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1.
An extracellular enzyme with glucose dehydrogenase activity was purified from liquid cultures of the basidiomycete Agaricus bisporus after growth with d-cellobiose or d-glucose as carbon source. The molecular mass was measured as 57 kDa by gel filtration and 55 kDa by sodiumdodecyl sulphate/polyacrylamide
gel electrophoresis, while the isoelectric point was at pH 3.6. By analysis of 1H-NMR spectra in D2O, the product of d-glucose oxidation was identified as 3-ketoglucose. The substrates oxidized included d-cellobiose, l-arabinose, d-xylose and sucrose, but the specificity parameter (k
cat/K
m) was highest for d-glucose. Two electron acceptors were identified, namely 2,6-dichloroindophenol and p-benzoquinone, but reduction of dioxygen, ferricyanide or cytochrome c was not detectable. The selective C-3 oxidation of d-glucose is well-characterized for Agrobacterium and Flavobacterium, but this is the first report for a fungus.
Received: 19 June 1998 / Received revision: 15 September 1998 / Accepted: 17 September 1998 相似文献
2.
Nawaz M. S. Zhang D. Khan A. A. Cerniglia C. E. 《Applied microbiology and biotechnology》1998,50(5):568-572
A gram-negative, rod-shaped bacterium capable of utilizing l-asparagine as its sole source of carbon and nitrogen was isolated from soil and identified as Enterobacter cloacae. An intracellularly expressed l-asparaginase was detected and it deaminated l-asparagine to aspartic acid and ammonia. High-pressure liquid chromatography analysis of a cell-free asparaginase reaction
mixture indicated that 2.8 mM l-asparagine was hydrolyzed to 2.2 and 2.8 mM aspartic acid and ammonia, respectively, within 20 min of incubation. High asparaginase
activity was found in cells cultured on l-fructose, d-galactose, saccharose, or maltose, and in cells cultured on l-asparagine as the sole nitrogen source. The pH and temperature optimum of l-asparaginase was 8.5 and 37–42 °C, respectively. The half-life of the enzyme at 30 °C and 37 °C was 10 and 8 h, respectively.
Received: 19 February 1998 / Received last revision: 4 June 1998 / Accepted: 10 July 1998 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.
J. Q. Liu S. Ito T. Dairi N. Itoh S. Shimizu H. Yamada 《Applied microbiology and biotechnology》1998,49(6):702-708
Low-specificity l-threonine aldolase, catalyzing the reversible cleavage/condensation reaction between l-threonine/l-allo-threonine and glycine plus acetaldehyde, was purified to homogeneity from Pseudomonas sp. NCIMB 10558. The enzyme has an apparent molecular mass of approximately 145 kDa and consists of four identical subunits
with a molecular mass of 38 kDa. The enzyme, requiring pyridoxal- 5′-phosphate as a coenzyme, is strictly l-specific at the α position, whereas it can not distinguish between threo and erythro forms at the β position. Besides the reversible cleavage/condensation of threonine, the enzyme also catalyzes the reversible
interconversion between glycine plus various aldehydes and l-β-hydroxy-α-amino acids, including l-β-(3,4-dihydroxyphenyl)serine, l-β-(3,4-met‐hylenedioxyphenyl)serine and l-β-phenylserine, providing a new route for the industrial production of these important amino acids.
Received: 10 November 1997 / Received revision: 7 January 1998 / Accepted 30 January 1998 相似文献
5.
d-Xylose/d-glucose isomerases from two strains, a newly isolated strain, Paenibacillus sp., and from Alcaligenes ruhlandii are described herein. The enzymes were purified to apparent homogeneity. Both of these d-xylose isomerases are homotetramers with relative subunit molecular masses of 45 000 and 53 000, respectively, as estimated
by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The native molecular masses determined on Superose 12 gel chromatography
are 181 kDa for the enzyme from Paenibacillus sp. and 199 kDa for that from A. ruhlandii. The activity of both enzymes shows a requirement for divalent metal ions; the d-xylose isomerase from Paenibacillus sp. has the highest activity with Mn2+, while the enzyme from A. ruhlandii prefers Mg2+. Both enzymes also accept Co2+ with a somewhat lower efficiency, while Cu2+ inhibits the enzyme reaction. The binding of the metal ions obeys a biphasic characteristic, indicating the presence of two
non-identical binding sites per subunit. d-Glucose is converted to d-fructose at a rate that is two- to three-fold slower than for the d-xylose isomerisation. d-Xylitol and d-lyxose are competitive inhibitors of both enzymes. Both enzymes have a pH optimum between 6.5 and 7.0, and they are active
up to 60 °C. The enzyme from Paenibacillus sp. retained 50% of its activity after 4 days at 55 °C, whereas that from A. ruhlandii still retained 50% of its activity after 6 days at 55 °C. Polyacrylamide entrapment and immobilisation to both controlled
pore glass and cyanogen-bromide-activated Sepharose were achieved for both enzymes with high efficiency.
Received: 14 May 1998 / Received last revision: 29 July 1998 / Accepted: 29 July 1998 相似文献
6.
S. Østergaard H. B. Aa. Theilgaard J. Nielsen 《Applied microbiology and biotechnology》1998,50(6):663-668
We have demonstrated that Penicillium chrysogenum possesses the l-cysteine biosynthetic enzyme O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence
of the direct sulphhydrylation pathway in P. chrysogenum, creates the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this
microorganism. Only O-acetyl-l-serine sulphhydrylase and O-acetyl-l-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-l-serine as substrate for the formation of l-cysteine. The purified␣enzyme did not catalyse the formation of l-homocysteine from O-acetyl-l-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyl-l-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity
93-fold in relation to the cell-free extract. Two bands, showing exactly the same intensity, were present on a sodium dodecyl
sulphate/polyacrylamide gel, and the molecular masses of these were estimated to be 59 kDa and 68 kDa respectively. The K
m value for O-acetyl-l-serine and V
max of O-acetyl-l-serine sulphhydrylase were estimated to be 1.3 mM and 14.9 μmol/mg protein−1 h−1 respectively. The activity of the purified enzyme had a temperature optimum of approximately 45 °C, which is much higher
than the actual temperature for penicillin synthesis. Furthermore, O-acetyl-l-serine sulphhydrylase activity was to have a maximum in the range of pH 7.0–7.4.
Received: 20 March 1998 / Received revision: 27 July 1998 / Accepted: 12 August 1998 相似文献
7.
B. L. García A. S. Ball J. Rodríguez M. I. Pérez-Leblic M. E. Arias J. L. Copa-Patiño 《Applied microbiology and biotechnology》1998,50(2):213-218
Streptomyces avermitilis CECT 3339 produces extracellular ferulic acid esterase (FAE) activity during growth on a range of lignocellulose substrates.
Maximal levels of FAE activity were detected in culture filtrates from S. avermitilis CECT 3339 grown in media containing wheat bran and yeast extract as carbon and nitrogen sources respectively. Biochemical
characterization of this enzyme activity revealed that it was 100-fold higher when wheat bran was pretreated with Celluclast
(a mix of hydrolytic enzymes). FAE was found to be end-product-inhibited. Characterization of the properties of the enzyme
showed that FAE exhibited an activity optimum pH at 6 with pH stability between pH 6 and 8. The optimum temperature was 50 °C
while the temperature stability was between 30 °C and 40 °C, with rapid inactivation at 60 °C and above. The characteristics
and stability of FAE from S. avermitilis CECT 3339 suggest a potential role for this enzyme in combination with endoxylanases for the upgrading of plant-residue silage
and for biopulping.
Received: 17 November 1997 / Received revision: 13 March 1998 / Accepted: 13 April 1998 相似文献
8.
C. Suresh A. K. Dubey S. Srikanta S. Umesh Kumar N. G. Karanth 《Applied microbiology and biotechnology》1999,51(5):673-675
A UV-induced mutant strain of Aspergillus niger (CFTRI-1105-U9) overproduced a starch-hydrolysing enzyme with properties characteristically different from the known amylases
of the fungus. The purified enzyme of 4.0 pI had an apparent molecular mass of 125 kDa and it dextrinised starch and then
saccharified the dextrins. Patterns of the enzyme activity on starch, resulting in glucose at 60 °C and glucose, maltose and
maltodextrins at 70 °C as primary products, suggested significant applications for the enzyme in starch-processing industries.
Received: 29 October 1998 / Received revision: 11 January 1999 / Accepted: 19 January 1999 相似文献
9.
Cloning and characterization of an endo-β-1,3(4)glucanase and an aspartic protease from Phaffia rhodozyma CBS 6938 总被引:1,自引:0,他引:1
We describe the identification and expression cloning of two novel enzymes, a β-glucanase and an aspartic protease, secreted
from the basidiomycetous yeast Phaffia rhodozyma. A cDNA library from P. rhodozyma CBS 6938 was constructed, and full-length cDNA encoding an endo-1,3(4)-β-glucanase (bg1) and an aspartic protease (pr1) were cloned by expression cloning in Saccharomyces cerevisiae W3124. The bg1 cDNA encodes a 424-residue precursor protein with a putative signal peptide. The pr1 cDNA encodes a 405-residue prepropolypeptide with an 81-residue leader peptide. The aspartic protease was purified and characterized.
It has a molecular mass of 36 kDa, an isoelectric point of pH 7.5, a pH activity optimum at 4.0–6.0, and a temperature activity
optimum around 40 °C. Both enzymes show only low sequence identity to other known enzymes.
Received: 6 August 1998 / Received revision: 29 October 1998 / Accepted: 30 October 1998 相似文献
10.
d-Hydantoinase from the lentil, Lens esculenta, seed is quite unstable, and has been immobilized on Diethyl amino ethyl (DEAE) cellulose by an adsorption and cross-linking
method. The immboilized d-hydantoinase exhibited 80% enzyme activity and contained 86% protein. The immobilization of the enzyme preparation does not
change its optimum pH, temperature or affinity constant, but increases its shelf-life, thermostability and stability in various
organic solvents. This immobilized d-hydantoinase can be used effectively for the production of d-amino acids from the corresponding hydantoins and may therefore be of use in the chemical and pharmaceutical industries.
Received: 28 April 1998 / Received last revision: 10 July 1998 / Accepted: 10 July 1998 相似文献
11.
J. Q. Liu M. Odani T. Dairi N. Itoh S. Shimizu H. Yamada 《Applied microbiology and biotechnology》1999,51(5):586-591
A new enzymatic resolution process was established for the production of l-threo-3-[4-(methylthio)phenylserine] (MTPS), an intermediate for synthesis of antibiotics, florfenicol and thiamphenicol, using
the recombinant low-specificity d-threonine aldolase from Arthrobacter sp. DK-38. Chemically synthesized dl-threo-MTPS was efficiently resolved with either the purified enzyme or the intact recombinant Escherichiacoli cells overproducing the enzyme. Under the optimized experimental conditions, 100 mM (22.8 g l−1) l-threo-MTPS was obtained from 200 mM (45.5 g l−1) dl-threo-MTPS, with a molar yield of 50% and a 99.6% enantiomeric excess.
Received: 2 September 1998 / Received revision: 27 October 1998 / Accepted: 29 November 1998 相似文献
12.
This is the first report describing the gene structure and the enzymatic properties of a β-fructosidase of a hyperthermophilic
organism. The bfrA gene of the ancestral bacterium Thermotoga maritima MSB8 codes for a 432-residue, polypeptide of about 50 kDa, with significant sequence similarity to other β-fructosidases.
On the basis of its primary structure, BfrA can be assigned to glycosyl hydrolase family 32. The bfrA gene was expressed in Escherichia coli and the recombinant enzyme was purified and characterised. BfrA was specific for the fructose moiety and the β-anomeric configuration
of the glycosidic linkages of its substrates. The enzyme released fructose from sucrose and raffinose, and the fructose polymer
inulin was hydrolysed quantitatively in an exo-type fashion. BfrA displayed similar catalytic efficiencies for the hydrolysis
of sucrose and inulin with k
cat/K
m values (at 75 °C, pH 5.5) of about 4.1 × 104 M−1s−1 and 3.1 × 104 M−1s−1 respectively. BfrA had an optimum temperature of 90–95 °C (10-min assay) and was extremely insensitive to thermo-inactivation.
During 5 h at temperatures up to 80 °C at pH 7, the enzyme retained at least 85% of its initial activity. Thus, BfrA is the
most thermostable β-fructosidase and also the most thermostable inulinase described to date. In conclusion, the T. maritima enzyme can be classified as an exo-β-d-fructofuranosidase (EC 3.2.1.26) with invertase and inulinase activity. Its catalytic properties along with the extreme thermostability
recommend it for use in biotechnology.
Received: 28 August 1997 / Received revision: 19 January 1998 / Accepted: 24 January 1998 相似文献
13.
Production of trehalose synthase from a basidiomycete, Grifola frondosa, in Escherichia coli 总被引:1,自引:0,他引:1
K. Saito H. Yamazaki Y. Ohnishi S. Fujimoto E. Takahashi S. Horinouchi 《Applied microbiology and biotechnology》1998,50(2):193-198
The genomic DNA and cDNA for a gene encoding a novel trehalose synthase (TSase) catalyzing trehalose synthesis from α-d-glucose 1-phosphate and d-glucose were cloned from a basidiomycete, Grifola frondosa. Nucleotide sequencing showed that the 732-amino-acid TSase-encoding region was separated by eight introns. Consistent with
the novelty of TSase, there were no homologous proteins registered in the databases. Recombinant TSase with a histidine tag
at the NH2-terminal end, produced in Escherichia coli, showed enzyme activity similar to that purified from the original G. frondosa strain. Incubation of α-d-glucose 1-phosphate and d-glucose in the presence of recombinant TSase generated trehalose, in agreement with the enzymatic property of TSase that
the equilibrium lay far in the direction of trehalose synthesis.
Received: 12 January 1998 / Received revision: 20 February 1998 / Accepted: 20 March 1998 相似文献
14.
M. A. Ferrero G. R. Castro C. M. Abate M. D. Baigorí F. Siñeriz 《Applied microbiology and biotechnology》1996,45(3):327-332
Bacillus licheniformis MIR 29 has been isolated and produces extracellular proteases. It is able to grow at temperatures up to 60 °C and at pH values
up to 9.0. Casein was the best carbon source for production of a thermostable protease activity which, in some conditions,
is 90% extracellular. The synthesis of alkaline protease is not constitutive; different levels of production were found with
different carbon and nitrogen sources. Casein was thought to be an inducer of enzyme synthesis. The optimal pH and temperature
of the enzyme activity were 12 °C and 60 °C, respectively. The enzyme was stable up to 60 °C in the absence of stabilizers.
The protease activity was inhibited with phenylmethylsulphonyl fluoride, indicating a serine-protease activity. The proteolytic
activity was lowered by molecules present in the culture supernatant, which include amino acids and peptides, indicating end-product
inhibition. Electrophoresis assay on denaturating gels showed two bands with alkaline protease activity, in the 25 to 40-kDa
molecular mass range.
Received: 7 June 1995/Received revision: 14 September 1995/Accepted: 20 September 1995 相似文献
15.
A putative N-acyl-d-glucosamine 2-epimerase from Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was identified as a cellobiose 2-epimerase by the analysis of the activity for substrates, acid-hydrolyzed
products, and amino acid sequence. The cellobiose 2-epimerase was purified with a specific activity of 35 nmol min–1 mg–1 for d-glucose with a 47-kDa monomer. The epimerization activity for d-glucose was maximal at pH 7.5 and 75°C. The half-lives of the enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 142, 71, 35,
18, and 4.6 h, respectively. The enzyme catalyzed the epimerization reactions of the aldoses harboring hydroxyl groups oriented
in the right-hand configuration at the C2 position and the left-hand configuration at the C3 position, such as d-glucose, d-xylose, l-altrose, l-idose, and l-arabinose, to their C2 epimers, such as d-mannose, d-lyxose, l-allose, l-gulose, and l-ribose, respectively. The enzyme catalyzed also the isomerization reactions. The enzyme exhibited the highest activity for
mannose among monosaccharides. Thus, mannose at 75 g l–1 and fructose at 47.5 g l–1 were produced from 500 g l–1 glucose at pH 7.5 and 75°C over 3 h by the enzyme. 相似文献
16.
K. M. J. Van Laere G. Beldman A. G. J. Voragen 《Applied microbiology and biotechnology》1997,47(3):231-235
An arabinofuranohydrolase (AXH-d3) was purified from a cell-free extract of Bifidobacterium adolescentis DSM 20083. The enzyme had a molecular mass of approximately 100 kDa as determined by gel filtration. It displayed maximum
activity at pH 6 and 30 °C. Using an arabinoxylan-derived oligosaccharide containing double-substituted xylopyranosyl residues
established that the enzyme specifically released terminal arabinofuranosyl residues linked to C-3 of double-substituted xylopyranosyl
residues. In addition, this arabinofuranohydrolase released arabinosyl groups from wheat flour arabinoxylan polymer but showed
no activity towards p-nitrophenyl α-l-arabinofuranoside or towards sugar-beet arabinan, soy arabinogalactan, arabino-oligosaccharides and arabinogalacto-oligosaccharides.
Received: 15 July 1996 / Received revision: 18 October 1996 / Accepted: 18 October 1996 相似文献
17.
A. Blanco P. Díaz J. Martínez T. Vidal A. L. Torres F. I. J. Pastor 《Applied microbiology and biotechnology》1998,50(1):48-54
The gene celA, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the celA gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44 803 Da. The deduced
amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases.
The celA gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the
presence of 10 mM Mg2+ and Ca2+ and showed its maximum at 40 °C and pH 4.0. Study of the performance of CelA on paper manufacture from agricultural fibres
showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. CelA treatment enhanced
the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished.
Electron-microscope analysis showed that the surface of straw fibres was modified by CelA.
Received: 11 February 1998 / Received revision: 20 March 1998 / Accepted: 20 March 1998 相似文献
18.
Liu JQ Odani M Yasuoka T Dairi T Itoh N Kataoka M Shimizu S Yamada H 《Applied microbiology and biotechnology》2000,54(1):44-51
The dtaAX gene encoding a pyridoxal 5′-phosphate (pyridoxal-P)-dependent low-specificity d-threonine aldolase was cloned from the chromosomal DNA of Alcaligenes xylosoxidans IFO 12669. It contains an open reading frame consisting of 1,134 nucleotides corresponding to 377 amino acid residues. The
predicted amino acid sequence displayed 54% identity with that of d-threonine aldolase from gram-positive bacteria Arthrobacter sp. DK-38, but showed no significant similarity with those of other known pyridoxal-P enzymes. This gram-negative bacterial enzyme was highly overproduced in recombinant Escherichia coli cells, and the specific activity of the enzyme in the cell extract was as high as 18 U/mg (purified enzyme 38.6 U/mg), which
was 6,000 times higher than that from the wild-type Alcaligenes cell extract. The recombinant enzyme was thus feasibly purified to homogeneity by ammonium sulfate fractionation and DEAE-Toyopearl chromatography
steps. The recombinant low-specificity d-threonine aldolase was shown to be an efficient biocatalyst for resolution of l-β-3,4-methylenedioxyphenylserine, an intermediate for production of a therapeutic drug for Parkinson's disease.
Received: 9 September 1999 / Received revision: 1 November 1999 / Accepted: 12 November 1999 相似文献
19.
A. Phongdara A. Merckelbach P. Keup G. Gellissen C. P. Hollenberg 《Applied microbiology and biotechnology》1998,50(1):77-84
A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA
fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with
a calculated M
r of 49400. The␣encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide
sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved
in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression.
Received: 15 January 1998 / Received revision: 2 March 1998 / Accepted: 4 March 1998 相似文献
20.
W. H. Schwarz K. Bronnenmeier B. Krause F. Lottspeich W. L. Staudenbauer 《Applied microbiology and biotechnology》1995,43(5):856-860
The gene arfB encoding α-L-arabino-furanosidase B of the cellulolytic thermophile Clostridium stercorarium was expressed in Escherichia coli from a 2.2-kb EcoRI DNA fragment. The recombinant gene product ArfB was purified by fast-performance liquid chromatography. It has a tetrameric
structure with a monomeric relative molecular mass of 52 00. The optima for temperature and pH are 70 °C and 5.0 respectively.
The enzyme appears to have no metal cofactor requirement and is sensitive to sulfhydryl reagents. It hydrolyzes aryl and alkyl
α-L-arabinofuranosides and cleaves arabinosyl side-chains from arabinoxylan (oat-spelt xylan) and from xylooligosaccharides produced
by recombinant endoxylanase XynA from the same organism. The identity of the N-terminal amino acid sequences indicates that
ArfB corresponds to the major α-arabinosidase activity present in the culture supernatant of C. stercorarium.
Received: 30 September 1994/Received revision: 24 November 1994/Accepted: 16 December 1994 相似文献