Optimization of glutaryl-7-aminocephalosporanic acid acylase expression in E. coli

Volontè, Federica; Marinelli, Flavia; Gastaldo, Luciano; Sacchi, Silvia; Pilone, Mirella S.; Pollegioni, Loredano; Molla, Gianluca

doi:10.1016/j.pep.2008.05.010

Cited by 72 publications

(48 citation statements)

References 22 publications

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“…For other proteins described in the literature [23], and pneumolysin from S. pneumoniae expressed in the same system as PsaA [14], tryptone had a positive effect on protein production in E. coli. The enhancement of recombinant protein expression in E. coli with a higher yeast extract concentration was also described in other works using similar shake flask conditions and in the same range of concentrations in the culture medium [4,29,30]. Chen et al [4] reported that yeast extract was effective as a nitrogen source to improve recombinant protein expression.…”

Section: Model Analysismentioning

confidence: 71%

“…Recently, researchers have given more attention to the effect of culture medium on protein expression, and more articles have been published that make use of experimental design tools. Experimental design has been used by other research groups to optimize the culture medium composition for recombinant protein expression in E. coli [4,6,9,11,16,19,20,23,29,30] or seed conditions and inoculum level/density [11,21,27]. Table 2, and estimated effects of each variable and p values in Table 3.…”

Section: First Designmentioning

confidence: 99%

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Optimization of medium formulation and seed conditions for expression of mature PsaA (pneumococcal surface adhesin A) in Escherichia coli using a sequential experimental design strategy and response surface methodology

Larentis

Nicolau

Argondizzo

et al. 2012

Journal of Industrial Microbiology and Biotechnology

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PsaA, a candidate antigen for a vaccine against pneumonia, is well-conserved in all Streptococcus pneumoniae serotypes. A sequence of two-level experimental designs was used to evaluate medium composition and seed conditions to optimize the expression of soluble mature PsaA in E. coli. A face-centered central composite design was first used to evaluate the effects of yeast extract (5 and 23.6 g/L), tryptone (0 and 10 g/L), and glucose (1 and 10 g/L), with replicate experiments at the central point (14.3 g/L yeast extract, 5 g/L tryptone, 5.5 g/L glucose). Next, a central composite design was used to analyze the influence of NaCl concentration (0, 5, and 10 g/L) compared with potassium salts (9.4 g/L K(2)HPO(4)/2.2 g/L KH(2)PO(4)), and seed growth (7 and 16 h). Tryptone had no significant effect and was removed from the medium. Yeast extract and glucose were optimized at their intermediate concentrations, resulting in an animal-derived material-free culture medium containing 15 g/L yeast extract, 8 g/L glucose, 50 μg/mL kanamycin, and 0.4% glycerol, yielding 1 g/L rPsaA after 16 h induction at 25°C in shake flasks at 200 rpm. All the seed age and salt conditions produced similar yields, indicating that no variation had a statistically significant effect on expression. Instead of growing the seed culture for 16 h (until saturation), the process can be conducted with 7 h seed growth until the exponential phase. These results enhanced the process productivity and reduced costs, with 5 g/L NaCl being used rather than potassium salts.

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Section: Model Analysismentioning

confidence: 71%

Section: First Designmentioning

confidence: 99%

Optimization of medium formulation and seed conditions for expression of mature PsaA (pneumococcal surface adhesin A) in Escherichia coli using a sequential experimental design strategy and response surface methodology

Larentis

Nicolau

Argondizzo

et al. 2012

Journal of Industrial Microbiology and Biotechnology

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“…variants were screened by a simplified 96-well microplate factorial design approach (10). Four different E. coli strains (E. coli BL21(DE3), BL21(DE3)pLysS, Origami2(DE3), and Rosetta2(DE3)pLysS strains, Novagen) and two media (LB or modified TB, to which 10 g/liter glycerol was added) were used.…”

Section: Screening Of Expression Conditions For Pmalaad Wild-type Andmentioning

confidence: 99%

“…The sequence alignment has been performed using the ClustalW program and manually corrected based on their three-dimensional structure comparisons. PmaLAAD-01N secondary structure elements are shown on the top of the alignment and shaded in gray (yellow for 3 10 helices) for all aligned proteins. Asterisks and circles indicate PmaLAAD-01N residues interacting with the FAD isoalloxazine ring and with anthranilate, respectively.…”

Section: յ008 S ϫ1mentioning

confidence: 99%

Structure-Function Relationships in l-Amino Acid Deaminase, a Flavoprotein Belonging to a Novel Class of Biotechnologically Relevant Enzymes

Motta

Molla

Pollegioni

et al. 2016

Journal of Biological Chemistry

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L-Amino acid deaminase from Proteus myxofaciens (Pma-LAAD) is a membrane flavoenzyme that catalyzes the deamination of neutral and aromatic L-amino acids into ␣-keto acids and ammonia. PmaLAAD does not use dioxygen to re-oxidize reduced FADH 2 and thus does not produce hydrogen peroxide; instead, it uses a cytochrome b-like protein as an electron acceptor. Although the overall fold of this enzyme resembles that of known amine or amino acid oxidases, it shows the following specific structural features: an additional novel ␣؉␤ subdomain placed close to the putative transmembrane ␣-helix and to the active-site entrance; an FAD isoalloxazine ring exposed to solvent; and a large and accessible active site suitable to bind large hydrophobic substrates. In addition, PmaLAAD requires substrate-induced conformational changes of part of the active site, particularly in Arg-316 and Phe-318, to achieve the correct geometry for catalysis. These studies are expected to pave the way for rationally improving the versatility of this flavoenzyme, which is critical for biocatalysis of enantiomerically pure amino acids.A variety of enzymes has been used to prepare chiral pharmaceutical and agricultural compounds containing enantiomeric amine or amino acid groups. Among these are aminotransferases (EC 2.6.4.X), lipases (EC 3.1.1.X), amine oxidases (EC 1.4.3.22), amino acid dehydrogenases (EC 1.4.1.X), and amino acid oxidases (EC 1.4.3.X) (1, 2).The deracemization of a racemic amino acid to obtain the L-configuration was achieved by using a stereoselective Damino acid oxidase (DAAO, 5 EC 1.4.3.3) followed by chemical reduction. The second step iteratively converts the imino acid produced (from the D-amino acid) back into a DL-mixture to obtain the full resolution of the racemic mixture into the L-enantiomer (1). This approach requires stable recombinant DAAOs possessing wide substrate specificity as well as variants engineered to act on synthetic amino acids (3). Amino acid oxidases with reverse stereoselectivity are also well known flavooxidases, mainly produced by snakes or by microorganisms. In particular, L-amino acid oxidases (LAAO, EC 1.4.3.2) catalyze the stereoselective oxidative deamination of L-amino acids into the corresponding ␣-keto acids and ammonia; the re-oxidation of FADH 2 by dioxygen then generates H 2 O 2 (4). These flavoenzymes catalyze an irreversible reaction (differently from aminotransferases) and do not require a specific step of cofactor regeneration, as otherwise required by the NAD-dependent dehydrogenases. However, because of the problems associated with overexpression of snake venom LAAOs in recombinant hosts and the limited substrate acceptance of the microbial counterparts, no appropriate LAAOs for biocatalysis are available (4).L-Amino acid deaminases (LAADs) represent a suitable alternative to LAAOs. LAAD (first identified in the genera Proteus, Providencia, and Morganella) catalyzes the deamination of the L-isomer of amino acids, yielding the corresponding ␣-keto acids and ammonia without any evide...

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“…Because of the environmental and safety concerns, enzymatic conversion of CPC has long been explored as a substitute for chemical methods [2]. The enzymatic conversion of CPC to 7-ACA was carried out by two methods [3]: (i) one-step process using cephalosporin C acylase which effectively uses CPC as substrate; however, the catalytic efficiency of these enzymes is weak thus their application is limited; (ii) two-step process comprising the conversion of CPC into GL 7-ACA, using DAAO, and its subsequent hydrolysis to 7-ACA by a GLA which have been widely applied in 7-ACA industrial production [4]. The genes coding for GLA from several Pseudomonas species have been expressed in E. coli and the enzymes were biochemically analyzed.…”

Section: Introductionmentioning

confidence: 99%

Expression of Glutaryl7-Aminocephalosporanic Acid Acylase in Escherichia Coli Bl21(de3) and Immobilization of Recombinant Enzyme on Nanoporous Materials

Hanh

2018

JST

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The synthesis of 7-ACA from cephalosporin C (CPC) by a two-step bioconversion using D-amino acid oxidase (DAAO) and glutaryl 7-ACA acylase (GLA) has been effectively and largely applied in pharmaceutical industry. In this study, the gene gla coding for 720-amino acid GLA from plasmid pUC57::gla was analyzed and successfully inserted into vector pET22b(+) to form expression vector pET22b(+)::gla. The newly constructed expression vector pET22b(+)::gla was cloned and then transformed into Escherichia coli BL21(DE3) to generate recombinant strain E. coli BL21(DE3)[pET22b(+)::gla]. The suitable conditions for expression of gla gene were in LB medium at 30 o C and induced by 0.4 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 hours. Under the chosen culturing parameters, expression of gla gene by E. coli BL21(DE3)/[pET22b(+)::gla] resulted in a recombinant GLA (rGLA) with molecular weight of 83 kDa and catalytic activity of 2.7 U/mg of total protein. Experimental research on immobilization of rGLA onto ten nanoporous materials were showed that, SBA-15 was the best one for immobilization of rGLA, reaching activity of immobilized enzyme of 22.2 U/g matrix. Furthermore, optimal conditions of procedure for immobilizing rGLA on nanomaterials (SBA-15) were determined as follows: temperature is 25 C, pH7.0 and immobilization time -60 minutes. Therefore the results reported in this study revealed the successfully heterologous expression of GLA in recombinant E. coli and potential immobilization of enzyme on inorganic nano-materials.

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Optimization of glutaryl-7-aminocephalosporanic acid acylase expression in E. coli

Cited by 72 publications

References 22 publications

Optimization of medium formulation and seed conditions for expression of mature PsaA (pneumococcal surface adhesin A) in Escherichia coli using a sequential experimental design strategy and response surface methodology

Optimization of medium formulation and seed conditions for expression of mature PsaA (pneumococcal surface adhesin A) in Escherichia coli using a sequential experimental design strategy and response surface methodology

Structure-Function Relationships in l-Amino Acid Deaminase, a Flavoprotein Belonging to a Novel Class of Biotechnologically Relevant Enzymes

Expression of Glutaryl7-Aminocephalosporanic Acid Acylase in Escherichia Coli Bl21(de3) and Immobilization of Recombinant Enzyme on Nanoporous Materials

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