Comparative genomics of a novel Erwinia species associated with the Highland midge (Culicoides impunctatus).

Erwinia (Enterobacterales: Erwiniaceae) are a group of cosmopolitan bacteria best known as the causative agents of various plant diseases. However, other species in this genus have been found to play important roles as insect endosymbionts supplementing the diet of their hosts. Here, I describe Candidatus Erwinia impunctatus (Erwimp) associated with the Highland midge Culicoides impunctatus (Diptera: Ceratopogonidae), an abundant biting pest in the Scottish Highlands. The genome of this new Erwinia species was assembled using hybrid long and short read techniques, and a comparative analysis was undertaken with other members of the genus to understand its potential ecological niche and impact. Genome composition analysis revealed that Erwimp is similar to other endophytic and ectophytic species in the genus and is unlikely to be restricted to its insect host. Evidence for an additional plant host includes the presence of a carotenoid synthesis operon implicated as a virulence factor in plant-associated members in the sister genus Pantoea. Unique features of Erwimp include several copies of intimin-like proteins which, along with signs of genome pseudogenization and a loss of certain metabolic pathways, suggests an element of host restriction seen elsewhere in the genus. Furthermore, a screening of individuals over two field seasons revealed the absence of the bacteria in Culicoides impunctatus during the second year indicating this microbe-insect interaction is likely to be transient. These data suggest that Culicoides impunctatus may have an important role to play beyond a biting nuisance, as an insect vector transmitting Erwimp alongside any conferred impacts to surrounding biota.

Production of vitamin A and vitamin E: expression of vitreoscilla hemoglobin gene in Erwinia herbicola.

Vitamin A prevents eye problems, blindness and skin problems by strengthening the immune system. Vitamin E is a nutrient that has important roles in many areas such as skin health, eye health and hormonal order. Vitreoscilla hemoglobin (VHb) gives an advantage in later phases of grown conditions to cells. In this study, the intracellular and extracellular production of vitamin A and E in E. herbicola and its recombinant strains (vgb- and vgb+) in the three different M9 mediums with supplemented 0.1% glucose, 0.1% fructose and 0.1% sucrose was investigated. Additionally, the viable cell number and total cell mass (OD600) were measured by the host and the recombinant bacteria in these mediums. The VHb gene expression in E. herbicola enhanced vitamin A under different carbon conditionals. Especially, in the vgb + strain (carrying vgb gene) the production of total vitamin in 0.1% glucose medium was recorded as 0.14 µg/ml, while the production in fructose and sucrose media was recorded as 0.07 µg/ml. The production of intracellular vitamin E in the host strain (0.025 µg/ml) was about 13-fold (0.002 µg/ml) higher than vgb + recombinant strain in 0.1% fructose. The vgb + strain showed about 2-fold higher extracellular vitamin E production than the host strain.

Microbial community dynamics in phyto-thermotherapy baths viewed through next generation sequencing and metabolomics approach.

Phyto-thermotherapy is a treatment consisting in immersing oneself in baths of self-heating alpine grass, to benefit of the heat and rich aromatic components released by the process. The aim of this study was to characterize the bacterial and fungal diversity of three phyto-thermal baths (PTB) performed in three different months, and to compare the data with the profile of the volatile organic compounds (VOCs) of the process. All the data collected showed that PTBs were structured in two stages: the first three days were characterised by an exponential rise of the temperature, a fast bacterial development, higher microbial diversity and higher concentrations of plant aliphatic hydrocarbons. The second stage was characterised by a stable high temperature, shrinkage of the microbial diversity with a predominance of few bacterial and fungi species and higher concentrations of volatiles of microbial origin. Erwinia was the dominant microbial species during the first stage and probably responsible of the self-heating process. In conclusion, PTBs has shown both similarities with common self-heating processes and important peculiarities such as the absence of pathogenic bacteria and the dominance of plant terpenoids with health characteristics among the VOCs confirming the evidence of beneficial effects in particular in the first three days.

Production, extraction and characterization of Chlorella vulgaris soluble polysaccharides and their applications in AgNPs biosynthesis and biostimulation of plant growth.

Chlorella vulgaris, like a wide range of other microalgae, are able to grow mixotrophically. This maximizes its growth and production of polysaccharides (PS). The extracted polysaccharides have a complex monosaccharide composition (fructose, maltose, lactose and glucose), sulphate (210.65 ± 10.5 mg g-1 PS), uronic acids (171.97 ± 5.7 mg g-1 PS), total protein content (32.99 ± 2.1 mg g-1 PS), and total carbohydrate (495.44 ± 8.4 mg g-1 PS). Fourier Transform infrared spectroscopy (FT-IR) analysis of the extracted polysaccharides showed the presence of N-H, O-H, C-H, -CH3, >CH2, COO-1, S=O and the C=O functional groups. UV-Visible spectral analysis shows the presence of proteins, nucleic acids and chemical groups (ester, carbonyl, carboxyl and amine). Purified polysaccharides were light green in color and in a form of odorless powder. It was soluble in water but insoluble in other organic solvents. Thermogravimetric analysis demonstrates that Chlorella vulgaris soluble polysaccharide is thermostable until 240°C and degradation occurs in three distinct phases. Differential scanning calorimetry (DSC) analysis showed the characteristic exothermic transition of Chlorella vulgaris soluble polysaccharides with crystallization temperature peaks at 144.1°C, 162.3°C and 227.7°C. The X-ray diffractogram illustrated the semicrystalline nature of these polysaccharides. Silver nanoparticles (AgNPs) had been biosynthesized using a solution of Chlorella vulgaris soluble polysaccharides. The pale green color solution of soluble polysaccharides was turned brown when it was incubated for 24 hours with 100 mM silver nitrate in the dark, it showed peak maximum located at 430 nm. FT-IR analysis for the biosynthesized AgNPs reported the presence of carbonyl, -CH3, >CH2, C-H,-OH and -NH functional groups. Scanning and transmission electron microscopy show that AgNPs have spherical shape with an average particle size of 5.76. Energy-dispersive X-ray (EDX) analysis showed the dominance of silver. The biosynthesized silver nanoparticles were tested for its antimicrobial activity and have positive effects against Bacillus sp., Erwinia sp., Candida sp. Priming seeds of Triticum vulgare and Phaseolus vulgaris with polysaccharides solutions (3 and 5 mg mL-1) resulted in significant enhancement of seedling growth. Increased root length, leaf area, shoot length, photosynthetic pigments, protein content, carbohydrate content, fresh and dry biomass were observed, in addition these growth increments may be attributed to the increase of antioxidant activities.

Comparative genomics of the Erwinia and Enterobacter olive fly endosymbionts.

The pestivorous tephritid olive fly has long been known as a frequent host of the obligately host-associated bacterial endosymbiont, Erwinia dacicola, as well as other facultative endosymbionts. The genomes of Erwinia dacicola and Enterobacter sp. OLF, isolated from a California olive fly, encode the ability to supplement amino acids and vitamins missing from the olive fruit on which the larvae feed. The Enterobacter sp. OLF genome encodes both uricase and ureases, and the Er. dacicola genome encodes an allantoate transport pathway, suggesting that bird feces or recycling the fly's waste products may be important sources of nitrogen. No homologs to known nitrogenases were identified in either bacterial genome, despite suggestions of their presence from experiments with antibiotic-treated flies. Comparisons between the olive fly endosymbionts and their free-living relatives revealed similar GC composition and genome size. The Er. dacicola genome has fewer genes for amino acid metabolism, cell motility, and carbohydrate transport and metabolism than free-living Erwinia spp. while having more genes for cell division, nucleotide metabolism and replication as well as mobile elements. A 6,696 bp potential lateral gene transfer composed primarily of amino acid synthesis and transport genes was identified that is also observed in Pseudomonas savastanoii pv savastanoii, the causative agent of olive knot disease.

Inhibición del crecimiento de Erwinia chrysanthemi a diferentes concentraciones de ácido fólico: posible uso del ácido fólico como agente bacteriostático y fortificante de la papa Solanum tuberosum / Inhibition of Erwinia chrysanthemi growth to different concentrations of folic acid: possible use of folic acid as bacteriostatic agent and fortifying of Solanum tuberosum potato

Resumen Introducción: Las enterobacterias del genero Erwinia spp producen enfermedades en la papa, un tubérculo de consumo masivo. La regulación de la metilación del DNA puede regular la proliferación de la Erwinia, de tal modo que las concentraciones del ácido fólico, pueden tener un efecto en la capacidad patógena del microorganismo. De otra parte, el ácido fólico previene la aparición de defectos del tubo neural en humanos. Objetivo: Evaluar al ácido fólico como un agente bacteriostático de la Erwinia y que a su vez sea parte de la fortificación de alimentos de consumo masivo como la papa. Materiales y métodos: Se llevó a cabo la caracterización bioquímica de la Erwinia chrysanthemi, se estudió su crecimiento frente a diferentes concentraciones de ácido fólico Resultados: Al aumentar las concentraciones de la vitamina, desde 0,3 µg/L hasta 6,8 µg/L se inhibe el crecimiento bacteriano de la Erwinia chrysanthemi. La vitamina inhibe el crecimiento en cultivo de Erwinia chrysanthemi y actúa como como agente bacteriostático, aspecto es de gran relevancia dado que teóricamente, si la papa estuviera fortificada con el micronutriente, este actuaría contra el agente infeccioso y al mismo tiempo contribuiría al consumo adecuado de la vitamina en la población general.

Abstract Introduction: The enterobacteria of the Erwinia spp genus produce disease in potatoes, which is a tuber of mass consumption. The regulation of DNA methylation can regulate the proliferation of Erwinia in such a way that the concentrations of folic acid may have an effect on the microorganism pathogenic ability. On the other hand, the folic acid prevents the appearance of neural tube defects in humans. Objective: To evaluate folic acid as a bacteriostatic agent of Erwinia and, at the same time, as part of the fortification of mass consumption food such as the potatoes. Materials and methods: The biochemical characterization of the Erwinia chrysanthemi was carried out and its growth compared to different concentrations of folic acid was studied. Results: When increasing the concentrations of the vitamin from 0.3 µg/L up to 6.8 µg/L, the bacterial growth of Erwinia chrysanthemi is inhibited. The vitamin inhibits the growth in cultivation of Erwinia chrysanthemi and acts as a bacteriostatic agent. This aspect is of great importance given that, theoretically, if potatoes were fortified with micro-nutrient, this would act against the infectious agent and, at the same time, contribute to the adequate intake of the vitamin in the general population.

Endophytic Detection in Selected European Herbal Plants.

A total of 181 cultivable endophytic bacterial isolates were collected from stems of 13 species of herbs inhabiting Europe (Poland): Chelidonium majus L., Elymus repens L., Erigeron annuus L., Euphrasia rostkoviana Hayne, Foeniculum vulgare L., Geranium pratense L., Humulus lupulus L., Matricaria chamomilla L., Mentha arvensis L., Papaver rhoeas L., Rosmarinus officinalis L., Solidago gigantea L. and Vinca minor L. The isolates were screened for their antifungal activity and fifty three were found to inhibit fungal growth. Of these, five had strong antifungal properties. These selected isolates were identified as: Pseudomonas azotoformans, P. cedrina, Bacillus subtilis group and Erwinia persicina.

Antimicrobial potentials of Catharanthus roseus by disc diffusion assay.

The present research work investigates the in vitro antimicrobial activity of different solvent extracted samples from the aerial parts (stem, leaf, fruit and flower) of C. roseus against different microbial species using disc diffusion assay at two different concentrations of 1 and 2 mg disc-1. Hexane extracted samples inhibited the growth of all tested microbial strains except S. typhi. Similarly, ethyl acetate extracted samples was effective to control the activity of all the tested microbial strains. E. coli and S. typhi showed resistance to chloroform extracted samples and the remaining eight microbial strains were susceptible to the same extract. Butanol extracted samples did not inhibit the growth of K. pneumonia and S. typhi at low concentration, however, at higher concentration the same extract reduced the growth of different microbes. Methanol extracted samples effectively controlled the growth of all tested microbes at both concentrations except for S. typhi. Water extracted samples did not inhibit the growth at low concentration except E. coli, K. pneumonia and S. aureus and were ineffective against P. aeroginosa at both concentration. C. albicans, showed resistance against chloroform and water extracted samples at low concentration and susceptible to other solvent extracted samples at both concentration. All fractions were effective against plant pathogens i.e. E. carotovora and A. tumefaciens.

Bioactivity of root endophytic freshwater Hyphomycetes Anguillospora longissima (Sacc. & Syd.) Ingold.

Anguillospora longissima, isolated from root as endophytic freshwater hyphomycetes, was evaluated for its bioactivity (antibacterial potential) against five bacterial strains, namely, Gram-positive (Bacillus subtilis MTCC 121) and Gram-negative (Agrobacterium tumefaciens MTCC 609, Escherichia coli MTCC 40, Erwinia chrysanthemum, and Xanthomonas pseudomonas). Antimicrobial activity was assessed by measuring the zone of inhibition with preliminary and secondary antimicrobial assays. The applied fungus was found significant for all tested bacterial strains as showen by their zone of inhibition. In preliminary antimicrobial assay, maximum zone of inhibition was recorded against Gram-negative human pathogenic bacterial strain Escherichia coli (23 mm) followed by Erwinia chrysanthemi (22 mm), Agrobacterium tumefaciens (21 mm), and Xanthomonas phaseoli (21 mm), while minimum zone of inhibition was observed against Bacillus subtilis (20 mm). In secondary antimicrobial assay, the maximum zone of inhibition was recorded against Erwinia chrysanthemi (11 mm) followed by Agrobacterium tumefaciens (10 mm), Xanthomonas phaseoli (10 mm), and Bacillus subtilis (9 mm) and minimum inhibition was found against Escherichia coli (8 mm).

ß-carotene-producing bacteria residing in the intestine provide vitamin A to mouse tissues in vivo.

Vitamin A deficiency (VAD) is an overwhelming public health problem that affects hundreds of millions of people worldwide. A definitive solution to VAD has yet to be identified. Because it is an essential nutrient, vitamin A or its carotenoid precursor ß-carotene can only be obtained from food or supplements. In this study, we wanted to establish whether ß-carotene produced in the mouse intestine by bacteria synthesizing the provitamin A carotenoid could be delivered to various tissues within the body. To achieve this, we took advantage of the Escherichia coli MG1655*, an intestine-adapted spontaneous mutant of E. coli MG1655, and the plasmid pAC-BETA, containing the genes coding for the 4 key enzymes of the ß-carotene biosynthetic pathway (geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and lycopene cyclase) from Erwinia herbicola. We engineered the E. coli MG1655* to produce ß-carotene during transformation with pAC-BETA (MG1655*-ßC) and gavaged wild-type and knockout mice for the enzyme ß-carotene 15,15'-oxygenase with this recombinant strain. Various regimens of bacteria administration were tested (single vs. multiple and low vs. high doses). ß-Carotene concentration was measured by HPLC in mouse serum, liver, intestine, and feces. Enumeration of MG1655*-ßC cells in the feces was performed to assess efficiency of intestinal colonization. We demonstrated in vivo that probiotic bacteria could be used to deliver vitamin A to the tissues of a mammalian host. These results have the potential to pave the road for future investigations aimed at identifying alternative, novel approaches to treat VAD.

Processive pectin methylesterases: the role of electrostatic potential, breathing motions and bond cleavage in the rectification of Brownian motions.

Pectin methylesterases (PMEs) hydrolyze the methylester groups that are found on the homogalacturonan (HG) chains of pectic polysaccharides in the plant cell wall. Plant and bacterial PMEs are especially interesting as the resulting de-methylesterified (carboxylated) sugar residues are found to be arranged contiguously, indicating a so-called processive nature of these enzymes. Here we report the results of continuum electrostatics calculations performed along the molecular dynamics trajectory of a PME-HG-decasaccharide complex. In particular it was observed that, when the methylester groups of the decasaccharide were arranged in order to mimic the just-formed carboxylate product of de-methylesterification, a net unidirectional sliding of the model decasaccharide was subsequently observed along the enzyme's binding groove. The changes that occurred in the electrostatic binding energy and protein dynamics during this translocation provide insights into the mechanism by which the enzyme rectifies Brownian motions to achieve processivity. The free energy that drives these molecular motors is thus demonstrated to be incorporated endogenously in the methylesterified groups of the HG chains and is not supplied exogenously.

Cold active pectinases: advancing the food industry to the next generation.

Pectinase has been an integral part of commercial food processing, where it is used for degradation of pectin and facilitates different processing steps such as liquefaction, clarification and juice extraction. The industry currently uses pectinases from mesophilic or thermophilic microorganisms which are well established, but recently, there has been is a new trend in the food industry to adopt low-temperature processing. This trend is due to the potential economic and environmental advantages which the industry envisages. In order to achieve this change, an alternative for the existing pectinases, which are mostly mesophilic and temperature-dependent, must be identified, which can function efficiently at low temperatures. Psychrophilic pectinases derived from cold-adapted microorganisms, are known to function at low to freezing temperatures and may be an alternative to address the problem. Psychrophilic pectinases can be obtained from the vast microflora inhabiting various cold regions on earth such as oceans, Polar Regions, snow-covered mountains, and glaciers. This article is intended to study the advantages of cold active pectinases, its sources, and the current state of the research.

Identification and genetics of 6-thioguanine secreted by Erwinia species and its interference with the growth of other bacteria.

We identified a compound in culture supernatants of Erwinia species, such as Erwinia amylovora, E. pyrifoliae, E. billingiae, E. tasmaniensis, E. persicina and E. rhapontici absorbing at 340 nm, which was associated before with the yellow pigment produced by E. amylovora on media containing copper ions. The compound was purified from E. tasmaniensis strain Et1/99 supernatants by chromatography on Dowex-1 and Dowex-50 columns and identified by HPLC/MS and NMR analysis as 6-thioguanine (6TG). Its signal at 167 Da matched with the expected molecular mass. By random mutagenesis with miniTn5, we obtained mutants defective in the genes for pyrimidine and purine metabolism. A specific gene cluster with ycf genes described by us before, absent in the corresponding region of Escherichia coli, was identified in the genome sequence of three Erwinia species and named tgs region for thioguanine synthesis. Clones of the tgs gene cluster promoted 6TG synthesis and secretion in E. coli, when the bacteria were grown in minimal medium supplemented with amino acids. 6TG was bacteriostatic for E. coli and Salmonella typhimurium strains, with cell growth resumed after prolonged incubation. Similar results were obtained with P. agglomerans strains. Bacteria from the genus Pectobacterium were barely and Rahnella or Gibbsiella species were not inhibited by 6TG. Adenine and guanine relieved the toxic effect of 6TG on E. coli. Non-producing strains were fully virulent on host plants. 6TG synthesis may help erwinias to interfere with growth of some microorganisms in the environment.

Analysis of supercooling activity of tannin-related polyphenols.

Based on the discovery of novel supercooling-promoting hydrolyzable gallotannins from deep supercooling xylem parenchyma cells (XPCs) in Katsura tree (see Wang et al. (2012) [38]), supercooling capability of a wide variety of tannin-related polyphenols (TRPs) was examined in order to find more effective supercooling-promoting substances for their applications. The TRPs examined were single compounds including six kinds of hydrolyzable tannins, 11 kinds of catechin derivatives, two kinds of structural analogs of catechin and six kinds of phenolcarboxylic acid derivatives, 11 kinds of polyphenol mixtures and five kinds of crude plant tannin extracts. The effects of these TRPs on freezing were examined by droplet freezing assays using various solutions containing different kinds of identified ice nucleators such as the ice nucleation bacterium (INB) Erwinia ananas, the INB Xanthomonas campestris, silver iodide and phloroglucinol as well as a solution containing only unintentionally included unidentified airborne ice nucleators. Among the 41 kinds of TRPs examined, all of the hydrolyzable tannins, catechin derivatives, polyphenol mixtures and crude plant tannin extracts as well as a few structural analogs of catechin and phenolcarboxylic acid derivatives exhibited supercooling-promoting activity (SCA) with significant differences (p>0.05) from at least one of the solutions containing different kinds of ice nucleators. It should be noted that there were no TRPs exhibiting ice nucleation-enhancing activity (INA) in all solutions containing identified ice nucleators, whereas there were many TRPs exhibiting INA with significant differences in solutions containing unidentified ice nucleators alone. An emulsion freezing assay confirmed that these TRPs did not essentially affect homogeneous ice nucleation temperatures. It is thought that not only SCA but also INA in the TRPs are produced by interactions with heterogeneous ice nucleators, not by direct interaction with water molecules. In the present study, several TRPs that might be useful for applications due to their high SCA in many solutions were identified.

Substrate dynamics in enzyme action: rotations of monosaccharide subunits in the binding groove are essential for pectin methylesterase processivity.

The dynamical behavior of biomacromolecules is a fundamental property regulating a large number of biological processes. Protein dynamics have been widely shown to play a role in enzyme catalysis; however, the interplay between substrate dynamics and enzymatic activity is less understood. We report insights into the role of dynamics of substrates in the enzymatic activity of PME from Erwinia chrysanthemi, a processive enzyme that catalyzes the hydrolysis of methylester groups from the galacturonic acid residues of homogalacturonan chains, the major component of pectin. Extensive molecular dynamics simulations of this PME in complex with decameric homogalacturonan chains possessing different degrees and patterns of methylesterification show how the carbohydrate substitution pattern governs the dynamics of the substrate in the enzyme's binding cleft, such that substrate dynamics represent a key prerequisite for the PME biological activity. The analyses reveal that correlated rotations around glycosidic bonds of monosaccharide subunits at and immediately adjacent to the active site are a necessary step to ensure substrate processing. Moreover, only substrates with the optimal methylesterification pattern attain the correct dynamical behavior to facilitate processive catalysis. This investigation is one of the few reported examples of a process where the dynamics of a substrate are vitally important.

Control of postharvest soft rot caused by Erwinia carotovora of vegetables by a strain of Bacillus amyloliquefaciens and its potential modes of action.

Erwinia carotovora subsp. carotovora (Ecc), the causal agent of bacterial soft rot, is one of the destructive pathogens of postharvest vegetables. In this study, a bacterial isolate (BGP20) from the vegetable farm soil showed strong antagonistic activity against Ecc in vitro, and its twofold cell-free culture filtrate showed excellent biocontrol effect in controlling the postharvest bacterial soft rot of potatoes at 25 °C. The anti-Ecc metabolites produced by the isolate BGP20 had a high resistance to high temperature, UV-light and protease K. Based on the colonial morphology, cellular morphology, sporulation, and partial nucleotide sequences of 16S rRNA and gyrB gene, the isolate BGP20 was identified as Bacillus amyloliquefaciens subsp. plantarum. Further in vivo assays showed that the BGP20 cell culture was more effective in controlling the postharvest bacterial soft rot of green peppers and Chinese cabbages than its twofold cell-free culture filtrate. In contrast, the biocontrol effect and safety of the BGP20 cell culture were very poor on potatoes. In the wounds of potatoes treated with both the antagonist BGP20 and the pathogen Ecc, the viable count of Ecc was 31,746 times that of BGP20 at 48 h of incubation at 25 °C. But in the wounds of green peppers, the viable count of BGP20 increased 182.3 times within 48 h, and that of Ecc increased only 51.3 %. In addition, the treatment with both BGP20 and Ecc induced higher activity of phenylalanine ammonia-lyase (PAL) than others in potatoes. But the same treatment did not induce an increase of PAL activity in green peppers. In conclusion, the present study demonstrated that the isolate BGP20 is a promising candidate in biological control of postharvest bacterial soft rot of vegetables, but its main mode of action is different among various vegetables.

Botanicals to control soft rot bacteria of potato.

Extracts from eleven different plant species such as jute (Corchorus capsularis L.), cheerota (Swertia chiraita Ham.), chatim (Alstonia scholaris L.), mander (Erythrina variegata), bael (Aegle marmelos L.), marigold (Tagetes erecta), onion (Allium cepa), garlic (Allium sativum L.), neem (Azadiracta indica), lime (Citrus aurantifolia), and turmeric (Curcuma longa L.) were tested for antibacterial activity against potato soft rot bacteria, E. carotovora subsp. carotovora (Ecc) P-138, under in vitro and storage conditions. Previously, Ecc P-138 was identified as the most aggressive soft rot bacterium in Bangladeshi potatoes. Of the 11 different plant extracts, only extracts from dried jute leaves and cheerota significantly inhibited growth of Ecc P-138 in vitro. Finally, both plant extracts were tested to control the soft rot disease of potato tuber under storage conditions. In a 22-week storage condition, the treated potatoes were significantly more protected against the soft rot infection than those of untreated samples in terms of infection rate and weight loss. The jute leaf extracts showed more pronounced inhibitory effects on Ecc-138 growth both in in vitro and storage experiments.

Immobilization of glucosyltransferase from Erwinia sp. using two different techniques.

Two different techniques of glucosyltransferase immobilization were studied for the conversion of sucrose into isomaltulose. The optimum conditions for immobilization of Erwinia sp. glucosyltransferase onto Celite 545, determined using response surface methodology, was pH 4.0 and 170 U of glucosyltransferase/g of Celite 545. Using this conditions more than 60% conversion of sucrose into isomaltulose can be obtained. The immobilization of glucosyltransferase was also studied by its entrapment in microcapsules of low-methoxyl pectin and fat (butter and oleic acid). The non-lyophilized microcapsules of pectin, containing the enzyme and fat, showed higher glucosyltransferase activity, compared with lyophilized microcapsules containing enzyme plus fat, and also lyophilized microcapsules containing enzyme without fat addition. The non-lyophilized microcapsules of pectin containing the glucosyltransferase and fat, converted 30% of sucrose into isomaltulose in the first batch. However the conversion decreased to 5% at the 10th batch, indicating inactivation of the enzyme.

Pinellia ternata agglutinin expression in chloroplasts confers broad spectrum resistance against aphid, whitefly, Lepidopteran insects, bacterial and viral pathogens.

Broad spectrum protection against different insects and pathogens requires multigene engineering. However, such broad spectrum protection against biotic stress is provided by a single protein in some medicinal plants. Therefore, tobacco chloroplasts were transformed with the agglutinin gene from Pinellia ternata (pta), a widely cultivated Chinese medicinal herb. Pinellia ternata agglutinin (PTA) was expressed up to 9.2% of total soluble protein in mature leaves. Purified PTA showed similar hemagglutination activity as snowdrop lectin. Artificial diet with purified PTA from transplastomic plants showed marked and broad insecticidal activity. In planta bioassays conducted with T0 or T1 generation PTA lines showed that the growth of aphid Myzus persicae (Sulzer) was reduced by 89%-92% when compared with untransformed (UT) plants. Similarly, the larval survival and total population of whitefly (Bemisia tabaci) on transplastomic lines were reduced by 91%-93% when compared with UT plants. This is indeed the first report of lectin controlling whitefly infestation. When transplastomic PTA leaves were fed to corn earworm (Helicoverpa zea), tobacco budworm (Heliothis virescens) or the beet armyworm (spodoptera exigua), 100% mortality was observed against all these three insects. In planta bioassays revealed Erwinia population to be 10,000-fold higher in control than in PTA lines. Similar results were observed with tobacco mosaic virus (TMV) challenge. Therefore, broad spectrum resistance to homopteran (sap-sucking), Lepidopteran insects as well as anti-bacterial or anti-viral activity observed in PTA lines provides a new option to engineer protection against biotic stress by hyper-expression of an unique protein that is naturally present in a medicinal plant.