Acetate-mediated pH-stat fed-batch cultivation of transconjugant <Emphasis Type="Italic">Enterobacter</Emphasis> sp. BL-2S over-expressing <Emphasis Type="Italic">glmS</Emphasis> gene for excretive production of microbial polyglucosamine PGB-1

A unique cationic polyglucosamine biopolymer PGB-1 comprising more than 95% D-glucosamine was excretively produced from a new bacterial strain Enterobacter sp. BL-2 under acetate-mediated culture conditions. Since the biopolymer PGB-1 could be synthesized from the UDP-N-acetylglucosamine monomer derived from the hexosamine pathway, three glmS, glmM, and glmU genes in the hexosamine pathway were cloned from Enterobacter sp. BL-2, and their molecular structures were elucidated. The cloned glmS, glmM, and glmU genes were reintroduced into the parent strain Enterobacter sp. BL-2 through a conjugative transformation for the overproduction of the biopolymer PGB-1. The biopolymer production increased 1.5-fold in the transconjugant Enterobacter sp. BL-2S over-expressing the first-step glmS gene encoding glucosamine-6-phosphate synthase. The transconjugant Enterobacter sp. BL-2S was cultivated pH-stat fed-batch widely, while intermittently feeding an acetate solution to maintain a constant pH level of 8.0 for 72 h, resulting in 1.15 g/L of the extracellular polyglucosamine biopolymer PGB-1.     

Proteomic analysis on acetate metabolism in Citrobacter sp. BL-4

Mass production of glucosamine (GlcN) using microbial cells is a worthy approach to increase added values and keep safety problems in GlcN production process. Prior to set up a microbial cellular platform, this study was to assess acetate metabolism in Citrobacter sp. BL-4 (BL-4) which has produced a polyglucosamine PGB-2. The LC-MS analysis was conducted after protein separation on the 1D-PAGE to accomplish the purpose of this study. 280 proteins were totally identified and 188 proteins were separated as acetate-related proteins in BL-4. Acetate was converted to acetyl-CoA by acetyl-CoA synthetase up-regulated in the acetate medium. The glyoxylate bypass in the acetate medium was up-regulated with over-expression of isocitrate lyases and 2D-PAGE confirmed this differential expression. Using (1)H-NMR analysis, the product of isocitrate lyases, succinate, increased about 15 times in the acetate medium. During acetate metabolism proteins involved in the lipid metabolism and hexosamine biosynthesis were over-expressed in the acetate medium, while proteins involved in TCA cycle, pentose phosphate cycle and purine metabolism were down-regulated. Taken together, the results from the proteomic analysis can be applied to improve GlcN production and to develop metabolic engineering in BL-4.     

Polymeric and Compositional Properties of Novel Extracellular Microbial Polyglucosamine Biopolymer from New Strain of Citrobacter sp. BL-4

A novel polyglucosamine polymer, PGB-2, was produced extracellularly from a new strain Citrobacter sp. BL-4 using pH-stat fed batch cultivation. It was composed of 97.3% glucosamine and 2.7% rhamnose; its average molecular weight, solubility in 2% acetic acid and viscosity were 20 kDa, 5 g l⁻¹ and 2.9 cps, respectively. FT-IR and ¹H NMR spectra of PGB-2 revealed a close identity with chitosan from crab shells. Received 20 September 2005; Revisions requested 6 October 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

Supplementation of a novel microbial biopolymer, PGB1, from new Enterobacter sp. BL-2 delays the deterioration of type 2 diabetic mice

Antidiabetic effects of a novel microbial biopolymer (PGB)1 excreted from new Enterobacter sp. BL-2 were tested in the db/db mice. The animals were divided into normal control, rosiglitazone (0.005%, wt/wt), low PGB1 (0.1%, wt/wt), and high PGB1 (0.25%, wt/wt) groups. After 5 weeks, the blood glucose levels of high PGB1 and rosiglitazone supplemented groups were significantly lower than those of the control group. In hepatic glucose metabolic enzyme activities, the glucokinase activities of PGB1 supplemented groups were significantly higher than the control group, whereas the PEPCK activities were significantly lower. The plasma insulin and hepatic glycogen levels of the low and high PGB1 supplemented groups were significantly higher compared with the control group. Specifically, the insulin and glycogen increases were dose-responsive to PGB1 supplement. PGB1 supplement did not affect the IPGTT and IPITT compared with the control group; however, rosiglitazone significantly improved IPITT. High PGB1 and rosiglitazone supplementation preserved the appearance of islets and insulin-positive cells in immunohistochemical photographs of the pancreas compared with the control group. These results demonstrated that high PGB1 (0.25% in the diet) supplementation seemingly contributes to preventing the onset and progression of type 2 diabetes by stimulating insulin secretion and enhancing the hepatic glucose metabolic enzyme activities.     

Isolation and identification of potential phosphate solubilizing bacteria from the rhizoplane of Oryza sativa L. cv. BR29 of Bangladesh

A total of 30 bacteria were isolated from the rhizoplane of rice cv. BR29 cultivated in Mymensingh, Bangladesh and from the seedlings obtained from surface-sterilized seeds of BR29. Upon screening, 6 isolates showed varying levels of phosphate solubilizing activity in both agar plate and broth assays using National Botanical Research Institute's phosphate medium. The bacterial isolates were identified based on their phenotypic and 16S rRNA genes sequencing data as Acinetobacter sp. BR-12, Klebsiella sp. BR-15, Acinetobacter sp. BR-25, Enterobacter sp. BR-26, Microbacterium sp. BRS-1 and Pseudomonas sp. BRS-2. The BR-25 exhibited highest phosphate solubilizing activity followed by BR-15. They grew rapidly in the liquid medium at pH 5 and 7 but almost no growth occurred at pH 3. The pH value of the culture medium was decreased with bacterial growth suggesting that they might secrete organic acids to solubilize insoluble phosphorus. Scanning electron microscope analysis of two-week-old rice seedlings germinated from seeds previously inoculated with BR-25 and BR-15 revealed dense colonization at the root surfaces presumably using fimbriae on the bacterial cells.     

光合细菌与产气肠杆菌协同产氢特性分析

对光合细菌(Rhodopseudomonas sp. DT)与产气肠杆菌(Enterobacter aerogenes)进行了发酵产氢试验, 考察了不同起始接种比例、培养温度及碳源条件下混合菌协同产氢特性。结果表明: 光合细菌与产气肠杆菌初始接种比例对协同产氢影响较大, 初始接种比例为1:1最有利于协同产氢, 产氢效率和产氢周期达到了3.1 mol H2/mol葡萄糖及81 h。进一步培养液pH动力学变化研究发现初始接种比例为1:1的混合菌培养液pH变化较小, 为pH 6~7, 利于混合菌协同产氢。28°     

rpoS基因在肠杆菌CGMCC 5087响应环境胁迫中的功能

[背景]苯乙醇(2-Phenylethanol,2-PE)是一种具有玫瑰香气味的高级香料添加剂,被广泛应用于香水、化妆品、食品和医药等领域.目前,利用工程菌合成苯乙醇有很好的应用前景.我们分离到一株肠杆菌(Enterobactersp.)CGMCC 5087,其可以通过苯丙酮酸途径合成2-PE.然而该菌的生长受到不同环...     

溶磷菌对4种难溶性磷酸盐溶解能力的初步研究

以4种难溶性磷酸盐为培养基,发现供试菌株溶解这些磷酸盐的特性差异很大,真菌溶磷能力普遍比细菌要高得多。以NO3-为氮源时的溶磷量通常高于以NH4+为氮源时的溶磷量,只有2TCiF2对氟磷灰石及4TCiF6对磷酸铝的溶解能力以NH4+为氮源时较高。大多数菌株较易溶解CaP(氟磷灰石和磷矿粉),其次为AlP(AlPO4),而溶解FeP(FePO4·4H2O的能力都比较弱,只有曲霉2TCiF2具有较强的溶解FeP能力,尤其是当供给NO3-时,溶解FeP的活性比供给NH4+时大幅度提高。欧文氏菌4TCRi22和肠杆菌1TCRi15能大量地溶解氟磷灰石,而两株节杆菌对磷矿粉的溶解能力最强。供试菌株的溶磷作用可能是由于分泌的有机酸与金属离子络合或螯合作用所致,欧文氏菌和肠杆菌溶解难溶性磷过程中,非有机酸物质可能在起主要作用。     

Purification, characterization and gene analysis of N-acetylglucosaminidase from Enterobacter sp. G-1.

Enterobacter sp. G-1 is a bacterium isolated previously as a chitinase-producing bacterium. We found this bacterium also produced N-acetylglucosaminidase and characterized that in this study. Extracellular N-acetylglucosaminidase of 92.0 kDa was purified near homogeneity by 8.57-fold from Enterobacter sp. G-1. The optimum temperature and the optimum pH of the purified N-acetylglucosaminidase was 45 degrees C and 6.0, respectively. The N-terminal amino acid sequence of 23 residues of N-acetylglucosaminidase was identified. Based on the N-terminal sequence, we amplified pieces of the DNA fragments by PCR. Using these PCR products as probes, we screened the genomic library and successfully isolated the entire N-acetylglucosaminidase gene (designated nag1) from Enterobacter sp. G-1. The nucleotide sequence of the nag1 gene was found to consist of 2,655 bp encoding a protein of 885 amino acid residues. Comparison of the deduced amino acid sequence from the nag1 gene found 97.3% identity with chitobiase from Serratia marcescens, 54.4% identity with N,N'-diacetylchitobiase from Vibrio harveyi, and 42.7% identity with N-acetylglucosaminidase (ExoI) from Vibrio furnissii. Enzymatic activity assay of N-acetylglucosaminidase indicated stronger activity toward PNP-GlcNAc than PNP-(GlcNAc)2 or PNP-(GlcNAc)3.     

Root colonization and growth promotion of sunflower (Helianthus annuus L.) by phosphate solubilizing Enterobacter sp. Fs-11

An Enterobacter sp. Fs-11 was isolated from sunflower rhizosphere, identified on the basis of 16S rRNA gene sequence analysis (GeneBank accession no. GQ179978) and studied for its root colonization and growth promotion ability in sunflower. Morphologically, it was rod shaped Gram-negative, motile bacterium, producing 4.5 μg mL(-1) indole acetic acid in tryptophan-supplemented medium. It utilized 27 out of 95 substrates in BIOLOG GN2 micro plate system. It was able to convert insoluble tri-calcium phosphate to soluble phosphorus up to 43.5 μg mL(-1) with decrease in pH of the medium up to 4.5 after 10 days incubation at 28 ± 2 °C in the Pikovskaya's broth. High performance liquid chromatography of cell free supernatant showed that Fs-11 produced malic acid and gluconic acid (2.43 and 16.64 μg mL(-1), respectively) in Pikovskaya's broth. Analysis of 900 bp fragment of pyrroloquinoline quinine pqqE gene sequence showed 98 % homology with that of E. cloacae pqqE gene. Confocal laser scanning microscope revealed strong colonization of fluorescently labeled Fs-11 with sunflower roots. Sunflower inoculation with Fs-11 and its rifampicin resistant derivative in sterile sand and natural soil showed that Fs-11 colonized sunflower roots up to 30 days after transplanting in both sterile sand as well as natural soil. Moreover, Fs-11 inoculation resulted in increased plant height, fresh weight, dry weight and total phosphorus contents as compared to un-inoculated plants. The data showed that Enterobacter sp. Fs-11 is an efficient phosphate solubilizing and plant growth promoting rhizobacterium and has great potential to be used as bio-inoculant for sunflower under phosphorus deficient conditions.     

Biopolymer flocculant produced by an Enterobacter sp.

A new biopolymer flocculant was produced by Enterobacter sp. BY-29. Flocculating activity increased in the presence of Al , Fe or Fe . The flocculant had flocculating activity not only in inorganic suspensions of kaolin and active carbon but also in organic suspensions of cellulose and yeast. The flocculant was an acidic polysaccharide consisting of glucose, galactose, xylose and galacturonic acid, and its MW was about 2.5 ¥ 10 6 Da.     

Isolation of endophytic diazotroph Pantoea agglomerans and nondiazotroph Enterobacter asburiae from sweetpotato stem in Japan

AIMS: To isolate and identify diazotrophic endophytes in the stem of Japanese sweetpotato cv. Koganesengan. METHODS AND RESULTS: Surface-sterilized and thinly sliced (1-2 mm) sweetpotato stem samples were incubated in test tubes with semi-solid modified Rennie (MR) medium. The test tubes were assayed for acetylene reduction activity (ARA) 5 days after incubation at 30 degrees C. Twelve isolates were obtained from MR plates inoculated with a loop of semi-solid MR medium from ARA+ tubes. However, ARA test showed that only nine isolates were diazotrophic and three were nondiazotrophic strains. Using the API 20E diagnostic kit, four diazotrophic isolates were identified as strains of Pantoea spp. and five isolates as Klebsiella spp. The nondiazotrophic bacteria were strains of Enterobacter spp. A diazotrophic isolate Pantoea sp. MY1 and nondiazotrophic isolate Enterobacter sp. MY2 were identified to the species level by full sequence analysis of 16S rRNA gene. The results showed that MY1 had 99.2% similarity to Pantoea agglomerans ATCC 27155 and MY2 had 99.5% similarity to Enterobacter asburiae ATCC 35953. CONCLUSION: The stem of sweetpotato cv. Koganesengan was colonized by diazotrophic endophyte P. agglomerans and nondiazotrophic endophyte E. asburiae. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is an essential step toward understanding the ecology and interaction between endophytic bacteria and sweetpotato.     

Unsaturated disaccharides in the enzymatic digests of heparan sulfates

High performance liquid chromatography was performed by an ion-pair reversed-phase method of six standard unsaturated disaccharides derived from heparan sulfate and heparin. Separation of delta Di-GlcNAc, delta Di-GlcN(2S), delta Di-GlcNAc(6S), delta Di-GlcN(2,6- or 2,2'-diS) and delta Di-GlcN(2,6,2'-triS) was achieved on a column of Jasco SC-02 with 10 mM tetrabutylammonium phosphate (pH 7.0) containing 30 or 47% methanol as a mobile phase. delta Di-GlcN(2,6-diS) and delta Di-GlcN(2,2'-diS) were separated on the same column with 35 mM triethylamine phosphate (pH 5.3). Four preparations (BL-1.0-1, BL-1.0-2, BL-1.0-3, and BL-1.25-1) separated from crude bovine lung heparan sulfate, a standard bovine lung heparan sulfate (BL-ST), bovine kidney heparan sulfate 1.0 M Fr and 1.25 M Fr (BK-1.0 and BK-1.25), and porcine kidney heparan sulfate 1.0 M Fr (PK-1.0) were digested with a mixture of heparinase, and heparitinases 1 and 2. The resulting foregoing unsaturated disaccharides in the digests were analyzed by the above HPLC procedures. The proportions of the unsaturated disaccharides in the digests of BL-1.25-1 and BL-ST were similar, but those of the others differed from each other. It is noteworthy that delta Di-GlcNAc plus delta Di-GlcNAc(6S) in the digest of BL-1.0-1 was approximately 95% of the total unsaturated disaccharides. Small amounts of delta Di-GlcN (2,6,2'-triS) were found in all the samples. It was found that delta Di-GlcN(2,2'-diS) was a prominent component in the disulfated unsaturated disaccharides from BL-1.25-1 and BK-1.25.     

鞘氨醇单胞菌TP-3合成新型生物聚合物Ss的发酵条件优化

鞘氨醇单胞菌(Sphingomonas sp.)TP-3能合成一种具有增稠性、假塑性、成凝胶特性和乳化性能的新型生物聚合物Ss。运用单因素实验和均匀设计法对菌株TP-3合成聚合物Ss的发酵条件进行优化, 实验结果表明, 培养基组成为葡萄糖41.2 g/L, 豆饼粉2.0 g/L, NaCl 0.85 g/L, K2HPO4 1.46 g/L, MgSO4 0.12 g/L, MnCl2 0.0075 g/L, FeSO4 0.002 g/L, 初始pH为7.0, 在27°C, 180 r/min的条件下摇床培养60 h, 聚合物Ss的产量达到21.5 g/L。该聚合物生产成本低, 在油田开发中极具应用前景。     

Purification and characterization of NAD-dependent n-butanol dehydrogenase from solvent-tolerant n-butanol-degrading Enterobacter sp. VKGH12

The solvent-tolerant bacterium Enterobacter sp. VKGH12 is capable of utilizing n-butanol and contains an NAD+-dependent n-butanol dehydrogenase (BDH). The BDH from n-butanol-grown Enterobacter sp. was purified from a cell-free extract (soluble fraction) to near homogeneity using a 3-step procedure. The BDH was purified 15.37-fold with a recovery of only 10.51, and the molecular mass estimated to be 38 kDa. The apparent Michaelis-Menten constant (Km) for the BDH was found to be 4 mM with respect to n-butanol. The BDH also had a broad range of substrate specificity, including primary alcohols, secondary alcohols, and aromatic alcohols, and exhibited an optimal activity at pH 9.0 and 40oC. Among the metal ions studied, Mg2+ and Mn2+ had no effect, whereas Cu2+, Zn2+, and Fe2+ at 1 mM completely inhibited the BDH activity. The BDH activity was not inhibited by PMSF, suggesting that serine is not involved in the catalytic site. The known metal ion chelator EDTA had no effect on the BDH activity. Thus, in addition to its physiological significance, some features of the enzyme, such as its activity at an alkaline pH and broad range of substrate specificity, including primary and secondary alcohols, are attractive for application to the enzymatic conversion of alcohols.     

Production of a biopolymer flocculant from Bacillus licheniformis and its flocculation properties

Bacillus licheniformis CCRC 12826 produced extracellularly an excellent biopolymer flocculant in a large amount when it was grown aerobically in a culture medium containing citric acid, glutamic acid and glycerol as carbon sources. The biopolymer flocculant was an extremely viscous material with a molecular weight over 2 x 10(6) by gel permeation chromatography. It could be easily purified from the culture medium by ethanol precipitation. It was shown to be a homopolymer of glutamic acid by amino acid analysis and thin layer chromatography and presumed to be poly-glutamic acid (PGA). This bioflocculant efficiently flocculated various organic and inorganic suspensions. It flocculated a suspended kaolin suspension without cations, although its flocculating activity was synergistically stimulated by the addition of bivalent or trivalent cations Ca2+, Fe3+ and Al3+. However, the synergistic effects of metal cations were most effective at neutral pH ranges. The comparison of the flocculating activity between the present biopolymer and a commercial lower molecular weight product showed that the biopolymer of the present study had much higher activity. The high productivity and versatile applications of PGA make its development as a new biodegradable, harmless, biopolymer flocculant economical and advantageous.     

Isolation and characterization of plant growth promoting bacteria from non-rhizospheric soil and their effect on cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp.) seedling growth

The efficacy of four potential phosphate solubilizing Enterobacter isolated from non-rhizospheric soil in Western ghat forest in India. Plant growth promoting ability of these isolates was evaluated in cowpea. All are gram negative, rod shaped, 0.8–1.6 mm in size, and psychrotrophic in nature, grow from 5 to 40°C (optimum temp. 28 ± 2°C). All isolates exhibits growth at a wide range of pH 6–12, optimum at pH 7.0 and tolerates up to 7% (w/v) salt concentration. 16S rRNA gene sequencing reveals the confirmation of isolates to Enterobacter aerogenes sp. (NII-0907 and NII-0929), Enterobacter cloacae subsp. cloacae sp. (NII-0931) and Enterobacter asburiae sp. (NII-0934) with which they share >99% sequence similarity. Under in vitro conditions, all the four isolates were found to produce indole acetic acid, P-solubilization and hydrogen cyanide. The P-solubilizing activity coincided with a concomitant decrease in pH of the medium (pH 7.0–<3.0). The plant growth promotion properties were demonstrated through a cow pea (Vigna unguiculata (L.) walp) based bioassay under greenhouse conditions. Although the bacterial inoculation was found to result in significant increment in root, shoot and biomass and it stimulated bacterial counts in the rhizosphere. Hence, these isolates can further formulated and used for field application.     

Formation of Methyl Mercury by Bacteria

Twenty-three Hg2+-resistant cultures were isolated from sediment of the Savannah River in Georgia; of these, 14 were gram-negative short rods belonging to the genera Escherichia and Enterobacter, six were gram-positive cocci (three Staphylococcus sp. and three Streptococcus sp.) and three were Bacillus sp. All the Escherichia, Enterobacter, and the Bacillus strain were more resistant to Hg2+ than the strains of staphylococci and streptococci. Adaptation using serial dilutions and concentration gradient agar plate techniques showed that it was possible to select a Hg2+-resistant strain from a parent culture identified as Enterobacter aerogenes. This culture resisted 1,200 mug of Hg2+ per ml of medium and produced methyl mercury from HgCl2, but was unable to convert Hg2+ to volatile elemental mercury (Hg0). Under constant aeration (i.e., submerged culture), slightly more methyl mercury was formed than in the absence of aeration. Production of methyl mercury was cyclic in nature and slightly decreased if DL-homocysteine was present in media, but increased with methylcobalamine. It is concluded that the bacterial production of methyl mercury may be a means of resistance and detoxification against mercurials in which inorganic Hg2+ is converted to organic form and secreted into the environment.     

蔗渣发酵菌剂培养基的筛选及发酵条件的优化

采用单因素和正交试验研究了蔗渣高效发酵菌剂(芽孢杆菌B-A、曲霉菌F-A、链霉菌A-B)的摇瓶发酵最佳工艺条件.结果表明:芽孢杆菌B-A的最佳培养基配方:牛肉膏0.3%、蛋白胨1%、葡萄糖1%、NaCl 0.5%、可溶性淀粉0.5%、3.08%浓度的MnSO4溶液0.1;最适发酵条件为pH7、装液量100 ml(250 ml三角瓶)、36℃培养27 h.曲霉F-A的最佳培养基配方:葡萄糖3%、豆饼粉3%、蛋白胨1.2%、酵母膏0.3%、K2HPO4 0.05%、KH2PO40.05%、CaCl20.08%、MgSO40.04%、MnSO40.04%、ZnSO40.02%;最适发酵条件为pH6、装液量50 ml、30℃培养3 d.链霉菌A-B的最佳培养基配方:可溶性淀粉4.5%、蔗糖1%、豆饼粉3%、NaNO30.2%、ZnSO40.01%、KH2PO40.001%;最适发酵条件为pH7、装液量50 ml、30℃培养3 d.     

Characteristics of a biopolymer flocculant produced by Bacillus sp. PY-90

A biopolymer flocculant-producing bacterium, strain PY-90, was isolated and considered to belong to Bacillus subtilis. For the production of biopolymer flocculant by strain PY-90, a medium containing 2 to 5% l-glutamic acid as a nitrogen source was suitable. The biopolymer flocculant was a homopolymer composed of glutamic acid residues and was presumed to be poly(γ-glutamic acid). In kaolin suspension, the highest flocculating activity was attained at the biopolymer flocculant concentration of 20 mg/l. The flocculating activity was increased by the addition of Ca²⁺, and the optimum concentration of which was about 2 to 8 mM. The flocculating activity was high in an acidic pH range of 3.0 to 5.0, and decreased upon heating at 100°C.