Oligomeric Structure of Yeast and Other Invertases Governs Specificity.

Invertases, or ß-fructofuranosidases, are metabolic enzymes widely distributed among plants and microorganisms that hydrolyze sucrose and release fructose from various substrates. Invertase was one of the earliest discovered enzymes, first investigated in the mid-nineteenth century, becoming a classical model used in the primary biochemical studies on protein synthesis, activity, and the secretion of glycoproteins. However, it was not until 20 years ago that a member of this family of enzymes was structurally characterized, showing a bimodular arrangement with a ß-propeller catalytic domain, and a ß-sandwich domain with unknown function. Since then, many studies on related plant and fungal enzymes have revealed them as basically monomeric. By contrast, all yeast enzymes in this family that have been characterized so far have shown sophisticated oligomeric structures mediated by the non-catalytic domain, which is also involved in substrate binding, and how this assembly determines the particular specificity of each enzyme. In this chapter, we will review the available structures of yeast invertases to elucidate the mechanism regulating oligomer formation and compare them with other reported dimeric invertases in which the oligomeric assembly has no apparent functional implications. In addition, recent work on a new family of invertases with absolute specificity for the α-(1,2)-bond of sucrose found in cyanobacteria and plant invertases is highlighted.

A novel ß-cyclodextrin-assisted enhancement strategy for portable and sensitive detection of miR-21 in human serum.

Benefiting from our discovery that ß-cyclodextrin (ß-CD) could enhance the catalytic activity of invertase through hydrogen bonding to improve detection sensitivity, a highly sensitive and convenient biosensor for the detection of miR-21 was proposed, which is based on the simplicity of reading signals from a personal glucose meter (PGM), combined with self-assembled signal amplification probes and the performance of ß-CD as an enhancer. In the presence of miR-21, magnetic nanoparticle coupled capture DNA (MNPs-cDNA) could capture it and then connect assist DNA/H1-invertase (aDNA/H1) and self-assembled signal amplification probes (H1/H2) in turn. As a result, a "super sandwich" structure was formed. The invertase on MNPs-cDNA could catalyze the hydrolysis of sucrose to glucose and this catalytic process could be enhanced by ß-CD. The PGM signal exhibited a linear correlation with miR-21 concentration within the range of 25 pmol L-1 to 3 nmol L-1, and the detection limit was as low as 5 pmol L-1 with high specificity. Moreover, the recoveries were 103.82-124.65% and RSD was 2.59-6.43%. Furthermore, the biosensor was validated for the detection of miR-21 in serum, and the results showed that miR-21 levels in serum samples from patients with Diffuse Large B-Cell Lymphoma (DLBCL) (n = 12) were significantly higher than those from healthy controls (n = 12) (P < 0.001). Therefore, the ingenious combination of PGM-based signal reading, self-assembled signal amplification probes and ß-CD as an enhancer successfully constructed a convenient, sensitive and specific biosensing method, which is expected to be applied to clinical diagnosis.

Tyramine-Invertase Bioconjugate-Amplified Personal Glucose Meter Signaling for Ultrasensitive Immunoassay.

Highly sensitive and facile detection of low levels of protein markers is of great significance for the early diagnosis and efficacy monitoring of diseases. Herein, aided by an efficient tyramine-signal amplification (TSA) mechanism, we wish to report a simple but ultrasensitive immunoassay with signal readout on a portable personal glucose meter (PGM). In this study, the bioconjugates of tyramine and invertase (Tyr-inv), which act as the critical bridge to convert and amplify the protein concentration information into glucose, are prepared following a click chemistry reaction. Then, in the presence of a target protein, the sandwich immunoreaction between the immobilized capture antibody, the target protein, and the horseradish peroxidase (HRP)-conjugated detection antibody is specifically performed in a 96-well microplate. Subsequently, the specifically loaded HRP-conjugated detection antibodies will catalyze the amplified deposition of a large number of Tyr-inv molecules onto adjacent proteins through highly efficient TSA. Then, the deposited invertase, whose dosage can faithfully reflect the original concentration of the target protein, can efficiently convert sucrose to glucose. The amount of finally produced glucose is simply quantified by the PGM, realizing the highly sensitive detection of trace protein markers such as the carcinoembryonic antigen and alpha fetoprotein antigen at the fg/mL level. This method is simple, cost-effective, and ultrasensitive without the requirement of sophisticated instruments or specialized laboratory equipment, which may provide a universal and promising technology for highly sensitive immunoassay for in vitro diagnosis of diseases.

Production and characterization of intracellular invertase from Saccharomyces cerevisiae (OL629078.1), using cassava-soybean as a cost-effective substrate.

The growing global market for industrial enzymes has led to a constant search for efficient, cost-effective methods for their production. This study reports the production of invertase using inexpensive and readily available agro-materials. Starch-digesting enzymes extracted from malted unkilned sorghum were used to hydrolyze cassava starch supplemented with 2% whole soybean. The production of intracellular invertase by Saccharomyces cerevisiae OL629078.1 in cassava-soybean and yeast sucrose broth was compared. The purification and characterization of invertase produced using the low-cost medium were also reported. The results showed that there was a 4.1-fold increase in the units of invertase produced in cassava-soybean medium (318.605 U/mg) compared to yeast sucrose broth medium (77.6 U/mg). The invertase produced was purified by chromatographic methods up to 5.53-fold with a recovery of 62.6%. Estimation of the molecular weight with gel filtration indicated a molecular weight of 118 kDa. The enzyme demonstrated its maximum activity at 50 °C and there was no decrease in its activity following a 1-h incubation at this temperature. At a pH of 5.0, the enzyme demonstrated optimal activity and it maintained over 60% of its activity in the acid range (pH 3-6). The Michalis-Menten constants Km and Vmax of intracellular invertase were 5.85 ± 1.715 mM and 6.472 ± 2.099 U/mg, respectively. These results suggest that Saccharomyces cerevisiae grown on cassava-soybean is a viable, cost-effective alternative for commercial invertase production, which can be explored for biotechnological processes.

Multiscale QM/MM Simulations Identify the Roles of Asp239 and 1-OH···Nucleophile in Transition State Stabilization in Arabidopsis thaliana Cell-Wall Invertase 1.

Arabidopsis thaliana cell-wall invertase 1 (AtCWIN1), a key enzyme in sucrose metabolism in plants, catalyzes the hydrolysis of sucrose into fructose and glucose. AtCWIN1 belongs to the glycoside hydrolase GH-J clan, where two carboxylate residues (Asp23 and Glu203 in AtCWIN1) are well documented as a nucleophile and an acid/base catalyst. However, details at the atomic level about the role of neighboring residues and enzyme-substrate interactions during catalysis are not fully understood. Here, quantum mechanical/molecular mechanical (QM/MM) free-energy simulations were carried out to clarify the origin of the observed decreased rates in Asp239Ala, Asp239Asn, and Asp239Phe in AtCWIN1 compared to the wild type and delineate the role of Asp239 in catalysis. The glycosylation and deglycosylation steps were considered in both wild type and mutants. Deglycosylation is predicted to be the rate-determining step in the reaction, with a calculated overall free-energy barrier of 15.9 kcal/mol, consistent with the experimental barrier (15.3 kcal/mol). During the reaction, the -1 furanosyl ring underwent a conformational change corresponding to 3E ↔ [E2]⧧ ↔ 1E according to the nomenclature of saccharide structures along the full catalytic reaction. Asp239 was found to stabilize not only the transition state but also the fructosyl-enzyme intermediate, which explains findings from previous structural and mutagenesis experiments. The 1-OH···nucleophile interaction has been found to provide an important contribution to the transition state stabilization, with a contribution of ∼7 kcal/mol, and affected glycosylation more significantly than deglycosylation. This study provides molecular insights that improve the current understanding of sucrose binding and hydrolysis in members of clan GH-J, which may benefit protein engineering research. Finally, a rationale on the sucrose inhibitor configuration in chicory 1-FEH IIa, proposed a long time ago in the literature, is also provided based on the QM/MM calculations.

Real-time assay of invertase activity using isoquinolinylboronic acid as turn-on fluorescent sensor.

Invertase is the key enzyme involved in several crucial biological processes by hydrolyzing sucrose for production of glucose and fructose. Invertase plays important roles in the fields of food, pharmacy, cosmetics, biofuels, and agriculture. Detection of invertase activity is urgently necessary for scientific research and industrial processes. Herein, a continuous fluorometric method was developed for real-time detection of invertase activity. 8-Isoquinolinylboronic acid responded to fructose by formation of a fluorescent complex in turn-on manner, and served as a fluorescent sensor to selectively recognize fructose in ternary enzymatic mixture containing sucrose and glucose. The limit of detection (LOD) for fructose was 0.07 mM. Progress curve for fructose production was established by directly and continuously monitoring the fluorescence for invertase reaction with sucrose as substrate. Initial velocity was obtained to characterize invertase activity. LOD for invertase assay was 0.10 U·mL-1. Km and υmax for invertase were determined as 7.70 mM and 0.86 mM·min-1, respectively. Copper ion was demonstrated to inhibit the invertase activity with IC50 of 33.61 mM. Applicability in high-throughput screening for inhibitor was demonstrated. The proposed method allows for real-time, simple, and rapidly monitoring the invertase activity. It has a broad range of potential applications for kinetics and screening inhibitor.

Cascade primer exchange reaction-based amplification strategy for sensitive and portable detection of amyloid ß oligomer using personal glucose meters.

Amyloid ß oligomer (AßO) is an important biomarker for Almerzheimer's disease (AD) early diagnosis. In present study, cascade primer exchange reaction (PER) based amplification strategy was proposed for sensitive and portable detection of AßO using personal glucose meters (PGM). Two PER processes were employed here. In the primary PER, the hairpin template 1 (HT1) was designed with a primer binding domain, a primer extending domain and a blocking extending domain. The primers were designed to be modified on magbeads surface. Initially, the primer binding domain in HT1 was locked by AßO aptamer. When target AßO was present, aptamer bound with AßO and dissociated from HT1 to initiate the primary PER. The products acted as the primer of the secondary PER to hybridize with another hairpin template 2 (HT2), initiating the secondary PER and producing numerous ssDNA with repeated DNA-invertase binding sites. After binding with DNA-invertase, the obtained conjugates were magnetically separation to catalyze the conversion of sucrose to glucose, which were detected by a PGM. The strategy achieved a limit of detection of 0.22 pM with a linear ranged from 1 pM to 250 pM. Satisfactory reproducibility results were obtained in actual samples. This strategy provided a superior tool for sensitive and convenient detection of AßO, and showing a great potential in the early diagnosis of AD.

Fully integrated sampler and dilutor in an electrochemical paper-based device for glucose sensing.

An electroanalytical platform capable to take and dilute the sample has been designed in order to fully integrate the different steps of the analytical process in only one device. The concept is based on the addition of glass-fiber pads for sampling and diluting to an electrochemical cell combining a paper-based working electrode with low-cost connector headers as counter and reference electrodes. In order to demonstrate the feasibility of this all-in-one platform for biosensing applications, an enzymatic sensor for glucose determination (requiring a potential as low as -0.1 V vs. gold-plated wire by using ferrocyanide as mediator) was developed. Real food samples, such as cola beverages and orange juice, have been analyzed with the bioelectroanalytical lab-on-paper platform. As a proof-of-concept, and trying to go further in the integration of steps, sucrose was successfully detected by depositing invertase in the sampling strip. This enzyme hydrolyzes sucrose into fructose and glucose, which was determined using the enzymatic biosensor. This approach opens the pathway for the development of devices applying the lab-on-paper concept, saving costs and time, and making possible to perform decentralized analysis with high accuracy.

Dietary Inulin Increases Lactiplantibacillus plantarum Strain Lp900 Persistence in Rats Depending on the Dietary-Calcium Level.

Synbiotics are food supplements that combine probiotics and prebiotics to synergistically elicit health benefits in the consumer. Lactiplantibacillus plantarum strains display high survival during transit through the mammalian gastrointestinal tract and were shown to have health-promoting properties. Growth on the fructose polysaccharide inulin is relatively uncommon in L. plantarum, and in this study we describe FosE, a plasmid-encoded ß-fructosidase of L. plantarum strain Lp900 which has inulin-hydrolyzing properties. FosE contains an LPxTG-like motif involved in sortase-dependent cell wall anchoring but is also (partially) released in the culture supernatant. In addition, we examined the effect of diet supplementation with inulin on the intestinal persistence of Lp900 in adult male Wistar rats in diets with distinct calcium levels. Inulin supplementation in high-dietary-calcium diets significantly increased the intestinal persistence of L. plantarum Lp900, whereas this effect was not observed upon inulin supplementation of the low-calcium diet. Moreover, intestinal persistence of L. plantarum Lp900 was determined when provided as a probiotic (by itself) or as a synbiotic (i.e., in an inulin suspension) in rats that were fed unsupplemented diets containing the different calcium levels, revealing that the synbiotic administration increased bacterial survival and led to higher abundance of L. plantarum Lp900 in rats, particularly in a low-calcium-diet context. Our findings demonstrate that inulin supplementation can significantly enhance the intestinal delivery of L. plantarum Lp900 but that this effect strongly depends on calcium levels in the diet.IMPORTANCE Synbiotics combine probiotics with prebiotics to synergistically elicit a health benefit in the consumer. Previous studies have shown that prebiotics can selectively stimulate the growth in the intestine of specific bacterial strains. In synbiotic supplementations the prebiotics constituent could increase the intestinal persistence and survival of accompanying probiotic strain(s) and/or modulate the endogenous host microbiota to contribute to the synergistic enhancement of the health-promoting effects of the synbiotic constituents. Our study establishes a profound effect of dietary-calcium-dependent inulin supplementation on the intestinal persistence of inulin-utilizing L. plantarum Lp900 in rats. We also show that in rats on a low-dietary-calcium regime, the survival and intestinal abundance of L. plantarum Lp900 are significantly increased by administering it as an inulin-containing synbiotic. This study demonstrates that prebiotics can enhance the intestinal delivery of specific probiotics and that the prebiotic effect is profoundly influenced by the calcium content of the diet.

DNAzyme Amplified Aptasensing Platform for Ochratoxin A Detection Using a Personal Glucose Meter.

Aptamer-based sensors have emerged as a major platform for detecting small-molecular targets, because aptamers can be selected to bind these small molecules with higher affinity and selectivity than other receptors such as antibodies. However, portable, accurate, sensitive, and affordable detection of these targets remains a challenge. In this work, we developed an aptasensing platform incorporating magnetic beads and a DNAzyme for signal amplification, resulting in high sensitivity. The biosensing platform was constructed by conjugating a biotin-labeled aptamer probe of small-molecular targets such as toxins and a biotin-labeled substrate strand on magnetic beads, and the DNAzyme strand hybridized with the aptamer probe to block the substrate cleavage activity. The specific binding of the small-molecular target by the aptamer probe can replace the DNAzyme strand and then induce the hybridization between the DNAzyme strand and substrate strand, and the iterative signal amplification reaction of hydrolysis and cleavage of the substrate chain occurs in the presence of a metal ion cofactor. Using invertase to label the substrate strand, the detection of small molecules of the toxin is successfully transformed into the measurement of glucose, and the sensitive analysis of small molecules such as toxins can be realized by using the household portable glucose meter as a readout. This platform is shown to detect ochratoxin, a common toxin in food, with a linear detection range of 5 orders of magnitude, a low detection limit of 0.88 pg/mL, and good selectivity. The platform is easy to operate and can be used as a potential choice for quantitative analysis of small molecules, at home or under point-of-care settings. Moreover, by changing and designing the aptamer probe and the arm of DNAzyme strand, it can be used for the analysis of other analytes.

Continuous production of fructooligosaccharides by recycling of the thermal-stable ß-fructofuranosidase produced by Aspergillus niger.

OBJECTIVE: To achieve continuous production of fructooligosaccharides (FOS) by recycling of the mycelial cells containing the thermal-stable ß-fructofuranosidase in Aspergillus niger without immobilization. RESULTS: The thermal-stable ß-fructofuranosidase FopA-V1 was successfully expressed in A. niger ATCC 20611 under the control of the constitutive promoter PgpdA. The engineered A. niger strain FV1-11 produced the ß-fructofuranosidase with improved thermostability, which remained 91.2% of initial activity at 50 °C for 30 h. Then its mycelial ß-fructofuranosidase was recycled for the synthesis of FOS. It was found that the enzyme still had 79.3% of initial activity after being reused for six consecutive cycles, whereas only 62.3% ß-fructofuranosidase activity was detected in the parental strain ATCC 20611. Meanwhile, the FOS yield of FV1-11 after six consecutive cycles reached 57.1% (w/w), but only 51.0% FOS yield was detected in ATCC 20611. CONCLUSIONS: The thermal-stable ß-fructofuranosidase produced by A. niger can be recycled to achieve continuous synthesis of FOS with high efficiency, providing a powerful and economical strategy for the industrial production of FOS.

Molecular insight into regioselectivity of transfructosylation catalyzed by GH68 levansucrase and ß-fructofuranosidase.

Glycoside hydrolase family 68 (GH68) enzymes catalyze ß-fructosyltransfer from sucrose to another sucrose, the so-called transfructosylation. Although regioselectivity of transfructosylation is divergent in GH68 enzymes, there is insufficient information available on the structural factor(s) involved in the selectivity. Here, we found two GH68 enzymes, ß-fructofuranosidase (FFZm) and levansucrase (LSZm), encoded tandemly in the genome of Zymomonas mobilis, displayed different selectivity: FFZm catalyzed the ß-(2→1)-transfructosylation (1-TF), whereas LSZm did both of 1-TF and ß-(2→6)-transfructosylation (6-TF). We identified His79FFZm and Ala343FFZm and their corresponding Asn84LSZm and Ser345LSZm respectively as the structural factors for those regioselectivities. LSZm with the respective substitution of FFZm-type His and Ala for its Asn84LSZm and Ser345LSZm (N84H/S345A-LSZm) lost 6-TF and enhanced 1-TF. Conversely, the LSZm-type replacement of His79FFZm and Ala343FFZm in FFZm (H79N/A343S-FFZm) almost lost 1-TF and acquired 6-TF. H79N/A343S-FFZm exhibited the selectivity like LSZm but did not produce the ß-(2→6)-fructoside-linked levan and/or long levanooligosaccharides that LSZm did. We assumed Phe189LSZm to be a responsible residue for the elongation of levan chain in LSZm and mutated the corresponding Leu187FFZm in FFZm to Phe. An H79N/L187F/A343S-FFZm produced a higher quantity of long levanooligosaccharides than H79N/A343S-FFZm (or H79N-FFZm), although without levan formation, suggesting that LSZm has another structural factor for levan production. We also found that FFZm generated a sucrose analog, ß-D-fructofuranosyl α-D-mannopyranoside, by ß-fructosyltransfer to d-mannose and regarded His79FFZm and Ala343FFZm as key residues for this acceptor specificity. In summary, this study provides insight into the structural factors of regioselectivity and acceptor specificity in transfructosylation of GH68 enzymes.

A Vacuolar Invertase CsVI2 Regulates Sucrose Metabolism and Increases Drought Tolerance in Cucumis sativus L.

Vacuolar invertase (VI) can irreversibly degrade sucrose into glucose and fructose and involve in plants abiotic-stress-tolerance. Cucumber (Cucumis sativus L.) is susceptible to drought stress, especially during the seedling stage. To date, the involvement of VI in drought tolerance in cucumber seedlings is in urgent need of exploration. In the present study, a cucumber vacuolar invertase gene, CsVI2, was isolated and functionally characterized. The results showed that (1) CsVI2 showed vacuolar invertase activity both in vivo and in vitro; (2) the transcript level of CsVI2, along with VI activity, was significantly induced by drought stress. Moreover, the expression of sucrose synthase 3 (CsSUS3) was increased and that of sucrose phosphate synthase 1 (CsSPS1) was decreased after exposure to drought stress, which was followed by an increase in sucrose synthase activity and a decrease in sucrose phosphate synthase activity; (3) CsVI2-overexpressing transformed cucumber seedlings showed enhanced vacuolar invertase activity and drought tolerance and 4) protein-protein interaction modelling indicated that a cucumber invertase inhibitor, CsINVINH3, can interact with CsVI2. In summary, the results indicate that CsVI2 as an invertase can regulate sucrose metabolism and enhance drought stress in cucumber seedlings.

Structural insight into the substrate specificity of Bombyx mori ß-fructofuranosidase belonging to the glycoside hydrolase family 32.

Sucrose-hydrolyzing enzymes are largely divided into ß-fructofuranosidase and sucrose α-glucosidase. The domestic silkworm Bombyx mori possesses both enzymes, BmSUC1 and BmSUH, belonging to the glycoside hydrolase family 32 (GH32) and GH13, respectively. BmSUC1 was presumed to be acquired by horizontal gene transfer from bacteria based on phylogenetic analysis and related to tolerance to sugar-mimic alkaloids contained in mulberry latex. Here we investigated the substrate specificity of recombinant BmSUC1 that can hydrolyze not only sucrose but also fructooligosaccharides and fructans, and revealed that the enzyme was competitively inhibited by 1,4-dideoxy-1,4-imino-D-arabinitol, one of the alkaloids. Moreover, the crystal structures of BmSUC1 in apo form and complex with sucrose were determined, and the active site pocket was shallow and suitable for shorter substrates but was related to more relaxed substrate specificity than the strict sucrose α-glucosidase BmSUH. Considering together with the distribution of BmSUC1-orthologous genes in many lepidopterans, our results suggest that BmSUC1 contributes to the digestion of fructooligosaccharides and fructans derived from feed plants.

An immobilized invertase enzyme for the selective determination of sucrose in fruit juices.

Poly(N-vinylpyrrolidone-co-butylacrylate-co-N-hydroxymethylacrylamide) has been synthesized by free radical polymerization at 70 °C. Copolymer were characterized by FT-IR, elemental analysis and viscometric methods. Invertase was immobilized onto poly(N-vinyl pyrrolidone-co-butyl acrylate-co-N-hydroxymethyl acrylamide) by entrapment method. Optimum parameters (pH, temperature, substrate concentration, amount of polymer) for immobilization to obtain maximum activity were investigated. Kinetic parameters, Km and Vmax, of the free and immobilized invertases were also assayed. Results showed that immobilization enhanced the enzyme stability against changes of pH and temperature and immobilized enzyme showed lower Km value than free enzyme. One of the most interesting results is that the optimum operational temperature of the immobilized enzyme was 15 °C higher than that of the free enzyme. The next is the activity of the immobilized enzyme at the optimum temperature (70 °C) was approximately the same as the activity of the free enzyme at its optimum temperature (55 °C). Finally, immobilized invertase were used for determination of sucrose in commercial fruit juices. A new method and equation based on immobilized invertase were derived for determination of sucrose in commercial cheryy and pomegranate juices.

Highly reusable invertase biocatalyst: Biological fibrils functionalized by photocrosslinking.

Here we report a novel strategy for the immobilization of invertase using amyloid-like fibrils as a support. Optimal conditions to get Tyr-Tyr covalent binding between invertase and the support were determined using a photocrosslinking approach. The biological fibrils with invertase activity turn into microstructured catalysts according to electron microscopy outcomes. Thermal and storage stability as well as optimal pH and temperature of the enzyme were conserved. Moreover, the immobilized enzyme recovered by low g-force centrifugation retained 83% of its initial enzymatic activity after 15 reuse cycles. Considering that enzyme cost is the most significant part of the overall fee of enzymatic biomass conversion, the highly efficient recovery/reuse strategy described herein becomes relevant. Besides, it can also be applied to the immobilization of other enzymes for industrial biocatalysis.

Comparative transcriptomic analysis of rhizomes, stems, and leaves of Polygonatum odoratum (Mill.) Druce reveals candidate genes associated with polysaccharide synthesis.

Polygonatum odoratum (Mill.) Druce is a well-known traditional Chinese herb. Polysaccharides are major bioactive components of Polygonatum odoratum, which can improve immunity, and are used to treat rheumatic heart disease, cardiovascular disease, and diabetes. This study identified potential genes and transcription factors (TFs) that regulate polysaccharide synthesis in Polygonatum odoratum (Mill.) Druce using RNA sequencing data from leaf, stem, and rhizome tissues. 76,714 unigenes were annotated in public databases. Analysis of KEGG annotations identified 18 key enzymes responsible for polysaccharide biosynthesis and the most of the upregulated expressed unigenes were enriched in rhizome tissue compared with leaf or stem tissue. 73 TFs involved in polysaccharide synthesis were predicted. In addition, key enzyme genes were verified by quantitative real-time PCR. This study substantially enlarged the public transcriptome datasets of this species, and provided insight into detection of novel genes involved in synthesis of polysaccharides and other secondary metabolites.

The influence of light combination on the physicochemical characteristics and enzymatic activity of soil with multi-metal pollution in phytoremediation.

Light irradiation with suitable quality and intensity could influence the success of phytoremediation by improving the biomass yield of plants. However, mechanisms involved in this influence on the contaminant accumulation and translocation ability of plants have rarely been studied. Five light combinations with different red (R) and blue (B) ratios (0, 10, 50, 75 and 100 % blue) at the same intensity (220 µmol m-2 s-1) were used to assist phytoremediation using Noccaea caerulescens, and the change in physicochemical characteristics and enzymatic activities of soils after phytoremediation were evaluated. Compared with the control, the light combinations and monochromic blue light significantly increased the activities of soil ureases, invertases, and phosphatases, whereas monochromic red light strongly inhibited the activities of these enzymes, because different light irradiations altered the formation and excretion of carbohydrates from plants for soil microorganism consumption. Plants under B50R50 treatment accumulated the highest concentrations of metals, but their chlorophyll concentrations and lipid peroxidation were similar to those other species with lower metal concentrations. Hence, light with a proper blue/red ratio can simultaneously improve the physicochemical characteristics and enzymatic activities of soils, increase the metal uptake capacity and oxidation resistance of plants, and reduce the leaching risk during phytoremediation processes.

Biosynthesis of polyhydroxyalkanoates from sucrose by metabolically engineered Escherichia coli strains.

Sucrose utilization has been established in Escherichia coli strains by expression of Mannheimia succiniciproducens ß-fructofuranosidase (SacC), which hydrolyzes sucrose into glucose and fructose. Recombinant E. coli strains that can utilize sucrose were examined for their abilities to produce poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] from sucrose. When recombinant E. coli strains expressing Ralstonia eutropha PhaCAB and SacC were cultured in MR medium containing 20 g/L of sucrose, all recombinant E. coli strains could produce P(3HB) from sucrose. Also, recombinant E. coli strains expressing Pseudomonas sp. MBEL 6-19 PhaC1437, Clostridium propionicum Pct540, R. eutropha PhaAB enzymes along with SacC could produce P(3HB-co-LA) from sucrose. Among the examined E. coli strains, recombinant E. coli XL1-Blue produced the highest contents of P(3HB) (53.60 ± 2.55 wt%) and P(3HB-co-LA) (29.44 ± 0.39 wt%). In the batch fermentations, recombinant E. coli XL1-Blue strains completely consumed 20 g/L of sucrose as the sole carbon source and supported the production of 3.76 g/L of P(3HB) and 1.82 g/L of P(3HB-co-LA) with 38.21 wt% P(3HB) and 20.88 wt% P(3HB-co-LA) contents, respectively. Recombinant E. coli strains developed in this study can be used to establish a cost-efficient biorefinery for the production of polyhydroxyalkanoates (PHAs) from sucrose, which is an abundant and inexpensive carbon source.

Preparation and application of silica nanoparticles-Ocimum basilicum seeds bio-hybrid for the efficient immobilization of invertase enzyme.

For efficient utilization of immobilized invertase enzyme, a novel bio-hybrid support comprising silica nanoparticles and Ocimum basilicum seed was synthesized. Ocimum basilicum seeds provide a natural fibrouspellicular structure which acts as template for assembly of silica nanoparticles. Bio-hybrids of two different morphologies have been obtained by changing the physico-chemical conditions of the assembly process. Developed bio-hybrids were characterized through small angle X-ray scattering (SAXS), scanning electron microscopy (SEM), Synchrotron radiation based X-ray micro-computed tomography (SRµCT), Brunauer-Emmett-Teller (BET) and Fourier transform infrared spectroscopy (FTIR). Incorporation of the nanoparticles results to a fourfold increase in the available surface area of the seeds which is one of the important criteria for an immobilizing support. Synthesized bio-hybrids were used for the immobilization of commercially applicable invertase enzyme and efficient loading of enzyme was realized. Enzyme immobilized bio-hybrids could be easily separated out and reused up to eight times with 82 % retention of enzyme activity. Present work suggests that the unique features of the bio-hybrid make it suitable candidate for immobilization of enzymes in general.