Cold adaptation of a mesophilic cellulase,EG Ⅲ from Trichoderma reesei,by directed evolution

Cold-active enzymes have received little research attention although they are very useful in industries. Since the structure bases of cold adaptation of enzymes are still unclear, it is also very difficult to obtain cold-adapted enzymes for industrial applications using routine protein engineering methods. In this work, we employed directed evolution method to randomly mutate a mesophilic cellulase, endoglucanase Ⅲ (EG Ⅲ) from Trichoderma reesei, and obtained a cold-adapted mutant, designated as w-3. DNA sequence analysis indicates that w-3 is a truncated form of native EG Ⅲ with a deletion of 25 consecutive amino acids at C-terminus. Further examination of enzymatic kinetics and thermal stability shows that mutant w-3 has a higher Kcat value and becomes more thermolabile than its parent. In addition, activation energies of w-3 and wild type EG Ⅲ calculated from Arrhenius equation are 13.3 kJ· mol-1 and 26.2 kJ ·mol-1, respectively. Therefore, the increased specific activity of w-3 at lower tempera     

Kinetics and thermodynamics of catalysis and thermal inactivation of a novel α-amylase (Tp-AmyS) from Thermotoga petrophila

Abstract

This research is focussed on kinetic, thermodynamic and thermal inactivation of a novel thermostable recombinant α-amylase (Tp-AmyS) from Thermotoga petrophila. The amylase gene was cloned in pHIS-parallel1 expression vector and overexpressed in Escherichia coli. The steady-state kinetic parameters (V_max, K_m, k_cat and k_cat/K_m) for the hydrolysis of amylose (1.39?mg/min, 0.57?mg, 148.6?s^?1, 260.7), amylopectin (2.3?mg/min, 1.09?mg, 247.1?s^?1, 226.7), soluble starch (2.67?mg/min, 2.98?mg, 284.2?s^?1, 95.4) and raw starch (2.1?mg/min, 3.6?mg, 224.7?s^?1, 61.9) were determined. The activation energy (E_a), free energy (ΔG), enthalpy (ΔH) and entropy of activation (ΔS) at 98?°C were 42.9?kJ mol^?1, 74?kJ mol^?1, 39.9?kJ mol^?1 and ?92.3 J mol^?1 K^?1, respectively, for soluble starch hydrolysis. While ΔG of substrate binding (ΔG_E-S) and ΔG of transition state binding (ΔG_E-T) were 3.38 and ?14.1?kJ mol^?1, respectively. Whereas, EaD, Gibbs free energy (ΔG*), increase in the enthalpy (ΔH*) and activation entropy (ΔS*) for activation of the unfolding of transition state were 108, 107, 105?kJ mol^?1 and ?4.1 J mol^?1 K^?1. The thermodynamics of irreversible thermal inactivation of Tp-AmyS revealed that at high temperature the process involves the aggregation of the protein.     

Exchange rates of pyridine on ferro- and ferriprotoporphyrin (IX) dimethylester in chloroform.

Nuclear magnetic resonance line-widths data have been used to determine the rate of solvent exchange from the first coordination sphere of ferro-and ferriprotoporphyrin(IX) dimethylester (Fe-PPD) in pyridine/chloroform. The average values of kinetic parameters for pyridine (PY) exchange indicate an SN2 mechanism tor Fe(III)-PPD(ΔH^&;#; = 36 kJ · mol⁻¹ ; ΔS^&;#; = −53 J·mol⁻¹K⁻¹; T_M(298 K) = 0.07 msec) and an SNI mechanism for Fe(II)-PPD (ΔH^&;#; = 67 kJ·mol⁻¹; ΔS^&;#; = 42 J · mol⁻¹K⁻¹; T_M(298 K) = 0.06 msec). Parallel to the accelerated ligand exchange rate at rising temperatures a redistribution of the electrons causing a transition of the metal porphyrin from the low-spin state to the high-spin state is observed. Enthalpy and entropy of the thermodynamic equilibrium between low- and high-spin Fe-PPD have been determined from experimental values of the average magnetic moment. A mean lifetime of low-spin Fe(III)-PPD was estimated from line. widths changes (T_L→H(298 K)≈ 20 msec) and the corresponding activation parameters have been obtained (ΔH^&;#;_L→H(298 K) = 26 kJ · mol⁻¹; ΔS^&;#;_L→H(298K) = −125 J · mol⁻¹K⁻¹).     

Purification and characterization of caprine kidney uricase, possessing novel kinetic and thermodynamic properties

Purified uricase from a caprine kidney, possessed K _m and V _max values of 1.1 mg ml⁻¹ and 3512 IU (mg protein)⁻¹ for uric acid hydrolysis, respectively. The optimum temperature and pH for catalytic activity were 40 °C and 8.5, respectively. The activation energy for formation of ES complex was 13.6 kJ mol⁻¹. Enthalpy (ΔH*), entropy of activation (ΔS*) and Gibbs free energy demand of uricase inactivation were 62.8 kJ mol⁻¹, −102 J mol⁻¹ K⁻¹ and 104.3 kJ mol⁻¹, respectively. Gibbs free enrgy demand for substrate binding and transition state stabilization were also determined which were comparable with those for themostable enzymes.     

Inter‐conversion of catalytic abilities in a bifunctional carboxyl/feruloyl‐esterase from earthworm gut metagenome

Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(k_cat/K_m)]_CE/[(k_cat/K_m)]_FAE factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys²⁸¹, Asp²⁸², Asn³¹⁶ and Lys³¹⁷) situated close to the catalytic core (Ser¹⁴³/Asp²⁷³/His³⁰⁵) and a deletion of a 34-AA–long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in k_cat/K_m values) and enzymes with inverted specificity ((k_cat/K_m)_CE/(k_cat/K_m)_FAE ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to −5.6 J mol⁻¹), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to −13.7 J mol⁻¹) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of ‘hot spot’ mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.     

Thermodynamics of base interaction in (A)n and (A.U)n

Using precision scanning microcalorimetry we studied (A)_n and (A·U)_n melting in highly diluted solutions (0.3 to 5.0 mm) with different Na⁺ activity. This permitted us to determine directly the thermodynamic functions of stacking interaction in (A)_n and base-pairing in (A·U)_n. For (A-A) stacking at (A)_n melting temperature we obtained ΔH^(A)n_m = 12.6 kJ mol^?1; ΔS^(A)n_m = 41 J K^?1 mol^?1. For A·U base-pairing at a standard temperature of 298 K and 0.1 m-Na⁺ we have: ΔH^(A·U) = 34 kJ mol^?1; ΔS^(A·U) = 102 J K^?1 mol^?1ΔG^(A·U) = ?3.5 kJ mol^?1.     

Kinetics of imidazole addition to the axial site of the iron(II) complex of 2, 3, 9,10-tetraphenyl-1,4, 8,11-tetraaza-1,3, 8,10-cyclotetradecatetraene in dimethyl sulfoxide. Evidence for a dissociative-interchange mechanism

The axial adduct formation of the iron(II) complex of 2,3,9,10-tetraphenyl-l,4,8,11-tetraaza-1,3,8,10-cyclotetradecatetraene (L) with imidazole in dimethyl sulfoxide has been investigated spectrophotometrically at various temperatures and pressures. In the presence of a large excess of imidazole the reaction with the two phases has been observed. The first faster reaction is the formation of the monoimidazole complex of FeL²⁺, and the second slower reaction corresponds to the formation of the bisimidazole complex. Activation parameters are as follows: for the first step with k₁ (25.0°C) = (6.8 ±0.2)×10⁵ mol⁻¹ kg s⁻¹, ΔH³₁ = 47.5 ± 4.9 kJ mol⁻¹, ΔS³₁ = 26±16 J K⁻¹ mol⁻¹, and ΔV³₁ (30.0°C) = 27.2±1.5 cm³ mol⁻¹; for the second step with k₂ (25.0°C) = 26.8±0.8 mol⁻¹ kg s⁻¹, ΔH³₂ = 91.6± 0.8 kJ mol⁻¹, ΔS³₂ = 90±3 J K⁻¹ mol⁻¹, and ΔV³₂ (35.0°C) = 21.8±0.9 cm³ mol⁻¹. The large positive activation volumes strongly indicate a dissociative character of the activation process.     

Conserved sequence motif DPPY in region IV of the phage T4 Dam DNA-[N-adenine]-methyltransferase is important for S-adenosyl-L-methionine binding

Comparison of the deduced amino acid sequences of DNA-[N⁶-adenine]-methyltransferases has revealed several conserved regions. All of these enzymes contain a DPPY-motif, or a variant of it. By site-directed mutagenesis of a cloned T4 dam gene, we have altered the first proline residue in this motif (located in conserved region IV of the T4 Dam-MTase) to alanine or threonine. The mutant enzymic forms, P172A and P172T, were overproduced and purified. Kinetic studies showed that compared to the wild-type (wt) the two mutant enzymic forms had: (i) an increased (6 and 23-fold, respectively) K_m for substrate, S-adenosyl-methionine (AdoMet) and an increased (6 and 23-fold) K_i for product, S-adenosyl-homocysteine (AdoHcy); (ii) a slightly reduced (1.5 and 3-fold lower) k_cat; (iii) a strongly reduced k_cat/K_m^AdoMet (10 and 80-fold); and (iv) the same K_m for substrate DNA. Equilibrium dialysis studies showed that the mutant enzymes had a reduced (3 and 7-fold lower) K_a for AdoMet; all forms bound two molecules of AdoMet. Taken together these data indicate that the P172A and P172T alterations resulted primarily in a reduced affinity for AdoMet. This suggests that the DPPY-motif is important for AdoMet-binding, and that region IV contains an AdoMet-binding site.     

The intracellular sucrase (SacA) of Zymomonas mobilis is not involved in sucrose assimilation

The intracellular sucrase SacA from Zymomonas mobilis was purified to homogeneity from a recombinant E. coli strain containing the SacA gene under an expression system. The protein was monomeric with a molecular mass of 58 kDa. The sucrase activity was maximal at 25 °C and thermal stability of the purified protein was low (50% recovery after 30 min at 46 °C ). The activation energy was low at 33 kJ mol^–1. Maximum activity was at pH 6.5. Activity was strongly inhibited (>99%) by SH blocking reagents and reducing agents slightly (10–60%) increased the activity of purified SacA. The sucrase showed a low K _M (42 mM) and k _cat (125 s^–1) which indicated its very low efficiency for sucrose hydrolysis. A mutant strain of Z. mobilis not able to grow on sucrose was isolated. This strain (ZM4S) lacked the two sucrases SacB and SacC but SacA was present in the intracellular fraction. Therefore, SacA alone is unable to allow growth Z. mobilis on sucrose.     

Thermodynamic profiles of penicillin G hydrolysis catalyzed by wild-type and Met→Ala168 mutant penicillin acylases from Kluyvera citrophila

The Met-168 residue in penicillin acylase from Kluyvera citrophila was changed to Ala by oligonucleotide site-directed mutagenesis. The Ala-168 mutant exhibited different substrate specificity than wild-type and enhanced thermal stability. The thermodynamic profiles for penicillin G hydrolysis catalyzed by both enzymes were obtained from the temperature dependence of the steady-state kinetic parameters K_m and k_cat. The high values of enthalpy and entropy of activation determined for the binding of substrate suggest that an induced-fit-like mechanism takes place. The Met→Ala168 mutation unstabilizes the first transition-state (E··S^≠) and the enzyme-substrate complex (ES) causing a decrease in association equilibrium and specificity constants in the enzyme. However, no change is observed in the acyl-enzyme formation. It is concluded that residue 168 is involved in the enzyme conformational rearrangements caused by the interaction of the acid moiety of the substrate at the active site.     

Thermodynamics and coordination characteristics of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 extraction complex

The extraction equilibrium of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 complex was carried out in the crown ether1,2-dichloroethaneHCl aqueous solution system at different temperatures. The extraction complex has the overall composition (L)₂·(H₃O⁺·χH₂O)₂·UO₂Cl₄²⁻ (L = dicyclohexano-24-crown-8). The values of the extraction equilibrium constants (K_ex) increase steadily with a decrease in temperature: 13.5 (298 K), 7.96 (301 K), 4.20 (303 K) and 2.07 (305 K). A plot of log K_ex against 1/T shows a straight line. The value of the enthalpy change, ΔH°, was calculated from the slope and equals −212 kJ mol⁻¹. The value of the entropy change, ΔS°, was calculated from ΔH° and K_ex and equals −690 J K⁻¹ mol⁻¹, whereas ΔG° = −6.45 kJ mol⁻¹. Comparing these thermodynamic parameters with those of the dicyclohexano-18-crown-6 isomer A [1] (ΔS° = −314 J K⁻¹ mol⁻¹, ΔH° = −101 kJ mol⁻¹ and ΔG° = −8.37 kJ mol⁻¹), it can be seen that ΔH° and ΔS° are more negative for the former than for the latter, and both are enthalpy-stabilized complexes. The molecular structure of the complex has the feature that there are two H₅O₂⁺ ions in it, in contrast to the H₃O⁺ ions in the dicyclohexano-18-crown-6 isomer A complex [1]. Each of the H₅O₂⁺ ions is held in the crown ether cavity by four hydrogen bonds. The H₅O₂⁺ ion has a central bond. The uranium atom forms UO₂Cl₄²⁻ as a counterion away from the crown ether. The formation of this complex is in good agreement with more negative entropy change and less negative free energy change, as mentioned above.     

Kinetics and thermodynamics of the P870+Q−A → P870+Q−B reaction in isolated reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides

The temperature dependences of the P870⁺Q^?_A → P870Q_A and P870⁺Q^?_B → P870Q_B recombination reactions were measured in reaction centers from Rhodopseudomonas sphaeroides. The data indicate that the P870⁺Q^?_B state decays by thermal repopulation of the P870⁺Q^?_A state, followed by recombination. ΔG° for the P870⁺Q^?_A → P870⁺Q^?_B reaction is ?6.89 kJ · mol^?1, while ΔH° = ?14.45 kJ · mol^?1 and ?TΔS° = + 7.53 kJ · mol^?1. The activation ethalpy, $\text{H}{}^3$, for the P870⁺Q^?_A Δ P870⁺Q^?_B reaction is +56.9 kJ · mol^?1, while the activation entropy is near zero. The results permit an estimate of the shape of the potential energy curve for the P870⁺Q^?_A → P870⁺Q^?_B electron transfer reaction.     

Activation of human plasma prekallikrein by Pseudomonas aeruginosa elastase. II. Kinetic analysis and identification of scissile bondof prekallikrein in the activation

Activation of human plasma prekallikrein by a bacterial metalloendopeptidase, Pseudomonas aeruginosa elastase, was reported (Shibuya et al. (1991) Biochim. Biophys. Acta 1097, 23–27). Details of the activation process were presently studied. The activation accompanied limited proteolysis of a peptide bond inside of a disulfide bridge of prekallikrein molecule. Amino acid sequencing analysis of the newly generated amino-terminal revealed that the cleavage site was Arg₃₇₁-Ile₃₇₂ bond which is the scissile bond in the activation of prekallikrein with trypsin-type proteinases. A pentapeptide substrate, 2-aminobenzoyl-Ser-Thr-Ile-Val-4-nitrobenzylamide, which contained the amino acid sequence identical to that around the scissile bond of prekallikrein was synthesized. Pseudomonal elastase, indeed, hydrolyzed the substrate at Arg-Ile bond with the kinetic parameters of K_m = 118 μM, k_cat = 1.56/s and k_cat/K_m = 1.33 · 10⁴/s M. These results indicated that the Arg₃₇₁-Ile₃₇₂ bond was sensitive not only to trypsin-type serine proteinases, but also a bacterial metalloproteinase. Kinetic analysis of the prekallikrein activation by psuedomonal elastase, however, revealed that the activation rate was show, though the K_m values was good enough to expect an occurence of this activation in vivo (K_m = 248 nM, k = 6.8 · 10^?4/s, and k_cat/K_m = 2.7 · 10³/s M. The activation rate of prekallikrein by pseudomonal elastase in Hageman factor deficient plasma was remarkably improved when the plasma was reconstituted with purified Hageman factor molecule. From the results, a biologuical significance of the proteinase cascade in the plasma kinin generation was also indicated. The present in vitro study might support the hypothesis that the Hageman factor/kallikrein-kinin system plays an important role in bacterial infection including the pseudomonal one.     

Stereodynamics of Small 1,2-Dialkyldiaziridines

Diaziridines are very interesting representatives of organic compounds containing stereogenic nitrogen atoms. In particular, 1,2-dialkyldiaziridines show extraordinarily high stereointegrity. The lone electron pairs of the nitrogen atoms are in trans configuration, avoiding a four-electron repulsive interaction. Furthermore, the trans configuration of the substituents at the nitrogen atoms is energetically favored because of reduced steric interactions. Therefore only two stereoisomers (enantiomers) are observed. At elevated temperatures the enantiomers are interconverting because of the limited stereointegrity of the chirotopic nitrogen atoms. The enantiomerization rate constants and the activation parameters of interconversion are of great interest. Here, we investigated the stereodynamics of a set of small 1,2-dialkyldiaziridines bearing short substituents (Me, Et, iPr, tBu), using enantioselective dynamic gas chromatography (DGC). Separation of enantiomers of all compounds, including the highly volatile 1,2-dimethyldiaziridine, was achieved using heptakis(2,3-di-O-ethyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin in 50% PS086 (w/w) as chiral stationary phase in fused silica capillaries with a length of up to 50 m. Measurements at variable temperatures were performed and reaction rate constants were determined using the unified equation of chromatography implemented in the software DCXplorer. The activation barriers at room temperature for 1-(tert-butyl)-2-ethyldiaziridine, ΔG^╪_298K = 123.8 kJ mol^–1 (ΔH^╪ = 115.5 ± 2.9 kJ mol^–1, ΔS^╪ = –28 ± 1 J mol^–1 K^–1), and 1-ethyl-2-isopropyldiaziridine, ΔG^╪_298K = 124.2 kJ mol^–1 (ΔH^╪ = 113.1 ± 2.4 kJ mol^–1, ΔS^╪ = –37 ± 2 J mol^–1 K^–1), were determined, representing some of the highest values observed for nitrogen inversion in diaziridines. Chirality 00:000–000, 2013. © 2013 Wiley Periodicals, Inc.     

Metal complexes of cyclic diamines,dissociation kinetics of bis(1,5-diazacyclo-octane)nickel(II) in acidic and basic solution,and electrochemical studies

The preparation of the planar yellow [Ni([8]aneN₂)₂](ClO₄)₂ is described. The complex dissociates in basic solution, with rate = k_OH[NiL][OH^?] (L = 1,5-diazacyclo-octane). At 25 °C, k_OH = 4.5 x 10^?2 M^?1 s^?1 and the corresponding activation parameters are ΔH^≠ = 69.2 kJ mol^?1 and ΔS₂₉₈^≠ = ?38.6 J K^?1 mol^?1. Acid catalysed dissociation in quite slow even in strongly acidic solutions. The kinetic data in this case can be fitted to the expression K_obs = k_o + K_H[H⁺], where k_o relates to a solvolytic pathway and k_H to the acid catalysed pathway. At 60 °C, K_o = 2 x 10^?5 s^?1 and k_H is 2 x 10^?5 M^?1 s^?1. Possible mechanisms for these reactions are considered.The Ni(II)/Ni(III) redox couple for NiLⁿ⁺ is irreversible on Pt using MeCN as solvent.     

Interaction between tryptophan‐Sm(III) complex and DNA with the use of a acridine orange dye fluorophor probe

The interaction of the Trp–Sm(III) complex with herring sperm DNA (hs‐DNA) was investigated with the use of acridine orange (AO) dye as a spectral probe for UV‐vis spectrophotometry and fluorescence spectroscopy. The results showed that the both the Trp–Sm(III) complex and the AO molecule could intercalate into the double helix of the DNA. The Sm(III)–(Trp)₃ complex was stabilized by intercalation into the DNA with binding constants: K^?_25°C = 7.14 × 10⁵ L·mol^?1 and K^?_37°C = 5.28 × 10⁴ L·mol^?1, and it could displace the AO dye from the AO–DNA complex in a competitive reaction. Computation of the thermodynamic functions demonstrates that Δ_rH_m^? is the primary driving power of the interaction between the Sm(III)(Trp)₃ complex and the DNA. The results from Scatchard and viscometry methods suggested that the interaction mode between the Sm(III)(Trp)₃ complex and the hs‐DNA is groove binding and weak intercalation binding. Copyright © 2015 John Wiley & Sons, Ltd.     

Thermodynamics of hexokinase-catalyzed reactions.

The enthalpies of the hexokinase-catalyzed phosphorylation or glucose, mannose, and fructose by ATP to the respective hexose 6-phosphates have been measured calorimetrically in TRIS/TRIS HCl buffer at 25.0, 28.5, and 32.0°C. The effects on the measured enthalpy of the glucose/hexokinase reaction due to variation of pH (over the range 6.7 to 9.0) and ionic strength (over the range 0.02 to 0.25) have been examined. Correction for enthalpy of buffer protonation leads to δH^o and δC_p^o values for the processes: eq-D-hexose + ATP⁴⁻ = eq-D-hexose 6-phosphate²⁻ + ADP³⁻+ H⁺. Results are δH^o = −23.8 ± 0.7 kJ · mol⁻¹ and δC_p^o = −156 ± 280 J·mol⁻¹·K⁻¹ for glucose. δH^o = −21.9 ± 0.7 kJ·mol⁻¹ and δC_p^o = 10 ± 140 J·mol⁻¹·K⁻¹ for mannose, and δH^o = −15.0 ± 0.9 kJ·mol⁻¹ and δC_p^o = −41 ± 160 J·mol⁻¹·K⁻¹ for fructose. Combination of these measured enthalpies with Gibbs energy data for hydrolysis of ATP⁴⁻ and that for the hexose 6-phosphates lead to δS^o values for the above hexokinase-catalyzed reactions.     

Catalytic Properties of Two Pyruvate Kinase Isoforms in Nordic Krill,Meganyctiphanes norvegica,With Respect to Seasonal Temperature Adaptation

Two isoforms of pyruvate kinase (PK I and PK II) were partly purified and characterized from the Nordic krill Meganyctiphanes norvegica. Both PK variants were present in summer and winter specimens with a tissue specificity in abdominal muscle (PK I) and cephalothorax (PK II). Obvious differences were found in chromatographic and kinetic characteristics. Enzymatic adaptations to low temperatures were found in PK I only, whereas PK II did not contribute to seasonal temperature adaptation. In winter specimens, the activation energy of PK I decreased significantly from 53.2 ± 1.5 to 50.2 ± 1.2 kJ·mol⁻¹. The affinity of PK I to phosphoenol-pyruvate was higher in winter (K_M = 0.024 ± 0.002 mmol·l⁻¹) compared to summer (K_M = 0.033 ± 0.003 mmol·l⁻¹). Both effects lead to an increased efficiency of this enzyme isoform in the cold. In contrast, K_M values of PK II showed no significant differences between summer (K_M = 0.181 ± 0.014 mmol·l⁻¹) and winter specimens (K_M = 0.193 ± 0.015 mmol·l⁻¹). The effects of cooperativity remained unchanged during the seasons with approximate values of n_Hill = 1.0 (PK I) and 1.5 (PK II). Fructose-1,6-bis-phosphate affected only PK II by shifting sigmoidal kinetics to hyperbolic curves resulting in a decrease of K_M to 0.027 mmol·l⁻¹. Further effectors were tested showing an inhibiting effect of oxalate on both isoforms with a reduction to 20% and 50% in PK I and PK II, respectively. Presumably, the ecophysiological effect of the capacity to regulate muscle PK is related to the necessity to increase motility during vertical migration and phases of feeding activity.     

Effect of process parameters on 3-hydroxypropionic acid production from glycerol using a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The stability and specific activity of endo-β-1,4-glucanase III from Trichoderma reesei QM9414 was enhanced, and the expression efficiency of its encoding gene, egl3, was optimized by directed evolution using error-prone PCR and activity screening in Escherichia coli RosettaBlue (DE3) pLacI as a host. Relationship between increase in yield of active enzyme in the clones and improvement in its stability was observed among the mutants obtained in the present study. The clone harboring the best mutant 2R4 (G41E/T110P/K173M/Y195F/P201S/N218I) selected in via second-round mutagenesis after optimal recombinating of first-round mutations produced 130-fold higher amount of mutant enzyme than the transformant with wild-type EG III. Mutant 2R4 produced by the clone showed broad pH stability (4.4–8.8) and thermotolerance (entirely active at 55°C for 30 min) compared with those of the wild-type EG III (pH stability, 4.4–5.2; thermostability, inactive at 55°C for 30 min). k _cat of 2R4 against carboxymethyl-cellulose was about 1.4-fold higher than that of the wild type, though the K _m became twice of that of the wild type.     

Purification,kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccharum officinarum L.)

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: k_m = 55 mg ml^?1, k_cat = 21 s^?1, k_cat/k_m = 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.