共查询到20条相似文献,搜索用时 15 毫秒
1.
Characterization of a Cell Envelope-Associated Proteinase Activity from Streptococcus thermophilus H-Strains 下载免费PDF全文
The production and biochemical properties of cell envelope-associated proteinases from two strains of Streptococcus thermophilus (strains CNRZ 385 and CNRZ 703) were compared. No significant difference in proteinase activity was found for strain CNRZ 385 when cells were grown in skim milk medium and M17 broth. Strain CNRZ 703 exhibited a threefold-higher proteinase activity when cells were grown in low-heat skim milk medium than when grown in M17 broth. Forty-one percent of the total activity of CNRZ 385 was localized on the cell wall. The optimum pH for enzymatic activity at 37°C was around 7.0. Serine proteinase inhibitors, such as phenylmethylsulfonyl fluoride and diisopropylfluorophosphate, inhibited the enzyme activity in both strains. The divalents cations Ca2+, Mg2+, and Mn2+ were activators, while Zn2+ and Cu2+ were inhibitors. β-Casein was hydrolyzed more rapidly than αs1-casein. The results of DNA hybridization and immunoblot studies suggested that the S. thermophilus cell wall proteinase and the lactococcal proteinase are not closely related. 相似文献
2.
Neutral proteinase was found in the leaves ofRicinus communie as assayed with α-casein and H-D-Val-Leu-Lys-pNA as substrates. The enzyme is maximally active at pH around 7.4. A selective
adsorbent for serine proteinase was prepared by attaching aprotinin to aminoalkyl-porous glass.
When partially purified leaf proteinase was passed through a column containing this adsorbent, the proteinase activity present
was bound to the porous glass. The proteinase eluted at IM NaCl was inhibited by aprotinin, leupeptin, DFP, phenylmethylsulfonyl
fluoride (PMSF) and serine proteinase inhibitor fromR, communis leaves, whereas pepstatin, EDTA, EGTA, and DTT had no effect on the enzyme. This inhibition profile suggests the leaf proteinase
is a neutral proteinase, such as a serine proteinase. 相似文献
3.
S. S. Sudge K. B. Bastawde D. V. Gokhale U. R. Kalkote T. Ravindranathan 《Applied microbiology and biotechnology》1998,49(5):594-599
About 1000 bacterial colonies isolated from sea water were screened for their ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine as a criterion for the determination of hydantoinase activity. The strain M-1, out of 11 hydantoinase-producing
strains, exhibited the maximum ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine. The strain M-1 appeared to be a halophilic Pseudomonas sp. according to morphological and physiological characteristics. Optimization of the growth parameters revealed that nutrient
broth with 2% NaCl was the preferred medium for both biomass and enzyme production. d-Hydantoinase of strain M-1 was not found to be inducible by the addition of uracil, dihydrouracil, β-alanine etc. The optimum
temperature for enzyme production was about 25 °C and the organism showed a broad pH optimum (pH 6.5–9.0) for both biomass
and hydantoinase production. The organism seems to have a strict requirement of NaCl for both growth and enzyme production.
The optimum pH and temperature of enzyme activity were 9–9.5 and 30 °C respectively. The biotransformation under the alkaline
conditions allowed the conversion of 80 g l−1
dl-5-phenylhydantoin to 82 g l−1
d(−)N-carbamoylphenylglycine within 24 h with a molar yield of 93%.
Received: 15 September 1997 / Received revision: 5 January 1998 / Accepted: 6 January 1998 相似文献
4.
The proteolytic system of thermophilic lactobacilli is considered important for bacterial nutrition as well as for the formation
of flavor and texture in fermented products. We investigated the influence of peptide content on the cell surface proteinase
and intracellular aminopeptidase activities from seven thermophilic lactobacilli strains. The proteinase activities were remarkably
reduced in cells grown in the peptide-rich medium MRS or in a chemically defined medium supplemented with Casitone compared
with those found in a synthetic medium. The degree of inhibition observed was strain dependent. When proteinase activities
were analyzed by their hydrolytic patterns of α- and β-casein degradation, four types of PIII-caseinolytic cleavage specificity were distinguished. Lactobacillus helveticus strains possessed aminopeptidase activities with broader specificity than those found in L. delbrueckii subsp. lactis strains. However, the aminopeptidase activities were not influenced by the peptide content of the medium.
Received: 1 February 2002 / Accepted: 27 February 2002 相似文献
5.
H D Paik S S Bae S H Park J G Pan 《Journal of industrial microbiology & biotechnology》1997,19(4):294-298
Bacillus thuringiensis subsp tochigiensis HD868 was identified as a bacteriocin producer which exhibited a bactericidal effect against closely related species. This
bacteriocin designated as tochicin, was partially purified by 75% ammonium sulfate precipitation followed by subsequent
dialysis. This partially purified tochicin showed a narrow antibacterial spectrum of activity against most of 20 typical
B. thuringiensis strains and a strain of B. cereus, but not against other bacteria and yeasts tested. The antibacterial activity of tochicin on sensitive indicator cells disappeared
completely by proteinase K treatment (1 mg ml−1), which indicates its proteinaceous nature. Tochicin was very stable throughout the range of pH 3.0–9.0 and was relatively
heat-stable at 90°C, but bacteriocin activity was not detected after boiling for 30 min. The relationship between cell growth
and bacteriocin production was studied in a semi-defined medium. Tochicin activity was detected at the mid-log growth phase,
reached the maximum at the early stationary phase, but decreased after the stationary phase. Direct detection of tochicin
activity on sodium dodecyl sulfate-polyacrylamide gel suggested it has an apparent molecular mass of about 10.5 kDa. Tochicin
exhibited a bactericidal activity against B. thuringiensis subsp thompsoni HD522 in phosphate buffer (pH 7.0).
Received 02 December 1996/ Accepted in revised form 25 August 1997 相似文献
6.
7.
Soybean-milk-coagulating activity of Bacillus pumilus derives from a serine proteinase 总被引:1,自引:0,他引:1
Aoyama M Yasuda M Nakachi K Kobamoto N Oku H Kato F 《Applied microbiology and biotechnology》2000,53(4):390-395
A proteolytic enzyme from Bacillus pumilus strain TYO-67, which was able to coagulate the protein in soybean milk, was characterized enzymologically. The optimum pH
and temperature for its activities were 9.0 and 50 °C, respectively. The enzyme was strongly believed to be a serine proteinase
because it was completely inhibited by the addition of diisopropyl fluorophosphate or phenylmethanesulfonyl fluoride. Hammerstein
milk casein, cytochrome c and soybean protein were good substrates for the enzyme. Seven cleavages were detected using the oxidized insulin B-chain
as peptide substrate for the proteolytic specificity test of the serine proteinase from B. pumilus. The bonds most susceptible to the action of the serine proteinase from B. pumilus were Leu-15–Tyr-16. The mode of action on soybean milk protein by the enzyme from B. pumilus was also investigated. The acidic subunit in glycinin and the α′-, α- and β-subunits in β-conglycinin were degraded during
the enzyme reaction. However, the basic subunit in glycinin could not be degraded by the enzyme. The formation of coagula
in soybean milk caused by the serine proteinase from B.
pumilus was mainly due to the hydrophobic interaction.
Received: 9 July 1999 / Received last revision: 22 October 1999 / Accepted: 5 November 1999 相似文献
8.
This study investigated the growth of Helicobacter (H.) pylori in Brucella broth supplemented with either IsoVitaleX (1% vol/vol), hemin (0.1% wt/vol), agar (0.3% wt/vol), or blood agar
blocks (1.5% wt/vol agar). IsoVitaleX was found to significantly shorten the lag phase, while hemin inhibited the growth within
the first 24 hours but later acted as a growth stimulant. There was a tendency toward stronger growth when blood agar blocks
were added to the medium. Subsequent electron microscopic evaluation revealed that cells of H. pylori were attached to blood agar block surfaces. In contrast, the supplementation of Brucella broth with agar did not significantly
increase the cell density. When H. pylori was grown in the presence of IsoVitaleX, strongly stainable electron-dense bodies (140–200 nm) were seen in the cytoplasms.
Incubation of cultures on rotary shakers at 120 rpm significantly enhanced growth. The addition of glycerol (15% vol/vol)
or fetal bovine serum (15% vol/vol) showed good ultrastructural preservation of bacteria with undamaged cell walls and cytoplasmic
membranes, and the cytoplasms were ribosome-dense. Cell counts revealed that cultures stored in glycerol or fetal bovine serum
had a significantly lower loss in viability when compared with cultures stored without cryopreservatives. Unprotected cells
of H. pylori showed on electron micrographs clumping, cell lysis, and flagellar damage. Finally, the survival rates of H. pylori after multiple thawing from storage at −80°C were best in Brucella broth/glycerol, Brucella broth/fetal bovine serum, and
Brucella broth without cryopreservative (in descending order).
Received: 10 November 1997 / Accepted: 29 January 1998 相似文献
9.
Summary Recovery of a bacterial alkaline proteinase after treatment of the fermentation broth with 16 different flocculating additives
has been investigated. The polyelectrolyte Sedipur TF 5 was the most effective at 150 ppm and pH 7.0–9.0, giving a 74% yield
of enzyme activity. 相似文献
10.
Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast 总被引:4,自引:0,他引:4
The enzyme 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) reductase is known as the rate-limiting enzyme in early sterol
biosynthesis in eukaryotic cells. To eliminate this regulation in the yeast Saccharomyces cerevisiae, a truncated HMG1 gene, producing a form of the enzyme that lacks the membrane-binding region (i.e. amino acids 1–552), was constructed and
overexpressed in this yeast. The transformed strains accumulated large amounts of the sterol precursor squalene, while the
levels of ergosterol and a number of other sterol compounds were only slightly elevated. These findings suggest that HMG-CoA
reductase is not the only rate-limiting step in sterol synthesis and its overexpression cannot significantly influence this
pathway beyond the sterol precursor squalene.
Received: 9 June 1997 / Received revision: 1 September 1997 / Accepted: 19 September 1997 相似文献
11.
A moderately halophilic bacterium LY6 with high proteolytic activity was isolated. Biochemical and physiological characterization,
along with 16S rDNA sequence analysis placed the isolate in the genus Halobacillus. The salinity of the culture medium strongly influenced the proteinase production of LY6. Maximum enzyme production was observed
in the medium containing 5% Na2SO4 or 10% NaCl. Proteinase production was synchronized with bacterial growth and reached a maximum level during the mid-stationary
phase. Enzyme purification was carried out by a simple approach including a combination of ammonium sulfate precipitation
and Sephacryl S-100 gel filtration chromatography. SDS-PAGE and gelatin zymography analysis revealed it was a monomer with
high molecular weight of 69 kDa. Optimal proteinase activity was obtained at pH 10.0, 40°C, and 10% NaCl. It was high active
over broad temperature (30–80°C), pH (6.0–12.0), and NaCl concentration (0–25%) ranges, indicating its thermostable, alkali-stable,
and halotolerant nature. Moreover, the enzyme activity was markedly enhanced by Ca2+ and Cu2+, but strongly inhibited by EDTA, PAO, and DEPC, indicating that it probably was a metalloproteinase with cysteine and histidine
residues located in its active site. 相似文献
12.
S. Halldórsdóttir E. T. Thórólfsdóttir R. Spilliaert M. Johansson S. H. Thorbjarnardóttir A. Palsdottir G. Ó. Hreggvidsson J. K. Kristjánsson O. Holst G. Eggertsson 《Applied microbiology and biotechnology》1998,49(3):277-284
A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified
and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid
sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan,
but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a
nickel nitrilotriacetate column. The enzyme had a pH optimum of 6–7 and its highest measured initial activity at 100 °C. The
heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after
8 h at 90 °C.
Received: 5 August 1997 / Received revision: 6 November 1997 / Accepted: 7 November 1997 相似文献
13.
Chymosin can specifically break down the Phe105–Met106 peptide bond of milk κ-casein to form insoluble para-κ-casein, resulting in milk coagulation, a process that is used in making cheese. In this study, in order to obtain an alternative
milk coagulant which is safe and efficient, and simultaneously can produce cheese with a good taste, bovine prochymosin B
was chosen and constitutively expressed to a high level in Pichia pastoris. The recombinant chymosin was expressed mainly as a secretory form, and it exhibited milk-clotting activity. It was purified
by ammonium sulfate fractionation, anion exchange, followed by cation exchange chromatography. A final yield of 24.2% was
obtained for the purified enzyme, which appeared as a single band in SDS–PAGE having a molecular mass of approximate 36 kDa.
Proteolysis assay showed that it specifically hydrolyzed κ-casein. It was stable at 25–50°C and had optimal activity at 37°C and pH 4.0. The activity of the recombinant chymosin was
activated by cations such as Mn2+, Fe3+, Mg2+ and Na+, but inhibited by K+, Co2+, Zn2+, Ni2+, and to a lesser extent by Cu2+. These results suggested that recombinant bovine chymosin is an acid milk coagulant, and it could be considered as a safe
and efficient enzyme suitable for use in cheese production. 相似文献
14.
Cephalosporin production by Streptomyces clavuligerus is known to be negatively regulated by carbon sources, e.g., glycerol and starch, and by phosphate at high concentrations.
Formation of lysine ɛ-aminotransferase (LAT) activity, the first enzyme of the biosynthetic pathway, was affected by a high
concentration of carbon source. Whereas 3% starch more than doubled LAT activity production as compared to 1% starch, 3% glycerol
repressed LAT activity formation by 20%–30%. LAT activity production was not affected by 100 mM K2HPO4. Our results thus show that the negative effects of 2% glycerol and 3% starch and 100 mM phosphate on cephalosporin production
are not due to an effect on production of LAT activity. However, repression of LAT activity by 3% glycerol would be expected
to play a negative role in antibiotic production.
Received: 13 June 1997 / Received revision: 20 August 1997 / Accepted: 25 August 1997 相似文献
15.
A moderately halophilic strain LY9 with high amylolytic activity was isolated from soil sample obtained from Yuncheng, China.
Biochemical and physiological characterization along with 16S rRNA sequence analysis placed the isolate in the genus Halobacillus. Amylase production started from the post-exponential phase of bacterial growth and reached a maximum level during the early-stationary
phase. The isolate LY9 was found to secrete the amylase, the production of which depended on the salinity of the growth medium.
Maximum amylase production was observed in the presence of 10% KCl or 10% NaCl. Maltose was the main product of soluble starch
hydrolysis, indicating a β-amylase activity. The enzyme showed optimal activity at 60°C, pH 8.0, and 10–12.5% of NaCl. It
was highly active over broad temperature (50–70°C), NaCl concentration (5.0–20.0%), and pH (4.0–12.0) ranges, indicating its
thermoactive and alkali-stable nature. However, activity dropped off dramatically at low NaCl concentrations, showing the
amylase was halophilic. Ca2+ was found to stimulate the β-amylase activity, whereas ethylenediaminetetraacetic acid (EDTA), phenylarsine oxide (PAO),
and diethyl pyrocarbonate (DEPC) strongly inhibited the enzyme, indicating it probably was a metalloenzyme with cysteine and
histidine residues located in its active site. Moreover, the enzyme exhibited remarkable stability towards sodium dodecyl
sulfate
(SDS) and Triton X-100. This is the first report of β-amylase production from moderate halophiles. The present study indicates
that the extracellular β-amylase of Halobacillus sp. LY9 may have considerable potential for industrial application owing to its properties. 相似文献
16.
Gioconda San-Blas Françoise Sorais Gustavo Niño-Vega Cioly Méndez Felipe San-Blas 《Current microbiology》1998,37(2):141-143
Cytosolic proteinases were assayed in both morphological phases of Paracoccidioides brasiliensis. Preparations from the mycelial phase were more active in vitro than those from the yeast cells. Optimal proteinase activities
for both phases occurred at pH's between 6.0 and 9.0, and at 45°C. Gelatin-SDS-PAGE electrophoresis separated several bands
(58–112 kDa) in mycelial preparations; a single band (70 kDa) was seen in yeast preparations. Enzymatic activities were inhibited
by antipain, phenyl methyl sulfonyl fluoride (PMSF), and chymostatin, suggestive of serine proteinases. Partial inhibition
of the mycelial enzymes by ethylene diamine tetraacetic acid (EDTA), 1,10-phenanthroline, and iodoacetamide, also suggested
the presence of cysteine- and metallo-proteinases. The enzymatic activity increased in preparations extracted from yeast cells
transforming to mycelia, and decreased in preparations obtained from the reverse process.
Received: 29 September 1997 / Accepted: 19 February 1998 相似文献
17.
A novel intracellular serine proteinase from the marine aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) that we designated pernilase was purified by ammonium sulfate precipitation, anionic-exchange chromatography,
affinity chromatography, and gel filtration chromatography. The purified enzyme was composed of a single polypeptide chain
with a molecular mass of 50 kDa as determined by SDS-PAGE. The proteinase had a broad pH profile (pH 5–10) with an optimum
pH of 9.0 for peptide hydrolysis. The optimum temperature for enzyme activity was 90°C. The enzyme was strongly inhibited
by diisopropyl fluorophosphate (DFP) and phenylmethyl sulfonylfluoride (PMSF), suggesting that it corresponds to a serine
proteinase. The enzyme was highly resistant to the reducing agents dithiothreitol and 2-mercaptoethanol but sensitive to the
denaturing reagents guanidine-HCl and urea and also to the detergent sodium dodecyl sulfate (SDS). Pernilase showed high substrate
specificity for Boc-Leu-Gly-Arg-MCA peptide. Thermostability of this enzyme showed half-lives of 85 min at 100°C and 12 min
at 110°C.
Received September 24, 1997 / Accepted May 20, 1998 相似文献
18.
Cell-Wall-Bound Proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: Characterization and Specificity for β-Casein 下载免费PDF全文
E. Tsakalidou R. Anastasiou I. Vandenberghe J. van Beeumen G. Kalantzopoulos 《Applied microbiology》1999,65(5):2035-2040
Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40°C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes β-casein mainly and α- and κ-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from β-casein, which have been identified. 相似文献
19.
The salinity tolerance of aRhizobium meliloti strain isolated from salt-affected soils was examined. Growth of the strain on yeast—mannitol broth containing 0–1.2% NaCl
exhibited in all cases the same generation time and simultaneous onset of the stationary phase while the total viable number
of cells was the same for three continuous generations. The nodulation, plant yield and elemental composition ofMedicago sativa plants grown on agar slopes, inoculated with cultures from the third generation grown on broth containing 0–1.2% NaCl responded
identically to all inocula. The salinity tolerance of the strain in fixing nitrogen was furthermore demonstrated withM. sativa plants grown on either nitrogen-free agar slopes containing 0.2–1.2% NaCl or soil-agar slopes with saline soil in which 0.15
and 0.3% additional NaCl was used. 相似文献
20.
W. J. Page N. Bhanthumnavin J. Manchak M. Ruman 《Applied microbiology and biotechnology》1997,48(1):88-93
Azotobacter salinestris, a sodium-dependent, microaerophilic N2-fixing soil bacterium, formed polyhydroxyalkanoate copolymers comprised of β-hydroxybutyric acid and 9–12 mol% β-hydroxyvaleric
acid (HV) during growth on sugars. Increased HV content was achieved by feeding valeric acid to the culture growing on glucose,
but propionic acid could be directed to HV formation only when it served as the sole C source. Polymer production in nitrogen-fixing
cells was increased at higher aeration, provided that a complex organic nitrogen source was also present, but there was no
HV in the polymer. HV production was increased to 28 mol% in nitrogen-fixing cells when aeration was lower and acetate was
provided with glucose in the medium. Enzymes leading to the production of polyhydroxyalkanoate copolymers were found to be
similar in A. salinestris and Azotobacter vinelandii, but A. vinelandii is unable to form HV from propionate or from sugars without valeric acid addition. A biochemical scheme is proposed for the
production of HV in A. salinestris, whereby the glyoxylate bypass assimilates acetate to generate succinate, which may be converted into propionyl-CoA for HV
synthesis. The results suggest that it may be possible to control the molar yield of HV formed from sugars by A. salinestris.
Received: 21 January 1997 / Received revision: 7 April 1997 / Accepted: 13 April 1997 相似文献