共查询到20条相似文献,搜索用时 46 毫秒
1.
The gram-negative bacterium Myxobacter sp. AL-1 produces chitosanase-cellulase activity that is maximally excreted during the stationary phase of growth. Carboxymethylcellulase
zymogram analysis revealed that the enzymatic activity was correlated with two bands of 32 and 35 kDa. Ion-exchange-chromatography-enriched
preparations of the 32-kDa enzyme were capable of degrading the cellulose fluorescent derivatives 4-methylumbelliferyl-β-d-cellobioside and 4-methylumbelliferyl-β-d-cellotrioside. These enzymatic preparations also showed a greater capacity at 70° C than at 42° C to degrade chitosan oligomers
of a minimum size of six units. Conversely, the β-1,4 glucanolytic activity was more efficient at attacking carboxymethylcellulose
and methylumbelliferyl-cellotrioside at 42° C than at 70° C. The 32-kDa enzyme was purified more than 800-fold to apparent
homogeneity by a combination of ion-exchange and molecular-exclusion chromatography. Amino-terminal sequencing indicated that
mature chitosanase-cellulase shares more than 70% identity with endocellulases produced by strains DLG, PAP115, and 168 of
the gram-positive microorganism Bacillus subtilis.
Received: 6 January 1997 / Accepted: 29 May 1997 相似文献
2.
Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately
chemolithoautotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite,
and Superdex-200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits
with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an αβγδ-structure.
The activity was detected with pyruvate, coenzyme A, and one of the following electron acceptors in substrate amounts: ferredoxin
isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective as the electron acceptor. The temperature optimum for pyruvate oxidation was approximately 80° C. The pH
optimum was 7.6–7.8. The apparent K
m values for pyruvate and coenzyme A at 70° C were 3.45 mM and 54 μM, respectively. The enzyme was extremely thermostable under
anoxic conditions; the time for a 50% loss of activity (t
50%) at 70° C was approximately 8 h.
Received: 9 September 1996 / Accepted: 27 December 1996 相似文献
3.
A thermostable homodimeric isocitrate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized. The mol. mass of the isocitrate dehydrogenase subunit was 42 kDa as determined by SDS-PAGE.
Following separation by SDS-PAGE, A. fulgidus isocitrate dehydrogenase could be renatured and detected in situ by activity staining. The enzyme showed dual coenzyme specificity
with a high preference for NADP+. Optimal temperature for activity was 90° C or above, and a half-life of 22 min was found for the enzyme when incubated at
90° C in a 50 mM Tricine-KOH buffer (pH 8.0). Based on the N-terminal amino acid sequence, the gene encoding the isocitrate
dehydrogenase was cloned. DNA sequencing identified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined
for the purified enzyme. The deduced amino acid sequence closely resembled that of the isocitrate dehydrogenase from the archaeon
Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, with 57% identity with isocitrate dehydrogenase from Escherichia coli. All the amino acid residues directly contacting substrate and coenzyme (except Ile-320) in E. coli isocitrate dehydrogenase are conserved in the enzyme from A. fulgidus. The primary structure of A. fulgidus isocitrate dehydrogenase confirmes the presence of Bacteria-type isocitrate dehydrogenases among Archaea. Multiple alignment
of all the available amino acid sequences of di- and multimeric isocitrate dehydrogenases from the three domains of life shows
that they can be divided into three distinct phylogenetic groups.
Received: 6 February 1997 / Accepted: 12 June 1997 相似文献
4.
Anne Siri Langelandsvik I. H. Steen Nils-Kåre Birkeland Torleiv Lien 《Archives of microbiology》1997,168(1):59-67
A thermostable l-malate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was isolated and characterized, and its gene was cloned and sequenced. The enzyme is a homodimer with a molecular mass of
70 kDa and catalyzes preferentially the reduction of oxaloacetic acid with NADH. A. fulgidus
l-malate dehydrogenase was stable for 5 h at 90° C, and the half-life at 101° C was 80 min. Thus, A. fulgidus
l-malate dehydrogenase is the most thermostable l-malate dehydrogenase characterized to date. Addition of K2HPO4 (1 M) increased the thermal stability by 40%. The primary structure shows a high similarity to l-lactate dehydrogenase from Thermotoga maritima and gram-positive bacteria, and to l-malate dehydrogenase from the archaeon Haloarcula marismortui and other l-lactate-dehydrogenase-like l-malate dehydrogenases.
Received: 20 November 1997 / Accepted: 28 February 1997 相似文献
5.
A dextranase (EC 3.2.1.11) was purified and characterized from the IP-29 strain of Sporothrix schenckii, a dimorphic pathogenic fungus. Growing cells secreted the enzyme into a standard culture medium (20 °C) that supports the
mycelial phase. Soluble bacterial dextrans substituted for glucose as substrate with a small decrease in cellular yield but
a tenfold increase in the production of dextranase. This enzyme is a monomeric protein with a molecular mass of 79 kDa, a
pH optimum of 5.0, and an action pattern against a soluble 170-kDa bacterial dextran that leads to a final mixture of glucose
(38%), isomaltose (38%), and branched oligosaccharides (24%). In the presence of 200 mM sodium acetate buffer (pH 5.0), the
K
m for soluble dextran was 0.067 ± 0.003% (w/v). Salts of Hg2+, (UO2)2+, Pb2+, Cu2+, and Zn2+ inhibited by affecting both V
max and K
m. The enzyme was most stable between pH values of 4.50 and 4.75, where the half-life at 55 °C was 18 min and the energy of
activation for heat denaturation was 99 kcal/mol. S. schenckii dextranase catalyzed the degradation of cross-linked dextran chains in Sephadex G-50 to G-200, and the latter was a good
substrate for cell growth at 20 °C. Highly cross-linked grades (i.e., G-10 and G-25) were refractory to hydrolysis. Most strains
of S. schenckii from Europe and North America tested positive for dextranase when grown at 20 °C. All of these isolates grew on glucose at
35 °C, a condition that is typically associated with the yeast phase, but they did not express dextranase and were incapable
of using dextran as a carbon source at the higher temperature.
Received: 29 December 1997 / Accepted: 4 March 1998 相似文献
6.
Bacteria that produced NAD+-dependent phenylalanine dehydrogenase (EC 1.4.1.20) were selected among l-methionine utilizers isolated from soil. A bacterial strain showing phenylalanine dehydrogenase activity was chosen and classified
in the genus Microbacterium. Phenylalanine dehydrogenase was purified from the crude extract of Microbacterium sp. strain DM 86-1 (TPU 3592) to homogeneity as judged by SDS-polyacrylamide disc gel electrophoresis. The enzyme has an
isoelectric point of 5.8 and a relative molecular weight (M
r) of approximately 330,000. The enzyme is composed of eight identical subunits with an M
r of approximately 41,000. The apparent K
m values for l-phenylalanine and NAD+ were calculated to be 0.10 mM and 0.20 mM, respectively. No loss of the enzyme activity was observed upon incubation at 55° C
for 10 min.
Received: 30 July 1997 / Accepted: 4 November 1997 相似文献
7.
Chemical mutagenesis of the nucleotide-producing strain Corynebacterium ammoniagenes ATCC 6872 with N-methyl-N-nitro-N-nitrosoguanidine followed by an enrichment protocol yielded 46 temperature-sensitive (ts) clones. A rapid assay for the allosterically
regulated Mn-ribonucleotide reductase (RRase) was developed with nucleotide-permeable cells of C. ammoniagenes in order to screen for possible defects in DNA precursor biosynthesis at elevated temperature. Three mutants (CH 31, CH 32,
and CH 33) grew well at 30° C but did not proliferate at 40° C because they did not reduce ribonucleotides to 2′-deoxyribonucleotides.
They were designated nrd
ts
(nucleotide reduction defective). When the cultures were shifted from 30 to 40° C, the nrd
ts
mutants immediately ceased to incorporate radiolabeled nucleic acid precursors into the DNA fraction, while DNA chain elongation
was barely affected. Thus, exhaustion of the deoxyribonucleotide pool ultimately inhibited cell division, leading to a filamentous
growth morphology. In contrast to the wild-type, all three nrd
ts
mutants displayed a distinctly enhanced sensitivity of ribonucleotide reduction towards hydroxyurea (in permeabilized cells
and in vitro) at 30° C. The results from assays for biochemical complementation of heat-inactivated (2 min, 37° C) mutant
enzyme with either the small or the large subunit of wild-type Mn-RRase located the mutational defect on the large subunit.
Received: 28 December 1995 / Revision received: 22 January 1997 / Accepted: 29 January 1997 相似文献
8.
R. N. Z. A. Rahman S. Fujiwara M. Takagi T. Imanaka 《Molecular & general genetics : MGG》1998,257(3):338-347
The gdhA gene encoding glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned and sequenced. Phylogenetic analysis was performed on an alignment of 25 GDH sequences including KOD1-GDH,
and two protein families were distinguished, as previously reported. KOD1-GDH was classified as new member of the hexameric
GDH Family II. The gdhA gene was expressed in Escherichia coli, and recombinant KOD1-GDH was purified. Its enzymatic characteristics were compared with those of the native KOD1-GDH. Both
enzymes had a molecular mass of 47 300 Da and were shown to be functional in a hexameric form (284 kDa). The N-terminal amino
acid sequences of native KOD1-GDH and the recombinant GDH were VEIDPFEMAV and MVEIDPFEMA, respectively, indicating that native
KOD1-GDH does not retain the initial methionine at the N-terminus. The recombinant GDH displayed enzyme characteristics similar
to those of the native GDH, except for a lower level of thermostability, with a half-life of 2 h at 100° C, compared to 4 h
for the native enzyme purified from KOD1. Kinetic studies suggested that the reaction is biased towards glutamate production.
KOD1-GDH utilized both coenzymes NADH and NADPH, as do most eukaryal GDHs.
Received: 6 May 1997 / Accepted: 23 September 1997 相似文献
9.
The coloration of cells of the cyanobacterium Synechococcus sp. PCC 7002 changed from normal blue-green to yellow-green when cells were grown at 15° C in a medium containing nitrate
as the sole nitrogen source. This change of coloration was similar to a general response to nutrient deprivation (chlorosis).
For the chlorotic cells at 15° C, the total amounts of phycobiliproteins and chlorophyll a decreased, high levels of glycogen accumulated, and growth was arithmetic rather than exponential. These changes in composition
and growth occurred in cells grown at low (50 μE m–2 s–1) as well as high (250 μE m–2 s–1) light intensity. After a temperature shift-up to 38° C, chlorotic cells rapidly regained their normal blue-green coloration
and normal exponential growth rate within 7 h. When cells were grown at 15° C in a medium containing urea as the reduced nitrogen
source, cells grew exponentially and the symptoms of chlorosis were not observed. The decrease in photosynthetic oxygen evolution
activity at low temperature was much smaller than the decrease in growth rate for cells grown on nitrate as the nitrogen source.
These studies demonstrate that low-temperature-induced chlorosis of Synechococcus sp. PCC 7002 is caused by nitrogen limitation and is not the result of limited photosynthetic activity or photodamage to
the photosynthetic apparatus, and that nitrogen assimilation is an important aspect of the low-temperature physiology of cyanobacteria.
Received: 24 April 1997 / Accepted: 5 August 1997 相似文献
10.
Miake F Satho T Takesue H Yanagida F Kashige N Watanabe K 《Archives of microbiology》2000,173(1):65-70
α-l-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had
a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca2+ and remained stable for several months when stored at –20 °C. The optimum pH was 7.8; the optimum temperature was 45 °C.
The K
m, V
max and k
cat for p-nitrophenyl α-l-rhamnopyranoside were 1.18 mM, 92.4 μM · min–1 and 117,000 · min–1, respectively. Examination of the substrate specificity using various synthetic and natural l-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far.
Received: 24 May 1999 / Accepted: 7 October 1999 相似文献
11.
Yoshihiro Hakamada Kenzo Koike Tadashi Yoshimatsu Hajime Mori Tohru Kobayashi Susumu Ito 《Extremophiles : life under extreme conditions》1997,1(3):151-156
Thermostable alkaline cellulase (endo-1,4-β-glucanase, EC 3.2.1.4) activity was detected in the culture medium of a strictly
alkaliphilic strain of Bacillus, designated KSM-S237. This novel enzyme was purified to homogeneity by a two-step column-chromatographic procedure with high
yield. The N-terminal amino acid sequence of the purified enzyme was Glu-Gly-Asn-Thr-Arg-Glu-Asp-Asn-Phe-Lys-His-Leu-Leu-Gly-Asn-Asp-Asn-Val-Lys-Arg.
The enzyme had a molecular mass of approximately 86 kDa and an isoelectric point of pH 3.8. The enzyme had a pH optimum of
8.6–9.0 and displayed maximum activity at 45°C. The alkaline enzyme was stable up to 50°C and more than 30% of the original
activity was detectable after heating at 100°C and at pH 9.0 for 10 min. The enzyme hydrolyzed carboxymethylcellulose, lichenan
(β-1,3;1,4-linkage), and p-nitrophenyl derivatives of cellotriose and cellotetraose. Crystalline forms of cellulose (Avicel and filter paper), H3PO4-swollen cellulose, NaOH-swollen cellulose, curdlan (β-1,3-linkage), laminarin (β-1,3;1,6-linkage), and xylan were barely
hydrolyzed at all.
Received: April 28, 1997 / Accepted: May 24, 1997 相似文献
12.
Isabella Di Lernia Alessandra Morana Antonio Ottombrino Stefania Fusco Mosè Rossi M. De Rosa 《Extremophiles : life under extreme conditions》1998,2(4):409-416
Enzymes that convert starch and dextrins to α,α-trehalose and glucose were found in cell homogenates of the hyperthermophilic
acidophilic archaeon Sulfolobus shibatae DMS 5389. Three enzymes were purified and characterized. The first, the S. shibatae trehalosyl dextrin-forming enzyme (SsTDFE), transformed starch and dextrins to the corresponding trehalosyl derivatives with
an intramolecular transglycosylation process that converted the glucosidic linkage at the reducing end from α-1,4 to α-1,1.
The second, the S. shibatae trehalose-forming enzyme (SsTFE), hydrolyzed the α-1,4 linkage adjacent to the α-1,1 bond of trehalosyl dextrins, forming
trehalose and lower molecular weight dextrins. These two enzymes had molecular masses of 80 kDa and 65 kDa, respectively,
and showed the highest activities at pH 4.5. The apparent optimal temperature for activity was 70°C for SsTDFE and 85°C for
SsTFE. The third enzyme identified was an α-glycosidase (SsαGly), which catalyzed the hydrolysis of the α-1,4 glucosidic linkages
in starch and dextrins, releasing glucose in a stepwise manner from the nonreducing end of the polysaccharide chain. The enzyme
had a molecular mass of 313 kDa and showed the highest activity at pH 5.5 and at 85°C.
Received: October 29, 1997 / Accepted: April 29, 1998 相似文献
13.
Toshio Sakamoto Gaozhong Shen Shoichi Higashi Norio Murata D. A. Bryant 《Archives of microbiology》1997,169(1):20-28
Cyanobacteria acclimate to low temperature by desaturating their membrane lipids. Mutant strains of Synechococcus sp. PCC 7002 containing insertionally inactivated desA (Δ12 acyl-lipid desaturase) and desB (ω3 acyl-lipid desaturase) genes were produced, and their low-temperature susceptibility was characterized. The desA mutant synthesized no linoleic acid or α-linolenic acid, and the desB mutant did not produce α-linolenic acid. The desA mutant grew more slowly than the wild-type at 22° C and could not grow at 15° C. The desB mutant could not continuously grow at 15° C, although no observable phenotype appeared at higher temperatures. It has been
shown that expression of the desA gene occurs at 38° C and is up-regulated at 22° C, and that the desB gene is only expressed at 22° C. These results indicate that the expression of the desA and desB genes occurs at higher temperatures than those at which a significant decline in physiological activities is caused by the
absence of their products. The temperature dependency of photosynthesis was not affected by these mutations. Since chlorosis
and inability to grow at 15° C with nitrate was suppressed by the substitution of urea as a nitrogen source, it is very likely
that the chilling susceptibility of the desaturase mutants is attributable to nutrient limitation.
Received: 24 April 1997 / Accepted: 5 August 1997 相似文献
14.
Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme 总被引:9,自引:0,他引:9
C. G. van Ginkel G. B. Rikken A. G. M. Kroon S. W. M. Kengen 《Archives of microbiology》1996,166(5):321-326
A novel enzyme that catalyzes the disproportionation of chlorite into chloride and oxygen was purified from a gram-negative
bacterium, strain GR-1 to homogeneity. A four-step purification procedure comprising Q-Sepharose, hydroxyapatite, and phenyl-Superose
chromatography and ultrafiltration resulted in a 13.7-fold purified enzyme with a final specific activity of 2.0 mmol min–1 (mg protein)–1. The dismutase obeyed Michaelis-Menten kinetics. The V
max and K
m calculated for chlorite were 2,200 U (mg protein)–1 and 170 μM, respectively. Dismutase activity was inhibited by hydroxylamine, cyanide, and azide, but not by 3-amino-1,2,4-triazole.
Chlorite dismutase had a molecular mass of 140 kDa and consisted of four 32-kDa subunits. The enzyme was red-colored and had
a Soret peak at 392 nm. Per subunit, it contained 0.9 molecule of protoheme IX and 0.7 molecule of iron. Chlorite dismutase
displayed maxima for activity at pH 6.0 and 30° C.
Received: 9 April 1996 / Accepted: 12 August 1996 相似文献
15.
Alkaline protease production by a newly isolated Bacillus species from laundry soil was studied for detergent biocompatibility. From its morphological and nucleotide sequence (about
1.5 kb) of its 16S rDNA it was identified as Bacillus species with similarity to Bacillus species Y (Gen Bank entry: ABO 55095), and close homology with Bacillus cohnii YN-2000 (Gen Bank entry: ABO23412). Partial purification of the enzyme by ammonium sulfate (50–70% saturation) yielded 8-fold
purity. Casein zymography and Sodium dodecylsulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) of the partially purified
enzyme revealed two isozymes of molecular sizes approximately 66 kDa and 18 kDa, respectively. The enzyme was most active
at pH 12 and 50°C. At pH 12 the enzyme was stable for 5 h and retained 60% activity. The enzyme retained 44% activity at 50°C
up to 2 h. The protease showed good hydrolysis specificity with different substrates tested. The presence of Mn2+, Co2+ and ethylenediaminetetracetic acid (EDTA) showed profound increase in protease activity. The protease of Bacillus species Y showed excellent stability and compatibility with three locally available detergents (Kite, Tide and Aerial) up
to 3 h retaining almost 70–80% activity and 10–20% activity at room temperature (30°C) and 50°C, respectively, indicating
the potential role of this enzyme for detergent application. 相似文献
16.
S. Shima Alexander Netrusov Melanie Sordel Michaela Wicke Gudrun C. Hartmann Rudolf K. Thauer 《Archives of microbiology》1999,171(5):317-323
Methanosarcina barkeri is a strictly anaerobic, cytochrome-containing, methane-forming archaeon. We report here that the microorganism contains
a catalase, which was purified and characterized. The enzyme with an apparent molecular mass of 190 kDa was shown to be composed
of four identical subunits of apparent molecular mass of 54 kDa. The heme-containing enzyme did not exhibit peroxidase activity,
which indicates that it is a monofunctional catalase. This is substantiated by the primary structure, which is related to
that of other monofunctional catalases rather than to that of bifunctional catalase-peroxidases. The enzyme showed an [S]0.5V for H2O2 of 25 mM and an apparent V
max of 200,000 U/mg; it was inhibited by azide ([I]0.5V = 1 μM) and cyanide ([I]0.5V = 5 μM) and inactivated by 1,2,4-aminotriazole. The activity was almost independent of the pH (between pH 4 and 10) and the
temperature (between 15 °C and 55 °C). Comparison of the primary structure of monofunctional catalases revealed that the enzyme
from M. barkeri is most closely related to the monofunctional catalase of Dictyostelium discoideum.
Received: 29 December 1998 / Accepted: 1 March 1999 相似文献
17.
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
18.
M. Graf A. Brunella M. Kittelmann K. Laumen O. Ghisalba 《Applied microbiology and biotechnology》1997,47(6):650-657
A new amidohydrolase deacetylating several N-acetyl-1-phenylethylamine derivatives (R)-specifically was found in Arthrobacter aurescens AcR5b. The strain was isolated from a wet haystack by enrichment culture with (R)-N-acetyl-1-phenylethylamine as the sole carbon source. (R) and (S )-N-acetyl-1-phenylethylamine do not serve as inducers for acylase formation. By improving the growth conditions the enzyme production
was increased 47-fold. The amidohydrolase was purified to homogeneity leading to a 5.2-fold increase of the specific activity
with a recovery of 67%. A molecular mass of 220 kDa was estimated by gel filtration. Sodium dodecyl sulfate/polyacrylamide
gel electrophorosis shows two subunits with molecular masses of 16 kDa and 89 kDa. The optimum pH and temperature were pH 8
and 50 °C, respectively. The enzyme was stable in the range of pH 7–9 and at temperatures up to 30 °C. The enzyme activity
was inhibited by Cu2+, Co2+, Ni2+, and Zn2+, and this inhibition was reversed by EDTA.M
Received: 20 September 1996 / Received version: 23 December 1996 / Accepted: 30 December 1996 相似文献
19.
《Bioscience, biotechnology, and biochemistry》2013,77(11):1764-1767
An NADP-specific glutamate dehydrogenase [L-glutamate: NADP+ oxidoreductase (deaminating), EC 1.4.1.4] from alkaliphilic Bacillus sp. KSM-635 was purified 5840-fold to homogeneity by a several-step procedure involving Red-Toyopearl affinity chromatography. The native protein, with an isoelectric point of pH 4.87, had a molecular mass of approximately 315 kDa consisting of six identical summits each with a molecular mass of 52 kDa. The pH optima for the aminating and deaminating reactions were 7.5 and 8.5, respectively. The optimum temperature was around 60°C for both. The purified enzyme had a specific activity of 416units/mg protein for the aminating reaction, being over 20-fold greater than that for deaminating reaction, at the respective pH optima and at 30°C. The enzyme was specific for NADPH (Km 44 μM), 2-oxoglutarate (Km 3.13 mM), NADP+ (Km 29 μM), and L-glutamate (Km 6.06 mM). The Km for NH4Cl was 5.96 mM. The enzyme could be stored without appreciable loss of enzyme activity at 5°C for half a year in phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol, although the enzyme activity was abolished within 20 h by freezing at ?20°C. 相似文献
20.
Two extracellular isoenzymes of polygalacturonase, isolated from the brown-rot fungus Postia placenta, were purified 342-fold by Mono S cation-exchange chromatography. The temperature optimum ranged from 25 °C to 37 °C, and the pH optimum ranged from 3.2 to 3.9. Apparent pI values of the isoenzymes (3.2 and 3.4) were lower than any previously reported. The estimated molecular mass from a single
band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (PAGE) was 34 kDa. Isoenzymes of polygalacturonase in native
PAGE and isoelectric focusing gels were identified by substrate/ agar overlays (zymograms). Comparison of viscosity reduction
rates with release of reducing sugars indicated that the enzyme from P. placenta is endo-acting. The objective of this study was to isolate polygalacturonase from the brown-rot fungus P. placenta and characterize the properties of the enzyme.
Received: 31 October 1995/Received revision: 12 February 1996/Accepted: 4 March 1996 相似文献