High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicability
Само за регистроване кориснике
2006
Аутори
Šenerović, LidijaStanković, Nada
Spizzo, P
Basso, A
Gardossi, L
Vasiljević, Branka
Ljubijankić, G
Tisminetzky, S
Degrassi, G
Чланак у часопису (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri (rPAC(P).(rett)) of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEN gene encoding for proteinase A, resulting in an increased rPAC(P.rett) activity of approximately 40% (3.8 U/mL vs. 2.7 U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPAC(P.rett) purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPAC(P.rett) expressed in P. pastoris were fully exploited and emphasized through the covalent immobilization of rPAC(P.rett). The enzyme was immob...ilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPAC(P.rett) performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability gt 3 years and specific activity ranging from 237 to 477 U/g (dry) based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPAC(P.rett) was finally evaluated by studying the kinetically controlled synthesis of P-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process.
Кључне речи:
recombinant penicillin G acylase / Providencia rettgeri / Pichia pastoris / enzymatic synthesis of cephalosporins / covalent immobilizationИзвор:
Biotechnology and Bioengineering, 2006, 93, 2, 344-354Издавач:
- Wiley, Hoboken
DOI: 10.1002/bit.20728
ISSN: 0006-3592
PubMed: 16259000
WoS: 000234773400016
Scopus: 2-s2.0-33645557358
Институција/група
Institut za molekularnu genetiku i genetičko inženjerstvoTY - JOUR AU - Šenerović, Lidija AU - Stanković, Nada AU - Spizzo, P AU - Basso, A AU - Gardossi, L AU - Vasiljević, Branka AU - Ljubijankić, G AU - Tisminetzky, S AU - Degrassi, G PY - 2006 UR - https://imagine.imgge.bg.ac.rs/handle/123456789/255 AB - A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri (rPAC(P).(rett)) of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEN gene encoding for proteinase A, resulting in an increased rPAC(P.rett) activity of approximately 40% (3.8 U/mL vs. 2.7 U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPAC(P.rett) purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPAC(P.rett) expressed in P. pastoris were fully exploited and emphasized through the covalent immobilization of rPAC(P.rett). The enzyme was immobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPAC(P.rett) performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability gt 3 years and specific activity ranging from 237 to 477 U/g (dry) based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPAC(P.rett) was finally evaluated by studying the kinetically controlled synthesis of P-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process. PB - Wiley, Hoboken T2 - Biotechnology and Bioengineering T1 - High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicability EP - 354 IS - 2 SP - 344 VL - 93 DO - 10.1002/bit.20728 ER -
@article{ author = "Šenerović, Lidija and Stanković, Nada and Spizzo, P and Basso, A and Gardossi, L and Vasiljević, Branka and Ljubijankić, G and Tisminetzky, S and Degrassi, G", year = "2006", abstract = "A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri (rPAC(P).(rett)) of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEN gene encoding for proteinase A, resulting in an increased rPAC(P.rett) activity of approximately 40% (3.8 U/mL vs. 2.7 U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPAC(P.rett) purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPAC(P.rett) expressed in P. pastoris were fully exploited and emphasized through the covalent immobilization of rPAC(P.rett). The enzyme was immobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPAC(P.rett) performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability gt 3 years and specific activity ranging from 237 to 477 U/g (dry) based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPAC(P.rett) was finally evaluated by studying the kinetically controlled synthesis of P-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process.", publisher = "Wiley, Hoboken", journal = "Biotechnology and Bioengineering", title = "High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicability", pages = "354-344", number = "2", volume = "93", doi = "10.1002/bit.20728" }
Šenerović, L., Stanković, N., Spizzo, P., Basso, A., Gardossi, L., Vasiljević, B., Ljubijankić, G., Tisminetzky, S.,& Degrassi, G.. (2006). High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicability. in Biotechnology and Bioengineering Wiley, Hoboken., 93(2), 344-354. https://doi.org/10.1002/bit.20728
Šenerović L, Stanković N, Spizzo P, Basso A, Gardossi L, Vasiljević B, Ljubijankić G, Tisminetzky S, Degrassi G. High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicability. in Biotechnology and Bioengineering. 2006;93(2):344-354. doi:10.1002/bit.20728 .
Šenerović, Lidija, Stanković, Nada, Spizzo, P, Basso, A, Gardossi, L, Vasiljević, Branka, Ljubijankić, G, Tisminetzky, S, Degrassi, G, "High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicability" in Biotechnology and Bioengineering, 93, no. 2 (2006):344-354, https://doi.org/10.1002/bit.20728 . .