Succinyl-CoA synthetase of Escherichia coli is an alpha 2 beta 2 protein containing active sites ... more Succinyl-CoA synthetase of Escherichia coli is an alpha 2 beta 2 protein containing active sites at the interfaces between alpha- and beta-subunits. The alpha-subunit contains a histidine residue that is phosphorylated during the reaction. The beta-subunit binds coenzyme A and probably succinate [see Nishimura, J. S. (1986) Adv. Enzymol. Relat. Areas Mol. Biol. 58, 141-172]. Chemical modification studies have been conducted in order to more clearly define functions of each subunit. Tryptophan residues of the enzyme were modified by treatment with N-bromosuccinimide at pH 7. There was a linear relationship between loss of enzyme activity and tryptophan modified. At one tryptophan residue modified per beta-subunit, 100% of the enzyme activity was lost. In this enzyme sample, one methionine residue in each alpha- and beta-subunit was oxidized to methionine sulfoxide, although loss of enzyme activity could not be related in a linear manner to the formation of this residue. Subunits were prepared from enzyme that was inactivated 50% by N-bromosuccinimide with 0.5 tryptophan modified per beta-subunit but with insignificant modification of methionine residues in either subunit. Small decreases in the tyrosine and histidine content were observed in the alpha-subunit but not in the beta-subunit. In this case, modified beta-subunit when mixed with unmodified alpha-subunit gave a population of molecules that was 50% as active as the refolded, unmodified control but was only slightly changed with respect to phosphorylation capacity and unchanged with respect to rate of phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
The variation of kinetic parameters of beta-glucosidase from Trichoderma reesei QM 9414 with pH w... more The variation of kinetic parameters of beta-glucosidase from Trichoderma reesei QM 9414 with pH was used to gain information about the chemical mechanism of the reaction catalysed by this enzyme. The pH-dependence of Vmax. and Vmax./Km for p-nitrophenyl beta-D-glucopyranoside showed that a group with a pK value of 4.3 must be unprotonated and a group with a pK value of 5.9 must be protonated for activity. Temperature and solvent-perturbation studies indicated that these groups are a histidine residue and a carboxy group respectively. Profiles of pKi for maltose as competitive inhibitor showed that binding is prevented when a group on the enzyme with a pK value of 4.5 becomes protonated.
Journal of Molecular Catalysis B-enzymatic, Mar 1, 2000
d-Amino acid oxidase was inactivated by N-bromosuccinimide (NBS) at 30°C and pH 8. The reaction f... more d-Amino acid oxidase was inactivated by N-bromosuccinimide (NBS) at 30°C and pH 8. The reaction followed pseudo-first order kinetics with second-order rate constants of 69.8 mM−1 min−1 for the apoenzyme and 0.63 mM−1 min−1 for the holoenzyme. The presence of substrates or benzoate protected the enzyme against inactivation. Difference absorption spectra at 280 nm, low consumption of NBS per mole of enzyme, the decrease in the fluorescence emission at 335 nm, integrity of the protein backbone and the absence of cysteine oxidation pointed to the modification of tryptophan residues. The statistical analysis of the residual fractional activity vs. the number of modified tryptophan residues led to the conclusion that one tryptophan residue is essential for the enzyme activity. This tryptophan residue was not involved in binding of FAD or dimerization of the enzyme.
The scope in improving enzyme productivities from the cellulose fermentation process is examined ... more The scope in improving enzyme productivities from the cellulose fermentation process is examined in laboratory‐scale fermentors. The maximum productivity (30 IU/liter hr) is attained in a continuous‐culture process with cell recycle using modified medium containing 0.5% cellulose. Optimum dilution rate and recycle ratio are determined as 0.025 hr−1 and 1.2, respectively, for the process. The system is analyzed and steady‐state equations for predicting enzyme protein concentrations in the fermentor are developed. In fed‐batch cultures, slow addition of cellulose at high concentrations can improve enzyme productivity by as much as 33% over a batch process. The scope and results of using modified medium for cellulase production are also presented.
Many Gram-negative bacteria produce N-acyl-homoserine lactones (AHLs), quorum sensing (QS) molecu... more Many Gram-negative bacteria produce N-acyl-homoserine lactones (AHLs), quorum sensing (QS) molecules that can be enzymatically inactivated by quorum quenching (QQ) processes; this approach is considered an emerging antimicrobial alternative. In this study, kinetic parameters of several AHLs hydrolyzed by penicillin acylase from Streptomyces lavendulae (SlPA) and aculeacin A acylase from Actinoplanes utahensis (AuAAC) have been determined. Both enzymes catalyze efficiently the amide bond hydrolysis in AHLs with different acyl chain moieties (with or without 3-oxo modification) and exhibit a clear preference for AHLs with long acyl chains (C12-HSL > C14-HSL > C10-HSL > C8-HSL for SlPA, whereas C14-HSL > C12-HSL > C10-HSL > C8-HSL for AuAAC). Involvement of SlPA and AuAAC in QQ processes was demonstrated by Chromobacterium violaceum CV026-based bioassays and inhibition of biofilm formation by Pseudomonas aeruginosa, a process controlled by QS molecules, suggesting the...
The mechanism of irreversible thermoinactivation of endoglucanase I from Trichoderma reesei has b... more The mechanism of irreversible thermoinactivation of endoglucanase I from Trichoderma reesei has been determined at 70 degrees C at the pH of maximum enzyme activity. The time-course of thermoinactivation did not follow first-order kinetics and kinetic constants of the process were dependent on enzyme concentration, suggesting that aggregation was the main process leading to irreversible inactivation. The enzyme was extremely resistant to urea, which in fact seemed to stabilize it against temperature. Disulphide exchange, deamidation and hydrolysis of peptide bonds were also responsible for the loss of enzyme activity at 70 degrees C.
n‐Propyl, n‐butyl and n‐pentyl β‐cellobiosides with a reactive ω‐epoxide in their aglycon complet... more n‐Propyl, n‐butyl and n‐pentyl β‐cellobiosides with a reactive ω‐epoxide in their aglycon completely and irreversibly inactivate endoglucanase III from Trichoderma reesei. The pentyl derivative was found to be most effective. From these affinity labeling experiments evidence was found for the implication of Glu329 in the reaction mechanism. This is discussed in relation to other structural/functional data known for endoglucanase III and several other family A glycanases.
Succinyl-CoA synthetase of Escherichia coli is an alpha 2 beta 2 protein containing active sites ... more Succinyl-CoA synthetase of Escherichia coli is an alpha 2 beta 2 protein containing active sites at the interfaces between alpha- and beta-subunits. The alpha-subunit contains a histidine residue that is phosphorylated during the reaction. The beta-subunit binds coenzyme A and probably succinate [see Nishimura, J. S. (1986) Adv. Enzymol. Relat. Areas Mol. Biol. 58, 141-172]. Chemical modification studies have been conducted in order to more clearly define functions of each subunit. Tryptophan residues of the enzyme were modified by treatment with N-bromosuccinimide at pH 7. There was a linear relationship between loss of enzyme activity and tryptophan modified. At one tryptophan residue modified per beta-subunit, 100% of the enzyme activity was lost. In this enzyme sample, one methionine residue in each alpha- and beta-subunit was oxidized to methionine sulfoxide, although loss of enzyme activity could not be related in a linear manner to the formation of this residue. Subunits were prepared from enzyme that was inactivated 50% by N-bromosuccinimide with 0.5 tryptophan modified per beta-subunit but with insignificant modification of methionine residues in either subunit. Small decreases in the tyrosine and histidine content were observed in the alpha-subunit but not in the beta-subunit. In this case, modified beta-subunit when mixed with unmodified alpha-subunit gave a population of molecules that was 50% as active as the refolded, unmodified control but was only slightly changed with respect to phosphorylation capacity and unchanged with respect to rate of phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
The variation of kinetic parameters of beta-glucosidase from Trichoderma reesei QM 9414 with pH w... more The variation of kinetic parameters of beta-glucosidase from Trichoderma reesei QM 9414 with pH was used to gain information about the chemical mechanism of the reaction catalysed by this enzyme. The pH-dependence of Vmax. and Vmax./Km for p-nitrophenyl beta-D-glucopyranoside showed that a group with a pK value of 4.3 must be unprotonated and a group with a pK value of 5.9 must be protonated for activity. Temperature and solvent-perturbation studies indicated that these groups are a histidine residue and a carboxy group respectively. Profiles of pKi for maltose as competitive inhibitor showed that binding is prevented when a group on the enzyme with a pK value of 4.5 becomes protonated.
Journal of Molecular Catalysis B-enzymatic, Mar 1, 2000
d-Amino acid oxidase was inactivated by N-bromosuccinimide (NBS) at 30°C and pH 8. The reaction f... more d-Amino acid oxidase was inactivated by N-bromosuccinimide (NBS) at 30°C and pH 8. The reaction followed pseudo-first order kinetics with second-order rate constants of 69.8 mM−1 min−1 for the apoenzyme and 0.63 mM−1 min−1 for the holoenzyme. The presence of substrates or benzoate protected the enzyme against inactivation. Difference absorption spectra at 280 nm, low consumption of NBS per mole of enzyme, the decrease in the fluorescence emission at 335 nm, integrity of the protein backbone and the absence of cysteine oxidation pointed to the modification of tryptophan residues. The statistical analysis of the residual fractional activity vs. the number of modified tryptophan residues led to the conclusion that one tryptophan residue is essential for the enzyme activity. This tryptophan residue was not involved in binding of FAD or dimerization of the enzyme.
The scope in improving enzyme productivities from the cellulose fermentation process is examined ... more The scope in improving enzyme productivities from the cellulose fermentation process is examined in laboratory‐scale fermentors. The maximum productivity (30 IU/liter hr) is attained in a continuous‐culture process with cell recycle using modified medium containing 0.5% cellulose. Optimum dilution rate and recycle ratio are determined as 0.025 hr−1 and 1.2, respectively, for the process. The system is analyzed and steady‐state equations for predicting enzyme protein concentrations in the fermentor are developed. In fed‐batch cultures, slow addition of cellulose at high concentrations can improve enzyme productivity by as much as 33% over a batch process. The scope and results of using modified medium for cellulase production are also presented.
Many Gram-negative bacteria produce N-acyl-homoserine lactones (AHLs), quorum sensing (QS) molecu... more Many Gram-negative bacteria produce N-acyl-homoserine lactones (AHLs), quorum sensing (QS) molecules that can be enzymatically inactivated by quorum quenching (QQ) processes; this approach is considered an emerging antimicrobial alternative. In this study, kinetic parameters of several AHLs hydrolyzed by penicillin acylase from Streptomyces lavendulae (SlPA) and aculeacin A acylase from Actinoplanes utahensis (AuAAC) have been determined. Both enzymes catalyze efficiently the amide bond hydrolysis in AHLs with different acyl chain moieties (with or without 3-oxo modification) and exhibit a clear preference for AHLs with long acyl chains (C12-HSL > C14-HSL > C10-HSL > C8-HSL for SlPA, whereas C14-HSL > C12-HSL > C10-HSL > C8-HSL for AuAAC). Involvement of SlPA and AuAAC in QQ processes was demonstrated by Chromobacterium violaceum CV026-based bioassays and inhibition of biofilm formation by Pseudomonas aeruginosa, a process controlled by QS molecules, suggesting the...
The mechanism of irreversible thermoinactivation of endoglucanase I from Trichoderma reesei has b... more The mechanism of irreversible thermoinactivation of endoglucanase I from Trichoderma reesei has been determined at 70 degrees C at the pH of maximum enzyme activity. The time-course of thermoinactivation did not follow first-order kinetics and kinetic constants of the process were dependent on enzyme concentration, suggesting that aggregation was the main process leading to irreversible inactivation. The enzyme was extremely resistant to urea, which in fact seemed to stabilize it against temperature. Disulphide exchange, deamidation and hydrolysis of peptide bonds were also responsible for the loss of enzyme activity at 70 degrees C.
n‐Propyl, n‐butyl and n‐pentyl β‐cellobiosides with a reactive ω‐epoxide in their aglycon complet... more n‐Propyl, n‐butyl and n‐pentyl β‐cellobiosides with a reactive ω‐epoxide in their aglycon completely and irreversibly inactivate endoglucanase III from Trichoderma reesei. The pentyl derivative was found to be most effective. From these affinity labeling experiments evidence was found for the implication of Glu329 in the reaction mechanism. This is discussed in relation to other structural/functional data known for endoglucanase III and several other family A glycanases.
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