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Volker Kasche - One of the best experts on this subject based on the ideXlab platform.

  • Improved A. faecalis Penicillin Amidase Mutant Retains the Thermodynamic and pH Stability of the Wild Type Enzyme
    The Protein Journal, 2010
    Co-Authors: Ruslan Yuryev, Zoya Ignatova, Volker Kasche, B Galunsky
    Abstract:

    Penicillin Amidase from Alacaligenes faecalis is an attractive biocatalyst for hydrolysis of Penicillin G for production of 6-aminopenicillanic acid, which is used in the synthesis of semi-synthetic β-lactam antibiotics. Recently a mutant of this enzyme with extended C-terminus of the A-chain comprising parts of the connecting linker peptide was constructed. Its turnover number for the hydrolysis of Penicillin G was 140 s^−1, about twice of the value for the wild-type enzyme (80 s^−1). At the same time the specificity constant was improved about three-fold. The wild-type and the mutant enzymes showed similar pH stability suggesting that the linker peptide fragment covalently attached to the A-chain does not alter the electrostatic interactions in the protein core. Although the global stability of A. faecalis wild-type enzyme and the T206GS213G variant does not differ, the presence of the linker fragment stabilizes the domains interface, as evidenced by the monophasic transition of the mutant enzyme from folded to unfolded state during urea-induced denaturation. The high stability and activity of the mutant enzyme provides a rationale to use it as a biocatalyst in the industrial processes, where the enzyme must be more robust to fluctuations in the operational conditions.

  • Direct Measurement of pH Profiles in Immobilized Enzyme Carriers during Kinetically Controlled Synthesis Using CLSM
    Biotechnology Progress, 2008
    Co-Authors: Antje C. Spiess, Volker Kasche
    Abstract:

    Confocal laser scanning microscopy was applied to measure the pH value in the carrier of immobilized enzymes during the enzyme-catalyzed synthesis. pH profiles with a high resolution are shown, with the pH increasing in the core of the particles. Significant differences occur for different carrier material, particle size, porosity and surface modification. The increased pH value is identified as one of the reasons leading to reduced enzyme selectivity in the Penicillin Amidase catalyzed synthesis of cephalosporins and Penicillins.

  • Enzyme catalysed reactions, enantioselectivity and stability under high hydrostatic pressure
    High Pressure Chemical Engineering Proceedings of the 3rd International Symposium on High Pressure Chemical Engineering, 2007
    Co-Authors: O. Guthmann, Volker Kasche, A Rieks, R. Schwerdtfeger, Garabed Antranikian, Gerd Brunner
    Abstract:

    Summary Pressure activation and inactivation is investigated for several enzymes like Penicillin Amidase ( E. coli ), glutamate dehydrogenase ( P. woesei ) and lipase ( Rhizopus arrhizus ) in the pressure range between 1 bar and 4000 bar. In dependance of pressure and temperature the enzymes are acivated or inactivated and hence their enantioselectivity can be directed. The activation of the enzymes corresponds to a decrease in the K M value which results in a higher substrate affinity.

  • effect of the increased stability of the Penicillin Amidase mrna on the protein expression levels
    FEBS Letters, 2005
    Co-Authors: Sandra C Viegas, Volker Kasche, Dorothea Schmidt, Cecilia M Arraiano, Zoya Ignatova
    Abstract:

    Several factors at transcriptional, post-transcriptional or post-translational level determine the fate of a target protein and can severely restrict its yield. Here, we focus on the post-transcriptional regulation of the biosynthesis of the periplasmic protein, Penicillin Amidase (PA). The PA mRNA stability was determined under depleted RNase conditions in strains carrying single or multiple RNase deletions. Single deletion of the endonuclease RNase E yielded, as the highest, a fourfold stabilization of the PA mRNA. This effect, however, was reduced twice at post-translational level. The RNase II, generating secondary exonucleolytic cleavages in the mRNA, although not significantly influencing the PA mRNA decay, led also to an increase of the amount of mature PA. The non-proportional correlation between increased mRNA longevity and amount of active enzyme propose that the rational strategies for yield improvement must be based on a simultaneous tuning of more than one yield restricting factor.

  • pro sequence and ca2 binding implications for folding and maturation of ntn hydrolase Penicillin Amidase from e coli
    Journal of Molecular Biology, 2005
    Co-Authors: Zoya Ignatova, Frank Wischnewski, Holger Notbohm, Volker Kasche
    Abstract:

    Penicillin Amidase (PA) is a bacterial periplasmic enzyme synthesized as a pre-pro-PA precursor. The pre-sequence mediates membrane translocation. The intramolecular pro-sequence is expressed along with the A and B chains but is rapidly removed in an autocatalytic manner. In extensive studies we show here that the pro-peptide is required for the correct folding of PA. Pro-PA and PA unfold via a biphasic transition that is more pronounced in the case of PA. According to size-exclusion chromatography and limited proteolysis experiments, the inflection observed in the equilibrium unfolding curves corresponds to an intermediate in which the N-terminal domain (A-chain) still possesses native-like topology, whereas the B-chain is unfolded to a large extent. In a series of in vitro experiments with a slow processing mutant pro-PA, we show that the pro-sequence in cis functions as a folding catalyst and accelerates the folding rate by seven orders of magnitude. In the absence of the pro-domain the PA refolds to a stable inactive molten globule intermediate that has native-like secondary but little tertiary structure. The pro-sequence of the homologous Alcaligenes faecalis PA can facilitate the folding of the hydrolase domain of Escherichia coli PA when added in trans (as a separate polypeptide chain). The isolated pro-sequence has a random structure in solution. However, difference circular dichroism spectra of native PA and native PA with pro-peptide added in trans suggest that the pro-sequence adopts an α-helical conformation in the context of the mature PA molecule. Furthermore, our results establish that Ca2+, found in the crystal structure, is not directly involved in the folding process. The cation shifts the equilibrium towards the native state and facilitates the autocatalytic processing of the pro-peptide.

Zoya Ignatova - One of the best experts on this subject based on the ideXlab platform.

  • Improved A. faecalis Penicillin Amidase Mutant Retains the Thermodynamic and pH Stability of the Wild Type Enzyme
    The Protein Journal, 2010
    Co-Authors: Ruslan Yuryev, Zoya Ignatova, Volker Kasche, B Galunsky
    Abstract:

    Penicillin Amidase from Alacaligenes faecalis is an attractive biocatalyst for hydrolysis of Penicillin G for production of 6-aminopenicillanic acid, which is used in the synthesis of semi-synthetic β-lactam antibiotics. Recently a mutant of this enzyme with extended C-terminus of the A-chain comprising parts of the connecting linker peptide was constructed. Its turnover number for the hydrolysis of Penicillin G was 140 s^−1, about twice of the value for the wild-type enzyme (80 s^−1). At the same time the specificity constant was improved about three-fold. The wild-type and the mutant enzymes showed similar pH stability suggesting that the linker peptide fragment covalently attached to the A-chain does not alter the electrostatic interactions in the protein core. Although the global stability of A. faecalis wild-type enzyme and the T206GS213G variant does not differ, the presence of the linker fragment stabilizes the domains interface, as evidenced by the monophasic transition of the mutant enzyme from folded to unfolded state during urea-induced denaturation. The high stability and activity of the mutant enzyme provides a rationale to use it as a biocatalyst in the industrial processes, where the enzyme must be more robust to fluctuations in the operational conditions.

  • effect of the increased stability of the Penicillin Amidase mrna on the protein expression levels
    FEBS Letters, 2005
    Co-Authors: Sandra C Viegas, Volker Kasche, Dorothea Schmidt, Cecilia M Arraiano, Zoya Ignatova
    Abstract:

    Several factors at transcriptional, post-transcriptional or post-translational level determine the fate of a target protein and can severely restrict its yield. Here, we focus on the post-transcriptional regulation of the biosynthesis of the periplasmic protein, Penicillin Amidase (PA). The PA mRNA stability was determined under depleted RNase conditions in strains carrying single or multiple RNase deletions. Single deletion of the endonuclease RNase E yielded, as the highest, a fourfold stabilization of the PA mRNA. This effect, however, was reduced twice at post-translational level. The RNase II, generating secondary exonucleolytic cleavages in the mRNA, although not significantly influencing the PA mRNA decay, led also to an increase of the amount of mature PA. The non-proportional correlation between increased mRNA longevity and amount of active enzyme propose that the rational strategies for yield improvement must be based on a simultaneous tuning of more than one yield restricting factor.

  • pro sequence and ca2 binding implications for folding and maturation of ntn hydrolase Penicillin Amidase from e coli
    Journal of Molecular Biology, 2005
    Co-Authors: Zoya Ignatova, Frank Wischnewski, Holger Notbohm, Volker Kasche
    Abstract:

    Penicillin Amidase (PA) is a bacterial periplasmic enzyme synthesized as a pre-pro-PA precursor. The pre-sequence mediates membrane translocation. The intramolecular pro-sequence is expressed along with the A and B chains but is rapidly removed in an autocatalytic manner. In extensive studies we show here that the pro-peptide is required for the correct folding of PA. Pro-PA and PA unfold via a biphasic transition that is more pronounced in the case of PA. According to size-exclusion chromatography and limited proteolysis experiments, the inflection observed in the equilibrium unfolding curves corresponds to an intermediate in which the N-terminal domain (A-chain) still possesses native-like topology, whereas the B-chain is unfolded to a large extent. In a series of in vitro experiments with a slow processing mutant pro-PA, we show that the pro-sequence in cis functions as a folding catalyst and accelerates the folding rate by seven orders of magnitude. In the absence of the pro-domain the PA refolds to a stable inactive molten globule intermediate that has native-like secondary but little tertiary structure. The pro-sequence of the homologous Alcaligenes faecalis PA can facilitate the folding of the hydrolase domain of Escherichia coli PA when added in trans (as a separate polypeptide chain). The isolated pro-sequence has a random structure in solution. However, difference circular dichroism spectra of native PA and native PA with pro-peptide added in trans suggest that the pro-sequence adopts an α-helical conformation in the context of the mature PA molecule. Furthermore, our results establish that Ca2+, found in the crystal structure, is not directly involved in the folding process. The cation shifts the equilibrium towards the native state and facilitates the autocatalytic processing of the pro-peptide.

  • Fragments of pro-peptide activate mature Penicillin Amidase of Alcaligenes faecalis.
    FEBS Journal, 2003
    Co-Authors: Volker Kasche, B Galunsky, Zoya Ignatova
    Abstract:

    Penicillin Amidase from Alcaligenes faecalis is a recently identified N-terminal nucleophile hydrolase, which possesses the highest specificity constant (kcat/Km) for the hydrolysis of benzylPenicillin compared with Penicillin Amidases from other sources. Similar to the Escherichia coli Penicillin Amidase, the A. faecalis Penicillin Amidase is maturated in vivo from an inactive precursor into the catalytically active enzyme, containing one tightly bound Ca2+ ion, via a complex post-translational autocatalytic processing with a multi-step excision of a small internal pro-peptide. The function of the pro-region is so far unknown. In vitro addition of chemically synthesized fragments of the pro-peptide to purified mature A. faecalis Penicillin Amidase increased its specific activity up to 2.3-fold. Mutations were used to block various steps in the proteolytic processing of the pro-peptide to obtain stable mutants with covalently attached fragments of the pro-region to their A-chains. These extensions of the A-chain raised the activity up to 2.3-fold and increased the specificity constants for benzylPenicillin hydrolysis mainly by an increase of the turnover number (kcat).

  • improvement of posttranslational bottlenecks in the production of Penicillin Amidase in recombinant escherichia coli strains
    Applied and Environmental Microbiology, 2003
    Co-Authors: Zoya Ignatova, A Mahsunah, M Georgieva, Volker Kasche
    Abstract:

    Escherichia coli is the most frequently used prokaryotic expression system for high-level expression of homologous and heterologous proteins. However, despite its advantages, it has been commonly observed that the overexpression of foreign proteins in E. coli triggers a large metabolic burden and leads to undesired metabolic responses, including changes in the central metabolism and regulatory functions (36), cell growth retardation (1), and ribosome destruction and cell death (8). The expression of heterologous genes in recombinant E. coli is associated with increased activity of the energy-generating dissimilatory pathway (41), and the fate of foreign proteins expressed in E. coli is determined in part by the degradative activities of the host cells (15). Moreover, the efficient expression of different genes in E. coli is not a routine matter, as the regulatory network of protein expression at the posttranslational level is very complex and must be considered in order to attain a high level of protein expression. Recent developments in DNA recombinant technology have contributed to significant progress in protein overexpression. Genetic manipulations improving biosynthesis at the transcriptional level or optimization of fermentation strategies developed to enhance the yield of recombinant proteins of interest lead to considerable increases in their production. Nevertheless, higher levels of production are still desired to satisfy the requirements for economically attractive process. Using an industrially important enzyme, Penicillin Amidase (PA), from E. coli ATCC 11105 as a model system, we investigated the posttranslational yield-limiting steps influencing its production and developed a biochemical engineering approach for enhancing its overexpression in recombinant E. coli strains. The nascent polypeptide precursor is synthesized as 96-kDa pre-pro-PA (ppPA), containing, at its N terminus, a signal peptide which mediates translocation into the periplasm via the Tat pathway (18). Thereafter, translocated pro-PA (pPA) is further processed into two chains (A, 23 kDa, and B, 63 kDa) by various intra- and intermolecular autoproteolytic reactions (16, 21). Due to this complex regulation mechanism for biosynthesis, PA serves as a good model system for studying the relative influences of all of these processes in order to develop efficient strategies for improving recombinant prokaryotic protein production. The emerging details of the structure and regulation of the pac gene (6, 32, 40) have allowed the manipulation of transcriptional and translational efficiencies via the development of appropriate host-vector systems (5). While PA activity was significantly improved, the processes leading to decreased yields posttranslationally were not completely understood. Yields of active PA are restricted by the following posttranslational processes: (i) nonspecific intracellular proteolytic degradation by host-specific cytoplasmic endopeptidases, (ii) inclusion body formation, (iii) transport through the cytoplasmic membrane, (iv) maturation, and (v) nonspecific proteolysis in the periplasm (17). The extent to which yields are affected by the posttranslational bottleneck steps mentioned above suggests strategies for improvement of the overexpression of a target protein. In the present report, both translocation and intracellular proteolysis were manipulated in order to increase PA expression at the molecular level in recombinant E. coli strains. Intracellular proteolysis was modulated either by the composition of the medium or by the host strain used for overexpression. The exchange of the original signal sequence of ppPA with the Sec-dependent OmpT signal peptide sequence varied in the different host strains. The overproduction of SecA, SecB, and SecF improved transport efficiency and enhanced up to threefold the level of expression of an OmpT-PA fusion in the periplasm. Parallel efforts were made to increase PA flux via coexpression with the kil gene (killing protein). The controlled release of PA into the extracellular medium, leading to increased PA production, was only temporary due to the decreased viability of the host cells.

B Galunsky - One of the best experts on this subject based on the ideXlab platform.

  • Improved A. faecalis Penicillin Amidase Mutant Retains the Thermodynamic and pH Stability of the Wild Type Enzyme
    The Protein Journal, 2010
    Co-Authors: Ruslan Yuryev, Zoya Ignatova, Volker Kasche, B Galunsky
    Abstract:

    Penicillin Amidase from Alacaligenes faecalis is an attractive biocatalyst for hydrolysis of Penicillin G for production of 6-aminopenicillanic acid, which is used in the synthesis of semi-synthetic β-lactam antibiotics. Recently a mutant of this enzyme with extended C-terminus of the A-chain comprising parts of the connecting linker peptide was constructed. Its turnover number for the hydrolysis of Penicillin G was 140 s^−1, about twice of the value for the wild-type enzyme (80 s^−1). At the same time the specificity constant was improved about three-fold. The wild-type and the mutant enzymes showed similar pH stability suggesting that the linker peptide fragment covalently attached to the A-chain does not alter the electrostatic interactions in the protein core. Although the global stability of A. faecalis wild-type enzyme and the T206GS213G variant does not differ, the presence of the linker fragment stabilizes the domains interface, as evidenced by the monophasic transition of the mutant enzyme from folded to unfolded state during urea-induced denaturation. The high stability and activity of the mutant enzyme provides a rationale to use it as a biocatalyst in the industrial processes, where the enzyme must be more robust to fluctuations in the operational conditions.

  • Kinetic comparison of Penicillin Amidase catalysed transfer of nonspecific and specific acyl moieties to 7-aminodeacetoxycephalosporanic acid
    Biocatalysis and Biotransformation, 2009
    Co-Authors: Nikolina Stambolieva, Zoia Mincheva, B Galunsky
    Abstract:

    Kinetic evidence for formation of an acylenzyme-nucleophile complex is presented for the Penicillin Amidase (EC 3.5.1.11) catalysed transfer of nonspecific (2-benzoxazolon-3-yl-acetyl) and specific (phenylacetyl) acyl moieties to 7-aminodeacetoxycephalosporanic acid. The specific and nonspecific acylenzymes differ in their binding constants for 7-aminodeacetoxy-cephalosporanic acid, in their values of transferase to hydrolase ratio, “specificity constants” and maximal yields in kinetically controlled cephem synthesis. The 2-benzoxazolon-3-yl-acetyl-Penicillin Amidase has a higher affinity for the nucleophile (KN=1.4mM) and the acylen-zyme-nucleophile complex formed is quantitatively converted into cephem, the deacylation by water being negligible. The value of transferase to hydrolase ratio is an order of magnitude higher compared to that for the specific acyl donor, while the “specificity constant” for the respective cephem synthesis is two orders of magnitude lower. The phenylacetyl-Penicillin Amidase h...

  • Fragments of pro-peptide activate mature Penicillin Amidase of Alcaligenes faecalis.
    FEBS Journal, 2003
    Co-Authors: Volker Kasche, B Galunsky, Zoya Ignatova
    Abstract:

    Penicillin Amidase from Alcaligenes faecalis is a recently identified N-terminal nucleophile hydrolase, which possesses the highest specificity constant (kcat/Km) for the hydrolysis of benzylPenicillin compared with Penicillin Amidases from other sources. Similar to the Escherichia coli Penicillin Amidase, the A. faecalis Penicillin Amidase is maturated in vivo from an inactive precursor into the catalytically active enzyme, containing one tightly bound Ca2+ ion, via a complex post-translational autocatalytic processing with a multi-step excision of a small internal pro-peptide. The function of the pro-region is so far unknown. In vitro addition of chemically synthesized fragments of the pro-peptide to purified mature A. faecalis Penicillin Amidase increased its specific activity up to 2.3-fold. Mutations were used to block various steps in the proteolytic processing of the pro-peptide to obtain stable mutants with covalently attached fragments of the pro-region to their A-chains. These extensions of the A-chain raised the activity up to 2.3-fold and increased the specificity constants for benzylPenicillin hydrolysis mainly by an increase of the turnover number (kcat).

  • Enzymic Synthesis Design and Enzymic Synthesis of Aspartame
    Tetrahedron, 2001
    Co-Authors: Ivanka Stoineva, B Galunsky, Valentin S. Lozanov, I. Ivanov, Dimiter D. Petkov
    Abstract:

    Abstract An enzymic synthesis of aspartame (H-Asp-Phe-OMe) has been designed and realized based on the structure-activity study of thermolysin and Penicillin Amidase hydrolysis of its p-substituted phenylacetyl derivatives. These compounds meet the structural and energetic requirements of two enzymic binding sites The peptide sweetener has been prepared by thermolysin - catalyzed condensation of the p-substituted phenylacetyl-Asp-OH and H-Phe-OMe followed by Penicillin Amidase - catalyzed deprotection of the resulted aspartame precursors.

  • ph dependence of Penicillin Amidase enantioselectivity for charged substrates
    Biochimica et Biophysica Acta, 1999
    Co-Authors: K Lummer, B Galunsky, A Rieks, Volker Kasche
    Abstract:

    Abstract The pH dependence of E (enantiomeric ratio or enantioselectivity, a quantitative measure for enzyme stereospecificity) was studied for Penicillin Amidase catalysed hydrolysis of charged enantiomeric substrates. Theoretical analysis shows that a pH dependence can only be observed around the pK values of groups in the active site whose ionisation control the enzyme activity. For charged substrates that may perturb these pK values, a pH dependence of E is also expected. This was experimentally verified around these pK values. The S′1-stereospecificity of Penicillin Amidase was studied for the hydrolysis of the enantiomeric phenylacetyl-S/R-Phe and for the racemic phenylacetyl-S,R-PhG. The S1-stereospecificity was investigated for the hydrolysis of the enantiomeric S/R-PhG-NH2. The observed pH modulation of E (more than 3-fold for the studied substrates in the pH range 4.5–9) was found to be a result of compensatory effects for binding and catalysis. The ratios kcat,S/kcat,R and Km,S/Km,R for the hydrolysis of the enantiomeric phenylacetyl-Phe were found to decrease from 1000 to 10 and from 0.1 to 0.01, respectively in the pH range 5–8. The dependence was stronger for the S′1- than for the S1-subsite. This is probably due to the stronger influence of the substrate carboxyl group in the S′1-subsite than that of the substrate amino group in the S1-subsite on the pK of the N-terminal Ser B1 that is essential for the activity. The observed pH dependence of E was used to discuss the importance of ground–state interactions for discrimination between enantiomers and for enzyme catalysis in general. The experimental results conform to the split site model according to which a better binding must not be fundamentally inhibitory.

Alexandra Cristina Blaga - One of the best experts on this subject based on the ideXlab platform.

  • Effect of bed configuration of immobilized biocatalysts on Penicillin G hydrolysis efficiency
    Korean Journal of Chemical Engineering, 2015
    Co-Authors: Ancairina Galaction, Ramona Mihaela Matran, Alexandra Cristina Blaga, Dan Cascaval
    Abstract:

    The external and internal mass transfer of Penicillin G in the process of its enzymatic hydrolysis to 6-Aminopenicillanic acid under competitive and non-competitive inhibitions have been comparatively analyzed for a bioreactor with mobile bed vs. a stationary basket bioreactor, both with Penicillin Amidase immobilized in Eupergit C. The Penicillin G mass transfer and hydrolysis enzymatic rates have been analyzed by means of the ratios’ values between the oxygen mass transfer coefficients, effectiveness factors, external mass flows and Penicillin G concentrations at the biocatalyst particle surface for the considered bioreactors. The results indicated that the bioreactor with mobile bed is more efficient especially for biocatalyst particles with diameter under 1.5 mm. For larger particles the performances of the two bioreactors become similar. Moreover, taking into consideration the external mass flow of Penicillin G and the number of enzymatic hydrolysis cycles, the basket bioreactor is recommended. The mathematical equations proposed are in good concordance with the experimental results, the average deviations varying from ±4.11% for the bioreactor with mobile bed of immobilized Penicillin Amidase to ±5.03% for the basket bioreactor.

  • engineering aspects of Penicillin g transfer and conversion to 6 aminopenicillanic acid in a bioreactor with a mobile bed of immobilized Penicillin Amidase
    Chemical Engineering Communications, 2014
    Co-Authors: Ancairina Galaction, Ramona Mihaela Matran, Marius Turnea, Alexandra Cristina Blaga, Dan Cascaval
    Abstract:

    This article presents studies on the external and internal mass transfers of Penicillin G for 6-aminopenicillanic acid enzymatic production using a bioreactor with a stirred bed of immobilized Penicillin Amidase. By means of the substrate mass balance for a single particle of biocatalyst and considering the kinetic model adapted for competitive and noncompetitive inhibitions, specific mathematical models were developed for describing the profiles of Penicillin G concentration in the outer and inner regions of biocatalyst and for estimating its mass flows in the liquid boundary layer surrounding the particle and inside the particle. The values of the mass flows are significantly influenced by the internal diffusion velocity and rate of the enzymatic conversion of substrate. These cumulated influences led to the appearance of an enzymatic inactive region near the particle center, its magnitude varying from 0 to 9.2% of the overall volume of particles.

  • 6 aminopenicillanic acid production in stationary basket bioreactor with packed bed of immobilized Penicillin Amidase Penicillin g mass transfer and consumption rate under internal diffusion limitation
    Biochemical Engineering Journal, 2012
    Co-Authors: Dan Cascaval, Ancairina Galaction, Marius Turnea, Alexandra Cristina Blaga
    Abstract:

    Abstract The external and internal mass transfers of Penicillin G in the process of its enzymatic hydrolysis to 6-Aminopenicillanic acid under competitive and non-competitive inhibitions using a bioreactor with stationary basket bed of immobilized Penicillin Amidase have been analyzed. By means of the Penicillin G mass balance for a single particle of biocatalysts, considering the specific kinetic model proposed by Warburton et al., mathematical expressions have been developed for describing the profiles of Penicillin G concentrations and mass flows in the outer and inner regions of biocatalyst particles, as well as for estimating the influence of internal diffusion on its hydrolysis rate. The results indicated that very low values of internal mass flow could be reached in the particles centre. The corresponding region was considered an “enzymatic inactive region”, its extent varying from 0 to 51% from the overall volume of each biocatalyst. By enzyme immobilization and using the basket bed, the rate of enzymatic reaction is reduced over 160 times compared to the process with free enzyme

  • 6-Aminopenicillanic acid production in stationary basket bioreactor with packed bed of immobilized Penicillin Amidase—Penicillin G mass transfer and consumption rate under internal diffusion limitation
    Biochemical Engineering Journal, 2012
    Co-Authors: Dan Cascaval, Ancairina Galaction, Marius Turnea, Alexandra Cristina Blaga
    Abstract:

    Abstract The external and internal mass transfers of Penicillin G in the process of its enzymatic hydrolysis to 6-Aminopenicillanic acid under competitive and non-competitive inhibitions using a bioreactor with stationary basket bed of immobilized Penicillin Amidase have been analyzed. By means of the Penicillin G mass balance for a single particle of biocatalysts, considering the specific kinetic model proposed by Warburton et al., mathematical expressions have been developed for describing the profiles of Penicillin G concentrations and mass flows in the outer and inner regions of biocatalyst particles, as well as for estimating the influence of internal diffusion on its hydrolysis rate. The results indicated that very low values of internal mass flow could be reached in the particles centre. The corresponding region was considered an “enzymatic inactive region”, its extent varying from 0 to 51% from the overall volume of each biocatalyst. By enzyme immobilization and using the basket bed, the rate of enzymatic reaction is reduced over 160 times compared to the process with free enzyme

T. Panda - One of the best experts on this subject based on the ideXlab platform.

  • Effect of mode of operation of bioreactors on the biosynthesis of Penicillin Amidase in Escherichia coli
    Bioprocess Engineering, 1996
    Co-Authors: T. Panda
    Abstract:

    Different operational mode of bioreactors influence the biosynthesis of the enzyme and related products as well as the growth of industrial microorganisms. This communication deals with the effect of mode of operation of various bioreactors with different geometric configurations, viz., batch (includes commercially available batch stirred tank, and custom-designed cylindrical and tapered reactors), batch-fed, continuous flow stirred tank reactors on the biosynthesis of Penicillin Amidase in Escherichia coli. Experimental findings show that the biosynthesis of Penicillin Amidase in E. coli show a little variation among batch reactor modes and significant variation on the continuous mode of operation. Further analysis show that the different reactor modes also influence periplasmic localization of the enzyme in the cell.

  • Release of periplasmic Penicillin Amidase from Escherichia coli by chloroform shock
    Bioprocess Engineering, 1996
    Co-Authors: K. Jagannadha Rao, T. Panda
    Abstract:

    Penicillin Amidase is a periplasmic enzyme in Escherichia coli. Conventionally, the periplasmic enzymes are released into the medium by osmotic shock which is tedious involving a number of centrifugation steps. The present communication deals with a simple technique for the release of Penicillin Amidase by chloroform shock. Experimental findings show that the periplasmic Penicillin Amidase does not show any variation by the chloroform treatment. This analysis was also extended to the E. coli cells grown at various concentrations of phenylacetic acid, optimal concentration of phenylacetic acid plus glucose and lactic acid.

  • studies on improved techniques for immobilizing and stabilizing Penicillin Amidase associated with e coli cells
    Enzyme and Microbial Technology, 1991
    Co-Authors: P S R Babu, T. Panda
    Abstract:

    Abstract A method for catalyst development has been suggested for immobilizing whole E. coli cells containing Penicillin Amidase. Conventional methods have limitations, such as permeation of substrate and product through cellular membranes, leaching of protein and other cellular components into the reaction phase, lower specific activity compared to immobilized enzyme system, etc. The whole cell immobilization technique has been optimized for different process parameters. The most suitable conditions for this process were pH, 4.25; cell concentration, 3.75%; concentration of glutaraldehyde, 1.5%; level of bovine serum albumin as additional support, 2 mg ml −1 . The reaction was continued for 2 h. The granular catalyst has good mechanical strength, low protein leachability, and high retention of Penicillin Amidase activity .

  • immobilization of whole escherichia coli containing Penicillin Amidase using cross linking agents and fillers
    Biotechnology Techniques, 1991
    Co-Authors: P S R Babu, T. Panda
    Abstract:

    To obtain a catalyst with good mechanical stress stability and other operational characteristics, cross-linked aggregates of whole Escherichia coli containing Penicillin Amidase were reinforced with surface modified precipitated silica and chitosan. The immobilized cells plus mixed fillers possess better performance characteristics i.e. higher stability at 4°C and 30°C, least protein leaching capacity and good settling characteristics. Most suitable conditions to prepare catalyst with mixed fillers were: chitosan, 0.3: silica, 0.2 g/g d.wt cell; and minimum moisture content in catalyst, 30% (w/w).

  • effect of recycling of fermentation broth for the production of Penicillin Amidase
    Process Biochemistry, 1991
    Co-Authors: P S R Babu, T. Panda
    Abstract:

    Abstract In general in any fermentation the volume of wastewater is too high. The excess water might be utilized to reduce consumption of process water. In this investigation cell-free culture filtrate was used instead of process water. In batch experiments, cell-free culture filtrate was used in ratios between 0·1 and 1·0 for eight subsequent batches. The specific growth rate was higher than that of the first batch for reusability ratios between 0·1 and 0·8 in all batches. Synthesis of Penicillin Amidase was found to be more suitable in the fourth batch with a recycle ratio between 0·6 and 1·0. An extracellular protein factor was found to influence the synthesis of Penicillin Amidase.