土壤氨基糖微生物转化过程与机理探讨

土壤氨基糖因其异源性和稳定性可用以指示微生物对土壤碳（C）氮循环的相对贡献。但由于氨基糖是微生物在土壤中长期残留的平衡结果，其数量变化无法准确反映在微生物作用下无机态氮（N）向氨基糖转化的动态过程和机制，从而使氨基糖对土壤氮内循环的指示作用受到限制。如果能够利用新的技术手段研究土壤氨基糖的微生物转化过程，将使土壤氮素内循环研究产生突破。同位素技术是研究土壤C，N转化过程的有效手段。但是研究特定化合物如氨基糖的微生物转化过程还需要新的技术支持，本研究首先建立了稳定同位素培养－气质联机技术测定土壤氨基糖同位素富集比例新方法。对于15N培养样品，由于氨基糖分子中只有一个N原子，15N富集比例可通过m/z（F+1）与F相对丰度的比值计算；对于13C培养样品，由于葡萄糖c整体掺入形成氨基糖c骨架，所以利用m／z（F＋n）与F相对丰度的比值计算13c在土壤氨基糖中的富集（n为质谱碎片中骨架C原子数）。同位素富集用原子百分超（APE）表示。EI和cI两种方式测得的APE有很好的同一性，且不受土壤基质的影响，表明方法的可靠性和广泛适用性。利用以上方法，进行了土壤样品的同位素培养与测定，以跟踪土壤氨基糖微生物合成动态，进行氨基糖的微生物转化与更新研究。主要结论如下：1．当以葡萄糖为碳源且每周施入底物时，NH4+和NO3-均可被微生物迅速同化并进行氨基糖的合成。但NO3-必须被还原成NH4＋才能被微生物利用，因而NO3．存在短暂的滞后期，之后被微生物快速利用。氨基葡萄糖（GluN）和胞壁酸（MurN）不同的同位素富集特征表明，N源形态对细菌增殖无显著影响，但真菌更倾向于利用NH4+。在NO3-培养中氨基糖的增量及微生物对C的截获均小于NH4＋。2．分别利用u一13c一glucose一NH4十和glLlcose一ISNH4＋进行样品培养时，同位素富集趋势相同，但APE（13C）大于APE（15N），这种差别反映了了土壤微生物利用C，N的时间特征及土壤有机质含量对C，N循环的影响。3．以gtucose-15NH4+为底物时，施入N素频率的改变也会影响微生物的活性。尤其是细菌的快速生长受到N素不足的限制，转而代之以细菌和真菌的持续的低速生长。微生物活性的降低减少了对有机c的截获。DCD的加入有效抑制了NH4+向NO3一的转化，但对氨基糖的合成无显著影响。4．土壤氨基糖反映的主要是土壤中已经死亡了的微生物的一种长期过程而产生的残留，同土壤微生物量无明显的相关性。但经外加底物培养后，氨基糖同位素富集比例的变化则来源于微生物的转化，因而与微生物量碳有直接的相关性。5．从原理上说，在氨基糖的微生物合成过程中，葡萄糖没有发生C骨架的断裂，而是直接转化成为氨基葡萄糖的骨架。但在复杂的土壤基质中，氨基葡萄糖的合成必然受到葡萄糖其他生物化学过程的影响。使少量葡萄糖经酵解后再次参与己糖胺的合成。以全取代葡萄糖为底物时，葡萄糖碳骨架的断裂与重排不影响13c同位素的富集。但对于单取代葡萄糖培养来说，必须要考虑因葡萄糖碳骨架断裂而产生的同位素的重新分配，Mass（F+1）和Mass（F+2）的丰度变化的总和真正代表了氨基葡萄糖的同位素富集。6．添加有机物料和N素进行土壤样品培养时，对外加氮素的同化远低于相应的葡萄糖培养。碳源尤其是能源不足限制了N的转化。微生物分解高C加的有机物料需吸收外加N源以满足自身生长需要。N素的加入频率影落响微生物对外加N素的利用。当加入的N不能满足微生物分解有机物料的需要，就会降低微生物对有机物料的分解速度，使无机N向氨基糖态N转化速度降低。应用稳定同位素技术，发现施到土壤中的无机氮素可被微生物快速转化成某种形态有机氮，这种有机态氮处于不断转化循环之中，构成土壤有效氮的暂存"过渡库"，其中氨基糖是重要成分之一。过渡库现象的发现为氮肥的有效利用提供了新的思路。根据土壤有效氮"过渡库"的模型，氮月巴高效利用调控实际上就是土壤氮素微生物转化过程的调控。提高土壤无机氮素向土壤有机氮的转化速率和转化强度可以有效减少无机氮在土壤中的积累，从而降低肥料和土壤氮素的硝化和反硝化损失，提高氮肥利用率。研究还发现，土壤有效氮"过渡库"容量与循环速率不仅取决于N源自身性质，碳源的可利用性显著影响施入土壤的N素微生物转化特征。只有适当提高可利用碳源即活性碳源的数量，才能提高氮素的微生物同化，土壤有效氮"过渡库"容量，从而减少氮素损失。

鲜甘薯快速乙醇发酵条件的初步研究

本文对不同菌种（酵母菌和运动发酵单胞菌）快速生产燃料乙醇的条件进行了研究，实现了鲜甘薯快速转化为燃料乙醇。全文分为两部分： 第一部分：酵母菌快速生产燃料乙醇的条件研究。通过单因素试验，酵母菌快速生产燃料乙醇的条件为：发酵方式采用边糖化边发酵（SSF），蒸煮温度为85 ℃，料水比2:1（初始糖浓度 210 g/kg），糖化酶用量0.75 AGU/g 鲜甘薯，接种量10%（v/w）。在最优条件下，经过24 h发酵，乙醇浓度可达97.44 g/kg, 发酵效率为92%，发酵强度为4.06 g/kg/h。由于采用了低温蒸煮和SSF，可以大大节约能耗，从而降低乙醇生产的成本。同时，利用摇瓶优化的条件，进行了10 L，100 L，500 L发酵罐的放大试验，由于发酵罐初期可以人为通氧，使菌体能迅速积累，发酵时间缩短2 h，发酵效率在90%以上。 第二部分：运动发酵单胞菌快速生产燃料乙醇条件研究。通过单因素试验和正交试验获得了发酵的最佳参数：初始pH值6.0-7.0，硫酸铵5.0 g/kg，糖化酶量1.6 AUG/kg淀粉，初始糖浓度200 g/kg，接种量12.5%（v/w）。经过21 h发酵，乙醇浓度为95.15 g/kg，发酵效率可达94%。同时对不灭菌发酵也进行了研究，发酵效率可达92%。为鲜甘薯运动发酵单胞菌燃料乙醇的工业化生产打下基础。 对发酵结束后的残糖进行了研究。通过薄层层析和葡萄氧化酶测定证明：无论是酵母菌还是运动发酵单胞菌发酵结束后的发酵液中都不含葡萄糖。经过HPLC进一步分析残糖说明：发酵液中已没有葡萄糖成分；经糖化酶水解后仍没有葡萄糖出现；但经酸水解后又出现了葡萄糖，说明结束后的残糖是一些低聚糖结构。有关残糖的结构需要进一步研究。可以通过开发高效的低聚糖水解酶来降低发酵液的残糖，提高原料的利用率。 A new technology for rapid production fuel ethanol from fresh sweet potato by different microorganisms (Saccharomyces cerevisiae and Zymomonas mobilis) was gained in this research. The paper involved two parts: Part 1: The study on fuel ethanol rapid production from fresh sweet potato by Saccharomyces cerevisiae. The following parameters of Saccharomyces cerevisiae was investigated by a series of experiments: fermentation models, cooking temperature, initial sugar concentration and glucoamylase dosage. The results showed that SSF (simultaneous saccharification and fermentation) not only reduced the fermentation time (from 30 to 24h) but also enhanced the ethanol concentration (from 73.56 to 95.96 g/kg). With low-temperature-cooking (85 ℃) using SSF, the Saccharomyces cerevisiae was able to produce ethanol 97.44 g/kg which the fermentation yield could reach to 92% and ethanol productivity 4.06 g/kg/h from sweet potato enzymatic hydrolysis. Furthermore, the savings in energy by carrying out the cooking (85 ℃) and saccharification (30 ℃) step at low temperature had been realized. The results were also verified in 10 L, 100 L and 500 L fermentor. The fermentation yield was no less than 90%. The fermentation time of fermenter was shorter than Erlenmeyer flask. This may be that the aeration in the early fermentation period is available, which lead to the rapidly commutations of biomass. Part 2: The technology of ethanol rapid production with simultaneous saccharification and fermentation ( SSF ) by Zymomonas mobilis，using fresh sweet potato as raw material was studied. The effects of various factors on the yield of ethanol were investigated by the single factor and the orthogonal experiments. As a result, the optimal technical conditions were obtained from those experiments：initial pH value 6.0-7.0, nitride 5.0 g/kg，(NH4)2SO4, glucoamylase 1.6 AUG/kg starch, inoculums concentration 12.5% (v/w). The Zymomonas mobilis was able to produce ethanol 95.15 g/kg, with 94% of the theoretical yield, from fresh sweet potato after 24 h fermentation. The fermentation efficiency of non-sterilized was also reach to 92%. We also analyzed the final fermentation residual sugars of Saccharomyces cerevisiae and Zymomonas mobilis. When the residual sugars were analyzed by thin-layer chromatogram and glucose oxidase, there was no glucose. The analysis of reducing sugars by HPLC showed that there was no glucose existed in the fermentation liquor. However, the glucose appeared after being hydrolyzed by acid. It is indicated that the residual sugars in the final fermentation liquor were the configuration of oligosaccharide, which was linked by the special glycosidic bonds. It was feasible for reducing residual sugars to develope the enzyme that can degradation the oligosaccharide.

AMPEROMETRIC DETECTION OF AMINO-ACIDS IN A FLOW-INJECTION SYSTEM WITH A NICKEL(II)-MODIFIED ELECTRODE WITH AN EASTMAN-AQ POLYMER FILM

Amperometic flow measurements were made at +0.55 V (vs. Ag/AgCl) in 0.1 mol l-1 KOH electrolyte with an Ni(II) chemically modified electrode (CME) with an Eastman-AQ polymer film. The use and characteristics of a Ni(II)-containing crystalline and polymer-modified electrode obtained by a double coating step as a detector for amino acids in a flow-injection system using reversed-phase liquid chromatography are described. The detection of these analytes is based on the higher oxidation state of nickel (NiOOH) controlled by the applied potential. The electroanalytical parameters and the detection current for a series of amines and amino acids were investigated. The use of such a CME in the flow-injection technique was found to be suitable in a solution at low pH. The linear range for glycine is 5 X 10(-6)-0.1 mol 1-1 with a detection limit of 1.0 X 10(-6) mol l-1. A 1 X 10(-4) mol 1-1 mixture of serine and tyrosine was also detected after separation on an Nucleosil C18 column.

AMPEROMETRIC FLOW-INJECTION ANALYSIS OF HYDRAZINE BY ELECTROCATALYTIC OXIDATION AT COBALT TETRAPHENYLPORPHYRIN MODIFIED ELECTRODE WITH HEAT-TREATMENT

Chemically modified electrodes prepared by treating the cobalt tetraphenylporphyrin modified glassy-carbon electrode at 750-degrees (HCME) are shown to catalyze the electrooxidation of hydrazine. The oxidation occurred at +0.63 V vs. Ag/AgCl (saturated potassium chloride) in pH 2.5 media. The catalytic response is evaluated with respect to solution pH, potential scan-rate, concentration dependence and flow-rate. The catalytic stability of the HCME is compared with that of the cobalt tetraphenylporphyrin adsorbed glassy-carbon electrode. The stability of the HCME was excellent in acidic solution and even in solutions containing organic solvent (50% CH3OH). When used as the sensing electrode in amperometric detection in flow-injection analysis, the HCME permitted sensitive detection of hydrazine at 0.5 V. The limit of detection was 0.1 ng. The linear range was from 50 ng to 2.4-mu-g. The method is very sensitive and selective.

Molecular cloning, characterization and expression analysis of a putative C-type lectin (Fclectin) gene in Chinese shrimp Fenneropenaeus chinensis

Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean. Knowledge on lectin at molecular level would help us to understand its regulation mechanism in crustacean immune system. A novel C-type lectin gene (Fclectin) was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1482 bp with an 861 bp open reading frame, encoding 287 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids. It also contains two carbohydrate recognition domains/C-type lectin-like domains (CRD1 and CRD2), which share 78% identity with each other. CRD1 and CRD2 showed 34% and 30% identity with that of mannose-binding lectin from Japanese lamprey (Lethenteron japonicum), respectively. Both CRD1 and CRD2 of Fclectin have I I amino acids residues, which are relatively invariant in animals' C-type lectin CRDs. Five residues at Ca2+ binding site I are conserved in Fclectin. The potential Ca2+/carbohydrate-binding (site 2) motif QPD, E, NP (Gln-Pro-Asp, Glu, Asn-Pro) presented in the two CRDs of Fclectin may support its ability to bind galactose-type sugars. It could be deduced that Fclectin is a member of C-type lectin superfamily. Transcripts of Fclectin were found only in hemocytes by Northern blotting and RNA in situ hybridization. The variation of mRNA transcription level in hemocytes during artificial infection with bacteria and white spot syndrome virus (WSSV) was quantitated by capillary electrophoresis after RT-PCR. An exploration of mRNA expression variation after LPS stimulation was carried out in primarily cultured hemocytes in vitro. Expression profiles of Fclectin gene were greatly modified after bacteria, LPS or WSSV challenge. The above-stated data can provide us clues to understand the probable role of C-type lectin in innate immunity of shrimp and would be helpful to shrimp disease control. (c) 2006 Elsevier Ltd. All rights reserved.

Polysaccharides from Ulva pertusa (Chlorophyta) and preliminary studies on their antihyperlipidemia activity

Polysaccharides from Ulva pertusa were isolated and prepared by extraction in hot water and precipitation by ethanol. The water-soluble polysaccharides were chemically well defined, containing 47.0% total carbohydrate, 23.2% uronic acids, 17.1% sulfate groups, 1.0% N and 29.9% ash. Gas chromatography analysis demonstrated that the neutral sugars were mainly composed of rhamnose, xylose and glucose and smaller amounts of mannose, galactose and arabinose. The FTIR and C-13-NMR spectra indicated that basic repeating units of the polysaccharides were (beta-D-GlcpA-(1->4)-alpha-L-Rhap 3S) and (alpha-L-IdopA-(1->4)-alpha-L-Rhap 3S). Fifty ICR mice were used to study the effect of water-soluble polysaccharides from Ulva pertusa on the level of plasma lipids, with inositol niacinate as positive control. The results indicated that the polysaccharides significantly lowered the contents of plasma total cholesterol, low-density lipoprotein cholesterol, triglyceride and markedly increased the contents of serum high-density lipoprotein cholesterol, compared with the hyperlipidemia control group (p<0.01). Moreover, administration of polysaccharides significantly decreased the atherogenic index. The present results suggest that the polysaccharides from Ulva pertusa have great potential for preventing ischemic cardiovascular and cerebrovascular diseases.

Purification and characterization of a natural lectin from the plasma of the shrimp Fenneropenaeus chinensis

A natural lectin from the plasma of the shrimp Fenneropenaeus chinensis was purified by singlestep affinity chromatography using fetuin-coupled agarose. The purified plasma lectin showed a strong affinity for human A/B/O erythrocytes (RBC), mouse RBC and chicken RBC. The hemagglutinating (HA) activity of the lectin was dependent on Ca2+ and reversibly sensitive to EDTA. This lectin was named FC-L and its inactive form had a molecular mass estimate of 168 kDa. Fifteen N-terminal amino acid sequences of this protein were determined. We performed HA-inhibition assays with several carbohydrates and glycoproteins. FC-L showed a distinct and unique specificity to N-acetylated sugars, particularly sialic acid and sialoproteins. The FC-L also has binding activity to some Gram-negative bacteria which caused disease in shrimp and fish. The activity of FC-L was inhibited at temperatures greater than 75 degrees C and at a pH less than 7 or greater than 11. These results suggest that FC-L may play a role as pattern recognition proteins in the reorganization and clearance of invaders in shrimp F. chinensis. Crown Copyright (c) 2008 Published by Elsevier Ltd. All rights reserved.

Jiangella gansuensis gen. nov., sp nov., a novel actinomycete from a desert soil in north-west China

A novel actinomycete strain, designated YIM 002(T), was isolated from a desert soil sample in Gansu Province, north-west China. This actinomycete isolate formed well-differentiated aerial and substrate mycelia. In the early stages of growth, the substrate mycelia fragmented into short or elongated rods. Chemotaxonomically, it contained LL-2,6-diaminopimelic acid in the cell wall. The cell-wall sugars contained ribose and glucose. Phospholipids present were phosphatidylinositol mannosides, phosphatidylinositol and diphosphatidylglycerol. MK-9(H-4) was the predominant menaquinone. The major fatty acids were anteiso C-15:0 (35.92%), anteiso C-17:0 (15.84%), iso C-15:0 (10.40%), iso C-16:0 (7.07%) and C(17:10)w8c (9.37%). The G+C content of the DNA was 70 mol%. Phylogenetic analysis and signature nucleotide data based on 16S rRNA gene sequences showed that strain YIM 002(T) is distinct from all recognized genera of the family Nocardioidaceae in the suborder Propionibacterineae. On the basis of the phenotypic and genotypic characteristics, it is proposed that isolate YIM 002(T) be classified as a novel species in a new genus, Jiangella gansuensis gen. nov., sp. nov. The type strain is YIM 002(T) (= DSM 44835(T) = CCTCC AA 204001(T) = KCTC 19044(T)).

NMR studies on synthesized diosgenyl saponin analogs

The identification of six synthesized diosgenyl saponin analogs with up to five sugars was accomplished by NMR studies. A combination of homo- and heteronuclear two-dimensional NMR techniques was utilized to achieve the complete H-1 and C-13 NMR assignments. Copyright (C) 2000 John Wiley & Sons, Ltd.

Formulation of a basal medium for primary cell culture of the marine sponge Hymeniacidon perleve

Marine sponge cell culture is a potential route for the sustainable production of sponge-derived bioproducts. Development of a basal culture medium is a prerequisite for the attachment, spreading, and growth of sponge cells in vitro. With the limited knowledge available on nutrient requirements for sponge cells, a series of statistical experimental designs has been employed to screen and optimize the critical nutrient components including inorganic salts (ferric ion, zinc ion, silicate, and NaCl), amino acids (glycine, glutamine, and aspartic acid), sugars (glucose, sorbitol, and sodium pyruvate), vitamin C, and mammalian cell medium (DMEM and RPMI 1640) using MTT assay in 96-well plates. The marine sponge Hymeniacidon perleve was used as a model system. Plackett-Burman design was used for the initial screening, which identified the significant factors of ferric ion, NaCl, and vitamin C. These three factors were selected for further optimization by Uniform Design and Response Surface Methodology (RSM), respectively. A basal medium was finally established, which supported an over 100% increase in viability of sponge cells.