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1.
Reyhan Gul Guven Alevcan Kaplan Kemal Guven Fatma Matpan Mehmet Dogru 《Biotechnology and Bioprocess Engineering》2011,16(1):114-119
β-Galactosidase purified from the thermoacidophilic Alicyclobacillus acidocaldarius subsp. rittmannii isolated from Antarctica is a member of the GH42 family. The enzyme was not effected by various concentrations of its reaction
product glucose, but was greatly inhibited by the other reaction product galactose using both substrates, ONPG and lactose.
Linewever-Burk plot analysis derived from both ONPG and lactose hydrolysis results showed that galactose is a mixed-type inhibitor
of the purified β-galactosidase. The enzyme was slightly activated by Mg2+ (13% at 20 mM), while inhibited at higher concentrations of Ca+2 (33% at 10 mM), Zn+2 (86% at 8 mM) and Cu+2 (87% at 4 mM). The enzyme activity was not significantly altered by the metal ion chelators EDTA and 1,10-phenanthroline
up to 20 mM, indicating that this enzyme is not a metalloenzyme. 2-Mercaptoethanol and DTT were found to enhance β-galactosidase
activity, while p-chloromercuribenzoic acid (PCMB) completely inhibited enzymatic activity (97% at 1 mM; 99.7% at 2 mM), indicating
at least one essential Cys residue modified by the reagents in the active site of β-galactosidase. Iodoacetamide and Nethylmaleimide
had little effect on the β-galactosidase. Phenylmethylsulfonyl fluoride (PMSF) inhibited the enzyme strongly (19.8% at 1 mM;
71.9% at 10 mM), also showing the participation of serine for enzyme activity. 相似文献
2.
Purification and Partial Characterization of a Prolyl-Dipeptidyl Aminopeptidase from Lactobacillus helveticus CNRZ 32 总被引:8,自引:6,他引:2
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X-prolyl-dipeptidyl aminopeptidase, which hydrolyzed Gly-Pro-p-nitroanilide (relative activity [RA] = 100%) and Arg-Pro-p-nitroanilide (RA, 130%), was purified to homogeneity from the cell extract of Lactobacillus helveticus CNRZ 32. The enzyme also hydrolyzed Ala-Pro-Gly (RA, 11%) and Ala-Ala-p-nitroanilide (RA, 2%) but was not active on Ala-Leu-Ala, dipeptides, and endopeptidase and carboxypeptidase substrates. The enzyme was purified 145-fold by streptomycin sulfate precipitation, ammonium sulfate fractionation, and a series of column chromatographies on DEAE-cellulose, arginine-Sepharose 4B, and glycyl-prolyl-AH-Sepharose 4B. The purified enzyme appeared as a single band on native polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoreses and had a molecular weight of 72,000. Optima for activity by the purified enzyme were pH 7.0 and 40°C. The enzyme was incubated at 40°C for 15 min with various metal ions. It was activated by Mg2+ (2.5 mM), Ca2+ (0.1 to 2.5 mM), Na+ (10 to 50 mM), and K+ (10 to 50 mM) and was inhibited by Hg2+ (0.1 to 2.5 mM), Cu2+ (0.1 to 2.5 mM), and Zn2+ (0.1 to 2.5 mM). Enzyme activity was partially inhibited by EDTA (1.0 mM, 20 h at 40°C), 1,10-phenanthroline (1.0 mM, 15 min at 40°C), phenylmethylsulfonyl fluoride (1.0 mM), N-ethylmaleimide (1.0 mM), and iodoacetate (1.0 mM). It was completely inhibited by diisopropyl fluorophosphate (1.0 mM, 2 h at 40°C) and p-chloromercuribenzoate (1.0 mM, 15 min at 40°C). The enzyme was not affected by dithioerythritol (1.0 to 10 mM). 相似文献
3.
Chitin synthetase (E.C.2.4.1.16) from mixed membrane fractions of the yeast and mycelial phases of Blastomyces dermatitidis were compared. The behavior of the enzyme from both phases was very similar: N-acetylglucosamine was stimulatory (Km 8.5 mM for yeast and 3.9 mM for mycelium); substrate Michaelis-Menten kinetics were sigmoidal; substrate Km of enzyme from yeast decreased from 3.0 mM at low N-acetylglucosamine (5 mM) levels to 1.4 mM at high (100 mM) levels; substrate Km of enzyme from mycelium was essentially unchanged at 1.4 mM; temperature optimum was 28 ° C; pH optimum was 7–7.5; Mg+2 optimum was 5–10 mM.The greatest difference was that enzyme from yeast was extracted in a mostly latent form that required trypsin treatment for maximal in vitro activity while enzyme from mycelium was extracted in an active form which was rapidly deactivated by trypsin treatment. 相似文献
4.
Judit M. Rezessy-Szabó Quang D. Nguyen Ágoston Hoschke Christophe Braet Gyöngyi Hajós Marc Claeyssens 《Biochimica et Biophysica Acta (BBA)/General Subjects》2007
High levels of an extracellular α-galactosidase are produced by the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b when grown in submerse culture and induced by sucrose. The enzyme was purified 114-fold from the culture supernatant by (NH4)2SO4 fractionation, and by chromatographical steps including Sepharose CL-6B gel filtration, DEAE-Sepharose FF anion-exchange, Q-Sepharose FF anion-exchange and Superose 12 gel filtration. The purified enzyme exhibits apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and iso-electric focusing (IEF). The native molecular weight of the monomeric α-galactosidase is 93 kDa with an isoelectric point of 3.9. The enzyme displays a pH and temperature optimum of 5–5.5 and 65 °C, respectively. The purified enzyme retains more than 90% of its activity at 45 °C in a pH range from 5.5 to 9.0. The enzyme proves to be a glycoprotein and its carbohydrate content is 5.3%. Kinetic parameters were determined for the substrates p-nitrophenyl-α-galactopyranoside, raffinose and stachyose and very similar Km values of 1.13 mM, 1.61 mM and 1.17 mM were found. Mn++ ions activates enzyme activity, whereas inhibitory effects can be observed with Ca++, Zn++ and Hg++. Five min incubation at 65° with 10 mM Ag+ results in complete inactivation of the purified α-galactosidase. Amino acid sequence alignment of N-terminal sequence data allows the α-galactosidase from Thermomyces lanuginosus to be classified in glycosyl hydrolase family 36. 相似文献
5.
A calcium-activated neutral proteinase was purified from myelin of bovine brain white matter. Myelin purified in the presence of EDTA (2 mM) was homogenized in 50 mM Trisacetate buffer at pH 7.5, containing 4 mM EDTA, 1 mM NaN3, 5 mM -mercaptoethanol and 0.1% Triton X-100 for two hours. After centrifugation at 87,000g for 1 hour, the supernatant was subjected to purification through successive column chromatography as follows: i) DEAE-cellulose, ii) Ultrogel (AC-34) filtration, iii) Phenyl-Sepharose, iv) a second DEAE-cellulose. The enzyme activity was assayed using azocasein as substrate. The myelin enzyme was purified 2072-fold and SDS-PAGE analysis of the purified enzyme revealed a major subunit of 72–76 K. The enzyme was inhibited by iodoacetate (1 mM), leupeptin (1 mM), E-64C (1.6 mM), EGTA (1 mM), antipain (2 mM) and endogenous inhibitor calpastatin (2 g). It required 0.8 mM Ca2+ for half-maximal activation and 5 mM Ca2+ for optimal activation. Mg2+ (5 mM) was ineffective while Zn2+ and Hg2+ were inhibitory. The pH optimum was ranged from 7.5–8.5. Treatment of myelin with Triton X-100 increased the enzyme activity by 10-fold suggesting it is membrane bound whereas the purufied enzyme was not activated by Triton X-100 treatment. The presence of CANP in myelin may mediate the turnover of myelin proteins and myelin breakdown in degenerative brain diseases. 相似文献
6.
Xuejun Hu Sylvain Robin Shane O’Connell Gary Walsh J. Gerard Wall 《Applied microbiology and biotechnology》2010,87(5):1773-1782
β-galactosidase is an enzyme administered as a digestive supplement to treat lactose intolerance, a genetic condition prevalent
in most world regions. The gene encoding an acid-stable β-galactosidase potentially suited for use as a digestive supplement
was cloned from Aspergillus niger van Tiegh, sequenced and expressed in Pichia pastoris. The purified recombinant protein exhibited kinetic properties similar to those of the native enzyme and thus was also competitively
inhibited by its product, galactose, at application-relevant concentrations. In order to alleviate this product inhibition,
a model of the enzyme structure was generated based on a Penicillium sp. β-galactosidase crystal structure with bound β-galactose. This led to targeted mutagenesis of an Asp258-Ser-Tyr-Pro-Leu-Gly-Phe amino acid motif in the A. niger van Tiegh enzyme and isolation from the resultant library of a mutant β-galactosidase enzyme with reduced sensitivity to
inhibition by galactose (K
i of 6.46 mM galactose, compared with 0.76 mM for the wildtype recombinant enzyme). The mutated enzyme also exhibited an increased
K
m (3.76 mM compared to 2.21 mM) and reduced V
max (110.8 μmol min−1 mg−1 compared to 172.6 μmol min−1 mg−1) relative to the wild-type enzyme, however, and its stability under simulated fasting gastric conditions was significantly
reduced. The study nevertheless demonstrates the potential to rationally engineer the A. niger van Tiegh enzyme to relieve product inhibition and create mutants with improved, application-relevant kinetic properties
for treatment of lactose intolerance. 相似文献
7.
Yuzbasheva EY Yuzbashev TV Laptev IA Konstantinova TK Sineoky SP 《Applied microbiology and biotechnology》2011,89(3):645-654
The gene encoding homodimeric β-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both
o-nitrophenyl-β-d-galactoside (oNPG) and lactose hydrolysis, were 50°C and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at
37°C and over a wide range of temperatures (4–42°C) at pH 6.5 for up to 1 month. The K
m values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose.
The monovalent ions Na+ and K+ in the concentration range of 1–100 mM as well as the divalent metal cations Mg2+, Mn2+, and Ca2+ at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for application in lactose hydrolysis
especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential
of this enzyme for the production of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L. 相似文献
8.
9.
A thermophilic Bacillus sp. was isolated that secreted an extracellular, thermostable lipolytic enzyme. The enzyme was purified to 58 folds with
a specific activity of 9730 units/mg of protein and yield of 10% activity by ammonium sulphate precipitation, Phenyl Sepharose
chromatography, gel-permeation followed by Q Sepharose chromatography. The relative molecular mass of the protein was determined
to be 61 kDa by SDS-PAGE and approximately 60 kDa by gel permeation chromatography. The enzyme showed optimal activity at
60–65 ∘C and retained 100% activity after incubation at 60 ∘C and pH 8.0 for 1 h. The optimum pH was determined to be 8.5. It exhibited 50% of its original activity after 65 min incubation
at 70 ∘C and 23 min incubation at 80 ∘C. Catalytic function of lipase was activated by Mg++ (10 mM), while mercury (10 mM) inactivated the enzyme completely. No effect on enzyme activity was observed with trypsin
and chymotrypsin treatment, while 50% inhibition was observed with thermolysin. It was demonstrated that PMSF, SDS, DTT, EDTA,
DEPC, βME (100 mM each) and eserine (10 mM) inhibited the activity of the lipolytic enzyme. With p-nitrophenyl laurate as
a substrate, the enzyme exhibited a K
m
and V
max of 0.5 mM and 0.139 μM/min/ml. The enzyme showed preference for short chain triacylglycerol and hydrolyzes triolein at all
positions. In contrast to other thermostable Bacillus lipases, this enzyme has very low content of hydrophobic amino acids (22.58 %). Immunological studies showed that the active
site and antigen-binding site of enzyme do not overlap. 相似文献
10.
El-Sayed AS 《Journal of microbiology (Seoul, Korea)》2011,49(1):130-140
L-Methioninase was purified to electrophoretic homogeneity from cultures of Aspergillus flavipes using anion-exchange and gel filtration chromatography by 12.1 fold compared to the crude enzyme preparation. The purified
enzyme had a molecular mass of 47 kDa under denaturing conditions and an isoelectric point of 5.8 with no structural glycosyl
residues. The enzyme had optimum activity at pH 7.8 and pH stability from 6.8–8.0 at 35°C. The enzyme appeared to be catalytically
stable below 40°C. The enzyme activity was strongly inhibited by DL-propargylglycine, hydroxylamine, PMSF, 2-mercaptoethanol,
Hg+, Cu2+, and Fe2+, with slight inhibition by Triton X-100. A flavipes L-methioninase has a higher catalytic affinity towards L-methionine (Km, 6.5 mM and Kcat, 14.1 S−1) followed by a relative demethiolating activity to L-homo-cysteine (Km, 12 mM and Kcat, 9.3 S−1). The enzyme has two absorption maxima at 280 and 420 nm, typical of other PLP-enzymes. Apo-L-methioninase has the ability
to reconstitute its structural catalytic state completely upon addition of 0.15 mM PLP. L-Methioninase has neither an appreciable
effect on liver function, platelet aggregation, nor hemolysis of human blood. The purified L-methioninase from solid cultures
of A. flavipes displayed unique biochemical and catalytic properties over the currently applied Pseudomonad enzyme. 相似文献
11.
An NAD+-dependent xylitol dehydrogenase (XDH) from Fusarium oxysporum, a key enzyme in the conversion of xylose to ethanol, was purified to homogeneity and characterised. It was homodimeric with a subunit of M
r 48 000, and pI 3.6. It was optimally active at 45 °C and pH 9–10. It was fully stable at pH 6–7 for 24 h and 30 °C. K
m values for d-xylitol and NAD+ were 94 mM and 0.14 mM, respectively. Mn2+ at 10 mM increased XDH activity 2-fold and Cu2+ at 10 mM inhibited activity completely. 相似文献
12.
Betaine-homocysteine methyl transferase (BHMT) from Aphanothece halophytica was purified to homogeneity by hydroxyapatite, DEAE-Sepharose CL-6B and Sephadex G-200 column chromatography. A 24-fold purification
and 11% overall yield were achieved with a specific activity of 595 nmol h−1 mg−1. The subunit molecular weight was determined to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
and the native enzyme was found to have a molecular weight of 350 kDa, suggesting an octameric structure of the enzyme. The
enzyme shows optimum activity at 37°C, pH 7.5. The apparent Km values for glycinebetaine and L-homocysteine were 4.3 mM and
1.3 mM, respectively. The enzyme was 70% inactivated by 5 mM dimethylglycine whereas the same concentration of sarcosine slightly
inactivated the enzyme. Two analogs of glycinebetaine were also tested for enzyme inactivation and it was found that 5 mM
choline inactivated 60% of the enzyme activity and 2.5 mM betaine aldehyde completely abolished the enzyme activity. NaCl
at 200 mM or higher also completely inactivated the enzyme.
Received: 6 December 2000 / Accepted: 10 January 2001 相似文献
13.
Purification and Characterization of Extracellular Phytase from a Marine Yeast <Emphasis Type="Italic">Kodamaea ohmeri</Emphasis> BG3 总被引:1,自引:0,他引:1
Li X Chi Z Liu Z Li J Wang X Hirimuthugoda NY 《Marine biotechnology (New York, N.Y.)》2008,10(2):190-197
The extracellular phytase in the supernatant of cell culture of the marine yeast Kodamaea ohmeri BG3 was purified to homogeneity with a 7.2-fold increase in specific phytase activity as compared to that in the supernatant
by ammonium sulfate fractionation, gel filtration chromatography (Sephadex™ G-75), and anion-exchange chromatography (DEAE
Sepharose Fast Flow Anion-Exchange). According to the data from sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
the molecular mass of the purified enzyme was estimated to be 98.2 kDa while the molecular mass of the purified enzyme was
estimated to be 92.9 kDa and the enzyme was shown to be a monomer according to the results of gel filtration chromatography.
The optimal pH and temperature of the purified enzyme were 5.0 and 65°C, respectively. The enzyme was stimulated by Mn2+, Ca2+, K+, Li+, Na+, Ba2+, Mg2+ and Co2+ (at a concentrations of 5.0 mM), but it was inhibited by Cu2+, Hg2+, Fe2+, Fe3+, Ag+, and Zn2+ (at a concentration of 5.0 mM). The enzyme was also inhibited by phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid (at
a concentration of 1.0 mM), and phenylgloxal hydrate (at a concentration of 5.0 mM), and not inhibited by EDTA and 1,10-phenanthroline
(at concentrations of 1.0 mM and 5.0 mM). The K
m, V
max, and K
cat values of the purified enzyme for phytate were 1.45 mM, 0.083 μmol/ml · min, and 0.93 s-1, respectively. 相似文献
14.
An enzyme synthesizing stachyose, galactinol-raffinose galactosyltransferase (EC2.4.1.67), has been purified ca 40-fold from mature leaves of Cucurbita pepo using ammonium sulphate precipitation, Sephadex gel filtration and DEAE-Sephadex gel chromatography. The purified enzyme fraction was separated from all but 2 % of the total,α-galactosidase activity extracted from the tissue. The enzyme was optimally active at pH 6.9 and was stable for at least a month at 4° in the presence of 20 mM 2-mercaptoethanol. The enzyme displayed high specificity for the donor galactinol (Km 7.7 mM) and the acceptor raffinose (Km 4.6 mM) and was unable to effect synthesis of any other member of the raffinose series of galactosyl-sucrose oligosaccharides. Co2+, Hg2+, Mn2+ and Ni2+ ions were particularly inhibitory; no metal ion promotion was observed and 5 mM EDTA was ineffective. Myo-inositol was strongly inhibitory (Ki 2 mM), melibiose weakly so. Tris buffer (0. 1 M) was also inhibitory. Galactinol hydrolysis occurred in the absence of the acceptor raffinose but there was no hydrolysis of either raffinose or stachyose in the absence of the donor galactinol. The reaction was readily reversible and exchange reactions were detected between substrates and products. It is proposed that the synthesis of stachyose in mature leaves ofC. pepo proceeds via this galactosyltransferase and not via α-galactosidase. 相似文献
15.
Agathe Garnier Ali Berredjem Bernard Botton 《Fungal genetics and biology : FG & B》1997,22(3):168-176
The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) fromLaccaria bicolorwas purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE–Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate–polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at −75°C, 4 days at 4°C, and 1 h at 50°C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at −75°C. NAD-GDH activity was stimulated by Ca2+and Mg2+but strongly inhibited by Cu2+and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 μM, 89 μM, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and itsKmvalue increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. 相似文献
16.
Trehalose-6-phosphate synthase, catalyzing the reaction between UDP-glucose and glucose 6-phosphate and forming trehalose 6-phosphate, was isolated and partially purified (30-fold) from the phototrophic, haloalkaliphilic bacteriumEctothiorhodospira halochloris. The activity is stabilized by 20mM MgCl2, 50mM NaCe and 2M glycine betaine. The molecular weight was 63000.The enriched enzyme had a MgCl2 optimum at 3–6mM, a pH optimum at 7.5 (in Tris-HCl buffer) and a temperature optimum at 50°C. The Km-values were 1.5×10–3M for UDP-glucose and 2×10–3M for glucose 6-phosphate. The enzyme showed a salinity dependence with optimal concentrations between 100 and 300mM salt. Higher concentrations of salt resulted in a decrease in activity. In the presence of inhibitory salt concentrations the compatible solute glycine betaine had a protective effect with a maximum between 0.5 and 2.0M. 相似文献
17.
A glucose-tolerant β-glucosidase was purified to homogeneity from prune (Prunus domestica) seeds by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography. The molecular mass of the enzyme was estimated to be 61 kDa by SDS-PAGE and 54 kDa by gel permeation chromatography. The enzyme has a pI of 5.0 by isoelectric focusing and an optimum activity at pH 5.5 and 55 °C. It is stable at temperatures up to 45 °C and in a broad pH range. Its activity was completely inhibited by 5 mM of Ag+ and Hg2+. The enzyme hydrolyzed both p-nitrophenyl β-d-glucopyranoside with a Km of 3.09 mM and a Vmax of 122.1 μmol/min mg and p-nitrophenyl β-d-fucopyranoside with a Km of 1.65 mM and a Vmax of 217.6 μmol/min mg, while cellobiose was not a substrate. Glucono-δ-lactone and glucose competitively inhibited the enzyme with Ki values of 0.033 and 468 mM, respectively. 相似文献
18.
F. Manganaro Y. Gaudette A. Pombo-Gentile K. Singh S. Rakhit 《Prostaglandins & other lipid mediators》1988,36(6)
Leukotriene A4 epoxide hydrolase from dog lung, a soluble enzyme catalyzing the hydrolysis of leukotriene A4 (LTA4) to leukotriene B4 (LTB4) was partially purified by anion exchange HPLC. The enzymatic reaction obeys Michaelis- Menten kinetics. The apparent Km ranged between 15 and 25 μM and the enzyme exhibited an optimum activity at pH 7.8. An improved assay for the epoxide hydrolase has been developed using bovine serum albumin and EDTA to increase the conversion of LTA4 to LTB4. This method was used to produce 700 mg of LTB4 from LTA4 methyl ester. The partial by purified enzyme was found to be uncompetitively inhibited by divalent cations. Ca2+, Mn+, Fe2+, Zn+2 and Cu+2 were found to have inhibitor constants (Ki) of 89 mM, 3.4 mM, 1.1 mM, 0.57 mM, and 28 μM respectively Eicosapentaenoic acid was shown to be a competitive inhibitor of this enzyme with a Ki of 200 μM. From these inhibition studies, it can be theorized that the epoxide hydrolae has at least one hydrophobic and one hydrophilic binding site. 相似文献
19.
An aminopeptidase from the mycelia of Streptomyces rimosus was isolated in an electrophoretically homogeneous form. It was shown to be a monomeric, acidic protein (pI = 4.4, mol. wt.
approx. 83,000), with optimal activity at pH 7.1–7.8 and at 35–41° C. The enzyme was fully inhibited by 0.1 mM EDTA or 1 mM
o-phenanthroline; the activity was restored upon addition of 0.05 mM Co2+, Zn2+, or Ni2+. Amastatin, bestatin, and puromycin also inhibited the enzyme. The aminopeptidase hydrolyzed amino-acid-2-naphthylamides
and various di- to heptapeptides. The highest catalytic coefficients (23 and 19 μM–1 s–1) were obtained with Arg- and Lys-2-naphthylamide, followed by Leu-, Phe- and Met-derivatives with one order of magnitude
lower catalytic coefficients. Basic or bulky hydrophobic amino acids at the P1 and/or P1′ position of peptide substrates were preferred. Acidic amino acids and proline were not accepted. The affinity of the enzyme
increased with the length of peptide. According to these properties, S. rimosus intracellular aminopeptidase is distinct from the extracellular leucine aminopeptidase of the same organism and can be classified
as an Arg(Lys)-preferring metalloaminopeptidase.
Received: 18 January 1996 / Accepted: 19 March 1996 相似文献
20.
Summary Partially purified fructose diphosphatase from the obligate chemolithotroph,Thiobacillus neapolitanus has been characterized, and some of its regulatory properties described. The enzyme had a high effinity for its substrate, but was inhibited by substrate at concentrations above 1 mM. The enzyme had an absolute requirement for a divalent cation. In the absence of EDTA there was a single pH optimum in the alkaline range between 8.5 and 9.5; in the presence of EDTA there was considerable was activity at both neutral and alkaline pH. This diphosphatase was inhibited by AMP at 10–4 M or greater-, the lower the pH, the greater the AMP inhibition. Treatment of the enzyme with 5×10–5 Mpara hydroxy mercuribenzoate allowed retention of full catalytic activity while abolishing considerable AMP inhibition. Exposure of the enzyme to several concentrations of urea had no effect on the AMP inhibition. Homocystine (0.06 mM) and coenzyme A (0.1 mM) had no effect. At 1 mM, PEP caused 60% inhibition, 2, 3-diphosphoglyceric acid produced 26% inhibition, and pyruvate had no effect. 相似文献