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1.
Cold and heat denaturation of the double mutant Arg 3→Glu/Leu 66→Glu of cold shock protein Csp of Bacillus caldolyticus was monitored using 1D 1H NMR spectroscopy in the temperature range from −12°C in supercooled water up to +70°C. The fraction of unfolded protein,
f
u, was determined as a function of the temperature. The data characterizing the unfolding transitions could be consistently
interpreted in the framework of two-state models: cold and heat denaturation temperatures were determined to be −11°C and
39°C, respectively. A joint fit to both cold and heat transition data enabled the accurate spectroscopic determination of
the heat capacity difference between native and denatured state, Δ C
p of unfolding. The approach described in this letter, or a variant thereof, is generally applicable and promises to be of
value for routine studies of protein folding. 相似文献
2.
Hansenula fabianii J640 highly expresses an extracellular glucoamylase (GA). Here, we purified the GA and showed that it has pH and temperature
optima of 5.0 and 50 °C, respectively, stable at temperatures up to 50 °C, and is inhibited by Ag 2+, Hg 2+, and Cu 2+. The gene was found in an expression library with anti-GA antibodies. A cDNA was found to encode 491 amino acids, including
a putative signal peptide of 21 amino acids. Because of the gene’s high expression, we used its promoter and terminator regions
to improve a previously developed H. fabianii J640 expression system. 相似文献
3.
A thermostable glucoamylase (TtcGA) from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli. The full-length gene (2112 bp) encodes a 703-amino acid polypeptide including a predicted signal peptide of 21 residues.
The recombinant mature protein was partially purified to 30-fold homogeneity by heat treatment and gel filtration chromatography.
The mature protein is a monomer with the molecular weight of 77 kD. The recombinant enzyme showed maximum activity at 75 °C
and pH 5.0. It is the most thermostable bacterial glucoamylase described to date with nearly no activity loss after incubation
at 75 °C for 6 h. TtcGA can hydrolyze both α-1, 4- and α-1, 6-glycosidic linkages in various α-glucans. It showed preference
for maltooligosaccharides over polysaccharides with specific activity of 80 U/mg towards maltose. Kinetic studies revealed
that TtcGA had the highest activity on maltooligosaccharide with four monosaccharide units. The cations Ca 2+, Mn 2+, Co 2+, Mg 2+, and reducing agent DTT showed no obvious effects on the action of TtcGA. In contrast, the enzyme was inactivated by Zn 2+, Pb 2+, Cu 2+, and EDTA. 相似文献
4.
Streptomyces luridiscabiei U05 was isolated from wheat rhizosphere. It produced chitinase, which showed in vitro antifungal properties. The crude enzyme inhibited the growth of Alternaria alternata, Fusarium oxysporum, F. solani, Botrytis cinerea, F. culmorum and Penicillium verrucosum. The chitinase enzyme of the molecular weight of 45 kDa was purified using affinity chromatography of chitin. Streptomyces luridiscabiei U05 produced different chitinolytic enzymes. The highest enzyme activity was observed with the use of 4‐MU‐(GlcNAc) , which points to the presence of an β‐N‐acetylhexosaminidase. The optimum activity was obtained at 35–40°C and pH 7–8. The enzyme showed thermostability at 35–40°C during 240 min of preincubation and lost its activity at 50°C and 60°C in 60 min. The chitinase activity from S. luridiscabei U05 was strongly inhibited by Hg 2+ and Pb 2+ ions, and sodium dodecyl sulphate (SDS). The Ca 2+, Cu 2+ and Mg 2+ ions stimulated the activity of the enzyme. 相似文献
5.
Xylaria regalis, a wood-grown ascomycete isolated in Taiwan, produces β-glucosidase (EC 3.2.1.21) extracellularly. The β-glucosidase was
purified to homogeneity by ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The molecular
mass of the purified enzyme was estimated to be 85 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. With
p-nitrophenyl β-D-glucopyranoside (PNPG) as the substrate at pH 5.0 and 50°C, the K
m
was 1.72 mM and V
max
was 326 μmol/min/mg. Optimal activity with PNPG as the substrate was at pH 5.0 and 50°C. The enzyme was stable at pH 5.0
at temperatures up to 50°C. The purified β-glucosidase was active against PNPG, cellobiose, sophorose, and gentiobiose, but
did not hydrolyze lactose, sucrose, Avicel, and o-nitrophenyl β-D-galactopyranoside. The activity of β-glucosidase was stimulated by Ca 2+, Mg 2+, Mn 2+, Cd 2+ and β-mercaptoethanol, and inhibited by Ag +, Hg 2+, SDS, and p-chloromercuribenzoate (PCMB).
Received: 30 March 1996 / Accepted: 3 May 1996 相似文献
6.
Glucoamylase produced by Scytalidium thermophilum was purified 80-fold by DEAE-cellulose, ultrafiltration and CM-cellulose chromatography. The enzyme is a glycoprotein containing
9.8% saccharide, p I of 8.3 and molar mass of 75 kDa (SDS-PAGE) or 60 kDa (Sepharose 6B). Optima of pH and temperature with starch or maltose
as substrates were 5.5/70 °C and 5.5/65 °C, respectively. The enzyme was stable for 1 h at 55 °C and for about 8 d at 4 °C,
either at pH 7.0 or pH 5.5. Starch, amylopectin, glycogen, amylose and maltose were the substrates preferentially hydrolyzed.
The activity was activated by 1 mmol/L Mg 2+ (27%), Zn 2+ (21%), Ba 2+ (8%) and Mn 2+ (5%). K
m and {ie11-1} values for starch and maltose were 0.21 g/L, 62 U/mg protein and 3.9 g/L, 9.0 U/mg protein, respectively. Glucoamylase
activity was only slightly inhibited by glucose up to a 1 mol/L concentration. 相似文献
7.
Fluorescence and circular dichroism data as a function of temperature were obtained to characterize the unfolding of nuclease A and two of its less stable mutants. These spectroscopic data were obtained with a modified instrument that enables the nearly simultaneous detection of both fluorescence and CD data on the same sample. A global analysis of these multiple datasets yielded an excellent fit of a model that includes a change in the heat capacity change, Δ Cp, between the unfolded and native states. This analysis gives a Δ Cp of 2.2 kcal/mol/·K for thermal unfolding of the WT protein and 1.3 and 1.8 kcal/mol/K for the two mutants. These Δ Cp values are consistent with significant population of the cold unfolded state at ∼0°C. Independent evidence for the existence of a cold unfolded state is the observation of a separately migrating peak in size exclusion chromatography. The new chromatographic peak is seen near 0°C, has a partition coefficient corresponding to a larger hydrodynamic radius, and shows a red-shifted fluorescence spectrum, as compared to the native protein. Data also indicate that the high-temperature unfolded form of mutant nuclease is relatively compact. Size exclusion chromatography shows the high temperature unfolded form to have a hydrodynamic radius that is larger than that for the native form, but smaller than that for the urea or pH-induced unfolded forms. Addition of chemical denaturants to the high-temperature unfolded form causes a further unfolding of the protein, as indicated by an increase in the apparent hydrodynamic radius and a decrease in the rotational correlation time for Trp140 (as determined by fluorescence anisotropy decay measurements). Proteins 28:227–240, 1997 © 1997 Wiley-Liss Inc. 相似文献
8.
In this study, two laccase isoenzymes (Lac1 and Lac2) from the culture supernatant of Trametes hirsuta MX2 were purified, and the genes (Lac1 and Lac2) coding the isoenzymes were cloned. Both Lac1 and Lac2 contained an open reading frame of 1563 bp with an identity of 79%. The two isoenzymes showed significant biochemical differences. The maximal activities of Lac1 and Lac2 were at pH 2.5 with 2-2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS), and the optimal temperatures for the activities of Lac1 and Lac2 were 60 and 50 °C, respectively. Lac1 exhibited excellent resistance to acidic conditions and retained 62.17% of its initial activity at pH 2.5 after a 72-h incubation. Lac2 was more thermostable than Lac1 with half-lives (t1/2) of 9.58 and 3.12 h at 50 and 60 °C, respectively; the t1/2 of Lac1 were only 4.19 and 0.88 h, respectively. Both Lac1 and Lac2 isoenzymes have a strong tolerance to Mg2+, Mn2+, Cu2+, and EDTA (50 mM). At a low concentration of 0.05 U mL?1, the enzymes could decolorize towards Remazol Brilliant Blue R, Acid Red 1, Crystal Violet, and Neutral Red in the presence of ABTS. These unusual properties demonstrated that the two laccases have strong potential for specific industrial applications. 相似文献
9.
AbstractTruffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22?±?2?°C) under static conditions which resulted in 1.41?U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50?°C. The enzyme showed good thermostability at 50?°C by retaining 98% of the maximal activity after 100?min of incubation. The amylase activity was marginally enhanced in presence of Cu 2+ and Na + and slightly reduced by K +, Ca 2+, Fe 2+, Mg 2+, Co 2+, Zn 2+, and Mn 2+ ions at 1?mM concentration. 相似文献
10.
A phytase from Penicillium oxalicum PJ3, PhyA, was purified near to homogeneity with 427-fold increase in specific phytase activity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatographies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis of the purified enzyme indicated an estimated molecular mass of 65 kD. The optimal pH and temperature of the purified enzyme were pH 4.5 and 55°C, respectively. The enzyme activity was strongly inhibited by Ca 2+, Cu 2+, Zn 2+, and phenylmethylsulfonyl fluoride (PMSF). The K m value for sodium phytate was 0.545 m M with a V max of 600 U/mg of protein. The phyA gene was cloned, and it contains an open reading frame of 1,383 with a single intron (118 bp), and encodes a protein of 461 amino acids. 相似文献
11.
EstA was purified from the supernatant by A. lwoffii 16C-1. Its molecular mass was determined to be 45 kDa, and the optimal activity occurred when the pH level was 8.0 at a temperature
of 37°C. The activation energies for the hydrolysis of p-nitrophenyl butyrate was determined to be 11.25 kcal/mol in the temperature range of 10–37°C. The enzyme was unstable at
temperatures higher than 50°C. The Michaelis constant ( K
m
) and V
max for p-nitrophenyl butyrate were 11 μ M and 131.6 μ M min −1 mg of protein-1, respectively. The enzyme was strongly inhibited by Hg 2−, Ca 2+, Mg 2+, Fe 2+, Cu 2+, Zn 2+, Mn 2+, Co 2+, ethylemediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), and diisopropyl fluorophosphate (DFP).
Received: 20 August 2001 / Accepted: 20 September 2001 相似文献
12.
Two thermostable xylanase isoforms T 60 and T 80 were purified to homogeneity from the cladodes of the xerophytic Cereus pterogonus plant species. After three consecutive purification steps, the specific activity of T 60 and T 80 isoforms were found to be 178.6 and 216.2 U mg −1 respectively. The molecular mass of both isoforms was determined to be 80 kDa. The optimum temperature for T 60 and T 80 xylanase isoforms were 60 and 80 °C respectively. The pH was 5.0 for both isoforms. The presence of divalent metal ions (10 mM
Co 2+) showed stimulatory effects of both catalytic activities, where as in the presence of Hg 2+, Cd 2+, Cu 2+ showed inhibitory effect on these activities at all concentrations studied. The thermodynamic analysis of xylanase activity
using denaturation kinetics and the presence divalent cations at 30–100 °C, showed lower Δ H, Δ S, and Δ G values at all the temperatures investigated. The melting temperature of purified T 80 xylanase isoform as determined by TG/DTA analysis and it showed the unfolding temperature was 80 °C. The g value and hyperfine
(A) value purified xylanase T 80 isoform was 2.017 and 10.80 respectively. Immunoblot analysis with antiserum raised against the purified T 80 xylanase isoforms revealed single immunolgically related polypeptides of 80 kDa, identical with the polypeptide band produced
on SDS-PAGE. The results of double immunodiffusion against the T 80 isoforms showed a single precipitin line indicating that the serum used was specific to these xylanase isoforms. The kinetic
and thermodynamic properties suggested that xylanase from C. pterogonus may have a potential usage in various industries. 相似文献
13.
The gene, AbfAC26Sari, encoding an α- l-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari, was isolated, cloned, sequenced, and characterizated. On the basis of amino acid sequence similarities, this 57-kDa
enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. Characterization of the purified recombinant
α- l-arabinofuranosidase produced in Escherichia coli BL21 revealed that it is active at a broad pH range (pH 4.5 to 9.0) and at a broad temperature range (45–85°C) and it has
an optimum pH of 5.5 and an optimum temperature of 65°C. Kinetic experiment at 65°C with p-nitrophenyl α- l-arabinofuranoside as a substrate gave a V
max and K
m values of 1,019 U/mg and 0.139 mM, respectively. The enzyme had no apparent requirement of metal ions for activity, and its
activity was strongly inhibited by 1 mM Cu 2+ and Hg 2+. The recombinant arabinofuranosidase released l-arabinose from arabinan, arabinoxylan, oat spelt xylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose.
Endoarabinanase activity was not detected. These findings suggest that AbfAC26Sari is an exo-acting enzyme. 相似文献
14.
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 Hg 2+, (UO 2) 2+, Pb 2+, Cu 2+, and Zn 2+ 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
15.
P 1 type nuclease, which hydrolyzes RNA and heat-denatured DNA completely into 5’-mononucleotides and also shows 3’-nucleotidase activity, was widely distributed among various species belonging to the genus Penicillium such as P. expansum, P. notatum, P. steckii and P. meleagrinum. P 1 type nucleases isolated from these strains were produced in a form of complex with malonogalactan when molds were grown on wheat bran. These enzymes showed similar characters in heat-stability (stable at 60°C), temperature optimum (60 to 70°C for RNA and heat denatured DNA, and 70°C for 3’-AMP) and sensitivity to EDTA. The enzymes from P. steckii and P. expansum were immunologically co-related to nuclease P 1.In addition, many strains of Penicillium produced base-nonspecific RNases forming 3’-mononucleotides via 2’: 3 ’-cyclic nucleotides. These RNases showed similarity in heat-lability (completely inactivated at 60°C), temperature optimum (45 to 50°C), sensitivity to Zn 2+ and Cu 2+, and relative hydrolysis rate toward 2’: 3’-cyclic nucleotides (A?C>U?G). 相似文献
16.
For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32
to generate pQE-LAPII. The His 6-tagged enzyme was overexpressed in IPTG-induced E. coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity
of 425 U/mg protein with a final yield of 76%. The subunit molecular mass of the purified protein was estimated to be 44.5
kDa by SDS-PAGE. The temperature and pH optima for the purified protein were 60°C and 8.0, respectively. Under optimal condition,
the purified enzyme showed a marked preference for Leu- p-nitroanilide, followed by Arg- and Lys-derivatives. The His 6-tagged enzyme was stimulated by Co 2+ ions, but was strongly inhibited by Cu 2+ and Hg 2+ and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co 2+ ions, indicating that it is a cobalt-dependent exopeptidase. Taking the biochemical characteristics together, we found that
the recombinant LAP II exhibits no important differences from those properties described for the native enzyme.
Received: 16 August 2002 / Accepted: 4 September 2002 相似文献
17.
Equilibrium unfolding of stem bromelain (SB) with urea as a denaturant has been monitored as a function of pH using circular
dichroism and fluorescence emission spectroscopy. Urea-induced denaturation studies at pH 4.5 showed that SB unfolds through
a two-state mechanism and yields Δ G (free energy difference between the fully folded and unfolded forms) of ∼5.0 kcal/mol and C
m (midpoint of the unfolding transition) of ∼6.5 M at 25°C. Very high concentration of urea (9.5 M) provides unusual stability
to the protein with no more structural loss and transition to a completely unfolded state. 相似文献
18.
AbstractStrain Bacillus amyloliquefaciens BH1 was evaluated for the generation of α-amylase. Culture conditions and medium components were optimized by a statistical approach for the optimal generation of α-amylase with response surface methodology (RSM) method. The Plackett–Burman (PB) design was executed to select the fermentation variables and Central composite design (CCD) for optimizing significant factors influencing production. The optimum levels for highest generation of α-amylase activity (198.26?±?3.54?U/mL) were measured. A 1.69-fold improve generation was acquired in comparison with the non-optimized. Partial characterization of the α-amylase indicated optimal pH and temperature at 7.0 and 40?°C, respectively. Crude α-amylase maintained a constant pH range 5.0–8.0 and 30–70?°C. The α-amylase was independent of Ca 2+, and the activity was inhibited by Fe 3+, Co 2+, Cu 2+, and Hg 2+. The thermo and pH stability of the α-amylase indicate its extensive application in the food and pharmaceutical industries. 相似文献
19.
A gene encoding cinnamoyl esterase (CE), which breaks down chlorogenic acid (ChA) into caffeic and quinic acids, was cloned from Lactobacillus helveticus KCCM 11223. The gene with an open reading frame of 759 nucleotides was expressed in Escherichia coli, which resulted in a 51.6-fold increase in specific activity compared to L. helveticus KCCM 11223. The recombinant CE exists as a monomeric enzyme having a molecular weight of 27.4?kDa. Although the highest activity was observed at pH 7, the enzyme showed stable activity at pH 4.0–10.0. Its optimum temperature was 65°C, and it also possessed a thermophilic activity: the half-life of CE was 24.4?min at 65°C. The half-life of CE was 145.5, 80.5, and 24.4?min at 60, 62, and 65°C, respectively. The Km and Vmax values for ChA were 0.153?mM and 559.6?µM/min, respectively. Moreover, the CE showed the highest substrate specificity with methyl caffeate among other methyl esters of hydroxycinnamic acids such as methyl ferulate, methyl sinapinate, methyl p-coumarate, and methyl caffeate. Ca 2+, Cu 2+, and Fe 2+ significantly reduced the relative activity on ChA up to 70%. This is the first report on a thermostable CE from lactic acid bacteria that can be useful to hydrolyze ChA from plant cell walls. 相似文献
20.
A maltooligosaccharide-forming amylase that hydrolyzes starch into maltotriose and maltopentaose was found in the culture
filtrate of a strain of Bacillus circulans GRS 313 isolated from local soil. The enzyme was purified by organic solvent fractionation, Sephadex G-100 gel filtration
and CM-Sephadex column chromatography. Optimum pH and temperature of amylase were evaluated using response surface methodology
(RSM) and were found to be 48°C and 4.9, respectively. The enzyme was stable up to 60°C and its pH stability was in the range
of 5.0–8.0. The K
m and V
max of the amylase with starch were 11.66 mg/ml and 68.97 U, respectively, and the energy of activation, E
a, was 7.52 kcal/mol. Dextrin inhibited the enzyme competitively, with a K
i of 6.1 mg/ml, and glucose caused noncompetitive inhibition with a K
i of 9.5 mg/ml. The enzyme was inhibited by Hg 2+, Mn 2+, Fe 3+ and Cu 2+ and enhanced by Co 2+ and Mg 2+. EDTA reversed the inhibitory effect of the metals. Paper chromatographic and high-performance liquid chromatography analysis
of the products of the amylolytic reaction showed the presence of maltotriose, maltotetraose, maltopentaose, maltose and glucose
in the starch hydrolysate. Journal of Industrial Microbiology & Biotechnology (2002) 28, 193–200 DOI: 10.1038/sj/jim/7000220
Received 11 December 2000/ Accepted in revised form 22 October 2001 相似文献
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