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1.
A new enzyme catalyzing the deamidation of seed storage proteins was found in germinating wheat grains and was partially purified. It also acts on egg lysozyme, horse hemoglobin and reduced RNAse, glutamine and Gly-L-Gln-L-Tyr. No activity was observed when using ovalbumin, serum albumin, RNAse, insulin, asparagine and an asparagine-containing peptide. Only glutaminyl residues appear to be deamidated by this enzyme. It differs from transglutaminase and proved to be a true protein deamidase. 相似文献
2.
A novel protein-deamidating enzyme was purified to homogeneity from Chryseobacterium proteolyticum and the gene encoding it was cloned. The enzyme is a monomer with a pI of 10.0, a measured M(r) of approximately 20,000 and a calculated M(r) of 19,860. Extensive comparison with Streptoverticillium transglutaminase showed that the protein-deamidating enzyme lacked transglutaminase activity in terms of hydroxamate-formation between benzyloxycarbonyl-Gln-Gly and hydroxylamine, or monodansylcadaverine incorporation into casein. The enzyme deamidated the two glutaminyl residues in the oxidized insulin A chain and deamidated both casein and the oxidized insulin B chain with higher catalytic efficiencies (k(cat)/K(m)) than with short peptides. The enzyme was active against several proteins, including insoluble wheat gluten, but did not deamidate asparaginyl residues in peptides, free glutamine or other amides. The enzyme was therefore named protein-glutaminase (EC 3.5.1). The gene encoding the protein was cloned and, when expressed in Escherichia coli, the protein product had protein-glutaminase activity and cross-reacted with antiserum raised against the purified enzyme. The protein-glutaminase was shown to be expressed as a prepro-protein with a putative signal peptide of 21 amino acids and a pro-sequence of 114 amino acids. The amino-acid sequence had no obvious homology to any published sequence and is therefore a novel protein-glutaminase. 相似文献
3.
GLUTAMYL, ASPARTYL AND AMIDE MOIETIES OF CEREBRAL PROTEINS: METABOLIC ASPECTS IN VITRO 总被引:1,自引:0,他引:1
Abstract— The total mixed proteins (excluding proteolipids) were isolated from cat cerebral cortex and subjected to acid and enzymic hydrolyses. Analyses on the hydrolysates were carried out by specific enzymic procedures to determine the glutamyl, glutaminyl, aspartyl and asparaginyl composition. The content of total glutamyl and total aspartyl residues was the same in all types of protein samples, with average values of 78 and 58 /miol/100 mg of protein, respectively. In biopsy samples approximately 45 per cent of each total was in the amide form. Preparation of slices of cerebral cortex for incubation was associated with deamidation in situ of 16 per cent of the protein-bound glutaminyl residues. The extent of deamidation was not increased by incubation or by prolonged hypoxia and was unaffected by prior anaesthesia or by incubation of slices with 10 mM-NH4Cl or 40 mM-malonate. Slices prepared from animals intoxicated with methionine sulphoximine exhibited no deamidation. No deamidation was observed for slices of subcortical white matter, liver, kidney, testis or diaphragm of the cat. Cortical proteins from other species appeared to behave similarly to those of the cat. The 5-4 μmol of NH3 released/g of fresh cortex could account for about 85 per cent of the endogenous free ammonia regularly encountered in such slices. Hence the labile fraction of protein-bound glutaminyl amide groups represents, as previously suspected, a major source of endogenous cerebral NH3. Proteins isolated from cerebral cortical slices incubated with L-[U-14C]glutamic acid or L-[U-14C]glutamine contained 105 (±0.095) per cent of the total 14C metabolized. The ratios (x 100) of protein to free pool specific radioactivities (c.p.m.μmol) of glutamic acid and of glutamine were in the range 0-22 to 0-42, or of the same order as previously reported for other amino acids. Comparable results were obtained with proteins isolated from cerebral cortical slices incubated with 10 mM-15NH4Cl or L-[amide-15N]glutamine or both. In the amide N of protein-bound glutaminyl residues the atoms per cent excess 15N ranged from 007 to 0-42. This degree of labelling could be accounted for completely by the turnover of the entire glutaminyl moiety, as indicated by the 14C studies. Simultaneous analyses of free pool NH3 and glutamine suggested that transfer of glutamine from medium to slice involves deamidation as it is taken up and reamidation after entry. 相似文献
4.
《Bioscience, biotechnology, and biochemistry》2013,77(5):960-965
Chitin deacetylase (CDA), the enzyme that catalyzes the hydrolysis of acetamido groups of GlcNAc in chitin, was purified from culture filtrate of the fungus Mortierella sp. DY-52 and characterized. The extracellular enzyme is likely to be a highly N-glycosylated protein with a pI of 4.2–4.8. Its apparent molecular weight was determined to be about 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and 67 kDa by size-exclusion chromatography. The enzyme had an optimum pH of 6.0 and an optimum temperature of 60 °C. Enzyme activity was slightly inhibited by 1–10 mM Co2+ and strongly inhibited by 10 mM Cu2+. It required at least two GlcNAc residues for catalysis. When (GlcNAc)6 was used as substrate, K m and V max were determined to be 1.1 mM and 54.6 μmol min?1 respectively. 相似文献
5.
Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP), the product of the PA5292 gene, is synthesized when the bacteria are grown with choline,
betaine, dimethylglycine, or carnitine. In the presence of Mg2+, PChP catalyzes the hydrolysis of both phosphorylcholine (PCh) and p-nitrophenylphosphate (p-NPP). PCh saturation curve analysis of the enzyme with or without the signal peptide indicated that the peptide was the fundamental
factor responsible for decreasing the affinity of the second site of PChP for PCh, either at pH 5.0 or pH 7.4. PChP contained
three conserved motifs characteristic of the haloacid dehalogenases superfamily. In the PChP without the signal peptide, motifs
I, II, and III correspond to the residues 31DMDNT35, 166SAA168, and K242/261GDTPDSD267, respectively. To determine the catalytic importance of the D31, D33, T35, S166, K242, D262, D265, and D267 on the enzyme
activity, site-directed mutagenesis was performed. D31, D33, D262, and D267 were identified as the more important residues
for catalysis. D265 and D267 may be involved in the stabilization of motif III, or might contribute to substrate specificity.
The substitution of T35 by S35 resulted in an enzyme with a low PChP activity, but conserves the catalytic sites involved
in the hydrolysis of PCh (Km1 0.03 mM, Km2 0.5 mM) or p-NPP (Km 2.1 mM). Mutating either S166 or K242 revealed that these residues are also important to catalyze the hydrolysis of both substrates.
The substitution of lysine by arginine or by glutamine revealed the importance of the positive charged group, either from
the amino or guanidinium groups, because K242Q was inactive, whereas K242R was a functional enzyme. 相似文献
6.
Crystal structure and inhibition studies of transglutaminase from Streptomyces mobaraense 总被引:1,自引:0,他引:1
Yang MT Chang CH Wang JM Wu TK Wang YK Chang CY Li TT 《The Journal of biological chemistry》2011,286(9):7301-7307
The crystal structure of the microbial transglutaminase (MTGase) zymogen from Streptomyces mobaraense has been determined at 1.9-Å resolution using the molecular replacement method based on the crystal structure of the mature MTGase. The overall structure of this zymogen is similar to that of the mature form, consisting of a single disk-like domain with a deep active cleft at the edge of the molecule. A major portion of the prosequence (45 additional amino acid residues at the N terminus of the mature transglutaminase) folds into an L-shaped structure, consisting of an extended N-terminal segment linked with a one-turn short helix and a long α-helix. Two key residues in the short helix of the prosequence, Tyr-12 and Tyr-16, are located on top of the catalytic triad (Cys-110, Asp-301, and His-320) to block access of the substrate acyl donors and acceptors. Biochemical characterization of the mature MTGase, using N-α-benzyloxycarbonyl-l-glutaminylglycine as a substrate, revealed apparent Km and kcat/Km values of 52.66 mm and 40.42 mm−1 min−1, respectively. Inhibition studies using the partial prosequence SYAETYR and homologous sequence SQAETYR showed a noncompetitive inhibition mechanism with IC50 values of 0.75 and 0.65 mm, respectively, but no cross-linking product formation. Nevertheless, the prosequence homologous oligopeptide SQAETQR, with Tyr-12 and Tyr-16 each replaced with Gln, exhibited inhibitory activity with the formation of the SQAETQR-monodansylcadaverine fluorophore cross-linking product (SQAETQR-C-DNS). MALDI-TOF tandem MS analysis of SQAETQR-C-DNS revealed molecular masses corresponding to those of NSQAETQC-C-DNS and C-DNS-NQRC sequences, suggesting the incorporation of C-DNS onto the C-terminal Gln residue of the prosequence homologous oligopeptide. These results support the putative functional roles of both Tyr residues in substrate binding and inhibition. 相似文献
7.
The sequence-ordered copolymer poly-(Lys-Ala-Ala) was synthesized by polycondensation of the N-hydroxysuccinimide ester of ε,Z-Lys-Ala-Ala and deprotection of the polymerization product. A fraction of molecular weight 13,000 obtained by ion-exchange chromatography was investigated. The polymer is freely soluble in water at all pH values, and is completely digested by trypsin and elastase. From CD and ORD data it was concluded that in water at 1°C the ionized form (at pH 6.5) of the polymer is helical. On heating, helix-coil transition curves were obtained with a midpoint, Tm, depending on salt concentration. In salt-free water Tm = 12.3°C and in 0.2M NaCl Tm = 28.5°C. Adding MeOH, causes an increase in the helical content of the polymer (half helicity at 20% MeOH, without salt, at 29°C). Guanidine·HCl was shown to decrease the helicity. At 1°C half helicity. The nonionized polymer helix is more stable (Tm~90°C). At the high pH, at 60°C, when concentration of the polymer is higher than 1.9 × 10-2M, a precipitate is formed which redissolves on cooling with the original helicity. This does not occur in the presence of 50% MeOH. By comparison with polylysine it was concluded that replacing two-thirds of the lysine residues in polylysine by alanine leads to a polymer forming a more stable α-helix, when fully ionized. This is essentially due to the diminished coulombic repulsion. Uncharged lysine residues are comparable to alanine residues in their helix-forming tendency since the sequential polymer as well as one-third ionized polylysine are helical to approximately the same extent at room temperature. 相似文献
8.
Poly(Lys48, His52), a random copolypeptide of L -lysine (48%) and L -histidine (52%), was used as a model protein for investigating the effects of protonation on the imidazole group of histidines on protein binding to DNA. The complexes formed between poly(Lys48, His52) and DNA were examined using absorbance, circular dichroism (CD), and thermal denaturation. Although increasing pH reduces the charges on histidine side chains in the model protein, the protein still binds the DNA with approximately one positive charge per negative charge in protein-bound regions. Nevertheless, CD and melting properties of poly(Lys48, His52)-DNA complexes still depend upon the solution pH which determines the protonation state of imidazole group of histidine side chains. At pH 7.0, the complexes show two characteristic melting bands with a tm (46–51°C) for free base pairs and a t′m (94°C) for protein-bound base pairs. The t′m of the complexes is reduced to 90°C at pH 9.2, although at this pH there is still one lysine per phosphate in protein-bound regions. Presumably, the presence of deprotonated histidine residues destabilizes the native structure of protein-bound DNA. The binding of this model protein to DNA causes a red shift of the crossover point and both a red shift and a reduction of the positive CD band of DNA near 275 nm. This phenomenon is similar to that caused by polylysine binding. These effects, however, are greatly diminished when histidine side chains in the model protein are deprotonated. The structure of already formed poly(Lys48, His52)·DNA complexes can be perturbed by changing the solution pH. However, the results suggest a readjustment of the complex to accommodate charge interactions rather than a full dissociation of the complex followed by reassociation between the model protein and DNA. 相似文献
9.
β-N-Acetyl-d-glucosaminidase (EC 3.2.1.30) was purified from the alimentary canal of the silkworm, Bombyx mori, by ammonium sulfate fractionation and chromatography with hydroxylapatite, DEAE Bio-Gel A, chromatofocusing, and Sephacryl S-200. The purified enzyme was a single band on disc-PAGE. The molecular weight was 119,000 by gel filtration and 125,000 by SDS-PAGE. The enzyme was separated into two peptides whose apparent molecular weights were 67,500 and 57,500 by SDS-PAGE. The pi was 4.86 by chromatofocusing. The optimum pH was 5.5 to 6.0 and the optimum temperature, 45°C, using pNp-β-GlcNAc as the substrate. The enzyme was stable from pH 5.5 to 8.5 and below 30°C. It was strongly inhibited by HgCl2. Small N-acetylchitooligomers were as good substrates as pNp-β-GlcNAc, and the enzyme cleaved colloidal chitin to GlcNAc, even though the relative velocity was slow. Smaller N-acetylchitooligomers were preferred substrates with Km 0.787 to 0.056mm, A:cat 1013 to 52sec–1, and kcat/Km 1690 to 754 mm–1 sec–1. The enzyme precipitated in as band with moulting fluid chitobiase antiserum, but not with haemolymph exo-β-N-acetylglucosaminidase antiserum. The results suggest that this enzyme is an exo-type enzyme which is involved in the degradation of chitin to GlcNAc. 相似文献
10.
One thermostable endoglucanase (CMCase) was purified to homogeneity from the culture supernatant of a new isolated thermophilic bacterium Caldibacillus cellulovorans. The molecular weight of the enzyme was 85.1 kDa as determined by SDS Polyacrylamide gel electrophoresis (PAGE) and 174 kDa by size-exclusion chromatography. The isoelectric point of the enzyme was at pH 4.12. The temperature for maximum activity was 80 °C, with half-lives of 32 min at 80 °C, and 2 min at 85 °C, and 83% activity remaining after 3 h at 70 °C. Thermostability of the enzyme was increased twofold by the addition of bovine serum albumin. Maximal activity was observed between pH 6.5 and 7.0. The enzyme activity was significantly inhibited by Zn2+, Hg2+, and p-chloromercuribenzenesulphonic acid. The enzyme showed high activity on carboxymethylcellulose (CMC) with much lower activity on Avicel; a low level of activity was also found against xylan. Cellobiose was the major product of hydrolysis of amorphous cellulose and CMC. Viscometric analysis indicated that the enzyme hydrolysed CMC in an exo-acting fashion. Cellotriose and cellobiose were not degraded and at least four contiguous glucosyl residues were necessary for degradation by the enzyme. The K
m and V
max of the enzyme for CMC were 3.4 mg ml–1 and 44.7 mol min–1 (mg protein)–1, respectively. 相似文献
11.
《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
12.
Christine App Jana Knop Thomas Huff Heinrich Sticht Ewald Hannappel 《The protein journal》2013,32(6):484-492
Thymosin β4 is the prototype of β-thymosins and is present in almost every mammalian cell. It is regarded to be the main intracellular G-actin sequestering peptide. Thymosin β4 serves as a specific glutaminyl substrate for guinea pig transglutaminase. In the absence of an appropriate additional aminyl donor an ε-amino group of thymosin β4 serves also as an aminyl substrate and an intramolecular bond is formed concomitantly NH3 (17 Da) is lost. The molecular mass of the product is 4,949.6 Da. This is 16.3 Da less than the molecular mass of thymosin β4 (4,965.9 Da). Digestion with endopeptidases and Edman degradation of the fragments identified the exact position of the ring forming isopeptide bond. In spite of 3 glutaminyl and 9 lysyl residues of thymosin β4 only one isopeptide bond between Lys16 and Gln36 was formed (cyclic thymosin β4). These two amino acid residues are conserved in all β-thymosins. Cyclic thymosin β4 still forms a complex with G-actin albeit the stability of the complex is about one fiftieth of the stability of the thymosin β4 × G-actin complex. 相似文献
13.
Summary An extracellular xylanase was purified to homogeneity from the culture filtrate of a thermophilic Bacillus sp. The molecular weight of the purified xylanase was 44 kDa, as analysed by SDS/PAGE. The enzyme reaction followed Michaelis–Menten kinetics with Kmapp and Vmax values of 0.025 mg/ml and 450 U/mg protein, respectively, as obtained from a Lineweaver–Burk plot. The xylanase contained no other enzyme activity except for the hydrolysis of xylan substrate. The optimal temperature of the enzyme assay was 50 °C. The optimum pH for the xylanase activity was at three peaks 6.5, 8.5 and 10.5, respectively and the enzyme was stable over a broad range of pH from pH 6 to 10.5. Metal ions tested with demetalized enzyme had no effect, with the exception of Hg2+ and Pb2+ (both strong inhibitors). Inhibition of the enzyme activity by N-bromosuccinimide (amino acid modifier) indicated the role of tryptophan residues in the catalytic function of the enzyme. Due to these outstanding properties, the xylanase of Bacillussp. finds potential applications in biopulping, biobleaching and de-inking of recycled paper and other industrial processes. 相似文献
14.
Kimiyasu Isobe Yoshiki Tani Hideaki Yamada 《Bioscience, biotechnology, and biochemistry》2013,77(12):2955-2960
Polyamine oxidase from Penicillium chrysogenum oxidized spermine rapidly and spermidine slightly at pH 7.5. The apparent Km values for spermine and spermidine were calculated to be 2.25 × 10?5 m and 9.54 × 10?6 m, respectively. The relative maximum velocities for spermine and spermidine were 3.37 × 10?3 m (H2O2) per min per mg of protein and 2.08 × 10?4 m (H2O2) per min per mg of protein, respectively. Spermine oxidation of the enzyme was competitively inhibited by spermidine and putrescine. The apparent Ki values by spermidine and putrescine were calculated to be 3.00 × 10?5 m and 1.80 × 10?8 m, respectively. On the other hand, polyamine oxidase from Aspergillus terreus rapidly oxidized both spermidine and spermine at pH 6.5. The apparent Km values for spermidine and spermine were 1.20 × 10?8 m and 5.37 × 10?7 m, respectively. The relative maximum velocities for spermidine and spermine were 1.55 × 10?2 m (H2O2) per min per mg of protein and 6.20 × 10?3 m (H2O2) per min per mg of protein, respectively.Differential determination of spermine and spermidine was carried out using the two enzymes. The initial rate was assayed with Penicillium enzyme and the end point was measured afte addition of Aspergillus enzyme. Small amounts of polyamines (25 to 200 nmol of spermine and 25 to 250 nmol of spermidine) were assayed by solving two simultaneous equations obtained from the rate assay method and the end point assay method. The calculated values were in close agreement with those obtained by an amino-acid analyzer. 相似文献
15.
Summary Investigations into the properties of 6-PG dehydrogenase in cell free extracts of Escherichia coli revealed a pH optimum at pH 9.5 with a sharp decline on both sides of the optimum. The addition of 1.0×10-2
m MgCl2 produced maximal activity, whereas higher concentrations caused inhibition. The K
m
values were 2.5×10-4
m for 6-phosphogluconate and 2.5×10-5
m for NADP+ as substrate. The enzyme was extremely stable for at least 5 hours if stored at 4°C in Tris–NaCl–MgCl2 buffer at pH 7.5. 6-PG dehydrogenase activity was shown to be proportional to cell free extract concentration over the range 0–0.3 mg protein. An assay method based on the new optimal conditions has been established and has been shown to be 33% more sensitive than a number of commonly used methods.Meinem hochverehrten Lehrer Herrn Professor A. Rippel zum 80. Geburtstage. 相似文献
16.
Y Kishimoto 《Journal of neurochemistry》1973,20(1):211-223
Abstract— –Enzymic transformation of [4-14C]dehydroepiandrosterone or [4-14C]dehydro-epiandrosterone sulphate to androstenediol or its sulphate occurred when incubated with a microsomal preparation of rat brain or a whole rat blood homogenate. The brain enzyme which appeared to cause this transformation had a pH optimum at 60, was NADPH2-dependent, and had an apparent Km of 4·6 × 10?6m . When the subcellular fractions of rat brain were compared for transformation, microsomes had the highest specific activity, followed by the cytosol. The crude nuclear and mitochondrial fractions had no significant activity. The level of enzymic activity in the brain microsomes increased from that for rats sacrificed at 7 days of postnatal age to a maximum for rats sacrificed at 1 month of age; then the activity appeared to level off in rats older than 1 month. Microsomes obtained from the cerebellum had the highest specific activity in comparison to that obtained from the cerebral cortex, the diencephalon, and the brain stem. The incubated preparations of rat brain also converted dehydroepiandrosterone sulphate to androstenediol sulphate without hydrolysis. The enzyme in rat blood which was similar to that in the brain was also partially characterized. The blood enzyme had a pH optimum at 6–5, was nearly exclusively present in erythrocytes, was also NADPH2-dependent, and had an apparent Km of 2·7 × 10?4m . The developmental pattern of the blood enzyme specific activity was similar to that of the rat brain enzyme. Upon haemolysis, most activity was recovered in the haemolysate. 相似文献
17.
Deimination of Human Myelin Basic Protein by a Peptidylarginine Deiminase from Bovine Brain 总被引:6,自引:1,他引:5
Abstract: A peptidylarginine deiminase (PAD; EC 3.5.3.15) has been isolated from bovine brain and some of its characteristics have been studied. The enzyme showed an absolute requirement for Ca2+, a temperature optimum at ~50°C, and two Kmvalues when benzoylarginine ethyl ester was used as substrate, 0.78 mMand 11.2 mM.The higher Kmhas not been reported previously. Protein substrates for the enzyme included polyarginine and myelin basic protein but not histones. Because one of the components of MBP contains six citrullinyl residues per mole, enzymic deimination appeared to be a likely mechanism. When the most cationic component (C-1) was subjected to PAD in solution, 17 of the 19 arginyl residues were modified. From sequence analyses we concluded that the nature of the amino acid residues adjacent to the deiminated arginine were not modifiers of the reaction as arginyl residues in a variety of environments were deiminated. This deimination was reflected in a large increase in random structure, as measured by [θ]200. At 5°C, the [θ]200of the deiminated protein was -70 × 103 compared with -30 × 103 deg cm2/dmol for the native protein. When the temperature was increased to 70°C, the [θ]200 was -44 × 103 for the deiminated protein and -20 × 107 deg cm2/ dmol for the native C-1. When plotted as a function of temperature, [θ]200 decreased linearly from 5°C to 50°C for both proteins and did not change from 50°C to 70°C. PAD provides a mechanism for deimination of arginyl residues of myelin basic protein. The selective deimination of the six arginyl residues that are consistently found deiminated in C-8 may be determined by the orientation of the protein in the membrane and/or the more complex lipid composition of myelin may affect the selectivity of the deimination. 相似文献
18.
The Effect of Several Protein Amino Acids and Some Inorganic Nitrogen Sources on the Growth of Sphagnum nemoreum 总被引:1,自引:0,他引:1
LIISA KAARINA SIMOLA 《Physiologia plantarum》1975,35(3):194-199
The nitrogen metabolism of a bog moss, Sphagnum nemoreum Scop., has been studied in aseptic cultures. The effect of several protein amino acids, especially those found in peat, has been investigated. NH4NO3 (1.25 mM) was the best nitrogen source but NH4+ ions were more effectively utilized than NO3− ions when given as the only nitrogen source. Some of the amino acids (2.5 mM) allowed fairly satisfactory growth (arginine and alanine) when given as the only nitrogen source, but some of them were not utilized at all (leucine, lysine, isoleucine and methionine). Given at low concentrations (0.001 and 0.25 mM) together with NH4NO3 (2.5 mM), most of the protein amino acids failed to reveal any growth-promoting or -inhibiting effect. Only lysine (0.25 mM) clearly inhibited growth under these conditions. The nitrogen metabolism of Sphagnum nemoreum seems to be rather flexible and this species is more tolerant of organic nitrogen, especially hydroxyproline, than the higher plants. 相似文献
19.
Lifang Cheng Wanmeng Mu Tao Zhang Bo Jiang 《Applied microbiology and biotechnology》2010,86(4):1089-1097
The araA gene encoding an L-arabinose isomerase (L-AI) from the acido-thermophilic bacterium Acidothermus cellulolytics ATCC 43068 was cloned and overexpressed in Escherichia coli. The open reading frame of the L-AI consisted of 1,503 nucleotides encoding 501 amino acid residues. The recombinant L-AI
was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The molecular mass of the
enzyme was estimated to be approximately 55 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified
enzyme was optimally active at 75°C and pH 7.5. It required divalent metal ions, either Mn2+ or Co2+, for both enzymatic activity and thermostability improvement at higher temperatures. The enzyme showed relatively high activity
and stability at acidic pH. It exhibited over 90% of its maximal activity at pH 6.0 and retained 80% of activity after 12 h
incubation at pH 6.0. Catalytic property study showed that the enzyme had an interesting catalytic efficiency. Its apparent
K
m, V
max, and catalytic efficiency (k
cat/K
m) for D-galactose was 28.9 mM, 4.9 U/mg, and 9.3 mM−1 min−1, respectively. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield over 50% after
12 h under optimal conditions, suggesting its potential in D-tagatose production. 相似文献
20.
Phytase activity in rabbit cecal bacteria 总被引:1,自引:0,他引:1
The presence of phytase activity was demonstrated in 26 strains of rabbit cecal bacteria. In 25 strains a low phytase activity,
0.10–0.62 μmol phosphate released per min per mg protein, was found. High activity (2.61 μmol/min per mg protein) was found
in the strain PP2 identified as Enterococcus hirae. Phytase activity was cell-associated, being higher in the cell extract than in the cell walls. Extracellular phytase activity
and cell-associated phosphatase activity were not detected. Phytase activity was optimal around pH 5.0, which is below the
physiological cecal pH range. The K
m determined using the Lineweaver-Burk plot was 0.19 μmol/mL. Cations Fe3+, Cu2+ and Zn2+ at 0.5 mmol/L decreased phytase activity in sonicated cells of E. hirae by 99.4, 90.7 and 96.5 %, respectively. In contrast, Mg2+ increased activity by 11.0 %. Characteristics of E. hirae phytase (pH optimum, K
m, cation sensitivity) were similar to those of other bacterial phytases reported in the literature. Other bacteria with a
high phytase activity may be present in the rabbit cecum but remain to be identified. 相似文献