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1.
M. O. Ilori O. O. Amund O. Omidiji 《World journal of microbiology & biotechnology》1995,11(5):595-596
An extracellular glucose-forming amylase was produced by Lactobacillus brevis isolated from Kagasok tea. The enzyme was purified 70-fold and had optimal activity at 55°C and pH 6.5. Its K
m value for starch was 0.27 mg ml-1 and its M
r was approx. 75,900 Da. The activity of the enzyme was enhanced by Ca2+, Mg2+, Na+ or K+ and inhibited by EDTA, KCN, citric acid and l-cysteine. 相似文献
2.
Two l-threonine (l-serine) dehydratases (EC 4.2.1.16) of the thermophilic phototrophic bacterium Chloroflexus aurantiacus Ok-70-fl were purified to electrophoretic homogeneity by procedures involving anion exchange and hydrophobic interaction chromatography. Only one of the two enzymes was sensitive to inhibition by l-isoleucine (K
i=2 M) and activation by l-valine. The isoleucine-insensitive dehydratase was active with l-threonine (K
m=20 mM) as well as with l-serine (K
m=10 mM) whereas the other enzyme, which displayed much higher affinity to l-threonine (K
m=1.3 mM), was inactivated when acting on l-serine. Both dehydratases contained pyridoxal-5-phosphate as cofactor. When assayed by gel filtration techniques at 20 to 25° C, the molecular weights of both enzymes were found to be 106,000±6,000. In sodium dodecylsulfate-polyacrylamide gel electrophoresis, the two dehydratases yielded only one type of subunit with a molecular weight of 55,000±3,000. The isoleucine-insensitive enzyme was subject to a glucose-mediated catabolite repression.Abbreviations A
absorbance
-
ile
isoleucine
- PLP
pyridoxal-5-phosphate
- SDS
sodium dodecyl sulfate
- TDH
threonine dehydratase
- U
unit 相似文献
3.
Subha Manna Amaresh Sinha Ramkrishna Sadhukhan S. L. Chakrabarty 《Current microbiology》1995,30(5):291-298
An l-asparaginase produced by Pseudomonas stutzeri MB-405 was isolated and characterized. After initial ammonium sulfate fractionation, the enzyme was purified by consecutive column chromatography on Sephadex G-100, Ca-hydroxylapatite, and DEAE-Sephadex A-50. The 665.5-fold purified enzyme thus obtained has the specific activity of 732.3 units mg protein-1 with an overall recovery of 27.2%. The apparent M
r of the enzyme under nondenaturing and denaturing conditions was 34 kDa and 33 kDa respectively, and the isoelectric point was 6.38±0.02. It displayed optimum activity at pH 9.0 and 37°C. The enzyme was very specific for l-asparagine and did not hydrolyze L-glutaminate. The K
m of the l-asparaginase was found to be 1.45×10-4
m towards l-asparagine and was competitively inhibited by 5-diazo-4-oxo-l-norvaline (DONV) with a K
i of 0.03mm. Metal ions such as Mn2+, Zn2+, Hg2+, Fe3+, Ni2+, and Cd2+ potentially inhibited the enzyme activity. The activity was enhanced in the presence of thiol-protecting reagents such as DTT, 2-ME, and glutathione (reduced), but inhibited by PCMB and iodoacetamide. The tumor inhibition study with Dalton's lymphoma tumor cells in vivo indicated that this enzyme possesses antitumor properties. 相似文献
4.
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
5.
J. J. P. Baars H. J. M. Op den Camp J. W. G. Paalman V. Mikeš C. van der Drift L. J. L. D. Van Griensven G. D. Vogels 《Current microbiology》1995,31(2):108-113
Agaricus bisporus glutamine synthetase, a key enzyme in nitrogen metabolism, was purified to apparent homogeneity. The native enzyme appeared to be a GS-II type enzyme. It has a molecular weight of 325 kDa and consists of eight 46-kDa subunits. Its pI was found at 4.9. Optimal activity was found at 30°C. The enzyme had low thermostability. Stability declined rapidly at temperatures above 20°C. The enzyme exhibits a K
m for glutamate, ammonium, and ATP of 22mm, 0.16mm and 1.25mm respectively in the biosynthetic reaction, with optimal activity at pH 7. The enzyme is slightly inhibited by 10mm concentrations of l-alanine, l-histidine, l-tryptophan, anthranilic acid, and 5-AMP and was strongly inhibited by methionine sulfoximine and phosphinothricine. For the transferase reaction K
i-values were 890 m and 240 m for methionine sulfoximine and phosphinothricine respectively. For the biosynthetic reaction K
i was 17 m for both methionine sulfoximine and phosphinothricine. 相似文献
6.
Bacillus stearothermophilus L1 was isolated by enrichment culture using an alkaline extract of pulp as the carbon source at 65°C and pH 9.0. The bacterium produced extracellular xylanase and -l-arabinofuranosidase (EC 3.2.1.55). The xylanase activity was high when the cells were grown in the presence of d-xylose, whereas the arabinofuranosidase activity was high when grown in media containing l-arabinose. The arabinofuranosidase was purified 59-fold with an 80% yield by DEAE Sephacel and Sephadex G-100 chromatography. The purified enzyme had an apparent molecular mass of 110 000 kDa and consisted of two subunits of 52 500 kDa and 57 500 kDa. Using p-nitrophenyl--l-arabinofuranosidase as the substrate, the enzyme had a Michaelis constant (K
m) of 2.2 × 10–4
m, maximum reaction velocity (Vmax) of 11o mol min–1 mg–1, temperature optimum of 70°C and pH optimum of 7.0 (50% activity at pH 8.0). The enzyme was specific for the furanoside configuration. The purified enzyme partially delignified softwood Kraft pulp. Treatment of the pulp with 38 units ml–1 of -l-arabinofuranosidase at 65°C for 2 h at pH 8.0 and 9.0 led to lignin releases of 2.3% and 2.1%, respectively. The enzyme acted synergistically with a thermophilic xylanase in the delignification process, yielding a 19.2% release of lignin.
Correspondence to: Eugene Rosenberg 相似文献
7.
Liu F Tachibana S Taira T Ishihara M Yasuda M 《Journal of industrial microbiology & biotechnology》2004,31(1):23-28
Carboxypeptidase produced by Monascus purpureus IFO 4478 was purified to homogeneity. The purified enzyme is a heterodimer with a molecular mass of 132 kDa and consists of two subunits of 64 and 67 kDa. It is an acidic glycoprotein with an isoelectric point of 3.67 and 17.0% carbohydrate content. The optimum pH and temperature were 4.0 and 40 °C, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 °C for 1 h, and up to 50 °C at pH 5.0 for 15 min. The enzyme was strongly inhibited by piperastatin A, diisopropylfluoride phosphate (DFP), phenylmethylsulfonylfluoride (PMSF), and chymostatin, suggesting that it is a chymotrypsin-like serine carboxypeptidase. Monascus purpureus carboxypeptidase was also strongly inhibited by p-chloromercuribenzoic acid (PCMB) but not by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, indicating that it requires cysteine residue but not metal ions for activity. Benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrate of the enzyme. The Km, Vmax, Kcat, and Kcat/Km values of the enzyme for Z-Tyr-Glu at pH 4.0 and 37 °C were 0.86 mM, 0.917 mM min–1, 291 s–1, and 339 mM–1 s–1, respectively. 相似文献
8.
Uridine 5-diphosphoglucose-dependent glucosyl-transferases (UDP-glucose:betanidin 5-O- and 6-O-glucosyltransferases; 5-GT and 6-GT; EC 2.4.1) catalyze the regiospecific transfer of glucose to the 5- and 6-hydroxy group of betanidin in the formation of betanin and gomphrenin I, respectively. Both GT activities were partially purified from cell suspension cultures of Dorotheanthus bellidiformis (Burm. f.) N.E. Br. Isoelectric focusing of crude protein extracts indicated the presence of three 5-GT isoforms and a single 6-GT form. The 5-GT isoforms were partially separated from each other and completely from the 6-GT. Studies of the glucosyltransferase activities were focused on the major isoform of the 5-GTs and the 6-GT, which displayed the same pH optimum near 7.5 in K-phosphate buffer. A 3- and 2.5-fold enrichment and 11% and 10% recovery of the 5-GT and 6-GT, respectively, were routinely achieved; however, a 3300-fold enrichment of the major 5-GT isoform and a 6-fold enrichment of the 6-GT were also achieved. Both enzymes are monomers and displayed apparent native Mrs near 55 000. The maxima of the reaction temperature were at 50 °C for the 5-GT and at 37°C for the 6-GT with respective apparent energies of activation of 51 and 53 kJ · mol–1. Kinetic studies indicated that the apparent Michaelis constants (apparent K
m) of the GTs for one substrate were dependent on the concentration of the second substrate. However, the relationship between the apparent K
m values and the dissociation constants (K
i) were different; m > K
i applies for the 5-GT and K
m < K
i for the 6-GT activity. Consequently, this results in a predominant formation of betanin at low substrate concentrations, but a predominant formation of gomphrenin I at high substrate concentrations, assuming that both enzymes may compete freely for their substrates. This might explain why we could not observe a correlation between extractable 5-GT and 6-GT activities and the in-vivo accumulation of the respective products from cell-suspension cultures of D. bellidiformis.Abbreviations BSA
bovine serum albumin
- DOPA
l-(3,4-dihydroxyphenyl)-alanine
- 5-GT
UDP-glucose:betanidin 5-O-glucosyltransferase
- 6-GT
UDP-glucose:betanidin 6-O-glucosyl-transferase
- Mr
molecular mass
This investigation was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. 相似文献
9.
Glycerate kinase (GK; EC 2.7.1.31) from maize (Zea mays L.) leaves was purified by a sequence of ammonium-sulfate precipitations and chromatography on diethylaminoethyl-cellulose, hydroxyapatite, Sephadex G-75SF and dye ligand (Green A) columns. The purest preparation was almost 1300-fold enriched and had a specific activity of 68 mol · min-1 · (mg protein) -1. The enzyme was a monomer of a relative molecular mass (Mr) of 44 kDa (kdalton) as determined by gel filtration, electrophoresis in dissociating conditions and by immunoblots. The enzyme was only weakly recognized by polyclonal antibodies against purified spinach GK, indicating substantial differences in molecular structure of the two proteins. Highly reducing conditions stabilized GK activity and were required for activation of crude leaf enzyme. The enzyme had a broad pH optimum of 6.8–8.5, and formed 3-phosphoglycerate and ADP as reaction products. Apparent K
ms for D-glycerate and Mg-ATP were 0.11 and 0.25 mM, respectively. The enzyme was strongly affected by a number of phosphoesters, especially by 3-phosphoglycerate (K
i= 0.36 mM), fructose bisphosphates and nucleoside bisphosphates. Inhibition by 3-phosphoglycerate was competitive to Mg-ATP and noncompetitive to D-glycerate. Pyruvate was found noncompetitive to D-glycerate (K
is=4 mM). The ratio of stromal concentration of Mg-ATP to phosphoesters, particularly to 3-phosphoglycerate, may be of importance in the regulation of GK during C4-photosynthesis.Abbreviations DEAE
diethylaminoethyl
- kDa
kdalton
- GAP-DH
glyceraldehyde phosphate dehydrogenase
- GK
glycerate kinase
- LDH
lactate dehydrogenase
- 2-ME
2-mercaptoethanol
- Mr
relative molecular mass
- PEP
phosphoenolpyruvate
- PGA(PK)
phosphoglycerate (phosphokinase)
- PK
pyruvate kinase
- SDS-PAGE
sodium dodecyl sulfatepolyacrylamide gel electrophoresis 相似文献
10.
Summary A thermostable NADP-dependent isocitrite dehydrogenase (IDH; EC. 1.1.1.42) was purified from the obligately thermophilic hydrocarbonoclastic bacterium Thermoleophilum minutum YS-4 (ATCC 35265). This was accomplished by affinity chromatography and electroelution from a nondenaturing polyacrylamide gel. The enzyme has an M
r of 60 000 and is composed of two identical subunits of M
r 30 500. The amino acid composition has an Arg/Lys ratio of 4:1 and very high levels of glycine. Under nondenaturing conditions, the enzyme has a distinct difference in electrophoretic mobility relative to IDHs obtained from other genera including the genus Thermus. The secondary strcuture consists of 16% -helix, 20% -sheet, 25% -turn and 37% random coil as determined by circular dichroism spectroscopy. The optimum pH and temperature for activity were 7.2 and 75° C respectively and the apparent K
mvalues for DL-isocitrate adn NADP+ were 33 M, and 48 M, respectively. The enzyme requires divalent cations, such as Mn2+ or Mg2+ for activity. NAD+ cannot substitute for NADP+. Oxaloacetate plus glyoxylate exert considerable inhibition on IDH activity while other glycolytic and tricarboxylic acid cycle intermediates have a lesser effect. p-Chloromercuribenzoic acid was inhibitory to the IDH although isocitrate and Mn2+ offered some protection from this inactivation. The enzyme is thermostable, retaining 84% and 57% of initial activity after incubation for 1 h at 60° and 70° C, respectively. Isocitrate provided protection from thermal inactivation allowing the IDH to maintain 21% activity after 1 h at 80° C.
Offprint requests to: J. J. Perry 相似文献
11.
V. Andreoni G. Baggi S. Bernasconi C. Foglieni F. Pelizzoni 《Applied microbiology and biotechnology》1990,32(6):633-636
Summary
N-(Benzyloxycarbonyl)-l-asparty-l-phenylalanine methyl ester, the precursor of the synthetic sweetener aspartame, was continuously synthesized in an immobilized thermolysin plug-flow type reactor at 25° C with the substrates (N-benzyloxycarbonyl-l-aspartic acid and l-phenylalanine methyl ester) dissolved in ethyl acetate. The immobilized enzyme was quite stable in ethyl acetate containing 2.5% 0.01 M 2-(N-morpholino)ethanesulphonic acid-NaOH buffer, pH 6.0, and 20 mM CaCl2 with or without the substrate at 25° C. By periodically washing the column, we could conduct a continuous reaction for over 500 h with an average yield of 95% and a space velocity of 1.85 h –1.Offprint requests to: K. Nakanishi 相似文献
12.
Purification and properties of thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60
Thermostable N-acylamino acid recemase from Amycolatopsis sp. TS-1-60, a rare actinomycete strain selected for its ability to grow on agar plates incubated at 40° C, was purified to homogeneity and characterized. The relative molecular mass (M
r) of the native enzyme and the subunit was estimated to be 300 000 and 40 000 on gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis respectively. The isoelectric point (pI) of the enzyme was 4.2. The optimum temperature and pH were 50° C and 7.5 respectively. The enzyme was stable at 55° C for 30 min. The enzyme catalyzed the racemization of optically active N-acylamino acids such as N-acetyl-l-or d-methionine, N-acetyl-l-valine, N-acetyl-l-tyrosine and N-chloroacetyl-l-valine. In addition, the enzyme also catalyzed the recemization of the dipeptide l-alanyl-l-methionine. By contrast, the optically active amino acids, N-alkyl-amino acids and methyl and athyl ester derivatives of N-acetyl-d- and l-methionine were not racemized. The apparent K
m values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 18.5 mM and 11.3 mM respectively. The enzyme activity was markedly enhanced by the addition of divalent metal ions such as Co2+, Mn2+ and Fe2+ and was inhibited by addition of EDTA and P-chloromercuribenzoic acid. The similarity between the NH2-terminal amino acid sequence of the enzyme and that of Streptomyces atratus Y-53 [Tokuyama et al. (1994) Appl Microbiol Biotechnol 40:835–840] was above 80%. 相似文献
13.
We used ion-sensitive, double-barrel microelectrodes to measure changes in hepatocyte transmembrane potential (V
m), intracellular K+, Cl-, and Na+ activities (a
i
k, a
Cl
i
and a
Na
i
), and water volume during l-alanine uptake. Mouse liver slices were superfused with control and experimental Krebs physiological salt solutions. The experimental solution contained 20 m
l-alanine, and the control solution was adjusted to the same osmolality (305 mOsm) with added sucrose. Hepatocytes also were loaded with 50 mm tetramethylammonium ion (TMA+) for 10 min. Changes in cell water volume during l-alanine uptake were determined by changes in intracellular, steady-state TMA+ activity measured with the K+ electrode. Hepatocyte control V
m was -33±1 mV. l-alanine uptake first depolarized V
m by 2±0.2 mV and then hyperpolarized V
m by 5 mV to-38±1 mV (n = 16) over 6 to 13 min. During this hyperpolarization, a
Na
i
increased by 30% from 19±2 to 25±3 mm (P < 0.01), and a
K
i
did not change significantly from 83±3 mm. However, with added ouabain (1 mm) l-alanine caused only a 2-mV increase in V
m, but now a
K
i
decreased from 61±3 to 54±5 mm (P < 0.05). Hyperpolarization of V
m by l-alanine uptake also resulted in a 38% decrease of a
Cl
i
from 20±2 to 12±3 mm (P < 0.001). Changes in V
m and V
Cl — V
m voltage traces were parallel during the time of l-alanine hyperpolarization, which is consistent with passive distribution of intracellular Cl– with the V
m in hepatocytes. Added Ba2+ abolished the l-alanineinduced hyperpolarization, and a
Cl
i
remained unchanged. Hepatocyte water volume during l-alanine uptake increased by 12±3%. This swelling did not account for any changes in ion activities following l-alanine uptake. We conclude that hepatocyte a
K
i
is regulated by increased Na+-K+ pump activity during l-alanine uptake in spite of cell swelling and increased V
m due to increased K+ conductance. The hyperpolarization of V
m during l-alanine uptake provides electromotive force to decrease a
Cl
i
. The latter may contribute to hepatocyte volume regulation during organic solute transport.This work was supported by grant AA-08867 from the Alcohol, Drug Abuse, and Mental Health Association. 相似文献
14.
Summary Ascorbic acid is essential for the formation of bone by osteoblasts, but the mechanism by which osteoblasts transport ascorbate has not been investigated previously. We examined the uptake ofl-[14C]ascorbate by a rat osteoblast-like cell line (ROS 17/2.8) and by primary cultures of rat calvaria cells. In both systems, cells accumulatedl-[14C]ascorbate during incubations of 1–30 min at 37°C. Unlike propionic acid, which diffuses across membranes in protonated form, ascorbic acid did not markedly alter cytosolic pH. Initial ascorbate uptake rate saturated with increasing substrate concentration, reflecting a high-affinity interaction that could be described by Michaelis-Menten kinetics (apparentK
m
=30±2 m andV
max=1460±140 nmol ascorbate/g protein/min in ROS 17/2.8 cells incubated with 138mm extracellular Na+). Consistent with a stereoselective carrier-mediated mechanism, unlabeledl-ascorbate was a more potent inhibitor (IC50=30±5 m) ofl-[14C]ascorbate transport than wasd-isoascorbate (IC50=380±55 m). Uptake was dependent on both temperature and Na+, since it was inhibited by cooling to 4°C and by substitution of K+, Li+ or N-methyl-d-glucamine for extracellular Na+. Decreasing the external Na+ concentration lowered both the affinity of the transporter for ascorbate and the apparent maximum velocity of transport. We conclude that osteoblasts possess a stereoselective, high-affinity, Na+-dependent transport system for ascorbate. This system may play a role in the regulation of bone formation. 相似文献
15.
Brian E. Peerce 《The Journal of membrane biology》1989,110(2):189-197
Summary The substrate stoichiometry of the intestinal Na+/phosphate cotransporter was examined using two measures of Na+-dependent phosphate uptake: initial rates of uptake with [32P] phosphate and phosphate-induced membrane depolarization using the potential-sensitive dye diSC3(5). Isotopic phosphate measures electrogenic and electroneutral Na+-dependent phosphate uptake, while phosphate-induced membrane depolarization measures electrogenic phosphate uptake. Using these measures of Na-dependent phosphate uptake, three parameters were compared: substrate affinity; phenylglyoxal sensitivity and labeling; and inhibiton by mono- and di-fluorophosphates. Na+/phosphate cotransport was found to have similar Na+ activations (apparentK
0.5's of 28 and 25mm), apparentK
m
's for phosphate (100 and 410 m), andK
0.5's for inhibition by phenylglyoxal (70 and 90 m) using isotopic phosphate, uptake and membrane depolarization, respectively. Only difluorophosphate inhibited Na+-dependent phosphate uptake below 1mm at pH 7.4.Difluorophosphate also protected a 130-kDa polypeptide from FITC-PG labeling in the presence of Na+ with apparentK
0.5 for phosphate of 200 m; similar to the apparentK
m
for phosphate uptake, andK
0.5 for phosphate protection against FITC-PG inhibition of Na+-dependent phosphate uptake and FITC-PG labeling of the 130-kDa polypeptide. These results indicate that the intestinal Na+/phosphate cotransporter is electrogenic at pH 7.4, that H2PO
4
–
is the transport-competent species, and that the 130-kDa polypeptide is an excellent candidate for the intestinal Na+/phosphate cotransporter. 相似文献
16.
《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
17.
Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula 总被引:1,自引:0,他引:1
Masahiro Nakao Masami Harada Yukiko Kodama Toru Nakayama Yuji Shibano Teruo Amachi 《Applied microbiology and biotechnology》1994,40(5):657-663
We purified an extracellular thermostable -galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted -d-galactopyranosides such as lactose [a Michaelis constant K
m=0.75 mm and molecular activity (k
cat)= 63.1 s–1 at pH 7.2 and 55° C] and p-nitrophenyl -d-galactopyranoside (K
m=0.04 mm
k
cat= 55.8 s–1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula -galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 m MnCl2) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 m MnCl2). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C <) for efficient production of the oligosaccharides from lactose.
Correspondence to: T. Nakayama 相似文献
18.
Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (Mr) of 600 000 is composed of monomeric sub-units of 60 000 (Mr). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K
m values (K
m
app
) in the biosynthetic reaction for glutamate, NH4Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K
m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF
phenylmethylsulfonylfluoride
- TCA
trichloroacetic acid
- GS
glutamine synthetase
- MSO
L-Methionine-D-L-sulfoximine
- SDS-PAGE
sodium dodecyl sulfatepolyacrylamide gel electrophoresis
- SVPDE
snake venum phosphodiesterase 相似文献
19.
Summary The benzoyl-CoA ligase from an anaerobic syntrophic culture was purified to homogeneity. It had a molecular mass of around 420 kDa and consisted of seven or eight subunits of 58 kDa. The temperature optimum was 37–40° C, the optimum pH around 8.0 and optimal activity required 50–100 mM TRIS-HCI buffer, pH 8.0 and 3–7 mM MgCl2; MgCl2 in excess of 10 mM was inhibitory. The activation energy for benzoate was 11.3 kcal/mol. Although growth occured only with benzoate as a carbon source, the benzoyl-coenzyme A (CoA) ligase formed benzoyl-CoA esters with benzoate, 2-, 3- and 4-fluorobenzoate, picolinate, nicotinate and isonicotinate. Acetate was activated to acetyl-CoA by an acetyl-CoA synthetase. The K
m values for benzoate, 2-, 3- and 4-fluorobenzoate were 0.04, 0.28, 1.48 and 0.32 mM, the V
max values 1.05, 1.0, 0.7 and 0.98 units (U)/mg, respectively. For reduced CoA (CoA-SH) a K
m of 0.17 mM and a V
max of 1.05 U/mg and for ATP a K
m of 0.16 mM and a V
max of 1.08 U/mg was determined. Benzoate activation was inhibited by more than 6 mM ATP, presumably by pyrophosphate generation from ATP. The inhibition constant (K
i) for pyrophosphate was 5.7 mM. No homology of the N-terminal amino acid sequence with that of a 2-aminobenzoyl-CoA ligase of a denitrifying Pseudomonas sp. was found.
Correspondence to: J. Winter 相似文献
20.
We have developed an enzymatic procedure for the enantiospecific synthesis ofN-acetyl-l-methionine with aminoacylase in an organic solvent.N-Acetyl-l-methionine was most effectively synthesized with a yield of about 90% (on the basis of thel-methionine used) when the reaction mixture, composed of 100 mm sodium acetate, 20 MMdl-methionine and aminoacylase (1000 units) immobilized on celite in 1 ml ethyl acetate saturated with 32 l 140mm sodium phosphate buffer (pH 7.0) containing 0.1 mm CoCl2, was incubated at 30°C for 24 h.N-Acetyl-l-methionine was isolated from the reaction mixture and the enantiomeric excess was 100%.d-Methionine was also isolated from the mixture with a yield of about 95% and 90% enantiomeric excess. The method is applicable to the synthesis of otherN-acetyl-l-amino acids. 相似文献