Therapeutic use of phage cocktail for controlling Escherichia coli O157:H7 in gastrointestinal tract of mice

To investigate the therapeutical use of phage mixture for controlling gastrointestinal Escherichia coli O157:H7 cells, in vitro and in vivo experiments were conducted. Three phages, SP15, SP21, and SP22 were selected from 26 phage stock screened from feces of stock animals and sewage influent. Addition of single or binary phage to the E. coli cell batch-culture reduced the turbidity of the culture. However, reascend of the turbidity due to the appearance of phage resistance cell was observed. On the other hand, addition of three phage mixture (SP15-21-22) did not produce reascend of culture turbidity under aerobic condition. Under anaerobic condition, slight reascend of culture turbidity was observed after SP15-21-22 addition. Chemostat continuous culture was operated under anaerobic condition to optimize the titer of phage cocktail and frequency of the addition for controlling E. coli cells. Five-log decrease of E. coli cell concentration after addition of phage cocktail of 10(9) Plaque forming unit (PFU)/ml was observed. However, reascend of cell concentration was observed after 1 d incubation. Repeated addition of phage cocktail was effective to reduce the cell concentration. Suspension of phage cocktail in the buffer containing 0.25% CaCO3 neutralized 9 times much more buffer of pH 2. Based on this in vitro experiment, phage cocktail (SP15-21-22) suspended in the buffer containing 0.25% CaCO3 was orally administrated to the mice in which E. coli O157:H7 cells was administrated in 2-d advance. E. coli and phage concentration in the feces was monitored for 9 d after phage addition. High titer of phage was detected in the feces when the phage cocktail administrated daily. E. coli O157:H7 concentration in the feces has been reduced according to the time period. However, difference of E. coli concentration in the feces of mice administrates with phage and in the control mice without phage addition became slight after 9-d test period. High titer of the phage settled down in the gastrointestinal tracts and reduced the concentration of E. coli cell. Repeated oral administration of SP15-21-22 was effective for rapid evacuation of E. coli O157:H7 from the feces and gastrointestinal tract of mice.     

Cloning and expression of NAD+-dependent L-arabinitol 4-dehydrogenase gene (ladA) of Aspergillus oryzae

A gene of Aspergillus oryzae, ladA, which encodes L-arabinitol 4-dehydrogenase (EC 1.1.1.12), and its cDNA were cloned in Escherichia coli. The gene consisted of a 1209-bp coding region, interrupted by a 59-bp intron, which encoded a 382-amino-acid polypeptide (40,812 Da). The protein showed 67% identity to a well-studied L-arabinitol 4-dehydrogenase (Lad1) of Hypocrea jecorina. The cell-free extract of E. coli, which expressed ladA cDNA, showed L-arabinitol dehydrogenase activity with NAD+. It was also reactive for ribitol and xylitol.     

Performance of a styrene-degrading biofilter inoculated with Pseudomonas sp. SR-5

Styrene removal was studied for 3 months in a laboratory-scale biofilter packed with a mixed packing material of peat and ceramic at a ratio of 1 to 1 on a dry-weight basis and inoculated with Pseudomonas sp. SR-5. More than 90% removal efficiency (RE) was attained at 1-140 g/m3/h styrene loads under nitrogen-source limitation. When RE decreased to 70% after 30 d with an increase in styrene load, readdition of SR-5 and washing of the filter packing material restored the RE to more than 90% by maintaining the population of SR-5 at 1-10% of the total cell number. The maximum elimination capacity (EC) by kinetic analysis was estimated to be 290 g/m3/h. High conversion of the removed styrene carbon to CO2, and significantly small production of cell mass from the removed carbon were confirmed.     

Removal and recovery of lithium using various microorganisms

The accumulation of lithium by microorganisms was examined. Among the 70 strains of the 63 species tested (20 bacteria, 18 actinomycetes, 18 fungi, and 14 yeasts), a high lithium accumulating ability was exhibited by strains of the bacteria, Arthrobacter nicotianae and Brevibacterium helovolum. Lithium accumulation by A. nicotianae cells was strongly affected by the pH of the solution. The amount of accumulated lithium was maximum at pH 6. Cells immobilized with polyacrylamide gel also adsorbed lithium. They could be reused during repeated adsorptions, and adsorbed 548 micromol of lithium/g dry wt. cells. The adsorbed lithium was quantitatively and easily desorbed with 1 M hydrochloric acid using a column system.     

Micrococcus luteus K-3-type glutaminase from Aspergillus oryzae RIB40 is salt-tolerant

Aspergillus oryzae RIB40 possesses the gene of glutaminase (Micrococcus luteus K-3-type glutaminase; AoGls), which has 40% homology with the salt-tolerant glutaminase from M. luteus K-3 (Micrococcus glutaminase). It was found that AoGls is a salt-tolerant enzyme, and its properties are similar to those of Micrococcus glutaminase.     

Two novel cyclodextrin-degrading enzymes isolated from thermophilic bacteria have similar domain structures but differ in oligomeric state and activity profile

In this paper, we present the expression and characterization of two novel enzymes from the alpha-amylase family exhibiting cyclomaltodextrinase specificity. The nucleotide sequences encoding the enzymes were isolated from the genomic DNA of two thermophilic bacterial strains originating from Icelandic hot springs and belonging to the genera Anoxybacillus (AfCda13) and Laceyella (LsCda13). The genes were amplified using a consensus primer strategy utilizing two of the four conserved regions present in glycoside hydrolase family 13. No identifiable signal peptides were present in open reading frames encoding the enzymes, indicating an intracellular location of both enzymes, and their physiological function to be intracellular cyclodextrin degradation. The domain structures of both enzymes were also similar, including an N-terminal domain, the catalytic module composed of the A- and B-domains, and a C-terminal domain. Despite the similarity in domain composition, the two enzymes displayed differences in the oligomeric state with AfCda13 being a dimeric protein, whereas LsCda13 was monomeric. The two enzymes also displayed significantly different activity profiles, despite being active on the same range of substrates. It was shown that the enzyme displaying the highest activity on cyclodextrin was dimeric (AfCda13). Moreover, a fraction of the dimeric enzyme could be converted to a monomeric state in the presence of KCl and this fraction retained only 23% of its activity on alpha-cyclodextrin while its activity on starch was not significantly affected, indicating that the oligomeric state is an important factor for a high activity on cyclodextrin substrates.     

Production of L-talitol from L-psicose by Metschnikowia koreensis LA1 isolated from soy sauce mash

A strain LA1 that can convert L-psicose to L-talitol was isolated from soy sauce mash and identified as Metschnikowia koreensis. The cells grown on L-arabitol were found to have relatively high conversion potential. Addition of D-sorbitol to the reaction mixture considerably accelerated the conversion rate of L-psicose to L-talitol. During the conversion reaction, D-sorbitol was added to the reaction mixture at 12-h intervals to maintain the concentration of D-sorbitol at 1.0%. The final conversion ratios were 81.4%, 75.2%, 73.0%, 60.4% and 43.5% using washed cells when the concentrations of L-psicose were 0.5%, 1.0%, 2.0%, 3.0% and 5.0%, respectively. The product from L-psicose was identified as L-talitol by HPLC analysis, and infrared spectroscopy, optical rotation and melting point measurements.     

A new microbial method for more efficient production of Nalpha-benzyloxycarbonyl-L-aminoadipate delta-semialdehyde and Nalpha-benzyloxycarbonyl-D-aminoadipate delta-semialdehyde

A new biochemical method for more efficient production of Nalpha-benzyloxycarbonyl-L-aminoadipate delta-semialdehyde (Nalpha-Z-L-AASA) and Nalpha-Z-D-AASA was developed with cells of Rhodococcus sp. AIU Z-35-1. Using the cells harvested after 1 d of cultivation, more than 95 mM Nalpha-Z-L-AASA was produced from 100 mM Nalpha-Z-L-lysine by incubating at pH 5.0 for 1 d at 30 degrees C or by incubating at pH 7.0 for 2 d at 10 degrees C. A similar conversion yield of Nalpha-Z-D-AASA was also obtained under the same conditions. These reaction times required were 1/4 and 1/2 of the respective ones by the method with amine oxidase, and the yields of Nalpha-Z-L-AASA and Nalpha-Z-D-AASA were 2 times higher than the respective ones by the method with amine oxidase. In addition, this method had the advantages of not requiring purification of enzyme and addition of catalase. Thus, the microbial method proposed here was superior to the chemical and other biochemical methods in simplicity, reaction rate, and yield.     

Production of L-sorbitol from L-fructose by Aureobasidium pullulans LP23 isolated from soy sauce mash

A strain LP23 that can convert L-fructose to L-sorbitol was isolated from soy sauce mash and identified as Aureobasidium pullulans. The cells grown on L-arabinose were found to have relatively high L-fructose to L-sorbitol conversion potential. Addition of erythritol to the reaction mixture considerably accelerated the conversion rate of L-fructose to L-sorbitol. During the conversion reaction, erythritol was added to the reaction mixture at 8-h intervals to maintain the concentration of erythritol at 1.0%. The final conversion ratios were 82.8%, 95.3%, 92.4%, and 42.6% using washed cells when the concentrations of L-fructose were 1.0%, 2.0%, 5.0% and 10.0%, respectively. The product from L-fructose was identified as L-sorbitol by HPLC analysis, infrared spectroscopy, optical rotation and melting point measurements.     

Vacuolar functions are involved in stress-protective effect of intracellular proline in Saccharomyces cerevisiae

Proline protects yeast cells from damage caused by various stresses. A yeast Saccharomyces cerevisiae mutant with high levels of intracellular proline grown in a minimal medium accumulated proline in its vacuole, but when grown in a nutrient medium, accumulated proline mainly in the cytosol. To understand the role of the proline pool in the vacuole, we examined the stress-protective effect of proline in proline-accumulating yeast cells deficient in vacuolar functions. The disruption of PEP3 encoding a vacuolar membrane protein required for vacuolar biogenesis caused hypersensitivity to heat shock and ethanol stresses, probably due to disappearance of normal vacuoles. The vph1-disrupted cells lacking vacuolar-ATPase activity showed resistance to heat shock without any change in proline localization, but showed severe growth defects in an ethanol-containing medium. These results indicate that vacuolar functions are involved in the stress-protective effect of proline in S. cerevisiae. Also, it appears that excess proline is transported to the vacuole in an ATP-independent manner.     

Characterization of d-succinylase from Cupriavidus sp. P4-10-C and its application in d-amino acid synthesis

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Analysis of essential carboxylic amino acid residues for catalytic activity of fungal chitosanases by site-directed mutagenesis

A comparison of amino acid sequences of fungal chitosanases, belonging to family 75 of glycosyl hydrolases, revealed three carboxylic amino acid residues completely conserved among all of the chitosanases. To study the role of these residues in catalysis, they were replaced with other residues by site-directed mutagenesis in the chitosanase gene of Fusarium solani. The mutated genes were expressed in the yeast Saccharomyces cerevisiae and the resulting recombinant chitosanases were used in kinetic analysis. Chitosanases with Asp-175-->Asn and Glu-188-->Gln mutations were essentially inactive, whereas those with Asp-175-->Glu and Glu-188-->Asp mutations retained 25-50% specific activity as compared with the wild-type enzyme. The mutation of Asp-212-->Asn did not decrease specific activity to a large extent. Circular dichroism analysis confirmed that the mutant chitosanases had similar secondary structures to that of the wild-type enzyme. These results indicate that Asp-175 and Glu-188 are essential residues for the catalytic activity of chitosanase. Time-dependent (1)H-NMR analysis for the hydrolysis of D-glucosamine hexamer revealed that a fungal chitosanase is an inverting enzyme producing only the alpha anomeric form of reaction products.     

γ-聚谷氨酸吸附金属离子性能的研究

研究了γ-聚谷氨酸的阻垢性能以及对六价铬离子的吸附性。试验表明,在pH8、温度为60℃、γ-聚谷氨酸的含量为0.32 mg/mL的条件下,吸附钙离子24 h,可达到最高阻垢率77.07%;pH6、温度为60℃,γ-聚谷氨酸的量为0.04 mg/mL条件下,γ-聚谷氨酸对24μg/mL铬离子液中六价铬的吸附率可达72.50%。     

Biological treatment of wastewater discharged from biodiesel fuel production plant with alkali-catalyzed transesterification

The biological treatment of wastewater discharged from a biodiesel fuel (BDF) production plant conducting alkali catalysis transesterification was investigated. BDF wastewater has a high pH and high hexane-extracted oil and low nitrogen concentrations, and inhibits the growth of microorganisms. The biological treatment of BDF wastewater is difficult because the composition of such wastewater is not suitable for microbial growth. To apply the microbiological treatment of BDF wastewater using an oil degradable yeast, Rhodotorula mucilaginosa, the pH was adjusted to 6.8 and several nutrients such as a nitrogen source (ammonium sulfate, ammonium chloride or urea), yeast extract, KH2PO4 and MgSO4.7H2O were added to the wastewater. The optimal initial concentration of yeast extract was 1 g/l and the optimal C/N ratio was between 17 and 68 when using urea as a nitrogen source. A growth inhibitor was also present in the BDF wastewater, and this growth inhibitor could be detected by measuring the solid content in an aqueous phase after the hexane extraction of the wastewater. Microorganisms could not grow at solid contents higher than 2.14 g/l in the wastewater. To avoid the growth inhibition, the BDF wastewater was diluted with the same volume of water. Oil degradation in the diluted BDF wastewater was observed and the best result was obtained under the determined optimal conditions. This treatment system is simple because no controllers, except for a temperature, are necessary. These results suggest that the biological treatment system developed for BDF wastewater is useful for small-scale BDF production plants.     

添加氧载体对γ-多聚谷氨酸发酵的影响

针对γ-多聚谷氨酸(Poly-γ-glutamic acid,γ-PGA)好氧发酵过程中存在的供氧不足而导致产量偏低的问题,探究了不同氧载体对γ-PGA发酵的影响,并对最适氧载体的添加量和添加时间进行了优化。结果表明,正十六烷为最适氧载体,其最佳添加条件为:添加量为0.5%(体积比),添加时间为0 h,发酵48 h,γ-PGA产量达到最高值为(18.62±0.84) mg/mL,相比对照组提高了30%以上。本研究为通过添加氧载体,增加溶氧来提高γ-PGA发酵产量提供了理论依据。     

雪茄烟叶（BESNO H382）不同组织器官及盐胁迫条件下内参基因的筛选

为了筛选适用于雪茄烟叶（BESNO H382）基因定量研究的内参基因,以温室栽培条件下雪茄烟叶的不同组织器官（旺长期根、茎、叶,盛花期花萼、花冠、柱头、子房、雄蕊、雌蕊及种子）及不同盐处理条件下（300 mmol/L NaCl处理不同时间）的样品为材料,采用实时荧光定量PCR技术对从雪茄烟叶转录组数据中筛选到的10个候选内参基因进行分析,同时使用geNorm、NormFinder和BestKeeper等3种软件综合评价这些候选基因的表达稳定性。结果表明,RPL14A和UBC27最适合作为雪茄烟叶不同组织的内参基因;UBC28最适合作为雪茄烟叶不同盐处理条件下的内参基因。分别以UBC27、UBC28和RPL14A为内参基因考察了胁迫相关基因脯氨酸合成酶基因（P5CS）在盐胁迫下的表达情况,发现P5CS上调表达且相对3个内参基因的表达量和变化趋势均较为一致。      

光强衰减对烟草叶片碳氮代谢关键基因表达的影响

通过电子显微镜和反转录 PCR方法研究了烟草在遮光处理(分别为 100%, 85%, 70%和 55%的自然光照强度)下烟叶移栽后 78天、85天、105天及 120天(采收当天)四个时期淀粉的积累和淀粉、糖、氮代谢途径关键基因表达量的变化。结果表明,移栽后 105天烟叶生理成熟期之前淀粉含量受光照影响最大,随着光的衰减含量逐渐降低。葡萄糖转化及蔗糖的合成途径中有 4个关键基因表达量随光强减少逐渐降低,且随着烟草的成熟也呈下降趋势。淀粉合成酶基因 GBSSI在叶片扩展期表达较弱,在叶片成熟期大量表达,随着光照强度的减弱表达量稍有减小。氮代谢途径关键基因 Nir、GS等同工酶基因的表达存在一定的互补性,总体基因表达趋势为随光强的减弱表达量逐渐增加,表明在弱光下烟叶氮代谢滞后。      

Short communication: The effect of increasing concentrations of different methionine forms and 2-hydroxy-4-(methylthio)butanoic acid on genes controlling methionine metabolism in primary bovine neonatal hepatocytes

The d-isomer of Met cannot be used directly by the mammary gland in dairy cows; instead, it is transformed into l-Met, the proteogenic isomer, in the liver and other extramammary tissues. It remains unclear whether different Met forms and a Met hydroxy analog, 2-hydroxy-4-(methylthio)butanoic acid (HMB), are metabolized and function similarly in the liver. The objective of the present study was to examine the regulation of key genes in Met regeneration, transulfuration, and transmethylation pathways in response to increasing doses of different Met forms. Hepatocytes isolated from 4 calves between 4 and 7 d old were maintained as monolayer cultures for 24 h before addition of treatments. Treatments of (0, 10, 20, 40 µM) d-Met, l-Met, dl-Met, dl-HMB, or a 1:1 mixture of dl-Met and dl-HMB were added to Met-free medium in triplicate. After 24 h, cell lysates were collected for quantification of gene expression by quantitative PCR, and mRNA abundance was normalized to the mean of 3 reference genes. Data were analyzed with PROC MIXED of SAS 9.3 (SAS Institute Inc., Cary, NC). Analyses of covariance confirmed equivalent slopes of Met form, and the final model included form, dose, and random effect of calf within form. Data are reported as least squares means ± standard error. No main effect of Met form was observed for any genes examined. The enzymes encoded by betaine-homocysteine methyltransferase (BHMT) and 5-methyltetrahydrofolate-homocysteine methyltransferase use betaine and 5-methyltetrahydrofolate, respectively, to regenerate Met from homocysteine. Increasing concentration of Met did not alter 5-methyltetrahydrofolate expression, but decreased BHMT expression. Expression of glycine N-methyltransferase, the enzyme that controls transmethylation flux from S-adenosyl-methionine, was not affected by Met concentration. Methionine concentration had no effect on expression of cystathionine β-synthase, a key enzyme for the transulfuration pathway. The decrease in BHMT expression indicates a decreased need for cellular Met regeneration with increasing Met concentration, independent of Met form. The lack of differences among Met forms on regulating genes examined indicates that all Met forms similarly reduced genes controlling Met regeneration and metabolism in primary bovine hepatocytes.     

普通烟草碳酸酐酶家族基因的生物信息学分析

为挖掘普通烟草碳酸酐酶基因的信息并探讨其功能,本研究利用生物信息学方法,在烟草基因组数据库中对普通烟草碳酸酐酶家族成员进行了检索,并对其理化性质、遗传进化、基因结构、蛋白保守基序、顺式作用元件和组织表达模式进行了分析。结果显示,在普通烟草中至少含有9个α和6个β亚家族成员。在进化关系上,不同亚家族成员之间,序列同源性较低;与水稻相比,拟南芥和普通烟草两个亚家族间的亲缘关系较近。亚细胞定位分析结果显示,烟草α亚家族成员在细胞壁、细胞膜、线粒体、叶绿体、细胞质等细胞器中均有分布,而β亚家族成员均存在于叶绿体中。基因结构和保守基序分析说明,各亚家族成员的基因结构和蛋白保守基序呈现较高的一致性。启动子顺式作用元件分析显示,烟草碳酸酐酶基因的启动子中,均含有多个参与光反应、激素响应、非生物胁迫和机械损伤等相关的顺式作用元件。基因组织表达模式分析表明,烟草的碳酸酐酶基因不同成员在不同组织表达水平存在差异,并呈现出时空特异性。本研究结果为烟草碳酸酐酶基因的功能研究奠定了基础。