the enzyme was inhibited by hydroxylamine and EDTA, which suggests that pyridoxal 5'-phosphate and divalent cations participate in the enzyme reaction. The NH(2)-terminal amino acid sequence showed significant similarity to the Saccharomyces cerevisiae YKL218c gene product, a hypothetical threonine dehydratase. However, the purified enzyme showed no threonine dehydratase activity.     

Purification and renaturation of recombinant human interleukin-2.

Recombinant human interleukin-2 (IL-2) expressed as Escherichia coli was isolated as insoluble aggregates of protein (inclusion bodies) after cell breakage. IL-2 and contaminants were dissolved in 6 M-guanidinium chloride/10 mM-dithiothreitol, pH 8.5, and further purified in reduced and denatured form by gel-permeation chromatography in the same solvent. Renaturation was effected by dilution and autoxidation; IL-2 of native specific activity was isolated at over 95% purity by reversed-phase h.p.l.c.; an additional peak of reduced protein was also observed. Most losses of native IL-2 occurred on refolding, probably because of an aggregation process; concentrations around 1 microgram/ml were necessary to achieve 30% recovery. It was essential to maintain the denatured protein in reduced form before renaturation and autoxidation, which was most efficient at pH 8.5 with 1.5 microM-CuSO4. A procedure based on these observations has been used to prepare IL-2 on the 50 micrograms scale.     

Cloning, expression, purification and characterization of patatin, a novel phospholipase A.

Patatin is the major protein constituent of potato tubers and displays broad esterase activity. The native enzyme actually belongs to a highly homologous multigene family of vacuolar glycoproteins. From these, the patB2 patatin gene was selected and cloned into pUC19 without its signal sequence but with an N-terminal histidine-tag. This patatin was overexpressed under the control of the lac promotor in Escherichia coli strain DH5alpha. The protein was recovered as inclusion bodies, folded into its native state by solubilization in urea and purified to homogeneity. Starting with one gram of inclusion bodies, 19 mg of pure and active recombinant patatin was isolated, with even higher specific activity than the glycosylated wild-type patatin purified from potato tubers. The purified enzyme showed esterolytic activity with p-nitrophenylesters dissolved in Triton X-100 micelles. The activity of patatin on p-nitrophenylesters with different carbon chain lengths showed an optimum for p-nitrophenylesters with 10 carbon atoms. Besides general esterolytic activity, the pure enzyme was found to display high phospholipase A activity in particular with the substrates 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC(8)PCho) (127 U.mg(-1)) and 1,2-dinonanoyl-sn-glycero-3-phosphocholine (diC(9)PCho) (109 U.mg(-1)). Recently, the structure of human cytosolic PLA(2) (cPLA(2)) was solved, showing a novel Ser-Asp active site dyad [1]. Based on a partial sequence alignment of patatin with human cPLA(2), we propose that patatin contains a similar active site dyad. To verify this assumption, conserved Ser, Asp and His residues in the family of patatins have been modified in patatin B2. Identification of active site residues was based on the observation of correctly folded but inactive variants. This led to the assignment of Ser54 and Asp192 as the active site serine and aspartate residues in patatin B2, respectively.     

Involvement of an extracellular protease in algicidal activity of the marine bacterium Pseudoalteromonas sp. strain A28

The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-M(w)-cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N-methyl-N'-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30 degrees C, respectively, by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium.     

Cloning and nucleotide sequence of the maltopentaose-forming amylase gene from Pseudomonas sp. KO-8940.

The gene coding for the maltopentaose-(G5)-forming amylase of Pseudomonas sp. KO-8940 was cloned into Escherichia coli and its nucleotides were sequenced. It was expected that a long open reading frame composed of 1,842-bp that encoded 614 amino acid residues for secretory precursor polypeptide including the typical signal sequence with an NH2-terminal was the gene. An extract of Escherichia coli carrying the cloned G5-forming amylase gene had amylolytic activity with which produced only G5 from starch, the same as that of the donor strain enzyme. In the deduced primary structure of this enzyme, the four conserved regions of many alpha-amylases were found, and the COOH-terminal portion of this enzyme showed high homology with other raw starch digesting amylases.