Mandelonitrile lyase MDL2-mediated regulation of seed amygdalin and oil accumulation of Prunus Sibirica.

BACKGROUND: The Prunus sibirica seeds with rich oils has great utilization, but contain amygdalin that can be hydrolyzed to release toxic HCN. Thus, how to effectively reduce seed amygdalin content of P. sibirica is an interesting question. Mandelonitrile is known as one key intermediate of amygdalin metabolism, but which mandelonitrile lyase (MDL) family member essential for its dissociation destined to low amygdalin accumulation in P. sibirica seeds still remains enigmatic. An integration of our recent 454 RNA-seq data, amygdalin and mandelonitrile content detection, qRT-PCR analysis and function determination is described as a critical attempt to determine key MDL and to highlight its function in governing mandelonitrile catabolism with low amygdalin accumulation in Prunus sibirica seeds for better developing edible oil and biodiesel in China. RESULTS: To identify key MDL and to unravel its function in governing seed mandelonitrile catabolism with low amygdalin accumulation in P. sibirica. Global identification of mandelonitrile catabolism-associated MDLs, integrated with the across-accessions/developing stages association of accumulative amount of amygdalin and mandelonitrile with transcriptional level of MDLs was performed on P. sibirica seeds of 5 accessions to determine crucial MDL2 for seed mandelonitrile catabolism of P. sibirica. MDL2 gene was cloned from the seeds of P. sibirica, and yeast eukaryotic expression revealed an ability of MDL2 to specifically catalyze the dissociation of mandelonitrile with the ideal values of Km (0.22 mM) and Vmax (178.57 U/mg). A combination of overexpression and mutation was conducted in Arabidopsis. Overexpression of PsMDL2 decreased seed mandelonitrile content with an increase of oil accumulation, upregulated transcript of mandelonitrile metabolic enzymes and oil synthesis enzymes (involving FA biosynthesis and TAG assembly), but exhibited an opposite situation in mdl2 mutant, revealing a role of PsMDL2-mediated regulation in seed amygdalin and oil biosynthesis. The PsMDL2 gene has shown as key molecular target for bioengineering high seed oil production with low amygdalin in oilseed plants. CONCLUSIONS: This work presents the first integrated assay of genome-wide identification of mandelonitrile catabolism-related MDLs and the comparative association of transcriptional level of MDLs with accumulative amount of amygdalin and mandelonitrile in the seeds across different germplasms and developmental periods of P. sibirica to determine MDL2 for mandelonitrile dissociation, and an effective combination of PsMDL2 expression and mutation, oil and mandelonitrile content detection and qRT-PCR assay was performed to unravel a mechanism of PsMDL2 for controlling amygdalin and oil production in P. sibirica seeds. These findings could offer new bioengineering strategy for high oil production with low amygdalin in oil plants.

Time-Separated Pulse Release-Activation of an Enzyme from Alginate-Polyethylenimine Hydrogels Using Electrochemically Generated Local pH Changes.

ß-Glucosidase (EC 3.2.1.21) from sweet almond was encapsulated into pH-responsive alginate-polyethylenimine (alginate-PEI) hydrogel. Then, electrochemically controlled cyclic local pH changes resulting from ascorbate oxidation (acidification) and oxygen reduction (basification) were used for the pulsatile release of the enzyme from the composite hydrogel. Activation of the enzyme was controlled by the very same pH changes used for ß-glucosidase release, separating these two processes in time. Importantly, the activity of the enzyme, which had not been released yet, was inhibited due to the buffering effect of PEI present in the gel. Thus, only a portion of the released enzyme was activated. Both enzymatic activity and release were monitored by confocal fluorescence microscopy and regular fluorescent spectroscopy. Namely, commercially available very little or nonfluorescent substrate 4-methylumbelliferyl-ß-d-glucopyranoside was hydrolyzed by ß-glucosidase to produce a highly fluorescent product 4-methylumbelliferone during the activation phase. At the same time, labeling of the enzyme with rhodamine B isothiocyanate was used for release observation. The proposed work represents an interesting smart release-activation system with potential applications in biomedical field.

Characteristics and Expression Analyses of Trehalose-6-Phosphate Synthase Family in Prunus mume Reveal Genes Involved in Trehalose Biosynthesis and Drought Response.

Trehalose and its key synthase (trehalose-6-phosphate synthase, TPS) can improve the drought tolerance of plants. However, little is known about the roles of trehalose and the TPS family in Prunus mume response to drought. In our study, we discovered that the trehalose content in leaf, root, and stem tissues significantly increased in P. mume in response to drought. Therefore, the characteristics and functions of the TPS family are worth investigating in P. mume. We identified nine TPS family members in P. mume, which were divided into two sub-families and characterized by gene structure, promoter elements, protein conserved domains, and protein motifs. We found that the Hydrolase_3 domain and several motifs were highly conserved in Group II instead of Group I. The distinctions between the two groups may result from selective constraints, which we estimated by the dN/dS (ω) ratio. The ω values of all the PmTPS family gene pairs were evaluated as less than 1, indicating that purity selection facilitated their divergence. A phylogenetic tree was constructed using 92 TPSs from 10 Rosaceae species, which were further divided into five clusters. Based on evolutionary analyses, the five clusters of TPS family proteins mainly underwent varied purity selection. The expression patterns of PmTPSs under drought suggested that the TPS family played an important role in the drought tolerance of P. mume. Combining the expression patterns of PmTPSs and the trehalose content changes in leaf, stem, and root tissues under normal conditions and drought stress, we found that the PmTPS2 and PmTPS6 mainly function in the trehalose biosynthesis in P. mume. Our findings not only provide valuable information about the functions of trehalose and TPSs in the drought response of P. mume, but they also contribute to the future drought breeding of P. mume.

The possible bottleneck effect of polyamines' catabolic enzymes in efficient adventitious rooting of two stone fruit rootstocks.

Adventitious rooting is an important plant physiological response utilized in cutting propagation, a procedure with high financial significance. Many endogenous factors are involved, such as plant growth regulators, carbohydrates, minerals, polyamines etc. The objective of the present study was to investigate the role of polyamines and polyamine catabolic enzymes in the bases of softwood cuttings of two Prunus rootstocks, during the early phases of rhizogenesis. An easy-to-root and a difficult-to-root rootstock were studied, concerning their polyamine content (in free, soluble conjugate and insoluble bound form), polyamine catabolic enzyme activities (polyamine oxidase, PAO and diamine oxidase, DAO) and catalase activity, with and without the effect of indole-3-butyric acid as rooting hormone, during the early phases of rhizogenesis. Putrescine, spermine and their catabolic product, H2O2, were applied to test their function to rescue the rooting percentage of the recalcitrant species. Spermine was not detected in the difficult to root rootstock, which exhibited higher titer of putrescine and spermidine, PAO and catalase activity, but lower DAO activity compared to the easy-to-root one. The rooting percentage of the recalcitrant species was doubled under spermine and H2O2 application. The results obtained, highlighted the role of polyamine catabolic enzymes and indirectly the role of the polyamine catabolic product H2O2 as more significant than the polyamine content per se in adventitious rooting of the specific stone fruit rootstocks.

Recognition of S-RNases by an S locus F-box like protein and an S haplotype-specific F-box like protein in the Prunus-specific self-incompatibility system.

KEY MESSAGE: S-RNase was demonstrated to be predominantly recognized by an S locus F-box-like protein and an S haplotype-specific F-box-like protein in compatible pollen tubes of sweet cherry. Self-incompatibility (SI) is a reproductive barrier that rejects self-pollen and inhibits self-fertilization to promote outcrossing. In Solanaceae and Rosaceae, S-RNase-based gametophytic SI (GSI) comprises S-RNase and F-box protein(s) as the pistil and pollen S determinants, respectively. Compatible pollen tubes are assumed to detoxify the internalized cytotoxic S-RNases to maintain growth. S-RNase detoxification is conducted by the Skp1-cullin1-F-box protein complex (SCF) formed by pollen S determinants, S locus F-box proteins (SLFs), in Solanaceae. In Prunus, the general inhibitor (GI), but not pollen S determinant S haplotype-specific F-box protein (SFB), is hypothesized to detoxify S-RNases. Recently, SLF-like proteins 1-3 (SLFL1-3) were suggested as GI candidates, although it is still possible that other proteins function predominantly in GI. To identify the other GI candidates, we isolated four other pollen-expressed SLFL and SFB-like (SFBL) proteins PavSLFL6, PavSLFL7A, PavSFBL1, and PavSFBL2 in sweet cherry. Binding assays with four PavS-RNases indicated that PavSFBL2 bound to PavS1, 6-RNase while the others bound to nothing. PavSFBL2 was confirmed to form an SCF complex in vitro. A co-immunoprecipitation assay using the recombinant PavS6-RNase as bait against pollen extracts and a mass spectrometry analysis identified the SCF complex components of PavSLFLs and PavSFBL2, M-locus-encoded glutathione S-transferase (MGST), DnaJ-like protein, and other minor proteins. These results suggest that SLFLs and SFBLs could act as predominant GIs in Prunus-specific S-RNase-based GSI.

Cloning and characterization of the Cerasus humilis sucrose phosphate synthase gene (ChSPS1).

Sucrose is crucial to the growth and development of plants, and sucrose phosphate synthase (SPS) plays a key role in sucrose synthesis. To understand the genetic and molecular mechanisms of sucrose synthesis in Cerasus humilis, ChSPS1, a homologue of SPS, was cloned using RT-PCR. Sequence analysis showed that the open reading frame (ORF) sequence of ChSPS1 is 3174 bp in length, encoding a predicted protein of 1057 amino acids. The predicted protein showed a high degree of sequence identity with SPS homologues from other species. Real-time RT-PCR analysis showed that ChSPS1 mRNA was detected in all tissues and the transcription level was the highest in mature fruit. There is a significant positive correlation between expression of ChSPS1 and sucrose content. Prokaryotic expression of ChSPS1 indicated that ChSPS1 protein was expressed in E. coli and it had the SPS activity. Overexpression of ChSPS1 in tobacco led to upregulation of enzyme activity and increased sucrose contents in transgenic plants. Real-time RT-PCR analysis showed that the expression of ChSPS1 in transgenic tobacco was significantly higher than in wild type plants. These results suggested that ChSPS1 plays an important role in sucrose synthesis in Cerasus humilis.

Phosphorylation of phosphoenolpyruvate carboxykinase (PEPCK) and phosphoenolpyruvate carboxylase (PEPC) in the flesh of fruits.

This study determined whether phosphoenolpyruvate carboxykinase (PEPCK) and phosphoenolpyruvate carboxylase (PEPC) are phosphorylated in the flesh of a range of fruits. This was done by incubating fruit flesh with 32P[P] (where 32P[P] = 32PO43-), then PEPCK and PEPC were immunoprecipitated from extracts using specific antisera. The incorporation of 32P[P] into these enzymes was then determined by autoradiography of SDS-PAGE gels. Both enzymes were subject to phosphorylation in vivo in the flesh of grape, tomato, cherry and plum. PEPCK was also subject to phosphorylation in vivo in developing grape seeds. Proteolytic cleavage of PEPCK showed that it was phosphorylated at a site(s) located on its N-terminal extension. Potentially phosphorylation of these enzymes could contribute to the coordinate regulation of their activities in the flesh of fruits and in developing seeds.

Optimization of Hyaluronidase Inhibition Activity from Prunus davidiana (Carriere) Franch Fruit Extract Fermented by its Isolated Bacillus subtilis Strain SPF4211.

Strain SPF4211, having hyaluronidase (HAase) inhibition activity, was isolated from P. davidiana (Carriere) Franch fruit (PrDF) sugar extract. The phenotypic and biochemical properties based on 16S rDNA sequencing and an API 50 CHB kit suggested that the organism was B. subtilis. To optimize the HAase inhibition activity of PrDF extract by fermentation of strain SPF4211, a central composite design (CCD) was introduced based on three variables: concentration of PrDF extract (X1: 1-5%), amount of starter culture (X2: 1-5%), and fermentation time (X3: 0-7 days). The experimental data were fitted with quadratic regression equations, and the accuracy of the equations was analyzed by ANOVA. The statistical model predicted the highest HAase inhibition activity of 37.936% under the optimal conditions of X1 = 1%, X2 = 2.53%, and X3 = 7 days. The optimized conditions were validated by observation of an actual HAase inhibition activity of 38.367% from extract of PrDF fermented by SPF4211. These results agree well with the predicted model value.

Drought Tolerance Is Correlated with the Activity of Antioxidant Enzymes in Cerasus humilis Seedlings.

Cerasus humilis, grown in the northern areas of China, may experience water deficit during their life cycle, which induces oxidative stress. Our present study was conducted to evaluate the role of oxidative stress management in the leaves of two C. humilis genotypes, HR (drought resistant) and ND4 (drought susceptible), when subjected to a long-term soil drought (WS). The HR plants maintained lower membrane injury due to low ROS and MDA accumulation compared to ND4 plants during a long-term WS. This is likely attributed to global increase in the activities of superoxide dismutase (SOD) isoenzymes and enzymes of the ascorbate-glutathione (AsA-GSH) cycle and maintenance of ascorbate (AsA) levels. Consistent closely with enzymes activities, the expression of cytosolic ascorbate peroxidase (cAPX) and dehydroascorbate reductase (DHAR) followed a significant upregulation, indicating that they were regulated at the transcriptional level for HR plants exposed to WS. In contrast, ND4 plants exhibited high ROS levels and poor antioxidant enzyme response, leading to enhanced membrane damage during WS conditions. The present study shows that genotypic differences in drought tolerance could be likely attributed to the ability of C. humilis plants to induce antioxidant defense under drought conditions.

Recognition of a wide-range of S-RNases by S locus F-box like 2, a general-inhibitor candidate in the Prunus-specific S-RNase-based self-incompatibility system.

Many species in the Rosaceae, the Solanaceae, and the Plantaginaceae exhibit S-RNase-based gametophytic self-incompatibility (GSI). This system comprises S-ribonucleases (S-RNases) as the pistil S determinant and a single or multiple F-box proteins as the pollen S determinants. In Prunus, pollen specificity is determined by a single S haplotype-specific F-box protein (SFB). The results of several studies suggested that SFB exerts cognate S-RNase cytotoxicity, and a hypothetical general inhibitor (GI) is assumed to detoxify S-RNases in non-specific manner unless it is affected by SFB. Although the identity of the GI is unknown, phylogenetic and evolutionary analyses have indicated that S locus F-box like 1-3 (or S locus F-box with low allelic sequence polymorphism 1-3; SLFL1-3), which are encoded by a region of the Prunus genome linked to the S locus, are good GI candidates. Here, we examined the biochemical characteristics of SLFL1-3 to determine whether they have appropriate GI characteristics. Pull-down assays and quantitative expression analyses indicated that Prunus avium SLFL1-3 mainly formed a canonical SCF complex with PavSSK1 and PavCul1A. Binding assays with PavS(1,3,4,6)-RNases showed that PavSLFL1, PavSLFL2, and PavSLFL3 bound to PavS(3)-RNase, all PavS-RNases tested, and none of the PavS-RNases tested, respectively. Together, these results suggested that SLFL2 has the appropriate characteristics to be the GI in sweet cherry pollen, while SLFL1 may redundantly work with SLFL2 to detoxify all S-RNases. We discuss the possible roles of SLFL1-3 as the GI in the Prunus-specific S-RNase-based GSI mechanism.

A Japanese plum α-l-arabinofuranosidase/ß-D-xylosidase gene is developmentally regulated by alternative splicing.

A full-length cDNA clone named PsARF/XYL was obtained from Prunus salicina Lindl., and determined to encode a putative α-l-arabinofuranosidase/ß-d-xylosidase belonging to glycoside hydrolase (GH, EC 3.2.1.-) family 3. Two related PsARF/XYL cDNAs were amplified, one from a fully-spliced transcript (PsARF/XYLa) and another one from an intron-retained transcript (PsARF/XYLb). The protein deduced from PsARF/XYLb is a truncated peptide at C-terminus that conserves the active-site amino acid sequence. High levels of PsARF/XYLa and PsARF/XYLb transcripts are detectable in several plant tissues. PsARF/XYLb transcripts accumulate progressively during the phase of exponential fruit growth but they become barely noticeable during on-tree ripening, or after a 6-h exposure of preclimacteric full-size plums to ethylene. In contrast, PsARF/XYLa is expressed throughout fruit development, and transcript accumulation parallels the climacteric rise in ethylene production during ripening. PsARF/XYLa expression is strongly induced in preclimacteric full-size plums after a 6-h treatment with physiologically active concentrations of ethylene. These findings suggest that PsARF/XYL gene is post-transcriptionally regulated by alternative splicing during development and that ethylene may be involved in this regulation. The isolation of a partial cDNA clone, PsARF1, is also reported. It encodes a putative cell-wall α-l-arabinofuranosidase, and its transcription is rapidly inhibited by ethylene in mature green plums.

Cloning and expression of UDP-glucose: flavonoid 3-O-glucosyltransferase gene in peach flowers.

To elucidate the connection between flower coloration and the expression of genes associated with anthocyanin biosynthesis, a gene encoding UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) was isolated, and the expression of the last four genes in the anthocyanin biosynthetic pathway during peach flower development was determined. The nucleotide sequence of the peach UFGT (GenBank accession No. JX149550) is highly similar to its homologs in other plants. Total anthocyanin content initially increased during peach flower development, and then decreased over time. Expression of the four anthocyanin biosynthesis genes increased until the full-bloom stage, and then decreased during late florescence. Expression of F3H, DFR, and UFGT increased dramatically at the full-bloom stage, coinciding with an increase in anthocyanin concentration. The UFGT gene may not be the only gene of the anthocyanin pathway to be differentially controlled in red peach flower tissues. Further studies are needed to genetically and physiologically characterize these genes and enzymes in peach flowers and to gain a better understanding of their functions and relationships with flower coloration.

Novel pyridine-2,4,6-tricarbohydrazide derivatives: design, synthesis, characterization and in vitro biological evaluation as α- and ß-glucosidase inhibitors.

A range of novel pyridine 2,4,6-tricarbohydrazide derivatives (4a-4h) were synthesized and its biological inhibition towards α- and ß-glucosidases was studied. Most of the compounds demonstrate to be active against α-glucosidase, and quite inactive/completely inactive against ß-glucosidase. A number of compounds were found to be more active against α-glucosidase than the reference compound acarbose (IC50 38.25±0.12µM); being compound 4d with the p-hydroxy phenyl motive the most active (IC50 20.24±0.72µM). Molecular modeling studies show the interactions of compound 4d with the active site of target α-glucosidase kinase.

Methylation of the S f locus in almond is associated with S-RNase loss of function.

Self-compatibility in almond (Prunus dulcis) is attributed to the presence of the S f haplotype, allelic to and dominant over the series of S-alleles controlling self-incompatibility. Some forms of the S f haplotype, however, are phenotypically self-incompatible even though their nucleotide sequences are identical. DNA from leaves and styles from genetically diverse almond samples was cloned and sequenced and then analyzed for changes affecting S f -RNase variants. Epigenetic changes in several cytosine residues were detected in a fragment of 4,700 bp of the 5' upstream region of all self-compatible samples of the S f -RNases, differentiating them from all self-incompatible samples of S f -RNases analyzed. This is the first report of DNA methylation in a Rosaceae species and appears to be strongly associated with inactivation of the S f allele. Results facilitate an understanding of the evolution of self-compatibility/self-incompatibility in almond and other Prunus species, and suggest novel approaches for future crop improvement.

Enhancing catalytic performance of ß-glucosidase via immobilization on metal ions chelated magnetic nanoparticles.

A novel magnetic Fe3O4 nanoparticles (MNPs) coupled with agarose (AMNPs) was synthesized using co-precipitation via alkaline condition and span-80 surfactants in organic solvent. Iminodiacetate was first attached to the MNPs through epichlorohydrin agent and then chelated with metal ions. The morphology and chemical properties of these prepared supports were characterized by scanning electron microscopy (SEM), X-ray power diffraction (XRD), vibrating sample magnetometer (VSM), and Fourier transform infrared spectroscopy (FT-IR). Among them, the Co(2+)-chelated AMNPs (AMNPs-ECH-IDA-Co(2+)) showed the second highest enzyme adsorption capacity of 1.81 mg/g particles, and achieved the largest activity recovery of 117% per protein gram in immobilization of ß-glucosidase (BGL). The Michaelis constant (Km) and Vmax of the immobilized BGL were 0.904 mM and 0.057 µmol/min, respectively, and its activation energy was much lower than the free form. Moreover, the immobilized enzyme exhibited enhanced thermostability and operational stability. It still retained more than 90% of its initial activity after being operated for 15 successive batches. This study demonstrates that the immobilized ß-glucosidase has a good prospect in industrial applications.

Identification and characterization of CYP79D16 and CYP71AN24 catalyzing the first and second steps in L-phenylalanine-derived cyanogenic glycoside biosynthesis in the Japanese apricot, Prunus mume Sieb. et Zucc.

Japanese apricot, Prunus mume Sieb. et Zucc., belonging to the Rosaceae family, produces as defensive agents the cyanogenic glycosides prunasin and amygdalin, which are presumably derived from L-phenylalanine. In this study, we identified and characterized cytochrome P450s catalyzing the conversion of L-phenylalanine into mandelonitrile via phenylacetaldoxime. Full-length cDNAs encoding CYP79D16, CYP79A68, CYP71AN24, CYP71AP13, CYP71AU50, and CYP736A117 were cloned from P. mume 'Nanko' using publicly available P. mume RNA-sequencing data, followed by 5'- and 3'-RACE. CYP79D16 was expressed in seedlings, whereas CYP71AN24 was expressed in seedlings and leaves. Enzyme activity of these cytochrome P450s expressed in Saccharomyces cerevisiae was evaluated by liquid and gas chromatography­mass spectrometry. CYP79D16, but not CYP79A68, catalyzed the conversion of L-phenylalanine into phenylacetaldoxime. CYP79D16 showed no activity toward other amino acids. CYP71AN24, but not CYP71AP13, CYP71AU50, and CYP736A117, catalyzed the conversion of phenylacetaldoxime into mandelonitrile. CYP71AN24 also showed lower conversions of various aromatic aldoximes and nitriles. The K m value and turnover rate of CYP71AN24 for phenylacetaldoxime were 3.9 µM and 46.3 min(−1), respectively. The K m value and turnover of CYP71AN24 may cause the efficient metabolism of phenylacetaldoxime, avoiding the release of the toxic intermediate to the cytosol. These results suggest that cyanogenic glycoside biosynthesis in P. mume is regulated in concert with catalysis by CYP79D16 in the parental and sequential reaction of CYP71AN24 in the seedling.

Crosslinked enzyme aggregates of hydroxynitrile lyase partially purified from Prunus dulcis seeds and its application for the synthesis of enantiopure cyanohydrins.

Hydroxynitrile lyases are powerful catalysts in the synthesis of enantiopure cyanohydrins which are key synthons in the preparations of a variety of important chemicals. The response surface methodology including three-factor and three-level Box-Behnken design was applied to optimize immobilization of hydroxynitrile lyase purified partially from Prunus dulcis seeds as crosslinked enzyme aggregates (PdHNL-CLEAs). The quadratic model was developed for predicting the response and its adequacy was validated with the analysis of variance test. The optimized immobilization parameters were initial glutaraldehyde concentration, ammonium sulfate saturation concentration, and crosslinking time, and the response was relative activity of PdHNL-CLEA. The optimal conditions were determined as initial glutaraldehyde concentration of 25% w/v, ammonium sulfate saturation concentration of 43% w/v, and crosslinking time of 18 h. The preparations of PdHNL-CLEA were examined for the synthesis of (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, (R)-4-bromomandelonitrile, (R)-4-fluoromandelonitrile, and (R)-4-nitromandelonitrile from their corresponding aldehydes and hydrocyanic acid. After 96-h reaction time, the yield-enantiomeric excess values (%) were 100-99, 100-21, 100-99, 83-91, 100-99, 100-72, and 100-14%, respectively, for (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, (R)-4-bromomandelonitrile, (R)-4-fluoromandelonitrile, and (R)-4-nitromandelonitrile. The results show that PdHNL-CLEA offers a promising potential for the preparation of enantiopure (R)-mandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, and (R)-4-bromomandelonitrile with a high yield and enantiopurity.

Characterization of an uncharacterized aldo-keto reductase gene from peach and its role in abiotic stress tolerance.

The aldo-keto reductase (AKR) superfamily is a large enzyme group of NADP-dependent oxidoreductases with numerous roles in metabolism, but many members in this superfamily remain uncharacterized. Here, PpAKR1, which was cloned from the rosaceous peach tree (Prunus persica), was investigated as a member of the superfamily. While PpAKR1 had amino acids that are important in AKRs and which belonged to the AKR4 group, PpAKR1 did not seem to belong to any of the AKR4 subgroups. PpAKR1 mRNA abundance increased with abscisic acid, oxidative stress, and cold and salt stress treatments in peach. NADP-dependent polyol dehydrogenase activity was increased in Arabidopsis thaliana transformed with PpAKR1. Salt tolerance increased in Arabidopsis transformed with PpAKR1. PpAKR1, which was a previously uncharacterized member of the AKR superfamily, could be involved in the abiotic stress tolerance.

Glucitol dehydrogenase from peach (Prunus persica) fruits is regulated by thioredoxin h.

Glucitol (Gol) is a major photosynthetic product in plants from the Rosaceae family. Herein we report the molecular cloning, heterologous expression and characterization of Gol dehydrogenase (GolDHase, EC 1.1.1.14) from peach (Prunus persica) fruits. The recombinant enzyme showed kinetic parameters similar to those reported for orthologous enzymes purified from apple and pear fruits. The activity of recombinant GolDHase was strongly inhibited by Cu(2+) and Hg(2+), suggesting that it might have cysteine residues critical for functionality. Oxidizing compounds (such as diamide, hydrogen peroxide and oxidized glutathione) inactivated the enzyme, whereas its activity was restored after incubation with reduced glutathione and thioredoxin from Escherichia coli. Recombinant thioredoxin h from peach fruits also recovered the activity of oxidized GolDHase. Our results suggest that peach fruit GolDHase could be redox regulated in vivo and this would be of relevance to determine carbon assimilation and partitioning in plants accumulating sugar alcohols.

Efficient magnetic recycling of covalently attached enzymes on carbon-coated metallic nanomagnets.

In the pursuit of robust and reusable biocatalysts for industrial synthetic chemistry, nanobiotechnology is currently taking a significant part. Recently, enzymes have been immobilized on different nanoscaffold supports. Carbon coated metallic nanoparticles were found to be a practically useful support for enzyme immobilization due to their large surface area, high magnetic saturation, and manipulatable surface chemistry. In this study carbon coated cobalt nanoparticles were chemically functionalized (diazonium chemistry), activated for bioconjugation (N,N-disuccinimidyl carbonate), and subsequently used in enzyme immobilization. Three enzymes, ß-glucosidase, α-chymotrypsin, and lipase B were successfully covalently immobilized on the magnetic nonsupport. The enzyme-particle conjugates formed retained their activity and stability after immobilization and were efficiently recycled from milliliter to liter scales in short recycle times.