Abstract:Aeromonas hydrophila, Outer membrane protein W gene, Cloning, Genetic engineering expression, Immunogenicity, IgM, Protection

Abstract:The complete gene sequence of the outer membrane protein W (OmpW) was amplified from the genomic DNA of Aeromonas hydrophila Wp3 strain which was isolated from grass carp suffered hemorrhage. The OmpW gene was 865 bp in length, containing an open reading frame (ORF) of 615 bp. The cloned gene possessed high similarity with that of the standard strain ATCC7966 (99.8%). The sequence encoding the mature peptide of OmpW was amplified and inserted into the expression vector pQE30. The recombinant vector was transformed into E. coli, and a 24.7 kD recombinant fusion protein His-W was expressed. Elisa analysis of the serum of grass carp immunized with His-W showed a positive immune reaction, suggesting production of the antibodies. Total RNA of the head kidney of the immunized grass carp was extracted and mRNA level of IgM was analyzed by qRT-PCR. Expression levels of IgM gene in the immunized group were higher than those of the control group, and significant difference (P<0.05) was found in the lowest protein concentration group (2 μg/g). These results showed that His-W was able to efficiently induce the immunized grass carp to produce antibodies. Protection experiments showed that the immunized groups had higher survival rates than those of the control groups (57%?86%). This study suggested that the recombinant protein His-W was a candidate vaccine for grass carp A. hydrophila disease.

Abstract:α-L-鼠李糖苷酶(α-L-rhamnosidase, EC 3.2.1.40)能特异性地水解许多糖苷类物质, 如柚皮苷(Naringin)、芦丁(Rutin)、橙皮苷(Hesperidin)等的末端α-L-鼠李糖基可用于去除柑桔类果汁的苦味、消除桔子汁的橙皮苷结晶、生物转化生产L-鼠李糖、切除糖苷类药物原料末端的L-鼠李糖以及改善酿造食品的风味等^[1?3]。国外已有利用微生物发酵生产α-L-鼠李糖苷酶的研究, 但尚未有工业化生产的报道, 主要原因在于缺乏适合于工业化生产的高产菌株。
虽然很多学者对α-L-鼠李糖苷酶高产菌株选育进行了研究, 但由于缺乏有效的筛选模型, 菌种选育效率低, 进展缓慢。本刊2009 年第7 期介绍了陈华根和蔡慧农发表的论文“黑曲霉α-鼠李糖苷酶高产菌株的选育”^[4], 作者利用α-L-鼠李糖苷酶高产菌株的透明圏筛选法有效地提高了α-L-鼠李糖苷酶高产育种的效率,选育得到了高产α-L-鼠李糖苷酶的黑曲霉DB056 菌株, 为实施高产育种及提高α-L-鼠李糖苷酶的发酵产量提供了基础。
最近, 蔡慧农等对黑曲霉DB056 菌株的α-L-鼠李糖苷酶发酵、分离纯化、酶制剂及应用技术进行了系统研究：(1) 发现黑曲霉DB056 在以柚皮苷为碳源的培养基中同时发酵产生了α-L-鼠李糖苷酶与β-D-葡萄糖苷酶, 这两个酶紧密结合形成柚苷酶^[5]；(2) 优化了黑曲霉DB056 发酵的培养基和培养条件, 在200 L 发酵罐上中试发酵α-L-鼠李糖苷酶获得了高产, 酶的产量达到1 400 U/mL 发酵液^[6]；(3) 制备了方便使用的酶制剂, 其活力不低于10 000 U/g 干粉, 可在4 °C 长期贮存；(4) 用α-L-鼠李糖苷酶对琯溪蜜柚果汁进行脱苦,在酶加量为10 U/mL 的条件下40 °C 处理60 min, 苦味就降低到阈值以下^[7]。这些研究不仅为α-L-鼠李糖苷酶的工业化生产奠定了基础, 而且也为食品加工行业提供了一种新的酶, 具有重要的应用前景。

Abstract:The Rhizopus chinensis lipase genes was cloned and expressed in Mut⁺ methylotrophic Pichia pastoris with different gene dosage. The high-density fermentation of the recombinant Pichia pastoris with gene dosage of three (XY RCL-3), five (XY RCL-5) and six (XY RCL-6) was investigated on 7-liter fermenter. The impact of methanol concentration on Rhizopus chinensis lipase expression was also explored. Our results indicated that more Rhizopus chinensis lipase was produced in XY RCL-5 produced than that of XY RCL-6 and XY RCL-3 under the same conditions. When methanol concentration was controlled at 0.10%±0.02%, the enzyme activity of Rhizopus chinensis lipase produced by XY RCL-5 could achieve 12 500 U/mL, and the dry weight of XY RCL-5 and protein concentration could reach 204 g/L and 8.02 g/L, respectively.

Abstract:In this study, a mixed culture of moderately thermophilic microorganisms was enriched from several acid mine drainage samples collected from copper mines in China at 45 °C. The pH and copper concentration in the leachate were monitored during bioleaching low-grade chalcopyrite by column reactor, and community structure and dynamics were investigated by restriction fragment length polymorphism (RFLP). The results show that variation of pH was more obvious, and pH value was always higher than 1.8 in the bioleaching process. And 13.6% of copper was recovered within 60 days. RFLP results show that microbial community was dominated by L. ferriphilum in the initial stage, which occupied 81% of total prokaryotes. With the bioleaching process continues, the proportion of L. ferriphilum decreased slowly, which only had a proportion of 13% in the final stage. The proportion of S. thermotolerans and A. caldus were increase gradually, and the proportion was 32% and 23% respectively in the middle stage. S. thermotolerans was the dominant microorganism in the final stage, and the proportion was up to 79%. The research results could promote to understand bioleaching characteristics and behavior of moderately thermophilic microorganisms and provide referential experience for industrial application.

Abstract:Intestinal microflora of grass carp (Ctenopharyngodon idellus) feeding with xylo-oligosaccharide for 56 days by concentrations of 0.1%, 0.2%, 0.4% and 0.6% respectively was investigated in this paper. Numbers of E. coli, Aeromonas and Bifidobacterium were analyzed before feeding and after 14, 28, 42, 56 days. The results showed that there was a certain influence on the intestinal microflora of grass carp after fed with different concentrations of xylo-oligosaccharides. The quantity of E. coli in the intestine of grass carps was smallest on 28th day, and compared with the control group, 0.4% group significantly different (P<0.05) has the highest decreasing magnitudes. The quantity of Aeromonas reduced but there was no significant difference with control. The quantity of Bifidobacterium increased and there was significant difference (P<0.05) between control and 0.4% group on 14th day. Therefore, feedstuffs with xylo-oligosaccharide, which were conducive to maintain a healthy state of intestinal microflora, group with 0.4% has the best effect.

Abstract:Clover plants (Trifolium repens L.) were inoculated with arbuscular mycorrhizal (AM) fungus, Glomus intraradices, in pot and split-root experiments. The mycorrhizal colonization and antioxidase activities in roots were measured for the influence of Glomus intraradices on the root antioxidase activities and the systematization of the influence. Results indicated that G. intraradices significantly enhanced the activities of SOD, POD, CAT in roots in the pot experiment, suggesting an increased antioxidase activity by AM fungus. For the plants with half roots inoculated with G. intraradices in the split-root experiment, the activities of SOD, POD also increased in the other half roots without G. intraradices, suggesting the systematization of the increase in antioxidase by AM fungus. Given that the antioxidase system is the physilogical and biochemical basis of the resistance to diverse stresses in plants, this systematical increase may contribute to the protection of the entire roots, not of the infected ones only.

Abstract:The selective media were used to isolate and screen the manganese-oxidizing bacteria. A high efficiency manganese-oxidizing bacterium (MN1405) was selected from manganese ores. According to the morphological features, physiological and biochemical characteristics and the sequence analysis of 16S rRNA gene, MN1405 was identified as Arthrobacter echigonensis. Under the cultural conditions, the manganese removal rate in the medium by MN1405 was reached 93.38%. The culture that was obtained from the bacterium also had a good manganese removal effects.

Abstract:A plant growth-promoting rhizobacteria (PGPR) strain, SZ7-1, was obtained from mangrove rhizosphere and tested previously for potential applications. To develop an economical and practical fermentation medium for the growth of SZ7-1, response surface method was used in this study. Plackett– Burman factorial design was used to examine the significance of 11 different media on the growth of SZ7-1. Experimental result demonstrated that media consisting of corn syrup, yeast extract and K₂HPO₄ significantly improved the growth process. The effect of different combinations of the three nutrients on the growth of SZ7-1 were further explored in a Box-Behnken design. The optimal concentration of each nutrient for maximum growth were corn syrup 28 g/L, yeast extract14 g/L and K₂HPO₄ 2.2 g/L. The optimized medium enhanced the growth of SZ7-1 by 12.4, 2.4 and 5.5 times more than the original medium, LB and cow meat extract protein peptone medium, respectively. This research indicates that it is possible to increase bacterial yield and reduce its costs using optimized growth medium.

Abstract:With the applying of completely mixed biological process, microbiological method of removing SO₂ was studied as pre-acidified waste molasses was used as carbon source. Under extensive experimental condition, Desulfovibrio desulfuricans’ utilization situation of organic acids in pre-acidified waste molasses as well as its desulfurization effect of sulfur dioxide in high concentration were researched, at the same time, the removal rate of the product H₂S in the second biological reactor was also determined. The results suggested that Desulfovibrio desulfuricans could exploit pyruvic acid and the lactic acid in pre-acidified waste molasses as its carbon source, with acetic acid as main product. When the inlet concentration of SO₂ ranged from 1 865 mg/m³ to 4 637 mg/m³, the removal rate of SO₂ was more than 91% in 1# biological reactor and the final removal rate of SO₂ was 95.5%, the produced H2S was nearly all transformed with the mean removal rate of 98%. pH and the concentration of the bacteria were quite stable and the system operated well.

Abstract:A fungal strain BFM-X1 was isolated from air environment on vegetable field, which had a strong ability to degrade poly (butylenes succinate, PBS). Morphological and phylogenetic analysis revealed that strain BFM-X1 was closely related to Bionectria ochroleuca. The optimum temperature for strain BFM-X1 degrading PBS film ranged from 25 °C to 30 °C and the initial pH of medium was 4.0. A high degradation rate for PBS film was observed with glucose, glycerole, soybean oil or PBS emulsion as a single carbon source for this strain. The curve of PBS film degradation rate with the increase of the PBS emulsion concentration showed a resupinate “U”, and the optimum PBS emulsion concentration was 1 g/L. Three phases were exhibited during PBS degradation by BFM-X1. Scanning electron micrographs showed that the surface of PBS film firstly became rough, then some holes appeared on the PBS film and the PBS film gradually disintegrated, at last the PBS film completely degraded.

Abstract:In order to improve the treatment effect of wastewater from pesticide enterprise, it is necessary to study the salt-tolerant characteristics and fermentation conditions of the degrading bacterium. A chlorpyrifos-degrading bacterium Bacillus cereus HY-1 was isolated in laboratory from pesticide wastewater. The optimal fermentation medium and fermentation conditions of HY-1 were selected by single factor and orthogonal test, and the amount of enzyme produced by HY-1 was estimated based on the specific activity of the enzyme. The experimental results were analyzed by the SPSS system. The optimal composition of the culture medium included glucose 6.0 g/L, tryptone 2.2 g/L, K₂HPO₄ 2.0 g/L, K₂HPO₄ 0.2 g/L, MgSO₄·7H₂O 0.1 g/L, NaCl 0.1 g/L, trace elements solution 2 mL/L. The optimal fermentation conditions of HY-1 were: incubation time of the seed culture 16 h, fermentation time 18 h, inoculum size 1% (V/V), initial pH 7.0. The specific activity of the degrading enzyme was not affected while the sodium chloride concentration of the fermentation medium increased from 0 g/L to 30 g/L. To the best of our knowledge, this is the most extremely salt-tolerant bacterium reported that could degrade chlorpyrifos.

Abstract:In order to study the effects of endophytically fungal extracts and plant growth regulators on soybean rhizosphere bacterial diversity, the dynamics of rhizosphere bacterial community in different developmental stages was studied based on PCR-DGGE approach. The results showed that they could increase some bacteria in populations; but the variation of rhizosphere bacterial diversity by fermentation extracts and plant growth regulator was no obvious, and the growth stages as an important factor that affects the rhizosphere bacteria. In addition, we found that the advantage bacteria mainly is Proteobacteria, Acidobacteria, Nitrospira, Bradyrhizobium, etc. These are the common bacterial species in the root of soybean.

Abstract:In order to screen and breed the strain with high fruiting body and cordycepin production, this study aims to investigate the effect of polymorphism on fruiting body and cordycepin production of C. militaris. Three progeny population were isolated from wild fruitbody of C. militaris, cultural characteristics of ascospores and fruitbody-producing ability were observed, and determined Cordycepin content in fruitbody and medium by HPLC. The results showed that the strains with colony reverse color of orange chrome could produce fruiting body easily. The sector mutation proportion of the strain produce few or no fruiting body. The shape and microstructure of fruiting body produced by original strain were similar to the wild strain in the nature. The original strain produced more fruiting body and cordycepin than the strains isolated from sector mutation proportion and the other parts without sector mutation. These results demonstrated the effect of polymorphism on fruiting body and cordycepin production of C. militaris.

Abstract:In the present paper we investigated the effects of specific bifidobacteria strain as supplementation on acute inflammatory bowel disease (IBD) induced by dextran sulfate sodium (DSS) and the possible mechanism was also discussed. Mice were divided into three groups as control, DSS, and experiment (DSS+Bf) group. The mice in DSS group were fed with 4% DSS (W/V) solution for 7 days to induce colitis, and the mice in DSS+Bf group, were then given bifidobacteria by oral gavage for next 4 weeks. Boththe colonic lengths and the magnitude of colonic inflammation were measured for three groups. Furter, expression of IL-10 mRNA in Peyer’s patch (PP) cells and secretion of IL-10 in intestinal mucosa were also assessed by reverse transcription- polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. It was found that the average colonic length observed in the DSS+Bf group was shorter than those in the control group but longer than those in the DSS group. Both macro-and micro-disease scorring showed that the values of the DSS+Bf goup significantly decreased compared with the DSS group, while both the expression of IL-10 mRNA in PP cells and the IL-10 positive cells in intestinal mucosa of the samples in the DSS+Bf group were also significantly higher than those of DSS group. It is obvious that the bifidobacteria strain involved in the present studies could prevent the DSS-induced murine colitis and improve the expression levels of anti-inflammatory cytokine IL-10 in intestinal mucosa.

Abstract:Poly (γ-glutamic acid) is a promising environmental friendly material with outstanding water solubility, biocompatibility and degradability. This review introduces the basal properties of γ-PGA, microbial production of γ-PGA, and the key factors affecting the yield of γ-PGA. Furthermore, the γ-PGA biosynthesis genes, γ-PGA synthetase complex, as well as the application prospects of γ-PGA in hydrogel and drug delivery, are also discussed.

Abstract:This paper describes a comprehensive from discovery to diagnosis and testing, from molecular biology to anti-viral genetic engineering of banana bunchy top virus (BBTV), and exploring history of one hundred years of BBTV. This is the bases of depth investigation and effective control of BBTV.

Abstract:Screening of effective Trametes strains with high laccase-productivity and optimization of conditions on laccase production by the diameters of colored rings by adding guaiacol to the solid medium and assaying the diameters of colored rings, we got the target strain Trametes orientalis Cui 6300. Meanwhile, the optimal conditions of strain Cui 6300 for laccase production were obtained by means of single factor analysis, orthogonal test and ABTS method: maltose 15 g/L, peptone 3 g/L, pH 4.8, Cu²⁺ 2.0 mmol/L, culture temperature 28 °C, inoculum 1.5 cm in diameter, then the highest activity was 19.923 U/mL. Besides, the influences of Cu²⁺ concentration and adding time on biomass and laccase activity were tested, and the optimal concentration and adding time were 2.0 mmol/L and on day 3 of culturing, respectively.

Abstract:To establish proteomic maps of Bifidobacterium longum subsp. longum BBMN68, applying two-dimensional gel electrophoresis, we built a 2-D reference map, by MALDI-TOF mass spectrometry. We identified a total number of 206 different proteins (11.4% of predicted total proteins in B. longum subsp. longum BBMN68). A total of 800±15 (exponential) and 800±20 (stationary) protein spots on each 2D–PAGE map were got, in which a total of 282 spots were successfully identified, represented 206 different proteins. Additionally, combined with analysis of pI and molecular masses, COG, CAI, GRVAVY and the cellular localizations of identified proteins in B. longum subsp. longum BBMN68. This study presented a proteome map of B. longum BBMN68 and provided a reference proteome database for comparative proteomic studied.

Abstract:The improved yeast strains obtained through classical breeding methods, such as the mutation and the acclimatization, easily lost their acquired characteristics during the process of passage or conservation. We investigated the epigenetic molecular mechanism of genetic instability about the yeast acquired traits. The relationship between the genetic stability of ethanol tolerance and the change of H3K4 methylation level of promoters of pro1, tps1, sod1 was studied in the process of yeast strain breeding for improving ethanol tolerance or passage of improved strains without ethanol stress. The results showed that the genetic stability of ethanol tolerance was regulated by epigenetic variation of some genes in the yeast. The genetic instability of acquired traits of yeast might result from its epigenetic variation of relative genes because many environmental factors influenced on the epigenetic molecular modification in cells.

Abstract:The detection period of Escherichia coli O157: H7 (E. coli O157: H7) using traditional testing methods is relatively long. Therefore a kind of rapid detection method, namely a loop-mediated isothermal amplification (LAMP) technology with two loop primers detection E. coli O157: H7 in meat, was developed to address this problem. Two sets LAMP primers with loop primers were respectively designed based on the target sequences of rfbE and fliC genes of E. coli O157: H7. The detection results were judged by observing the white precipitate at simultaneous detection of single-tube. The specificity of the improved LAMP was verified using 36 strains of bacteria. DNA extracted by pyrolysis was amplified 20 min by the LAMP system. The detecting sensitivity of E. coli O157: H7 was 1.4 CFU/mL. The detection limit of E. coli O157: H7 from artificial contamination meat was 1.8 CFU/g. One false positive of 137 meat samples was detected, which can obtain compliance rate of 99.3% with the industry standard SNT0973-2000.