Codon optimized Thermobifida fusca hydrolase secreted by Bacillus megaterium
ABSTRACT Production and secretion of a 28,172 Da hydrolase from Thermobifida fusca (TFH) in Bacillus megaterium MS941 and WH323 was investigated in shake flask and pH controlled bioreactors. Successful production of heterologous TFH was achieved by adapting the original tfh gene to the optimal codon usage of B. megaterium. A codon adaption index close to one was reached. The codon optimized tfh was cloned into an open reading frame with DNA sequence for the N-terminal signal peptide of B. megaterium lipase A and a C-terminal His(6)-tag, all under the control of a xylose inducible promoter. Successful TFH production and secretion were observed using batch reactor cultivations with complex medium. Expression of the tfh gene from the P(xylA) promoter and secretion of produced TFH were compared in detail to batch reactor cultivations with semi-defined growth medium. For the first time, significant TFH secretion was achieved using a semi-defined medium in glucose limited fed batch cultivations yielding 10-fold higher cell densities compared to LB medium cultivation. Comparable volumetric TFH activities were obtained for both cultivation strategies. Surprisingly, measured specific TFH activities exhibited drastic discrepancies between preparations from LB and semi-defined medium grown B. megaterium. TFH recovery by Ni-chelate affinity chromatography resulted in higher purification factors when LB medium was used. These results indicated that secreted TFH is favorably produced by batch cultures of B. megaterium WH323 in LB medium.
- SourceAvailable from: Ren Wei
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- "E. coli BL21- Gold(DE3) pET- 20b(+) α- hemolysin secretion pathway Tfu_0883 (Su et al., 2012) Bacillus megaterium MS941 / WH323 pYYBm LipA of B. megaterium TfH C-terminal (Fürch et al., 2007; Yang et al., 2007 "
ABSTRACT: Abstract Thermophilic actinomycetes produce enzymes capable of hydrolyzing synthetic polyesters such as polyethylene terephthalate (PET). In addition to carboxylesterases, which have hydrolytic activity predominantly against PET oligomers, esterases related to cutinases also hydrolyze synthetic polymers. The production of these enzymes by actinomycetes as well as their recombinant expression in heterologous hosts is described and their catalytic activity against polyester substrates is compared. Assays to analyze the enzymatic hydrolysis of synthetic polyesters are evaluated, and a kinetic model describing the enzymatic heterogeneous hydrolysis process is discussed. Structure鈥揻unction and structure鈥搒tability relationships of actinomycete polyester hydrolases are compared based on molecular dynamics simulations and recently solved protein structures. In addition, recent progress in enhancing their activity and thermal stability by random or site-directed mutagenesis is presented.Advances in applied microbiology 08/2014; 89:267-305. DOI:10.1016/B978-0-12-800259-9.00007-X · 2.24 Impact Factor
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- "The thermophiles Thermobifida fusca and T. alba have been isolated from composts and are potent degraders of polyesters such as aliphatic-co-aromatic polyesters (Kleeberg et al. 1998; Hu et al. 2008; Sinsereekul et al. 2010). The genes for polyesterhydrolyzing enzymes from T. fusca and T. alba have been cloned and determined to encode serine hydrolases (Kleeberg et al. 2005; Dresler et al. 2006; Yang et al. 2007: Chen et al. 2008; Hu et al. 2010) that belong to the lipase/esterase family (Arpigny and Jäger 1999). The complete genome of T. fusca YX has been disclosed (Lykidis et al. 2007), and the genome includes two tandem genes that encode extracellular lipases. "
ABSTRACT: Recombinant polyesterase (Est119) from Thermobifida alba AHK119 was purified by two chromatography steps. The final protein was observed as a single band in SDS-PAGE, and the specific activity of Est119 for p-nitrophenyl butyrate was 2.30 u/mg. Purified Est119 was active with aliphatic and aliphatic-co-aromatic polyesters. Kinetic data indicated that p-nitrophenyl butyrate (pNPB) or hexanoate was the best substrate for Est119 among p-nitrophenyl acyl esters. Calcium was required for full activity and thermostability of Est119, which was stable at 50 °C for 16 h. Three-dimensional modeling and biochemical characterization showed that Est119 is a typical cutinase-type enzyme that has the compact ternary structure of an α/β-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second β-sheets (A68V had the greatest effect). Introduction of a proline residue (S219P) in a predicted substrate-docking loop increased the thermostability. The specific activity of the A68V/S219P mutant on pNPB was increased by more than 50-fold over the wild type. The mutant was further activated by 2.6-fold (299 u/mg) with 300 mM Ca(2+) and was stable up to 60 °C with 150 mM Ca(2+). Another identical gene was located in tandem in the upstream of est119.Applied Microbiology and Biotechnology 12/2011; 95(2):419-30. DOI:10.1007/s00253-011-3781-6 · 3.81 Impact Factor
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- "For particular stages of medium optimization different media compositions were used. For carbon source screening, the medium composition was based on A5 medium  with the following composition: "
ABSTRACT: Bacillus megaterium was used as an alternative high potential microbial production system for the production of antibody fragment D1.3 scFv. The aim of the study was to follow a holistic optimization approach from medium screening in small scale microtiter platforms, gaining deeper process understanding in the bioreactor scale and implementing advanced process strategies at larger scales (5-100 L). Screening and optimization procedures were supported by statistical design of experiments and a genetic algorithm approach. The process control relied on a soft-sensor for biomass estimation to establish a μ-oscillating time-dependent fed-batch strategy. Several cycles of growth phases and production phases, equal to starving phases, were performed in one production. Flow cytometry was used to monitor and characterize the dynamics of secretion and cell viability. Besides the biosynthesis of the product, secretion was optimized by an appropriate medium design considering different carbon sources, metal ions, (NH(4))(2)SO(4), and inductor concentrations. For bioprocess design, an adapted oscillating fed-batch strategy was conceived and successfully implemented at an industrially relevant scale of 100 L. In comparison to common methods for controlling fed-batch profiles, the developed process delivered increased overall productivities. Thereby measured process parameters such as growth stagnation or productivity fluctuations were directly linked to single cell or population behavior leading to a more detailed process understanding. Above all, the importance of single cell analysis as key scale-free tool to characterize and optimize recombinant protein production is highlighted, since this can be applied to all development stages independently of the cultivation platform.Biotechnology Journal 12/2011; 6(12):1516-31. DOI:10.1002/biot.201000417 · 3.71 Impact Factor