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1.
We report for the first time kinetic and thermodynamic properties of soluble acid invertase (SAI) of sugarcane (Saccharum officinarum L.) salt sensitive local cultivar CP 77-400 (CP-77). The SAI was purified to apparent homogeneity on FPLC system. The crude enzyme was about 13 fold purified and recovery of SAI was 35%. The invertase was monomeric in nature and its native molecular mass on gel filtration and subunit mass on SDS-PAGE was 28 kDa. SAI was highly acidic having an optimum pH lower than 2. The acidic limb was missing. Proton transfer (donation and receiving) during catalysis was controlled by the basic limb having a pKa of 2.4. Carboxyl groups were involved in proton transfer during catalysis. The kinetic constants for sucrose hydrolysis by SAI were determined to be: km = 55 mg ml?1, kcat = 21 s?1, kcat/km = 0.38, while the thermodynamic parameters were: ΔH* = 52.6 kJ mol?1, ΔG* = 71.2 kJ mol?1, ΔS* = ?57 J mol?1 K?1, ΔG*E–S = 10.8 kJ mol?1 and ΔG*E–T = 2.6 kJ mol?1. The kinetics and thermodynamics of irreversible thermal denaturation at various temperatures 53–63 °C were also determined. The half -life of SAI at 53 and 63 °C was 112 and 10 min, respectively. At 55 °C, surprisingly the half -life increased to twice that at 53 °C. ΔG*, ΔH* and ΔS* of irreversible thermal stability of SAI at 55 °C were 107.7 kJ mol?1, 276.04 kJ mol?1 and 513 J mol?1K?1, respectively.  相似文献   

2.
Anoxybacillus beppuensis TSSC-1 (GenBank Number, EU710556), a thermophilic bacterium isolated from a hot spring reservoir, was found to optimally secrete a monomeric α-amylase at 55 °C and pH 7. The enzyme was purified to homogeneity by a single-step purification on phenyl sepharose 6FF, achieving a 58% yield, 10,000 U/mg specific activity and 19.5 fold purification. The molecular weight, Km and Vmax were 43 kD, 0.5 mg ml?1 and 3571.42 μmol ml?1 m?1, respectively. The enzymatic catalysis of soluble starch was optimum at 80 °C and pH 7. The thermodynamic parameters, Kd, t1/2, ΔH*, ΔS*, E and ΔG*, were consistent. The very compact structure of the enzyme and the transitional enzyme–substrate complex resisted denaturation at extreme temperatures and alkaline pH. The Kd and t1/2 measurements were consistent with the high thermostability and pH tolerance observed. The structural stability of the enzyme was also reflected by the values of ΔH*, ΔS*, E and ΔG*. While the enzyme did not exhibit metal ion dependency, it was resistant to chemical denaturation. The broad thermo- and pH-tolerance of this enzyme suggests potential commercial opportunities.  相似文献   

3.
Energetics of the catalysis of Class II α-mannosidase (E.C.3.2.1.24) from Aspergillus fischeri was studied. The enzyme showed Kcat/Km for Man (α1-3) Man, Man (α1-2) Man and Man (α1-6) Man as 7488, 5376 and 3690 M?1 min?1, respectively. The activation energy, Ea was 15.14, 47.43 and 71.21 kJ/mol for α1-3, α1-2 and α1-6 linked mannobioses, respectively, reflecting the energy barrier in the hydrolysis of latter two substrates. The enzyme showed Kcat/Km as 3.56 × 105 and 4.61 × 105 M?1 min?1 and Ea as 38.7 and 8.92 kJ/mol, towards pNPαMan and 4-MeUmbαMan, respectively. Binding of Swainsonine to the enzyme is stronger than that of 1-deoxymannojirimycin.  相似文献   

4.
Thirty two morphologically different bacterial were isolated from different soil samples and screened for their ability to produce lipolytic enzymes. Among all isolates, the isolate coded AZ1 was selected due to its high potency to produce lipase at elevated temperature up to 65 °C. Phylogenetic analysis based on 16SrDNA sequence revealed its close relationship to Geobacillus thermodenitrificans. The effect of ten culture variable on lipase production was evaluated by implementing Plackett–Burman statistical design. d-sucrose, peptone and soy bean flour were the most significant variables affecting lipase production. A pre-optimized medium based on this experiment yielded an enzyme activity of 260 U min?1 ml?1. For further optimization, a fourteen trials’ multi-factorial Box–Behnken experimental design was applied to find out the optimum level of each of the significant variables. The tested variables, namely: d-sucrose (X1); peptone (X2) and soy bean flour (X3) were examined, each at three different levels coded ?1, 0, +1. The optimal levels of the three components were founded to be (g/L): d-sucrose, 6.56; peptone, 6.35; and soy bean flour, 6.92, with a predicted activity of approximately 610 U min?1 ml?1. According to the results of the Plackett–Burman and Box–Behnken designs the following medium composition is expected to be optimum (g/L): d-sucrose 6.56, peptone 6.35, soy bean flour 6.92, CaCl2 0.02, Y.E. 2.5, K2HPO4 1.0, MgSO4.7H2O 0.2 and Fe2 (SO4)3 0.02; pH, 8; cultivation temperature 55 °C and incubation time 24 h, the enzyme activity measured in the medium was approximately 593 U min?1 ml?1.  相似文献   

5.
Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg2+ is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca2+ (alone) only supports DNA binding. To investigate the role of Mg2+ in DNA cleavage by restriction endonucleases, we have studied the Mg2+ and Mn2+ concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg2+ and Mn2+ concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20 mM while no activity was detected in the presence of Mn2+ and in the presence of Ca2+ cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn2+ than in the presence of Mg2+ and can be activated by Ca2+. Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a Km value for pTrcHisB DNA of 6.15 nM and a Vmax of 1.79 × 10?2 nM min?1, while EhoI had a Km for pUC19 plasmid of 8.66 nM and a Vmax of 2 × 10?2 nM min?1.  相似文献   

6.
Xylitol-2-dehydrogenase from Candida albicans was cloned and overexpressed in Escherichia coli. The purified recombinant XDH has an apparent molecular weight of 40 kDa which belongs to the medium chain alcohol dehydrogenase family and exclusively uses NAD+ as a cofactor. The recombinant caXDH has a KM of 8.8 mM and 37.7 μM using the substrate xylitol and NAD+, respectively, and its catalytic efficiency is 53,200 min?1 mM?1. Following site-directed mutagenesis, one of the engineered caXDHs with six mutations at Ser95Cys, Ser98Cys, Tyr101Cys, Asp206Ala, Ile207Arg, and Phe208Ser shifted its cofactor dependence from NAD+ to NADP+ in which the KM and kcat/KM towards NADP+ are 119 μM and 26,200 min?1 mM?1, respectively.  相似文献   

7.
Enzymes in the newly described rumen bacterium, Treponema zioleckii strain kT, capable of digesting Timothy grass fructan, inulin, and sucrose were identified and characterized. Two specific endolevanases and one non-specific β-fructofuranosidase were found in a cell-free extract. The molecular weight of the endolevanases were estimated to be 60 and 36 kDa, whereas that of β-fructofuranosidase, 87 kDa. The former of the specific enzymes was associated with the outer membrane, while the latter and the non-specific β-fructofuranosidase, with the periplasm or cytosol. The Km and Vmax for Timothy grass fructan degradation by endolevanase were 0.27% and 15.75 μM fructose equivalents × mg protein?1 × min?1, those for sucrose and inulin digestion by β-fructofuranosidase were 1.35 × 10?3 M and 1.73 μM hexoses × mg protein?1 × min?1 and 1.77% and 1.83 μM hexoses × mg protein?1 × min?1, respectively.  相似文献   

8.
Extracellular laccase produced by the wood-rotting fungus Cerrena unicolor was immobilized covalently on the mesostructured siliceous cellular foams (MCFs) functionalised using various organosilanes with amine and glycidyl groups. The experiments indicated that laccase bound via glutaraldehyde to MCFs modified using 2-aminoethyl-3-aminopropyltrimethoxysilane remains very active. In the best biocatalyst activity was about 42,700 U mL?1 carrier (66,800 U mg?1 bound protein), and hence significantly higher than ever reported before. Optimisation of the immobilization procedure with respect to protein concentration, pH of coupling mixture and the enzyme purity afforded the biocatalyst with activity of about 90,980 U mL?1. For the best preparation, thermal- and pH-stability, and activity profiles were determined. Experiments carried out in a batch reactor showed that kcat/Km for immobilized enzyme (0.88 min?1 μM?1) was acceptable lower than the value obtained for the native enzyme (2.19 min?1 μM?1). Finally, potentials of the catalysts were tested in the decolourisation of indigo carmine without redox-mediators. Seven consecutive runs with the catalysts separated by microfiltration proved that adsorption of the dye onto the carrier and enzymatic oxidation contribute to the efficient decolourisation without loss of immobilized enzyme activity.  相似文献   

9.
This work reports the purification and biochemical characterization of angiotensin I-converting enzyme (ACE) from ostrich (Struthio camelus) lung. The molecular weight of the purified enzyme was approximately evaluated to be 200 kDa and the maximum enzyme activity was observed at pH 7.5. The enzyme activity was increased by detergents of Triton X-100 (0.01%), cetyltrimethylammonium bromide (CTAB) (0.1 and 1 mM) and sodium dodecyl sulfate (SDS) (0.1 mM), while decreased by Triton X-100 (1% and 10%) and SDS (1 mM and 10 mM). The secondary and tertiary structure and activity of ACE in the absence and presence of trifluoroethanol (TFE) were investigated using circular dichroism, fluorescence quenching and UV–visible spectroscopy, respectively. Our results revealed that TFE stabilizes ACE at low concentrations, while acts as a denaturant at higher concentration (20%). The Km, Kcat and Kcat/Km values of ostrich ACE towards FAPGG were 0.8 × 10?4 M, 59,240 min?1 and 74 × 107 min?1 M?1, respectively. The values of IC50 and Ki for captopril were determined to be 36.5 nM and 16.6 nM, respectively. In conclusion, ostrich lung ACE is a new enzyme which could be employed as a candidate for studying ACE structure and its natural or synthetic inhibitors.  相似文献   

10.
The intrinsic and inducible o-diphenoloxidase (o-diPO) activity of Cancer pagurus hemocyanin (CpH) and Limulus polyphemus hemocyanin (LpH) were studied using catechol, l-Dopa and dopamine as substrates. The kinetic analysis shows that dopamine is a more specific substrate for CpH than catechol and l-Dopa (Km value of 0.01 mM for dopamine versus 0.67 mM for catechol, and 2.14 mM for l-Dopa), while kcat is highest for catechol (2.44 min? 1 versus 0.67 min? 1 for l-Dopa and 0.71 min? 1 for dopamine). On treatment with 4 mM sodium dodecyl sulfate (SDS) or by proteolysis the o-diPO activity of CpH increases about twofold. In contrast, native LpH shows no o-diPO activity, and exhibits only a slight activity after incubation with SDS. Neither CpH nor LpH show intrinsic mono-PO activity with l-tyrosine and tyramine as substrates. To explore the possible correlation between the degree of PO activity and protein stability of arthropod hemocyanins, the thermal stability of CpH and LpH was investigated by differential scanning calorimetry and Fourier transform infrared spectroscopy. CpH is found to be less thermostable (Tm ~ 80 °C), suggesting that the dicopper active sites are more accessible, thereby allowing the hemocyanin to show PO activity in the native state. The LpH, on the other hand, is more thermostable (Tm ~ 92 °C), suggesting the existence of a correlation between the thermal stability and the intrinsic PO activity of arthropod hemocyanins.  相似文献   

11.
To reduce CO2 emissions from alcoholic fermentation, Arthrospira platensis was cultivated in tubular photobioreactor using either urea or nitrate as nitrogen sources at different light intensities (60 μmol m?2 s?1?≤?I?≤?240 μmol m?2 s?1). The type of carbon source (pure CO2 or CO2 from fermentation) did not show any appreciable influence on the main cultivation parameters, whereas substitution of nitrate for urea increased the nitrogen-to-cell conversion factor (Y X/N ), and the maximum cell concentration (X m ) and productivity (P X ) increased with I. As a result, the best performance using gaseous emissions from alcoholic fermentation (X m ?=?2,960?±?35 g m?3, P X ?=?425?±?5.9 g m?3 day?1 and Y X/N ?=?15?±?0.2 g g?1) was obtained at I?=?120 μmol m?2 s?1 using urea as nitrogen source. The results obtained in this work demonstrate that the combined use of effluents rich in urea and carbon dioxide could be exploited in large-scale cyanobacteria cultivations to reduce not only the production costs of these photosynthetic microorganisms but also the environmental impact associated to the release of greenhouse emissions.  相似文献   

12.
A steady-state kinetic analysis of the activation of bovine Factor X, by bovine Factor Xa, was undertaken. The activation was found to be dependent on the presence of divalent cations; Ca2+ showing the greatest stimulatory effect and Mn2+ exhibiting a lower degree of activity for this reaction. Although Sr2+ and Mg2+ were ineffective when present alone, each contributed synergistically to the activation rate at suboptimal levels of Ca2+. The effect of phospholipid (phosphatidylcholine:phosphatidylserine, 4:1, w:w) on the rate of activation and on the activation pathway was investigated. Phospholipid (PL) concentrations of up to 40 μm had no effect on the activation rate; whereas, concentrations of 40–180 μm were slightly inhibitory. In the absence of PL, the major product of the activation was Factor α-Xa, while in the presence of PL, lower-molecular-weight forms of Factor X (Factor β-X) and Factor Xa (Factor β-Xa were produced. At saturating levels of Ca2+, the Km app for the activation, at pH 7.4 and 37 °C, in the absence of PL, was found to be 0.6 ± 0.1 μm and the V was 1.7 ± 0.3 mol Factor X cleaved min?1 mol?1 Factor Xa. The corresponding values, in the presence of 90 μm PL, were 1.4 ± 0.2 μm and 2.2 ± 0.2 mol Factor X cleaved min?1 mol?1 Factor Xa.  相似文献   

13.
An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2-103 (Δ-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Δ-domain is thought to resemble a phage scaffolding protein, by virtue of its highly α-helical secondary structure and a tertiary fold that projects inward from the interior surface of the shell. Here, we employ factor analysis of temperature-dependent Raman spectra to characterize the thermostability of the Δ-domain secondary structure and to quantify the thermodynamic parameters of Δ-domain unfolding. The results are compared for the Δ-domain within the prohead I architecture (in situ) and for a recombinantly expressed 111-residue peptide (in vitro). We find that the α-helicity (∼ 70%), median melting temperature (Tm = 58 °C), enthalpy (ΔHm = 50 ± 5 kcal mol− 1), entropy (ΔSm = 150 ± 10 cal mol− 1 K− 1), and average cooperative melting unit (〈nc〉 ∼ 3.5) of the in situ Δ-domain are altered in vitro, indicating specific interdomain interactions within prohead I. Thus, the in vitro Δ-domain, despite an enhanced helical secondary structure (∼ 90% α-helix), exhibits diminished thermostability (Tm = 40 °C; ΔHm = 27 ± 2 kcal mol− 1; ΔSm = 86 ± 6 cal mol− 1 K− 1) and noncooperative unfolding (〈nc〉 ∼ 1) vis-à-vis the in situ Δ-domain. Temperature-dependent Raman markers of subunit side chains, particularly those of Phe and Trp residues, also confirm different local interactions for the in situ and in vitro Δ-domains. The present results clarify the key role of the gp5 Δ-domain in prohead I architecture by providing direct evidence of domain structure stabilization and interdomain interactions within the assembled shell.  相似文献   

14.
The pool of thiamine diphosphate (TDP), available for TDP-dependent enzymes involved in the major carbohydrate metabolic pathways, is controlled by two enzyme systems that act in the opposite directions. The thiamine pyrophosphokinase (TPK) activates thiamine into TDP and the numerous phosphatases perform the reverse two-step dephosphorylation of TDP to thiamine monophosphate (TMP) and then to free thiamine. Properties and a possible cooperation of those enzymes in higher plants have not been extensively studied. In this work, we characterize highly purified preparations of TPK and a TDP/TMP phosphatase isolated from 6-day Zea mays seedlings. TPK was the 29-kDa monomeric protein, with the optimal activity at pH 9.0, the Km values of 12.4 μM and 4.7 mM for thiamine and ATP, respectively, and the Vmax value of 360 pmol TDP min?1 mg?1 protein. The enzyme required magnesium ions, and the best phosphate donor was GTP. The purified phosphatase was the dimer of 24 kDa subunits, showed the optimal activity at pH 5.0 and had a rather broad substrate specificity, although TDP, but not TMP, was one of the preferable substrates. The Km values for TDP and TMP were 36 μM and 49 μM, respectively, and the Vmax value for TDP was significantly higher than for TMP (164 versus 60 nmoles min?1 mg?1 protein). The total activities of TPK and TDP phosphatases were similarly decreased when the seedlings were grown under the illumination, suggesting a coordinated regulation of both enzymes to stabilize the pool of the essential coenzyme.  相似文献   

15.
A Metarhizium anisopliae spore surface lipase (MASSL) strongly bound to the fungal spore surface has been purified by ion exchange chromatography on DEAE sepharose followed by ultrafiltration and hydrophobic interaction chromatography on phenyl sepharose. Electrophoretic analyses showed that the molecular weight of this lipase is ~66 kDa and pI is 5.6. Protein sequencing revealed that identified peptides in MASSL shared identity with several lipases or lipase-related sequences. The enzyme was able to hydrolyze triolein, the animal lipid cholesteryl stearate and all ρNP ester substrates tested with some preference for esters with a short acyl chain. The values of Km and Vmax for the substrates ρNP palmitate and ρNP laurate were respectively 0.474 mM and 1.093 mMol min?1 mg?1 and 0.712 mM and 5.696 mMol min?1 mg?1. The optimum temperature of the purified lipase was 30 °C and the enzyme was most stable within the most acid pH range (pH 3–6). Triton X-100 increased and SDS reduced enzyme lipolytic activity. MASSL activity was stimulated by Ca2+, Mg2+ and Co2+ and inhibited by Mn2+. The inhibitory effect on activity exerted by EDTA and EGTA was limited, while the lipase inhibitor Ebelactone B completely inhibited MASSL activity as well as PMSF. Methanol 0.5% apparently did not affect MASSL activity while β-mercaptoethanol activated the enzyme.  相似文献   

16.
Tetrastigma hemsleyanum Diels et Gilg was grown under full sunlight and moderate and high levels of shade for one month to evaluate its photosynthetic and chlorophyll fluorescence response to different light conditions. The results showed that T. hemsleyanum attained greatest leaf size and Pn when cultivated with 67% shade. Leaves of seedlings grown with 90% shade were the smallest. Leaf color of plants grown under full sunlight and 50% shade was yellowish-green. The Pn value increased rapidly as PPFD increased to 200 μmol m?2 s?1 and then increased slowly to a maximum, followed by a slow decrease as PPFD was increased to 1000 μmol m?2 s?1. Pn was highest for the 67% shade treatment and the LSP for this shade treatment was 600 μmol m?2 s?1. Full sunlight and 50% shade treatments resulted in significant reduction of ETR and qP and increased NPQ. Chl a, Chl b and total chlorophyll content increased and Chl a/b values decreased with increased shading. Results showed that light intensity greater than that of 50% shade depressed photosynthetic activity and T. hemsleyanum growth. Irradiance less than that of 75% shade limited carbon assimilation and led to decreased plant growth. Approximately 67% shade is suggested to be the optimum light irradiance condition for T. hemsleyanum cultivation.  相似文献   

17.
Arthrospira (Spirulina) platensis (Nordstedt) Gomont was autotrophically cultivated for biomass production in repeated fed-batch process using urea as nitrogen source, with the aim of making large-scale production easier, increasing cell productivity and then reducing the production costs. It was investigated the influence of the ratio of renewed volume to total volume (R), the urea feeding time (tf) and the number of successive repeated fed-batch cycles on the maximum cell concentration (Xm), cell productivity (Px), nitrogen-to-cell conversion yield (Yx/n), maximum specific growth rate (μm) and protein content of dry biomass. The experimental results demonstrated that R = 0.80 and tf = 6 d were the best cultivation conditions, being able to simultaneously ensure, throughout the three fed-batch cycles, the highest average values of three of the five responses (Xm = 2101 ± 113 mg L?1, Px = 219 ± 13 mg L?1 d?1 and Yx/n = 10.3 ± 0.8 g g?1).  相似文献   

18.
An efficient synthesis of a 5-fluorouracil-cephalosporin prodrug is described for use against colorectal and other cancers in antibody and gene-directed therapies. The compound shows stability in aqueous media until specifically activated by β-lactamase (βL). The kinetic parameters of the 5-fluorouracil-cephalosporin conjugate were determined in the presence of Enterobacter cloacae P99 βL (ECl βL) revealing a Km = 95.4 μM and Vmax = 3.21 μMol min?1 mg?1. The data compare favorably to related systems that have been reported and enable testing of this prodrug against cancer cell lines in vitro and in vivo.  相似文献   

19.
The molecular mechanism responsible for the regulation of the mitochondrial membrane proton conductance (G) is not clearly understood. This study investigates the role of the transmembrane potential (ΔΨm) using planar membranes, reconstituted with purified uncoupling proteins (UCP1 and UCP2) and/or unsaturated FA. We show that high ΔΨm (similar to ΔΨm in mitochondrial State IV) significantly activates the protonophoric function of UCPs in the presence of FA. The proton conductance increases nonlinearly with ΔΨm. The application of ΔΨm up to 220 mV leads to the overriding of the protein inhibition at a constant ATP concentration. Both, the exposure of FA-containing bilayers to high ΔΨm and the increase of FA membrane concentration bring about the significant exponential Gm increase, implying the contribution of FA in proton leak. Quantitative analysis of the energy barrier for the transport of FA anions in the presence and absence of protein suggests that FA remain exposed to membrane lipids while crossing the UCP-containing membrane. We believe this study shows that UCPs and FA decrease ΔΨm more effectively if it is sufficiently high. Thus, the tight regulation of proton conductance and/or FA concentration by ΔΨm may be key in mitochondrial respiration and metabolism.  相似文献   

20.
Pectinesterase isolated from Malatya apricot pulp was covalently immobilized onto glutaraldehyde-containing amino group functionalized porous glass beads surface by chemical immobilization at pH 8.0. The amount of covalently bound apricot PE was found 1.721 mg/g glass support. The properties of immobilized enzyme were investigated and compared to those of free enzyme. The effect of various parameters such as pH, temperature, activation energy, heat and storage stability on immobilized enzyme were investigated. Optimum pH and temperature were determined to be 8.0 and 50 °C, respectively. The immobilized PE exhibited better thermostability than the free one. Kinetic parameters of the immobilized enzyme (Km and Vmax values) were also evaluated. The Km was 0.71 mM and the Vmax was 0.64 μmol min?1 mg?1. No drastic change was observed in the Km and Vmax values. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. Thermal and storage stability experiments were also carried out. It was observed that the immobilized enzyme had longer storage stability and retained 50% of its initial activity during 30 days.  相似文献   

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