Efficient extracellular recombinant production and purification of a Bacillus cyclodextrin glucanotransferase in Escherichia coli

Background: Cyclodextrin glucanotransferases (CGTases) catalyze the synthesis of cyclodextrins, cyclic oligosaccharides composed of glucose monomers that find applications in the pharmaceutical, food, and cosmetic industries. An economic application of these industrially important enzymes requires their efficient production and recovery. In this study, the effect of Sec-type signal peptides on the recombinant expression of a CGTase derived from Bacillus sp. G825-6 was investigated in Escherichia coli BL21(DE3) using a codon-adapted gene. In addition, a novel purification method for the CGTase using starch adsorption was developed. Results: Expression vectors encoding N-terminal PelB, DacD, and the native Bacillus sp. G825-6 CGTase signal peptides (SP) were constructed for the recombinant CGTase. With the DacD SP derived from E. coli, a 3.9- and 3.1-fold increase in total enzyme activity was obtained compared to using the PelB and the native CGTase SP, respectively. DacD enabled a 7.3-fold increase of activity in the extracellular fraction after induction for 24 h compared to the native CGTase SP. After induction for 48 h, 75% of the total activity was detected in the extracellular fraction. By a batch wise adsorption to starch, the extracellular produced CGTase could be purified to homogeneity with a yield of 46.5% and a specific activity of 1637 U/mg. Conclusions: The signal peptide DacD promoted the high-level heterologous extracellular expression of a recombinant CGTase from Bacillus sp. G825-6 with a pET20b(+) vector in E. coli BL21(DE3). A protocol based on starch adsorption enabled a fast and efficient purification of the recombinant enzyme

Effect of tomato variety, cultivation, climate and processing on Sola l 4, an allergen from <i>Solanum lycopersicum</i>

<div><p>Tomatoes (<i>Solanum lycopersicum</i>) are one of the most consumed vegetables worldwide. However, tomato allergies in patients suffering from birch pollen allergy occur frequently. Due to highly similar protein structures of the tomato allergen Sola l 4 and the major birch pollen allergen Bet v 1, patients cross-react with allergenic proteins from tomato as well as other fruits or vegetables. The aim of this study was to quantify Sola l 4 in various tomatoes differing in color, size and shape for identification of varieties with a reduced allergen level. Therefore, an indirect competitive ELISA using a specific polyclonal Sola l 4 antibody was developed. In addition, two varieties, both cultivated either conventionally or organically and furthermore dried with different methods, were analyzed to investigate the influence of the cultivation method and processing techniques on Sola l 4 level. Within 23 varieties, Sola l 4 content varied significantly between 0.24 and 1.71 μg Sola l 4/g FW. The tomato cultivars Rugantino and Rhianna showed the significantly lowest level, whereas in cultivars Farbini and Bambello the significantly highest concentration was determined. Drying of tomatoes in the oven and by sun resulted in a significant decrease. The thermal instability was verified for the recombinant Sola l 4 emphasizing the results for the native protein in dried tomato samples. Overall, the Sola l 4 content is cultivar-dependent and no correlation between color and Sola l 4 amount was found. During the drying process of tomatoes Sola l 4 level was significantly reduced due to thermal instability. Growing conditions have a minor effect whereas seasonal effects show a more pronounced impact. These findings could extend the knowledge about the allergen level of different tomato varieties and may help to improve food safety to potentially increase the life quality of patients suffering from birch pollen allergy.</p></div

Heat stability of soluble recombinant Sola l 4.02.

<p>(A) SDS-PAGE and (B) Western-Blot analysis of purified pooled elution fractions of the recombinant Sola l 4.02 protein heated for 10, 20, 30, 60 and 90 min at 99 °C. Untreated protein served as control (0). SDS-PAGE was performed under reducing conditions. Coomassie Brilliant Blue G250 was used for protein staining. For Western blot analysis specific polyclonal Sola l 4-antibody was used. M: PageRuler Prestained Protein Ladder.</p

SDS-PAGE analysis of the recombinant Sola l 4.02 protein.

<p>(A) Purification from soluble protein fraction, SDS-PAGE under reducing condition with ß-mercaptoethanol (A1 crude extract; A2 flow through; A3 washing; A4 elution 1; A5 elution 2; A6 elution 3). (B) insoluble protein fractions, SDS-PAGE under non-reducing conditions (B1 denaturation; B2 refolding; B3 flow through; B4 elution 1; B5 elution 2; B6 elution 3). (C) insoluble protein fraction (C1 pooled elution 1–3). Under non-reducing conditions, (B) two distinct protein bands were visible in the elution fractions at 18 kDa and 36 kDa. Under reducing condition with ß-mercaptoethanol (A, C) only one band at approximately 18 kDa appeared. Five μg protein per lane were visualized by Coomassie Brilliant Blue G250. M: PageRuler Plus Prestained Protein Ladder.</p

Sola l 4 content in dried and fresh tomatoes.

<p>(A) Allergen content in μg Sola l 4/g fresh weight (FW) of tomato cultivars SAAB and (B) Perbruzzo determined with indirect competitive ELISA. Plants were grown in Italy in 2015 and 2016 conventionally with artificial mulch (conv), organically with artificial mulch (org) and organically with natural mulch (norg). Tomato fruits were dried via freeze-drying (freeze), in the oven (oven) and in the sun (solar). Allergen content of dried tomato samples was referred to μg Sola l 4/g FW and compared with fresh tomatoes (fresh). Significant differences for each group were calculated at a significance level of 5%.</p

Sola l 4 content in different tomato cultivars.

<p>(A) Diversity of tomato cultivars (bar = 2 cm) and (B) corresponding Sola l 4 content in μg/g fresh weight (FW) determined with indirect competitive ELISA. The color of the box plots corresponds to the color of the ripe tomato fruit. Significant differences for each cultivar were calculated at a significance level of 5%.</p

Meteorological data at the growing location Monsampolo del Tronto, Italy for the years 2015 and 2016.

<p>Meteorological data at the growing location Monsampolo del Tronto, Italy for the years 2015 and 2016.</p

Effect of cultivation and drying method on Sola l 4 content in dried tomatoes.

<p>(A) Allergen content in μg Sola l 4/g dry weight (DW) of tomato cultivars SAAB and (B) Perbruzzo determined with indirect competitive ELISA. Plants were grown in Italy in 2015 and 2016 conventionally with artificial mulch (conv), organically with artificial mulch (org) and organically with natural mulch (norg). Tomato fruits were dried via freeze-drying (freeze), in the oven (oven) and in the sun (solar). Significant differences for each group were calculated at a significance level of 5%.</p

The chloroplastic UGT72A2 glycosylating naringenin is involved in leaf redox homeostasis in poplar

Flavonoids scavenge reactive species produced during photosynthesis and protect plant cells against deleterious oxidative damages. Their biosynthesis and conjugations are therefore under tight homeostasis at several levels. Glycosylation modifies the subcellular repartition of these metabolites, regulating the flux of their formation. This contribution presents a functional analysis of a poplar chloroplastic UDP-glycosyltransferase (UGT) involved in the homeostasis of flavonoids. Recombinant UGT72A2 specifically glucosylates naringenin, a flavanone precursor of most other flavonoids. Molecular docking of UGT72A2 with various flavonoids highlights key residues interacting with UDP-glucose and naringenin, paving the way for future targeted mutagenesis. UGT72A2 is trans-activated by flavonoid regulators MYB119 and TT8. Leaves of transgenic poplar lines with reduced expression of UGT72A2 are characterised by necrosis and premature abscission typical of oxidative damages under standard growing conditions. Lower pool of phenolics and flavonoids, lower soluble peroxidase activity and impaired homeostasis of NADPH and NADP+ pools in the leaves of RNAi lines reflect the decreased efficiency of systems scavenging reactive species. Poplar lines overexpressing UGT72A2 accumulate more phenolics and flavonoids. Finally, leaves of RNAi lines are less sensitive to oxidative stress caused by methyl viologen, which may suggest a retrograde signalling from damaged chloroplasts to mitigate the consequences of deficient redox homeostasis. This study highlights naringenin glucosylation as a metabolic node in the biosynthesis of flavonoids and points to its important role in buffering redox status under standard growing conditions in poplar.Présentation oraleinfo:eu-repo/semantics/nonPublishe

The chloroplastic UGT72A2 glycosylating naringenin is involved in leaf redox homeostasis in poplar

Flavonoids in leaves scavenge reactive species produced during photosynthesis and protect plant cells against deleterious oxidative damages. Their biosynthesis and conjugations are therefore under tight homeostasis at several levels. Glycosylation modifies the subcellular repartition of these metabolites, regulating the flux of their formation. This contribution presents a functional in planta analysis of a poplar chloroplastic UDP-glycosyltransferase (UGT) involved in the homeostasis of flavonoids. Recombinant UGT72A2 specifically glucosylates naringenin, a flavanone precursor of most other flavonoids. Molecular docking of UGT72A2 with various flavonoids highlights key residues interacting with UDP-glucose and naringenin, paving the way for future targeted mutagenesis. Leaves of transgenic poplar lines with reduced expression of UGT72A2 are characterised by necrosis and premature abscission typical of oxidative damages under normal growing conditions. Lower pool of phenolics and flavonoids, lower soluble peroxidase activity and impaired homeostasis of NADPH and NADP+ pools in the leaves of RNAi lines reflect the decreased efficiency of systems scavenging reactive species. Poplar lines overexpressing UGT72A2 accumulate more phenolics and flavonoids. Finally, leaves of RNAi lines are less sensitive to oxidative stress caused by methyl viologen, which may suggest a retrograde signalling from damaged chloroplasts to mitigate the consequences of deficient redox homeostasis. This study highlights naringenin glucosylation as a regulation node in the biosynthesis of flavonoids and points to its important role in buffering redox status under normal growing conditions in poplar.info:eu-repo/semantics/nonPublishe