Levan Formation Research Articles

First report of bacterial leaf spot on Hami melon caused by Pseudomonas aeruginosa in China.

Hami melon (Cucumis melon var. saccharinus) is an economically important crop all over the world. It is being extensively planted in greenhouse in the southwest part of Hainan province, China. A new bacterial leaf spot was observed in a 20 hm2 Hami melon plantation in Huangliu town, Ledong county, Hainan province, in January 2022. The incidence of the disease was approximately 5%. Symptoms were irregularly shaped, brown lesions with yellow haloes on mature leaves, and first appeared as small, dark-green, water-soaking spots. Specimens from the lesion margin were disinfected by submersion in 0.1% mercuric chloride for 1 min, then soaked with 75% alcohol for 30 s, and rinsed with sterilized distilled water. The tissues were then ground in 2 ml of sterile water and allowed to stand for 5min. The supernatant was streaked onto nutrient agar (NA) and incubated for 48h at 30°C. Colonies were round, smooth, colorless, nearly transparent, bead-shaped at first, and then became lightly blue. After being cultured for days on NA at 30℃, the bacteria can turn the media brown. Yellow green pigments (pyoverdin) that fluoresce under ultraviolet light could be produced by the isolates in the Luria Broth. The bacteria were gram-negative, rod shaped with a single polar flagellum, 0.4 to 1.1 × 1.4 to 3.4 µm. Its physiological and biochemical characteristics were as follows: positive for the oxidase, aerobic, arginine dihydrolase, gelatin liquefaction, denitrification, lipase, growth at 41℃, utilization of mannitol, and production of pyocyanin tests; negative for the hydrolysis of starch, levan formation, lecithinase, growth at 4℃, growth in media supplemented with 8.5% NaCl, and utilization of maltose, xylose, and ethylene glycol tests. The 16S rRNA (1,437 bp), gyrB (1,181 bp), and rpoB genes (1,510 bp) were amplified with 27F/1492R (Zhang et al. 2016）, UP-1s/UP-2sr（Hannula M，2007）, and rpoB-F/rpoB-R (Ogier, JC. et al., 2019) primer sets respectively. One of the 5 isolates collected was sequenced. A BLASTn search of GenBank revealed that the sequence of 16S rRNA gene (OQ918303) had 99.7% identity and 98% coverage comparing with the best hit Pseudomonas aeruginosa strain DSM 50071(NR_117678.1), and both gyrB (OR261077) and rpoB (OR261078) had 99.9% identity and over 98% coverage comparing with P. aeruginosa E90 (CP044006.1). A pathogenicity test was conducted by spraying a suspension of the bacteria (108 CFU/mL) onto 10 Hami melon seedlings with two true leaves. Controls were inoculated with sterile water. All inoculated plants were maintained at 28℃ with 80 to 85% relative humidity in a greenhouse. Dark-green, water soaking spots appeared on the cotyledon and stems of treated seedlings 3-5 days after inoculation, and dark green lesions with halos were observed on the true leaves at the same time. Symptoms did not occur on the control plants. The bacteria which were re-isolated from the inoculated plants were confirmed as P. aeruginosa based on the 16S rRNA gene sequence. The bacterium was not isolated from control plants. P. aeruginosa has been reported to cause disease on a variety of plants including tomato (Zhang et al., 2021), poplar (Liu, et al., 2019), ginseng (Gao et al., 2014), tinda (Mondal et al., 2012), onion (Abd-Alla et al., 2011), tobacco (Yu et al., 2008) and sweet basil (Walker et al., 2004). As far as we know, this is the first report of P. aeruginosa causing leaf spot on Hami melon in China.. This report will contribute to the recognition and diagnosis of the new disease for the Hami melon growers.

First Report of Bacterial Canker of Pecan Caused by Pseudomonas syringae pv. syringae in China.

Pecan (Carya illinoinensis) is a world-famous nut tree that is widely cultivated in China, especially in Jiangsu Province (Zhang et al. 2015). In April 2022, cankers on trunks were recorded in pecan (cv. Pawnee) fields located in Taizhou (32°27'58″ N, 120°0'49″ E), Jiangsu. Cankers on the trunks resulted in wilt of the plants. Usually, the color of infected bark on the trunk became darker than the healty bark. When the outer bark was peeled away, the inner tissues were water-soaked, often with reddish streaks. In the surveyed orchards, disease incidence ranged from 10 to 20% among young saplings (about 200 three-year-old trees). While no fungal mycelium or spores were found in the diseased areas by microscope, bacterial colonies were isolated by surface-sterilizing small fragments (25 mm2) of symptomatic tissue in 0.5% NaOCl, rinsing the sections twice in sterilized water, and then streaking them on Luria-Bertani (LB) plates. More than 20 bacterial isolates were obtained and all isolates induced a hypersensitive response on Nicotiana tabacum. All isolates were fluorescent on King's medium B, and were gram-negative based on lysis by KOH. Isolates were positive for levan formation, negative for oxidase and arginine dihydrolase, and did not cause soft rot on potato slices. Based on above information, the isolates thus belonged to Lelliot's LOPAT group 1, P. syringae (Lelliott and Stead 1988). The 16S rRNA sequences of five representative isolates (accession numbers OP175939-OP175943) were amplified by PCR, sequenced, and compared with the NCBI GenBank database (Weisburg et al. 1991; Sarkar and Guttman 2004), finding a 99.92% genetic similarity with a previously reported 16S rRNA sequence of a Pseudomonas syringae pv. syringae (Pss) isolate (accession numbers NW389777). Additional housekeeping genes gap1(accession numbers OP186937-OP186941), rpoD (accession numbers OP186952-OP186956), gyrB (accession numbers OP186947-OP186951), and gltA (accession numbers OP186942-OP186946) were PCR-amplified and sequenced as reported by Hwang et al. (2005), followed by multilocus sequence typing analysis (MLSA). Molecular phylogenetic trees (MEGA vesion 6.0, maximum likelihood with Jukes-Cantor model, 1,000 bootstraps) were generated based on each of these five DNA regions and revealed that all five isolates were clustered together with the strains in P. syringae genomospecies 2, and grouped these isolates with Pss in the PAMDB database (Hwang et al. 2005). As a result, these isolates were identified as Pss. Pathogenicity on pecan (cv. Pawnee) was confirmed by cutting the trunks of two-year-old pecan trees with sterilized blades dipped in cell suspensions containing 107 CFU/ml of each isolate. Plants inoculated in a similar manner with sterile water served as negative controls. The inoculated plants were incubated in a greenhouse maintained at 25°C and 80% relative humidity. After 7 to 8 days, all inoculated plants showed the symptoms of necrosis previously described for the original field plants, while the control plants did not show symptoms. The bacteria reisolated from the inoculated plants were identified as Pss using the LOPAT tests. These results and the sequence analysis of the 16S rRNA and four housekeeping genes described above, fulfilled Koch's postulates. No target bacteria were isolated from the control plants. To our knowledge, this is the first report of Pseudomonas syringae pv. syringaecausing bacterial canker of pecan worldwide. The identification of this pathogen will allow the study of strategies for managing the disease.

Biosynthesis and Structural Characterization of Levan by a Recombinant Levansucrase from Bacillus subtilis ZW019

PurposeThe yield of levan extracted from microbial fermentation broth is low, so in vitro catalytic synthesis of levan by levansucrase is expected to be one of the industrial production approaches of levan.MethodsThe recombinant plasmid pET-28a-AcmA-Z constructed in a previous study was used to produce levansucrase. The effects of temperature, pH, and metal ions on the levan formation activity of the levansucrase were investigated. The polymer was analyzed by means of HPIC, FTIR, and NMR techniques.ResultsThe recombinant levansucrase could be easily purified in one step and the purified enzyme had a single band clearly visible in SDS-PAGE. The conditions for enzymatic reactions were optimal at pH 5.2 and 40 ℃, and the activity of enzymes was stimulated by K+ and Ca2+. The yield of levan biosynthesis from 10% (w/v) sucrose with 6.45 U/g sucrose of levansucrase was 30.6 g/L. The molecular weight of the levan was approximately 1.56 × 106 Da, as measured by GPC. HPIC analysis showed that the monosaccharide composition of the levan was fructose and glucose. The results of FTIR and NMR analysis indicated that the polymer produced by the recombinant levansucrase was β-(2,6) levan.ConclusionsThe results of this study provide a basis for the large-scale production of levan by enzymatic methods.Graphical Abstract

Konya İli Böğürtlen, Ahududu ve Kuşburnu Yetiştiriciliğinde Potansiyel Bir Tehdit: Ateş Yanıklığı Hastalığı

In the present study, totally 49 samples, which showed the symptoms of leaf and shoot blight and cankers with brown discoloration of necrotic tissues on mature branches, were collected from 22 districts and areas of Konya Province between 2017 and 2019. Presence rate of E. amylovora in collected samples, showing symptoms of the disease, from the province was determined to be 40% for blackberry and raspberry and 33% rosehip for rosehip in three years. Bacteria consistently isolated from the diseased tissues were identified on the basis of biochemical, physiological, and molecular tests, comparing with a reference strain of E. amylovora, isolated from blackberry (Kbb 371). Twenty seven representative bacterial strains were gram-negative, rod-shaped, mucoid, fermentative, positive for levan formation and acetoin production, no growth at 36°C, positive for gelatin hydrolysis, and negative for esculin hydrolysis, indole, urease, catalase, oxidase, arginine dehydrolase, reduction of nitrate, acid production from lactose, and inositol. All strains induced a hypersensitive response in tobacco (Nicotiana tobacum cv. White Burley) 24 h after inoculation with a 108 CFU ml-1 bacterial suspension in sterile distilled water. The strains were identified as E. amylovora using the species-specific primers set A/B (1), which amplified a 1-kb DNA fragment in PCR, and the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method. In order to fulfill the Koch postulates, pathogenicity test was confirmed by injecting bacterial suspensions of 108 CFU ml-1 in sterile distilled water into the shoot tips of 3-year-old blackberry R. fruticosus cv. Chester, raspberry R. idaeus cv. Heritage and rosehip R. canina. All tests were repeated three times. The bacterium was re-isolated from inoculated plants and identified as E. amylovora. Phytosanitary measures are needed to prevent any further spread of the bacterium as potential inoculum sources to new blackberry, raspberry and rosehip growing areas.

Revitalizing the ethanologenic bacterium Zymomonas mobilis for sugar reduction in high-sugar-content fruits and commercial products

The excessive consumption of sugars can cause health issues. Different strategies have been developed to reduce sugars in the diets. However, sugars in fruits and commercial products may be difficult to reduce, limiting their usage among certain populations of people. Zymomonas mobilis is a generally recognized as safe (GRAS) probiotic bacterium with the capability to produce levan-type prebiotics, and thrives in high-sugar environments with unique characteristics to be developed for lignocellulosic biofuel and biochemical production. In this study, the sugar reduction capabilities of Z. mobilis ZM4 were examined using two fruits of pear and persimmon and three high-sugar-content commercial products of two pear pastes (PPs) and one Chinese traditional wine (CTW). Our results demonstrated that Z. mobilis ZM4 can utilize sugars in fruits with about 20 g/L ethanol and less than 5 g/L sorbitol produced within 22 h using pears, and about 45 g/L ethanol and 30 g/L sorbitol produced within 34 h using persimmons. When PPs made from pears were used, Z. mobilis can utilize nearly all glucose (ca. 60 g/L) and most fructose (110 g/L) within 100 h with 40 ~ 60 g/L ethanol and more than 20 g/L sorbitol produced resulting in a final sorbitol concentration above 80 g/L. In the high-sugar-content alcoholic Chinese traditional wine, which contains mostly glucose and ethanol, Z. mobilis can reduce nearly all sugars with about 30 g/L ethanol produced, resulting in a final ethanol above 90 g/L. The ethanol yield and percentage yield of Z. mobilis in 50 ~ 60% CTW were 0.44 ~ 0.50 g/g and 86 ~ 97%, respectively, which are close to its theoretical yields—especially in 60% CTW. Although the ethanol yield and percentage yield in PPs were lower than those in CTW, they were similar to those in fruits of pears and persimmons with an ethanol yield around 0.30 ~ 0.37 g/g and ethanol percentage yield around 60 ~ 72%, which could be due to the formation of sorbitol and/or levan in the presence of both glucose and fructose. Our study also compared the fermentation performance of the classical ethanologenic yeast Saccharomyces cerevisiae BY4743 to Z. mobilis, with results suggesting that Z. mobilis ZM4 had better performance than that of yeast S. cerevisiae BY4743 given a higher sugar conversion rate and ethanol yield for sugar reduction. This work thus laid a foundation for utilizing the advantages of Z. mobilis in the food industry to reduce sugar concentrations or potentially produce alcoholic prebiotic beverages.Graphical

First Report of Pseudomonas aeruginosa Causing Basal Stem Rot of Solanum lycopersicum in China.

Tomato (Solanum lycopersicum L.) is economically important crop in China and elsewhere where it is grown. A previously unreported basal rot of tomato affecting as much as 15% of the stems was firstly observed in greenhouses in Northwest China (Jingyuan County, Baiyin City, Gansu Province) in March of 2015, and then the same disease of tomato was observed every year from 2016 -2021 in the region. The loss of local agricultural income caused by this plant disease in greenhouse is up to 2 million ¥ (CNY) each year. Symptoms on the basal stems were initially characterized by water-soaked lesions, which and developed rapidly into a soft, watery, decayed mass within 5 to 10 days after their appearance. Ten symptomatic stem samples were collected from basal stems exhibiting decay and were processed for microbiological analysis. Three strains of the bacteria (BBSR1~3) isolated from symptomatic samples were gram-negative, rods with a single polar flagellum, 0.5 to 1.0 × 1.5 to 3.5 µm in size. Colonies on nutrient agar (NA) medium were round, smooth, translucent, with flat edges and elevated center ("fried egg" appearance), and yellowish green to light purple in color. All isolates were positive for oxidase, gelatin liquefaction, denitrification, lipase, arginine dihydrolase, growth at 41 oC; utilization of mannitol, and production of pyocyanin. In contrast, they were negative for levan formation, lecithinase, hydrosis of starch, growth at 4 oC, utilization of maltose, xylose, γ-aminobutyrate and ethylene glycol; and growth in media supplemented with 8.5% NaCl. The representative bacterium (BBSR1) was identified as Pseudomonas aeruginosa using Biolog based Bacterial Identification System version 4.2 (Biolog Inc., Hayward, CA), revealing 0.88 similarities to P. aeruginosa in the Omnilog Biolog database. The identity was further confirmed as P. aeruginosa based on 16S rRNA sequence (1,387 bp) analysis with universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). A BlastN search of GenBank revealed a 100% identity to that of Pseudomonas aeruginosa strains JCM5962 (LC069033), SM-1 (KP794924), and KUN4 (KR025537). The 16S rRNA gene sequence of strain BBSR1 (1,387 bp) was deposited in GenBank (KT454095). The representative strain BBSR1 was further evaluated in pathogenicity tests. 1-month-old tomato (cv. Tomo608) potted plants were spray inoculated with BBSR1 bacterial suspensions (≈ 3×108 CFU/ml) and wounded with a sterilized needle prior to inoculation. Controls were wounded similarly and inoculated with sterile water. This experiment was maintained at 28°C with 80 to 85% relative humidity in a greenhouse. Three replicates were used for each treatment and three mature plants in each pot. Symptoms developed within 14 days and were similar to those originally observed, whereas symptoms did not occur on the control plants. Strains recovered on NA were identified as P. aeruginosa based on physiological and biochemical characteristics and Biolog (Biolog, Hayward, CA). Although Pseudomonas aeruginosa is generally regarded as a human conditional pathogen, it has been reported to cause disease on a variety of plants including ginseng (Gao et al. 2014), tinda (Mondal et al. 2012), onion (Abd-Alla and Bashandy 2012) and sweet basil (Walker et al. 2004). This is the first report of P. aeruginosa causing basal stem rot on tomato in Gansu Province, China. Under the globalization, human beings should give great importance to the bacterial pathogens that cross kingdom to cause infection and become co-pathogenic in plant and animal.

Lowering whole cost for sugarcane‐based ethanol production by engineered Zymomonas mobilis

AbstractLowering cost will prompt the sustainable development of sugarcane‐based ethanol industry. In this work, we developed a low‐cost process for ethanol production from sugarcane by a genetically engineered Zymomonas mobilis. Fermentation media were first optimized, resulting in a 15.54% increase in ethanol fermentation efficiency as compared to control media. To further reduce the byproduct levan formation, a levansucrase‐encoding gene of Z. mobilis, sacB, was deleted through the type I‐F CRISPR‐Cas system, which resulted in a further elevation of both ethanol conversion ratio and productivity comparing with the starting strain ZMS912 (87.50% vs. 76.77%, 1.95 g/L/h vs. 1.71 g/L/h). Moreover, we conducted fed‐batch fermentation for ethanol production using sugarcane juice in 5 L bioreactors and employing the optimized media and engineered strain. The results showed that maximum ethanol titer of 81.59 g/L and productivity of 5.83 g/L/h were achieved. Finally, preliminary techno‐economic assessment demonstrated that our efforts to modify media and strain could reduce the processing cost of ethanol production from sugarcane juice, which provides the feasibility for economic ethanol production in the future.

РАЗРАБОТКА СХЕМЫ ВЫДЕЛЕНИЯ И БАКТЕРИОЛОГИЧЕСКОЙ ИДЕНТИФИКАЦИИ БАКТЕРИЙ PSEUDOMONAS SYRINGAE И ЕЕ АПРОБАЦИИ

The article presents results of studies on development of isolation scheme and bacteriological identification of Pseudomonas syringae bacteria and its approbation. The introduction of the article describes the objects of Pseudomonas syringae contamination - fruit trees and shrubs, agricultural plants, which proves the relevance of the research in the field of expanding of laboratory methods for identifying phytopathogenic microorganisms. The author’s bacteriological scheme includes the use of King B Medium (Pseudomonas F Agar; Pronadisa 1532) as a selective medium. Initially, the isolated bacteria are differentiated to Pseudomonas genus, the following paramters are studied: anaerobic fermentation, production of enzymes catalase, lecithinase, lipase; hydrolysis of starch and gelatin; fermentation of glucose and lactose, also, a test for maceration is put. The second stage of the research includes the study of the growth of bacterial cultures on meat-and-peptone agar at 41 ° C and at 5% of NaCl; oxidase production, arginine hydrolase; fermentation of mannose and sorbitol; formation of levan, hydrogen sulfide and indole, esculin, a hypersensitivity reaction is set. The determined parametres allow to type the representatives of the genus to Pseudomonas syringae species within 192 hours. During the research, a collection of 12 strains of Pseudomonas syringae bacteria was formed, isolated from 97 objects of phytosanitary supervision and identified according to the developed technique. The proposed bacteriological scheme allows to differentiate the above microorganisms on the basis of the analysis of 25 parameters. The application of a phage biological product as a diagnosticum (according to the Otto method) expands the spectrum of the analyzed biological properties of the isolated and identified Pseudomonas syringae bacteria.

Identification and Characterization of a New Quorum-Quenching N-acyl Homoserine Lactonase in the Plant Pathogen Erwinia amylovora.

Quorum quenching (QQ) is the ability to interfere with bacterial cell to cell communication, known as quorum sensing (QS). QQ enzymes that degrade or modify acyl homoserine lactones (AHLs) have been attracting increasing interest as promising agents for inhibiting QS-mediated bacterial pathogenicity. Plant pathogens from the genus Erwinia cause diseases in several economically important crops. Fire blight is a devastating plant disease caused by Erwinia amylovora, affecting a wide range of host species within the Rosaceae and posing a major global threat for commercial apple and pear production. While QS has been described in Erwinia species, no AHL-degrading enzymes were identified and characterized. Here, phylogenetic analysis and structural modeling were applied to identify an AHL lactonase in E. amylovora (dubbed EaAiiA). Following recombinant expression and purification, the enzyme was biochemically characterized. EaAiiA lactonase activity was dependent on metal ions and effectively degraded AHLs with high catalytic efficiency. Its highest specific activity (kcat/KM value) was observed against one of the AHLs (3-oxo-C6-homoserine lactone) secreted from E. amylovora. Exogenous addition of the purified enzyme to cultures of E. amylovora reduced the formation of levan, a QS-regulated virulence factor, by 40% and the transcription level of the levansucrase-encoding gene by 55%. Furthermore, preincubation of E. amylovora cultures with EaAiiA inhibited the progress of fire blight symptoms in immature Pyrus communis fruits. These results demonstrate the ability of the identified enzyme from E. amylovora to act as a quorum-quenching lactonase.

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

Glycoside hydrolase family 68 (GH68) enzymes catalyze β-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, β-fructofuranosidase (FFZm) and levansucrase (LSZm), encoded tandemly in the genome of Zymomonas mobilis, displayed different selectivity: FFZm catalyzed the β-(2→1)-transfructosylation (1-TF), whereas LSZm did both of 1-TF and β-(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 β-(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, β-D-fructofuranosyl α-D-mannopyranoside, by β-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.

Insights into the pH-dependent, extracellular sucrose utilization and concomitant levan formation by Gluconobacter albidus TMW 2.1191

Many bacteria and archaea produce the polydisperse fructose polymer levan from sucrose upon biofilm formation via extracellular levansucrases (EC 2.4.1.10). We have investigated levansucrase-release and -activities as well as molecular size of the levan formed by the acetic acid bacterium Gluconobacter albidus TMW 2.1191 at varying environmental pH conditions to obtain insight in the ecological role of its constitutively expressed levansucrase and the produced levan. A buffer system was established enabling the recovery of levansucrase-containing supernatants from preincubated cell suspensions at pH 4.3–pH 5.7. The enzyme solutions were used to produce levans at different pH values and sucrose concentrations. Finally, the amounts and size distributions of the produced levans as well as the corresponding levansucrase activities were determined and correlated with each other. The data revealed that the levansucrase was released into the environment independently of its substrate sucrose, and that more levansucrase was released at pH ≥ 5.0. The glucose release and formation of high molecular weight levans (> 3.5 kDa) from 0.1 M initial sucrose was comparable between pH ~ 4.3–5.7 using equal amounts of released levansucrase. Hence, this type of levansucrase appears to be structurally adapted to changes in the extracellular pH and to exhibit a similar total activity over a wide acidic pH range, while it produced higher amounts of larger levan molecules at higher production pH and sucrose concentrations. These findings indicate the physiological adaptation of G. albidus TMW 2.1191 to efficient colonisation of sucrose-rich habitats via released levansucrases despite changing extracellular pH conditions in course of acid formation.

Effect of exopolysaccharides produced by Lactobacillus sanfranciscensis on the processing properties of wheat doughs

The effect of crude exopolysaccharides (EPS) of Lactobacillus sanfranciscensis strains on processing properties of wheat doughs was investigated and compared to the commercial available hydrocolloids guar gum and xanthan using straight dough method. In addition, the effect of swelling process occurring during fermentation and of in situ formation of levan on dough and bread properties was determined. The doughs supplemented with levan showed higher water absorption and lower stickiness than those supplemented with guar gum and xanthan. However, F(max) and l(max) resulting from extensibility measurements as well as peak viscosity during gelatinization were less affected. The use of chemically acidified sponge doughs resulted in increased stickiness and changes in the extension properties of the wheat doughs with decreasing pH. These negative effects were compensated by the addition of crude levan extracts. Addition of sourdoughs markedly decreased their stickiness but not their F(max) and l(max) resulting from extensibility measurements, as compared with chemically acidified sponge doughs. Enhanced in situ production of levan increased dough stickiness as well as increased resistance and decreased extensibility. This effect could be traced back to an enrichment of sugar or metabolites due to an altered metabolism of the bacteria. Breads produced from straight doughs supplemented with levan showed higher volumes as compared to commercial hydrocolloids.

Understanding the transfer reaction network behind the non-processive synthesis of low molecular weight levan catalyzed by Bacillus subtilis levansucrase

Under specific reaction conditions, levansucrase from Bacillus subtilis (SacB) catalyzes the synthesis of a low molecular weight levan through the non-processive elongation of a great number of intermediates. To deepen understanding of the polymer elongation mechanism, we conducted a meticulous examination of the fructooligosaccharide profile evolution during the levan synthesis. As a result, the formation of primary and secondary intermediates series in different reaction stages was observed. The origin of the series was identified through comparison with product profiles obtained in acceptor reactions employing levanbiose, blastose, 1-kestose, 6-kestose, and neo-kestose, and supported with the isolation and NMR analyses of some relevant products, demonstrating that all of them are inherent products during levan formation from sucrose. These results allowed to establish the network of fructosyl transfer reactions involved in the non-processive levan synthesis. Overall, our results reveal how the relaxed acceptor specificity of SacB during the initial steps of the synthesis is responsible for the formation of several levan series, which constitute the final low molecular weight levan distribution.

Insights into hydrolysis versus transfructosylation: Mutagenesis studies of a novel levansucrase from Brenneria sp. EniD312

Levan is a kind of fructan that composing of fructose by β-(2, 6) linkage and has been already applied as thickening agent and colloidal stabilizer in the cosmetic, medicinal and food industries. Microbial levansucrase is a key enzyme catalyzing the formation of levan from sucrose by transfructosylation. Here, a gene encoding levansucrase from Brenneria sp. EniD312 was cloned and expressed in Escherichia coli. The recombinant levansucrase showed the optimal pH and temperature at pH 6.5 and 45 °C. The enzyme produced 85 g/L levan from 250 g/L sucrose at pH 6.5 and 45 °C for 6 h. The residues D68, D225 and E309 of this levansucrase were speculated to be the nucleophile, the transition stabilizer and the general acid respectively by homology modelling, site-directed mutagenesis and molecular docking. Particularly, the residues in position 154 and 327 were found to play a significant role in determining the ratio of hydrolysis activity to transfructosylation activity.

Biosynthesis of levan from sucrose using a thermostable levansucrase from Lactobacillus reuteri LTH5448

Levan, a versatile fructan, possesses promising prospects for application in the food and pharmaceutical industries and in many other fields due to the superior physicochemical properties and physiological functions of this fructan. In this study, a thermostable levansucrase was identified from Lactobacillus reuteri LTH5448. This enzyme shared higher identity with reported inulosucrases than levansucrases but, unexpectedly, yielded a levan-type fructan. Structural analysis of the produced polysaccharide revealed that the glycosidic bonds were β-(2, 6) linkages, and the average molecular weight was 3.924×107Da under optimized conditions. The optimal pH and temperature for levan formation were 6.0 and 35°C, respectively. The total activity was enhanced to 135% in the presence of Ca2+. After optimizing the production of levan, 13U/g sucrose of levansucrase and 500g/L sucrose were utilized to obtain 183g/L levan after a 12-h reaction.

Expression and Characterization of Levansucrase from Clostridium acetobutylicum.

The Clostridium acetobutylicum gene Ca-SacB encoding levansucrase was cloned and expressed in Escherichia coli. Ca-SacB is composed of 1287 bp and encodes 428 amino acid residues, which could convert 150 mmol/L sucrose to levan with the liberation of glucose. The optimum pH and temperature of this enzyme for levan formation were pH 6 and 60 °C, respectively. Levansucrase activity of Ca-SacB was completely abolished by 5 mmol/L Ag+ and Hg2+. The Km and Vmax values for levansucrase were calculated to be 64 mmol/L and 190 μmol/min/mg, respectively. Interestingly, Ca-SacB was found to have high product specificity, and no fructooligosaccharide was identified in the product, indicating that Ca-SacB may be valuable for industrial production of levan. In addition, Ca-SacB is the first characterized levansucrase isolated from an anaerobic bacterium, which should be valuable for exploring new enzyme resources and deepening the understanding of the catalytic mechanisms of levansucrases.

Characterization of Erwinia amylovora strains from Hungary

Erwinia amylovora is the causative agent of fire blight, which is a destructive bacterial disease of rosaceous plants. In Hungary Erwinia amylovora (Burrill) Winslow et al. was first detected in 1996. Since the appearance of fire blight, E. amylovora samples have been collected from different host plats from various geographic locations. A motif of eight nucleotides (ATTACAGA) is repeated 3–15 times in the PstI fragment of the pEa29 plasmid in Erwinia amylovora strains, and represents a valuable tool for strain classification. The number of short-sequence DNA repeats in plasmid pEa29 of 30 Hungarian isolates were determined by PCR assays and they ranged from five to ten. The SSR test is suitable for distinguishing the individual strains between the E. amylovora isolates. The examined isolates showed high pathogenicity on immature pear fruits. Several biochemical techniques, such as miniaturized API 20E, were applied on the samples. Differences were also revealed in microbiological assays like levan formation and colony morphology on semi-selective media. Examining the Hungarian Erwinia amylovora population by molecular analysis we can draw the conclusion that the population consists of different strains, which shows great diversity. E. amylovora is a widespread pathogen in Hungary, which is supported by the 30 strains isolated from various host plants from many parts of the country. The phenotypic diversity-evaluation of the E. amylovora strains showed, that they differ metabolically, like other plant pathogenic bacteria as reported by several authors. This is the first report on the diversity of E. amylovora strains isolated from Hungary.

MICROBIAL PRODUCTION OF LEVANSUCRASE AND SYNTHESIZED BIOPOLYMERS

Levansucrases (E.C.2. 4. 1. 10) are fructosyltransferase (Ftase) which belong to GH68 glycoside hydrolaseenzyme family and catalyze the formation of levan and fructooligosaccharides (FOS) by using sucroseas substrate. Levansucrases are produced by several microorganisms including Bacillus subtilis,Streptococcus mutansand Zymomonas mobilis. The importance of levansucrase enzyme which catalyzesthe formation of levan biopolymer and fructooligosaccharides is increasing day by day and the industrialproduction of this enzyme is usually performed through microbial bioprocesses using differentmicroorganisms. The fructan polymers levan and FOS catalyzed by levansucrases have several industrialapplications in food, cosmetic, medicine biomedical and nanotechnology. In this review, the functionalproperties of levansucrases, their microbial production, purification strategy and biopolymers synthesizedby levansucrases was summarized

Biochemical Characterization of Native Fluorescent Pseudomonads and its Suitable Carrier Material for Mass Multiplication in Kuttanad Ecosystem

Bacterial antagonist fluorescent pseudomonads for sheath blight disease were isolated from different locations in Kuttanad region. Three effective strains viz., PF 43, PF 46 and PF 47 were tested along with standard culture P 1 against sheath blight disease of rice under glass house condition. The confirmation tests viz., physiological and biochemical characterization of the efficient isolates were carried out at Rice Research Station, Kerala Agricultural University, Moncompu. Various physiological tests on growth at different pH, Iron toxicity and Aluminium toxicity level showed that the isolate PF 43 grew at pH ranging from 1.0 to 14 and tolerated upto 1000 ppm of iron toxicity and 90 ppm of aluminium toxicity level. The biochemical tests indicated that three efficient isolates were confirmed as gram negative, rod shaped, fluorescent in King's B medium and positive response for growth at 4°C, Levan formation, Gelatin liquefaction and Catalase tests. However, there was a negative response for growth at 41°C, Methyl red, VogesProskaur and Indole tests. Thus, based on morphological and biochemical characteristics, the isolated strains were identified to be Pseudomonas fluorescens. P. fluorescens PF 43 product survived upto 150 days with required population of 1.01 to 1.63 × 10 8 cfu per g in talc, dolomite and gypsum based formulations. At 240 days of storage, 1 × 10 7 cfu were detected in talc, dolomite and gypsum based formulations. The cheap and easily available carrier material, gypsum and dolomite can be used for mass production of native P. fluorescens and is recommended to 66,000 ha rice growing tracts of Lower, Upper and Karilands of Kuttanad regions like Alleppey, Kottayam and Pathanamthitta District for ecofriendly management of diseases.

Levan versus fructooligosaccharide synthesis using the levansucrase from Zymomonas mobilis: Effect of reaction conditions

This work addresses the effect of sucrose concentration and temperature on the three activities displayed by the levansucrase from Zymomonas mobilis: formation of levan (polymerization), production of short-chain fructooligosaccharides (FOS), and sucrose hydrolysis. Of the conditions tested, levan formation reached the highest value at 4°C and 100g/L sucrose. The increase of temperature (40°C) and sucrose concentration (600g/L) caused a significant decrease of the levan concentration and a higher production of FOS. However, an increase of the temperature also caused an enhancement of the undesired hydrolytic activity. Several inulin-type FOS (1-kestose, nystose, 1F-fructosylnystose), neoFOS (blastose, neokestose, neonystose) and levan-type FOS (6-kestose, 6,6-nystose) were synthesized by levansucrase. The latter compound was purified and characterized by mass spectrometry and 2D NMR. Using 600g/L sucrose at 40°C, the maximum yield of FOS was reached at 85% sucrose conversion; at this point, the reaction mixture contained (in weight basis) 31% glucose, 14% fructose, 15% sucrose and 40% FOS (including a small contribution of levan).