Genome-wide analysis of sucrose synthase family in soybean and their expression in response to abiotic stress and seed development.

The sucrose synthase (SS) is an important enzyme family which play a vital role in sugar metabolism to improve the fruit quality of the plants. In many plant species, the members of SS family have been investigated but the detailed information is not available in legumes particularly and Glycine max specifically. In the present study, we found thirteen SS members (GmSS1-GmSS13) in G. max genome. High conserved regions were present in the GmSS sequences that may due to the selection pressure during evolutionary events. The segmental duplication was the major factor to increase the number of GmSS family members. The identified thirteen GmSS genes were divided into Class I, Class II and Class III with variable numbers of genes in each class. The protein interaction of GmSS gave the co-expression of sucrose synthase with glucose-1-phosphate adenylyltransferase while SLAC and REL test found number of positive sites in the coding sequences of SS family members. All the GmSS family members except GmSS7 and few of class III members, were highly expressed in all the soybean tissues. The expression of the class I members decreased during seed development, whireas, the class II members expression increased during the seed developing, may involve in sugar metabolism during seed development. Solexa sequencing libraries of acidic condition (pH 4.2) stress samples showed that the expression of class I GmSS genes increased 1- to 2-folds in treated samples than control. The differential expression pattern was observed between the members of a paralogous. This study provides detailed genome-wide analysis of GmSS family in soybean that will provide new insights for future evolutionary and soybean breeding to improve the plant growth and development.

Effects of Engineered Saccharomyces cerevisiae Fermenting Cellobiose through Low-Energy-Consuming Phosphorolytic Pathway in Simultaneous Saccharification and Fermentation.

Until recently, four types of cellobiose-fermenting Saccharomyces cerevisiae strains have been developed by introduction of a cellobiose metabolic pathway based on either intracellular ß-glucosidase (GH1-1) or cellobiose phosphorylase (CBP), along with either an energy-consuming active cellodextrin transporter (CDT-1) or a non-energy-consuming passive cellodextrin facilitator (CDT-2). In this study, the ethanol production performance of two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-2 (N306I) with GH1-1 or CBP were compared with two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-1 (F213L) with GH1-1 or CBP in the simultaneous saccharification and fermentation (SSF) of cellulose under various conditions. It was found that, regardless of the SSF conditions, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the best ethanol production among the four strains. In addition, during SSF contaminated by lactic acid bacteria, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the highest ethanol production and the lowest lactate formation compared with those of other strains, such as the hydrolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-1 with GH1-1, and the glucose-fermenting S. cerevisiae with extracellular ß-glucosidase. These results suggest that the cellobiose-fermenting yeast strain exhibiting low energy consumption can enhance the efficiency of the SSF of cellulosic biomass.

Characterization and expression pattern of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase gene families in Populus.

Trehalose plays an important role in plant metabolism, growth development, and stress tolerance. Trehalose-6-phosphate synthase gene (TPS) and trehalose-6-phosphate phosphatase gene (TPP) are vital for the synthesis of trehalose. Populus is a prominent perennial woody plant, in which systematic genome-wide analysis of the TPS and TPP family is limited. In this study, 13 PtTPS and 10 PtTPP genes were identified in the Populus genome. Phylogenetic analysis indicated PtTPS and PtTPP genes were both divided into two subfamilies, and gene members of each subfamily have highly conserved intron structures. Analysis of cis-acting elements showed that PtTPS and PtTPP genes were involved in plant hormones and environmental stress responses. Expression profiles also found PtTPSs and PtTPPs expressed differently in response to salt stress, cold, mechanical damage, salicylic acid, and methyl jasmonate treatment. Furthermore, reverse transcription quantitative real-time PCR results found PtTPSs and PtTPPs displayed a specific expression pattern in the seven developmental stages of Populus male and female floral buds. This work will not only lead a foundation on reveal the functions of PtTPS and PtTPP gene families in trehalose regulation of poplar but also provide references to related trehalose research in other perennial plants.

Molecular cloning and biochemical characterization of a trehalose synthase from Myxococcus sp. strain V11.

The tresI gene of Myxococcus sp. strain V11 was cloned, and found to encode a trehalose synthase comprising 551 amino acids. The deduced molecular weight of the encoded TreS I protein 64.7 kDa and the isoelectric point (pI) was predicted to be 5.6. The catalytic cleft consists of the Asp202-Glu244-Asp310 catalytic triad and additional conserved residues. The recombinant (His)6-tag enzyme was expressed in Escherichia coli BL21(DE3) and purified by Ni2+-affinity chromatography, resulting in a specific activity of up to 172.7 U/mg. TLC and HPLC results confirmed that rTreS I can convert maltose into trehalose, with a yield of 61%. The KM and Vmax values of recombinant TreS I for maltose were 0.62 mM and 25.5 mM min-1 mg-1 protein, respectively. TreS I was optimally active at 35° and stable at temperatures of <25 °C. TreS I was stable within a narrow range of pH values, from 6.0 to 7.0. The enzymatic activity was slightly stimulated by Mg2+ and strongly inhibited by Fe3+, Co2+ and Cu2+. TreS I was also strongly inhibited by SDS and weakly by EDTA and TritonX-100.

ß-Sitosterol 3-O-D-glucoside increases ceramide levels in the stratum corneum via the up-regulated expression of ceramide synthase-3 and glucosylceramide synthase in a reconstructed human epidermal keratinization model.

ß-Sitosterol 3-O-d-glucoside (BSG) is known to act as an agonist by binding to estrogen receptors, and estrogen has been reported to enhance the activity of ß-glucocerebrosidase, an epidermal ceramide metabolizing enzyme. In this study, we determined whether BSG up-regulates ceramide levels in the stratum corneum (SC) of a reconstructed human epidermal keratinization (RHEK) model. Treatment with BSG significantly increased the total ceramide content by 1.2-fold compared to that in the control in the SC of the RHEK model, accompanied by a significant increase of the ceramide species, Cer[EOS] by 2.1-fold compared to that in the control. RT-PCR analysis demonstrated that BSG significantly up-regulated the mRNA expression levels of serine palmitoyltransferase (SPT)2, ceramide synthase (CerS)3, glucosylceramide synthase (GCS) and acid sphingomyelinase by 1.41-1.89, 1.35-1.44, 1.19 and 2.06-fold, respectively, compared to that in the control in the RHEK model. Meanwhile, BSG significantly down-regulated the mRNA expression levels of sphingomyelin synthase (SMS)2 by 0.87-0.89-fold. RT-PCR analysis also demonstrated that BSG significantly up-regulated the mRNA expression levels of CerS3 and GCS by 1.19-1.55 and 1.20-fold, respectively, but not of SPT2 and significantly down-regulated that of SMS2 by 0.74-fold in HaCaT keratinocytes. Western blotting analysis revealed that BSG significantly increased the protein expression levels of CerS3 and GCS by 1.78 and 1.28-1.32-fold, respectively, compared to that in the control in HaCaT cells. These findings indicate that BSG stimulates ceramide synthesis via the up-regulated expression levels of CerS3 and GCS in the glucosylceramide pathway, which results in a significantly increased level of total ceramides in the SC accompanied by significantly increased levels of acylceramide species such as Cer[EOS].

Combined Optimization of Codon Usage and Glycine Supplementation Enhances the Extracellular Production of a ß-Cyclodextrin Glycosyltransferase from Bacillus sp. NR5 UPM in Escherichia coli.

Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM ß-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of ß-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. ß-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant ß-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular ß-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly ß-CGTase by the recombinant E. coli.

Enhancing soluble expression of sucrose phosphorylase in Escherichia coli by molecular chaperones.

Sucrose phosphorylase (SPase, EC 2.4.1.7) has a wide range of application in food, cosmetics, and pharmaceutical industries because of its broad substrate specificity. However, low SPase yields produced by wild-type strains cannot meet industrial requirements due to their complex metabolic regulation mechanisms. In this study, spase gene from Thermoanaerobacterium thermosaccharolyticum was cloned and expressed in Escherichia coli BL21 (DE3), leading to 7.05 U/mL (3.71 U/mg) of T. thermosaccharolyticum SPase (TtSPase) under optimum conditions. Co-expression of molecular chaperone teams pGro7 (GroES-GroEL), pG-KJE8 (DnaK-DnaJ-GrpE and GroES-GroEL), and pG-TF2 (GroES-GroEL-Tig) significantly enhanced the TtSPase activities to 18.5 U/mg (59.2 U/mL), 9.52 U/mg (28.6 U/mL), and 25.7 U/mg (64.5 U/mL), respectively. Results suggested that GroES-GroEL chaperone combination could regulate protein folding processes and protect misfolded proteins from aggregation. The enzymatic characterization results showed that TtSPase had an optimal temperature of 60 °C and optimal pH of 6.5. In particular, it had high thermostability of T5030 = 67 °C and half-life (t1/2 at 70 °C) of 19 min. Furthermore, purified TtSPase was used for hydroquinone transglycosylation and 21% of molar production yield of α-arbutin was obtained. This study provides a TtSPase with high thermostability for potential industrial applications, and develops an effective strategy for improving soluble TtSPase production in E. coli.

Improved production of cyclodextrin glycosyltransferase from Bacillus stearothermophilus NO2 in Escherichia coli via directed evolution.

Cyclodextrin glycosyltransferases (CGTases) are widely used in starch deep processing, so reducing their cost by improving their production is of significant industrial interest. The CGTase from Bacillus stearothermophilus NO2 possesses excellent catalytic properties but suffers from low production in E. coli. In this study, directed evolution was used to create three point mutants (I631T, I641T and K647E) that were produced in E. coli with shake-flask yields 1.7-, 2.1-, and 2.2-fold higher than that of wild-type, respectively. The wild-type and K647E were then produced in a 3-L fermenter. The CGTase activity of the K647E (1904 U mL-1) was 2.0-fold higher than that of the wild-type. The K647E fermentation supernatant could be used directly to prepare 2-O-α-D-glucopyranosyl-L-ascorbic acid, reducing the costs associated with its production. Structural modeling of the three mutants suggested that hydrophilicity, hydrogen bonding, and negative charge may be responsible for their improved production. Since K647 is conserved in the CGTase family, the corresponding residues in the CGTases from Bacillus circulans 251, Paenibacillus macerans, and Anaerobranca gottschalkii were changed to glutamic acid. Productions of the resulting K647E mutants were 2.0-, 1.5-, and 1.0-fold higher than those of their respective wild-types. Electrostatic protein surface analysis suggested that mutations occurring at low negative surface charge may increase CGTase production.

A multidomain α-glucan synthetase 2 (AmAgs2) is the key enzyme for pullulan biosynthesis in Aureobasidium melanogenum P16.

Pullulan, a biological macromolecule, has many applications. However, it is completely unknown how and where it is synthesized. In this study, it was found that the multidomain AmAgs2 (α-glucan synthase 2) encoded by an AmAGS2 gene in Aureobasidium melanogenum P16 contained the amylase domain (Amy_D), the glycogen synthetase domain (Gys_D) and the transmembrane regions in which the exopolysaccharide transporter domain (EPST_D) was embedded. Removal of the AmAGS2 gene in A. melanogenum P16 rendered the disruptants not to synthesize any pullulan and complementation of the AmAGS2 gene in the disruptants restored pullulan synthesis. Overexpression of the gene in Aureobasidium melanogenum CBS105.22, a non-pullulan producer, resulted in the transformants producing pullulan. Therefore, the AmAGS2 gene was the key gene responsible for pullulan biosynthesis in A. melanogenum P16. It was speculated that the short α-1,4-glucosyl chains (pullulan primers) were elongated by the Gys_D of the AmAgs2 to form long α-1,4-glucosyl chains (precursors of pullulan). All the precursors were transported to outside plasma membrane by the EPST_D in the transmembrane regions of the AmAgs2. Then, the Amy_D of the AmAgs2 was responsible for both hydrolysis of the endo-α-1,4-linkages in the precursors to release maltotriose and transfer of the maltotriose to Lph-glucose to form α-1,6 glucosidic bonds between maltotrioses in pullulan molecule. This is the first time to report that the AmAgs2 can play the key role in pullulan biosynthesis.

Enhanced secretion of cyclodextrin glucanotransferase (CGTase) by Lactococcus lactis using heterologous signal peptides and optimization of cultivation conditions.

In previous studies of Lactococcus lactis, the levels of proteins secreted using heterologous signal peptides were observed to be lower than those obtained using the signal peptide from Usp45, the major secreted lactococcal protein. In this study, G1 (the native signal peptide of CGTase) and the signal peptide M5 (mutant of the G1 signal peptide) were introduced into L. lactis to investigate the effect of signal peptides on lactococcal protein secretion to improve secretion efficiency. The effectiveness of these signal peptides were compared to the Usp45 signal peptide. The highest secretion levels were obtained using the G1 signal peptide. Sequence analysis of signal peptide amino acids revealed that a basic N-terminal signal peptide is not absolutely required for efficient protein export in L. lactis. Moreover, the introduction of a helix-breaking residue in the H-region of the M5 signal peptide caused a reduction in the signal peptide hydrophobicity and decreased protein secretion. In addition, the optimization of cultivation conditions for recombinant G1-CGTase production via response surface methodology (RSM) showed that CGTase activity increased approximately 2.92-fold from 5.01 to 16.89 U/ml compared to the unoptimized conditions.

Heterologous co-expression of two ß-glucanases and a cellobiose phosphorylase resulted in a significant increase in the cellulolytic activity of the Caldicellulosiruptor bescii exoproteome.

The ability to deconstruct plant biomass without conventional pretreatment has made members of the genus Caldicellulosiruptor the target of investigation for the consolidated processing of plant lignocellulosic biomass to biofuels and bioproducts. To investigate the synergy of enzymes involved and to further improve the ability of C. bescii to degrade cellulose, we introduced CAZymes that act synergistically with the C. bescii exoproteome in vivo and in vitro. We recently demonstrated that the Acidothermus cellulolyticus E1 endo-1,4-ß-D-glucanase (GH5) with a family 2 carbohydrate-binding module (CBM) increased the activity of C. bescii exoproteome on biomass, presumably acting in concert with CelA. The ß-glucanase, GuxA, from A. cellulolyticus is a multi-domain enzyme with strong processive exoglucanase activity, and the cellobiose phosphorylase from Thermotoga maritima catalyzes cellulose degradation acting synergistically with cellobiohydrolases and endoglucanases. We identified new chromosomal insertion sites to co-express these enzymes and the resulting strain showed a significant increase in the enzymatic activity of the exoproteome.

Sequencing, cloning, and heterologous expression of cyclomaltodextrin glucanotransferase of Bacillus firmus strain 37 in Bacillus subtilis WB800.

Bacillusfirmus strain 37 produces the cyclomaltodextrin glucanotransferase (CGTase) enzyme and CGTase produces cyclodextrins (CDs) through a starch cyclization reaction. The strategy for the cloning and expression of recombinant CGTase is a potentially viable alternative for the economically viable production of CGTase for use in industrial processes. The present study used Bacillus subtilis WB800 as a bacterial expression host for the production of recombinant CGTase cloned from the CGTase gene of B. firmus strain 37. The CGTase gene was cloned in TOPO-TA® plasmid, which was transformed in Escherichia coli DH5α. The subcloning was carried out with pWB980 plasmid and transformation in B. subtilis WB800. The 2xYT medium was the most suitable for the production of recombinant CGTase. The enzymatic activity of the crude extract of the recombinant CGTase of B. subtilis WB800 was 1.33 µmol ß-CD/min/mL, or 7.4 times greater than the enzymatic activity of the crude extract of CGTase obtained from the wild strain. Following purification, the recombinant CGTase exhibited an enzymatic activity of 157.78 µmol ß-CD/min/mL, while the activity of the CGTase from the wild strain was 9.54 µmol ß-CD/min/mL. When optimal CDs production conditions for the CGTase from B. firmus strain 37 were used, it was observed that the catalytic properties of the CGTase enzymes were equivalent. The strategy for the cloning and expression of CGTase in B. subtilis WB800 was efficient, with the production of greater quantities of CGTase than with the wild strain, offering essential data for the large-scale production of the recombinant enzyme.

Production and characterization of ß-cyclodextrin glucanotransferase from Bacillus sp. ND1.

A potent ß-CGTase producing bacterium ND1 has been isolated from sugarcane field soil in India. The biochemical, physiologicaland phylogenetic analyses based on 16S rRNA gene suggest that the isolate belongs to Bacillus cereus group. The enzyme ß-CGTase produced from isolate ND1 catalyzes production of ß-cyclodextrin utilizing starch as a substrate which has diverse applications in various fields. The enzyme production parameters pH, temperature, and substrate concentration were optimized using Central Composite Design (CCD) of Response Surface Methodology (RSM) and were found to be 8.9, 30.55 °C, and 1.88%, respectively for optimal enzyme activity. The crude enzyme was partially purified (29-fold) using ammonium sulphate precipitation followed by ion exchange chromatography. The specific activity of the purified enzyme was found to be 63.53 U mg-1 . The enzyme is monomeric in nature with a molecular weight of 97.4 kD as determined by SDS-PAGE. It is stable in a wide range of pH (6-10) and temperature (40-60 °C) values. The maximum CGTase activity was observed at pH 9 and temperature 50 °C. The Km value was found to be 2.613 ± 0.5 and Vmax was 0.309 ± 0.05 µg min-1 indicating high substrate specificity. Together; these results suggest that the enzyme may be of wide commercial value in various industrial processes.

Synthetic repetitive extragenic palindromic (REP) sequence as an efficient mRNA stabilizer for protein production and metabolic engineering in prokaryotic cells.

In prokaryotic cells, 3'-5' exonucleases can attenuate messenger RNA (mRNA) directionally from the direction of the 3'-5' untranslated region (UTR), and thus improving the stability of mRNAs without influencing normal cell growth and metabolism is a key challenge for protein production and metabolic engineering. Herein, we significantly improved mRNA stability by using synthetic repetitive extragenic palindromic (REP) sequences as an effective mRNA stabilizer in two typical prokaryotic microbes, namely, Escherichia coli for the production of cyclodextrin glucosyltransferase (CGTase) and Corynebacterium glutamicum for the production of N-acetylglucosamine (GlcNAc). First, we performed a high-throughput screen to select 4 out of 380 REP sequences generated by randomizing 6 nonconservative bases in the REP sequence designed as the degenerate base "N." Secondly, the REP sequence was inserted at several different positions after the stop codon of the CGTase-encoding gene. We found that mRNA stability was improved only when the space between the REP sequence and stop codon was longer than 12 base pairs (bp). Then, by reconstructing the spacer sequence and secondary structure of the REP sequence, a REP sequence with 8 bp in a stem-loop was obtained, and the CGTase activity increased from 210.6 to 291.5 U/ml. Furthermore, when this REP sequence was added to the 3'-UTR of glucosamine-6-phosphate N-acetyltransferase 1 ( GNA1), which is a gene encoding a key enzyme GNA1 in the GlcNAc synthesis pathway, the GNA1 activity was increased from 524.8 to 890.7 U/mg, and the GlcNAc titer was increased from 4.1 to 6.0 g/L in C. glutamicum. These findings suggest that the REP sequence plays an important function as an mRNA stabilizer in prokaryotic cells to stabilize its 3'-terminus of the mRNA by blocking the processing action of the 3'-5' exonuclease. Overall, this study provides new insight for the high-efficiency overexpression of target genes and pathway fine-tuning in bacteria.

Characterization of Cellulose Synthase A (CESA) Gene Family in Eudicots.

Cellulose synthase A (CESA) is a key enzyme involved in the complex process of plant cell wall biosynthesis, and it remains a productive subject for research. We employed systems biology approaches to explore structural diversity of eudicot CESAs by exon-intron organization, mode of duplication, synteny, and splice site analyses. Using a combined phylogenetics and comparative genomics approach coupled with co-expression networks we reconciled the evolution of cellulose synthase gene family in eudicots and found that the basic forms of CESA proteins are retained in angiosperms. Duplications have played an important role in expansion of CESA gene family members in eudicots. Co-expression networks showed that primary and secondary cell wall modules are duplicated in eudicots. We also identified 230 simple sequence repeat markers in 103 eudicot CESAs. The 13 identified conserved motifs in eudicots will provide a basis for gene identification and functional characterization in other plants. Furthermore, we characterized (in silico) eudicot CESAs against senescence and found that expression levels of CESAs decreased during leaf senescence.

Combined Drug Resistance Mutations Substantially Enhance Enzyme Production in Paenibacillus agaridevorans.

This study shows that sequential introduction of drug resistance mutations substantially increased enzyme production in Paenibacillus agaridevorans The triple mutant YT478 (rsmG Gln225→stop codon, rpsL K56R, and rpoB R485H), generated by screening for resistance to streptomycin and rifampin, expressed a 1,100-fold-larger amount of the extracellular enzyme cycloisomaltooligosaccharide glucanotransferase (CITase) than the wild-type strain. These mutants were characterized by higher intracellular S-adenosylmethionine concentrations during exponential phase and enhanced protein synthesis activity during stationary phase. Surprisingly, the maximal expression of CITase mRNA was similar in the wild-type and triple mutant strains, but the mutant showed greater CITase mRNA expression throughout the growth curve, resulting in enzyme overproduction. A metabolome analysis showed that the triple mutant YT478 had higher levels of nucleic acids and glycolysis metabolites than the wild type, indicating that YT478 mutant cells were activated. The production of CITase by the triple mutant was further enhanced by introducing a mutation conferring resistance to the rare earth element, scandium. This combined drug resistance mutation method also effectively enhanced the production of amylases, proteases, and agarases by P. agaridevorans and Streptomyces coelicolor This method also activated the silent or weak expression of the P. agaridevorans CITase gene, as shown by comparisons of the CITase gene loci of P. agaridevorans T-3040 and another cycloisomaltooligosaccharide-producing bacterium, Paenibacillus sp. strain 598K. The simplicity and wide applicability of this method should facilitate not only industrial enzyme production but also the identification of dormant enzymes by activating the expression of silent or weakly expressed genes.IMPORTANCE Enzyme use has become more widespread in industry. This study evaluated the molecular basis and effectiveness of ribosome engineering in markedly enhancing enzyme production (>1,000-fold). This method, due to its simplicity, wide applicability, and scalability for large-scale production, should facilitate not only industrial enzyme production but also the identification of novel enzymes, because microorganisms contain many silent or weakly expressed genes which encode novel antibiotics or enzymes. Furthermore, this study provides a new mechanism for strain improvement, with a consistent rather than transient high expression of the key gene(s) involved in enzyme production.

Is there still room to explore cyclodextrin glycosyltransferase-producers in Brazilian biodiversity?

In the present work, different Brazilian biomes aiming to identify and select cyclodextrin glycosyltransferase-producer bacteria are explored. This enzyme is responsible for converting starch to cyclodextrin, which are interesting molecules to carry other substances of economic interest applied by textile, pharmaceutical, food, and other industries. Based on the enzymatic index, 12 bacteria were selected and evaluated, considering their capacity to produce the enzyme in culture media containing different starch sources. It was observed that the highest yields were presented by the bacteria when grown in cornstarch. These bacteria were also characterized by sequencing of the 16S rRNA region and were classified as Bacillus, Paenibacillus, Gracilibacillus and Solibacillus.

Enzymatic Synthesis of a Novel Pterostilbene α-Glucoside by the Combination of Cyclodextrin Glucanotransferase and Amyloglucosidase.

The synthesis of a novel α-glucosylated derivative of pterostilbene was performed by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. The reaction was carried out in a buffer containing 20% (v/v) DMSO to enhance the solubility of pterostilbene. Due to the formation of several polyglucosylated products with CGTase, the yield of monoglucoside was increased by the treatment with a recombinant amyloglucosidase (STA1) from Saccharomyces cerevisiae (var. diastaticus). This enzyme was not able to hydrolyze the linkage between the glucose and pterostilbene. The monoglucoside was isolated and characterized by combining ESI-MS and 2D-NMR methods. Pterostilbene α-d-glucopyranoside is a novel compound. The α-glucosylation of pterostilbene enhanced its solubility in water to approximately 0.1 g/L. The α-glucosylation caused a slight loss of antioxidant activity towards ABTS˙⁺ radicals. Pterostilbene α-d-glucopyranoside was less toxic than pterostilbene for human SH-S5Y5 neurons, MRC5 fibroblasts and HT-29 colon cancer cells, and similar for RAW 264.7 macrophages.

Cloning, expression and characterization of the maltooligosyl trehalose synthase from the archaeon Sulfolobus tokodaii.

The maltooligosyl trehalose synthase gene from the hyperthermophilic archaeon Sulfolobus tokodaii strain 7 was cloned and the recombinant peotein was expressed in E. coli. The protein was purified to homogeneity by nickel column chromatography. The archaeal enzyme could catalyze an intramolecular transglycosylation reaction and convert the glycosidic bond at the reducing end of dextrins from α-1, 4 (reducing end) into α-1, 1 (non-reducing end). The most suitable temperature was 75°C and the optimal pH was 5. Substrate specificity investigation revealed that maltodextrin and maltooligosaccharide were used as substrates by the enzyme but maltose, chitooligosaccharide, sucrose and ß-cyclodextrin weren't used.

The iron-stress activated RNA 1 (IsaR1) coordinates osmotic acclimation and iron starvation responses in the cyanobacterium Synechocystis sp. PCC 6803.

In nature, microorganisms are exposed to multiple stress factors in parallel. Here, we investigated the response of the model cyanobacterium Synechocystis sp. PCC 6803 to simultaneous iron limitation and osmotic stresses. Iron is a major limiting factor for bacterial and phytoplankton growth in most environments. Thus, bacterial iron homeostasis is tightly regulated. In Synechocystis, it is mediated mainly by the transcriptional regulator FurA and the iron-stress activated RNA 1 (IsaR1). IsaR1 is an important riboregulator that affects the acclimation of the photosynthetic apparatus to iron starvation in multiple ways. Upon increases in salinity, Synechocystis responds by accumulating the compatible solute glucosylglycerol (GG). We show that IsaR1 overexpression causes a reduction in the de novo GG synthesis rate upon salt shock. We verified the direct interaction between IsaR1 and the 5'UTR of the ggpS mRNA, which in turn drastically reduced the de novo synthesis of the key enzyme for GG synthesis, glucosylglycerol phosphate synthase (GgpS). Thus, IsaR1 specifically interferes with the salt acclimation process in Synechocystis, in addition to its primary regulatory function. Moreover, the salt-stimulated GgpS production became reduced under parallel iron limitation in WT - an effect which is, however, attenuated in an isaR1 deletion strain. Hence, IsaR1 is involved in the integration of the responses to different environmental perturbations and slows the osmotic adaptation process in cells suffering from parallel iron starvation.