不对称还原α-氨基苯乙酮菌种的筛选及转化条件优化

芳香基手性胺醇是许多手性药物合成的重要手性砌块,生物催化不对称还原前手性酮是合成该类醇的重要方法之一.以α-氨基苯乙酮盐酸盐为模型底物从土壤中筛选获得两株能分别高立体选择性催化底物产生R型、S型相应醇的菌株,对映体过量值(e.e.)分别为99%和77%,编号为1403和4802,鉴定菌株所属为镰刀菌属和地霉属.对两株菌培养时期和转化条件的研究表明镰刀菌1403最适生长时间为24 h,最优菌体浓度20 g/L,最优底物浓度5 g/L;地霉4802最适生长时间24 h,最优菌体浓度80 g/L,最优底物浓度3 g/L.底物特异性研究表明,菌株1403和4802均可转化α-氯代苯乙酮、α-溴代苯乙酮、α-羟基苯乙酮和苯乙酮为相应醇,且以α-羟基苯乙酮为底物时,其产物均为S型,e.e.值达99%.     

浓香型白酒中优势乳酸菌和酵母菌间的相互关系

为了更深入地认识白酒发酵机理,探究多粮浓香型白酒中优势乳酸菌和优势酵母菌的相互作用关系及代谢模型,利用五粮粉为培养基,测定分离自酒醅中的2株优势乳酸菌和2株优势酵母菌纯培养及其共培养中菌株的理化指标、活菌数以及主要挥发性代谢产物的变化.结果显示,Saccharomyces cerevisiae J7产乙醇(56.7 g/L)和酯类物质多,Lactobacillus buchneri R62和Lactobacillus acetotolerans R73产乳酸较多,峰值分别达到11.79和11.64 g/L;共培养时,S.cerevisiae J7显著抑制两株乳酸菌的生长和乳酸合成;两株乳酸菌对Pichia membranifaciens J42的生长和乙醇合成有抑制作用;从挥发性代谢产物可知,P.membranifaciens S42与乳酸菌共培养时,乳酸乙酯含量显著增加.本研究表明浓香型白酒酒醅中的乳酸菌和酵母菌存在相互作用,并且共培养时生成了乳酸乙酯;结果可为白酒发酵提供理论基础.(图5表2参22)     

紫外诱变选育高产丁二酮乳酸菌株及其低成本发酵优化

2,3-丁二酮是一种常用的安全食品添加剂,为了提高微生物发酵中菌株的丁二酮产量,从泡菜水样品中分离筛选出一株野生型高产丁二酮菌株,并进行紫外诱变选育以及发酵条件优化.筛选获得高产丁二酮野生型乳酸菌株(1)-2,产量为67.02 mg/L,经16S r DNA分子鉴定为植物乳杆菌(Lactobacillus plantarum).紫外诱变获得高产丁二酮突变株U13,产量为127.88 mg/L.对突变前后菌株几种丁二酮代谢相关酶酶活变化的比较结果显示,突变株丁二酮产量的提高是因为乳酸脱氢酶减少及乙酰乳酸合成酶增加.正交实验优化突变株U13的最佳丁二酮发酵条件为接种量3%,初始pH 6.6,葡萄糖30 g/L,组合氮源(蛋白胨:酵母粉:牛肉膏=2:1:2)20 g/L,柠檬酸氢二铵3 g/L,乙酸钠2 g/L,吐温-80 1 m L/L,K_2HPO_4 2 g/L,Mg~(2+)2 mmol/L,Mn~(2+)0.7 mmol/L,Cu~(2+)2 mmol/L,温度为37℃.利用廉价碳氮源淀粉及小麦麸皮替代原有碳氮源,淀粉替代率不超过20%、小麦麸皮替代率不超过40%时,丁二酮产量降幅比较低.本研究通过诱变选育和发酵条件优化,提高了菌株丁二酮产量,并通过廉价碳氮源替换降低了成本,可为丁二酮的微生物工业发酵提供参考.     

氧化微杆菌C3催化3,5-双三氟甲基苯乙酮的不对称反-Prelog还原(英文)

(R)-3,5-双三氟甲基-1-苯乙醇是合成神经激肽1受体拮抗剂阿瑞匹坦的关键手性醇中间体.通过筛选得到一株氧化微杆菌C3(Microbacterium oxydans C3),其能不对称还原底物3,5-双三氟甲基苯乙酮为(R)-3,5-双三氟甲基-1-苯乙醇.在底物浓度为5 g/L时,生物转化反应40 h,能达到>99%的ee值和95%的底物转化率.底物浓度提高到50 g/L时,生物转化的ee值依然保持99%,并能达到56%的底物转化率.利用该氧化微杆菌C3对其他含氟苯乙酮衍生物进行生物转化反应,同样得到了较高的立体选择性和底物转化率.     

一株高效不对称还原产生(R)-苯基乙二醇的菌株筛选、鉴定及全细胞催化体系

(R)-苯基乙二醇是合成许多光学活性药物的重要手性中间体,其制备具有重要的现实意义.以α-羟基苯乙酮为底物,从土壤中筛选得到一株能够立体选择性催化α-羟基苯乙酮产生(R)-苯基乙二醇的细菌菌株HBU-SI7.经形态学观察和16S r DNA序列分析,鉴定此转化菌株为红球菌属菌株Rhodococcus sp.菌株全细胞催化体系研究表明在邻苯二甲酸二丁酯-磷酸盐缓冲液(V:V=1:3)的两相催化体系下,菌体转化α-羟基苯乙酮的最优浓度为3.0 g/L,转化率高达96.2%,e.e值为99.3%.本研究建立的红球菌全细胞催化还原体系对(R)-苯基乙二醇的高效制备具有潜在的研究和应用价值.     

酶催化奎宁酮不对称还原合成(S)-3-奎宁醇

光学纯的3-奎宁醇是一种重要的手性医药中间体,目前已报道的通过酶催化3-奎宁酮的不对称还原几乎都是得到(R)-3-奎宁醇.在前期的工作中我们筛选获得一株能催化还原得到(S)-3-奎宁醇的红串红球菌(Rhodococcus erythropolis WY1406),其ee值达到99%.本研究从该红串红球菌中克隆得到6个3-奎宁酮还原酶基因,并将其转入到大肠杆菌BL21(DE3)中诱导表达,利用粗酶液检测它们催化3-奎宁酮还原的活性及立体选择性.结果发现其中两种酶Re QR-13和Re QR-25催化3-奎宁酮还原生成(S)-3-奎宁醇,ee值大于99%.此外还发现了能催化生成(S)-3-奎宁醇的ee值分别为83%、46%和57%的奎宁酮还原酶Re QR-18、Re QR-27和Re QR-28.其中表达Re QR-25的整细胞催化5 g/L的底物3-奎宁酮还原,转化率达93%.本研究为合成光学纯的(S)-3-奎宁醇提供了一种新的方法.     

产琥珀酸重组大肠杆菌的构建和发酵性能

琥珀酸是一种重要的化工产品,微生物发酵法生产琥珀酸具有广阔的应用前景.野生大肠杆菌厌氧发酵产物中琥珀酸含量低,且有大量副产物.为构建高产琥珀酸的重组大肠杆菌,阻断代谢旁路,利用Xer/dif重组酶系统对Escherichia coli CICIM B0013进行代谢途径的调控研究.结果显示,敲除了包括乙酸激酶和磷酸乙酰转移酶基因(ackA-pta)、乳酸脱氢酶基因(ldhA)、丙酮酸甲酸裂解酶基因(pflB)、乙醇脱氢酶基因(adhE)等琥珀酸合成竞争途径中关键酶基因的重组大肠杆菌菌株E.coli CICIM B0013-1040,其发酵液中琥珀酸成为主要产物得到积累;进一步对其PTS系统进行修饰并适当增强PEP羧化酶基因(ppc)表达剂量,成功获得一株具备较强琥珀酸生产能力的菌株E.coli CICIM B0013-1050(pTH-ppc),该菌株厌氧转化葡萄糖36 h,琥珀酸产量达到36.2 g/L,生产强度为1.01 g L-1 h-1,葡萄糖-琥珀酸转化率为64.3%,发酵液经高效液相色谱(HPLC)检测,无乳酸、乙酸、甲酸、乙醇生成.     

四川盆地大豆根瘤内生细菌的分离鉴定及促生效果

以四川盆地大豆根瘤为材料,采用划线法分离内生细菌、16S rDNA PCR-RFLP分析其遗传多样性,并结合菌株促生特性和盆栽试验筛选优良促生菌.从分离获得的130株内生细菌中选取了40株细菌作为供试菌株,16S rDNA序列表明分属于芽孢杆菌属(Bacillus)、肠杆菌属(Enterobacter)、假单胞菌属(Pseudomonas)、中华根瘤菌属(Sinorhizobium)和慢生根瘤菌属(Bradyrhizobium).以大豆为供试作物筛选出了12株具有促生能力的细菌,所有菌株均能分泌吲哚-3-乙酸(IAA),浓度达到0.353-32.404μg/mL;7株能产铁载体,活性单位为7.35%-34.31%;有11株具有溶磷能力,溶磷量达到4.26-10.6μg/mL;6株具有固氮能力.接种12株供试菌株后,玉米的农艺性状、植株全氮和全磷含量均优于单施化肥处理,其中菌株DA16-5效果最好,表现出良好的促生潜力.综上,四川盆地大豆根瘤内生菌遗传多样性丰富并且普遍具有促生能力,是重要的生物资源.     

（R）—酮基布洛芬乙酯对映选择性水解酶产生菌的筛选及其特性

以酮洛芬乙酯为唯一碳源,经过两轮富集培养从土壤中分离得到45株优先生成（S）-酮洛芬和25株优先生成（R）-酮洛芬的菌株,其中产物对映体过量值高于85%的分别有13株和9株。在25株优先选择（R）构型的菌株中,G13号菌的活性较高而且选择性最好,对G13号菌的培养和催化特性研究表明：在培养基中添加吐温-80可以显著提高细菌的催化活力;静息细胞的最适反应温度为40℃,最适pH范围为7.0-8.0,在具有档板和磁力搅拌的三角板中,用G13号菌的静息细胞水解酮洛芬乙酯（50mmol/L）,68h的转化率为33.7%,产物（R）-酮洛芬的对映体过量值达到93%。     

草酸青霉菌Penicillium oxalicum IM-3对氯代吡啶烟碱类杀虫剂的代谢作用

从土壤中分离出1株可降解氯代吡啶烟碱类杀虫剂啶虫脒(AAP)的丝状真菌菌株IM-3,经形态观察和18S rDNA序列比对,IM-3菌株被鉴定为草酸青霉菌(Penicillium oxalicum).培养14 d,P. oxalicum IM-3在矿物盐培养基中可降解41.6%的啶虫脒、14.1%的吡虫啉(IMI),但不降解噻虫啉(THI)和烯啶虫胺(NIT).HPLC和LC-MS/MS分析显示,啶虫脒降解途径为N-脱甲基生成IM 2-1和氧化断裂氰基亚胺基生成IM 1-3.     

Nukleinsäure- und substratgehalte in der dorsalen rumpfmuskulatur von teleosteern während eines „biologischen stress”

Two teleosts were subjected to extreme biological stimuli: Idus idus L. (6 to 7 cm) to predators (perches or a catfish, 15 to 22 cm) or to an alarm pheromone (skin extract), and male Betta splendens Regan (6 to 8 cm) to fighting each other. Uniform alterations occurred in nucleic acid contents (I. idus) and substrate levels (both species) of the epaxial muscle. In acute stress situations, the nucleic acid content and lactate: pyruvate (L:P) ratio remained unchanged; lactate, pyruvate and glucose-6-phosphate (G-6-P) levels increased to about 200% of those in controls. After an insignificant reduction (or a few minutes constancy), glucose increased to the same extent. In chronic stress situations, lactate, glucose and G-6-P levels remained elevated, L:P increased to about 200%. The pyruvate level and ribonucleic acid:deoxyribonucleic acid (RNA:DNA) ratio decreased significantly (by a maximum of 20%), DNA per gramme wet weight fell insignificantly by about 6%. Substrate alterations, especially the increase in L:P, were similar to those observed post mortem in fish muscle (I. idus), indicating an insufficient oxygen supply in chronic stress situations. Nucleic acid and substrate alterations, probably induced by an increased discharge of catecholamines and glucocorticoids, are interpreted in terms of stress effects.     

L-Lactate dehydrogenase in the longitudinal muscle of the sea cucumber Sclerodactyla briareus (Echinodermata: Holothuroidea)

The infaunal holothurian Sclerodactyla briareus (Thyone briareus) is able to tolerate exposure to hypoxic conditions for over 2 days. Since the in vitro anaerobic degradation of glucose-U-C¹⁴ by longitudinal muscle preparations leads to an accumulation of labeled lactic acid, it is apparent that lactate dehydrogenase plays a key role during anoxia. Disc electrophoresis resolved one major band of lactate dehydrogenase (LDH) activity in longitudinal muscle extracts. Muscle extracts of s. briareus oxidized L-lactate only, as is the case with most other echinoderms. The apparent Michaelis constants for lactate oxidation and pyruvate reduction are dissimilar from values reported for other marine invertebrates. The LDH activity in this holothurian is higher than the activities reported for other marine invertebrates with the exception of some active, large arthropods. Substrate inhibition of LDH maximal activity occurs at substrate concentrations (pyruvate) exceeding 10 mM, and then to only a minor extent. The characteristics of lactate dehydrogenase from the longitudinal muscle of S. briareus support the hypothesis that it functions to maintain glycolytic flux during prolonged periods of exposure to anoxic or hypoxic conditions.     

乳酸氧化酶转化乳酶产丙酮酸

从土壤样品中富集、筛选和纯化获得了3株能产生乳酸氧化酶的菌株,其中SM-10#菌株的产酶活力最高,经初步鉴定,该菌株属腐生葡萄球菌（Staphylococcus saprophyticus）,对SM-10#菌株还进行了破壁条件的优化、生长曲线的分析和酶液稳定性研究,并初步探讨了其酶液的底物转化效率,4h最高转化率达到80%,该研究结果为将这种有价值的酶源推向生产打下基础。     

Inhibitory effect of the plant Boerhavia diffusa l. against the dermatophytic fungus Microsporum fulvum

Antifungal activity (reduction in colony diameter) of various extracts (pt. ether, chloroform, ethyl acetate, ethyl alcohol and aqueous) of aerial and root parts of Boerhavia diffusa (Nictaginaceae) was screened against dermatophytic fungi Microsporum fulvum. Statistically significant increase has been recorded in the % inhibition of the target fungal species with increasing test concentrations (1000-5000 ppm) of chloroform, ethyl acetate and ethyl alcohol extracts of the root. The maximum % inhibition observed in various solvent extracts of root was about 26% (chloroform), 46% (ethyl alcohol) and 57% (ethyl acetate) at 5000 ppm concentration with time exposure of 10 days. The colony diameter of the target mycelial colony decreased with increasing supplementation of the phytoextract, showing the presence of significant amount of some antifungal phytochemical moiety.     

Effect of monsoonal climate on sulfate reduction in coastal sediments of the central Great Barrier Reef lagoon

Rates of sulfate reduction were measured during the spring dry and summer wet seasons of 1992 in shallow coastal sediments of the central Great Barrier Reef lagoon. In the dry season, sulfate reduction rates, integrated to 18–20 cm depth, ranged from 23.8 to 30.8 mmol S m^-2 d^-1. In the wet season, heavy monsoonal rains and wind-induced waves caused severe disturbance of sediments leading to less anoxic conditions, and to a 2- to 3-fold decline in rates of sulfate reduction (10.2 to 12.8 mmol S m^-2 d^-1). The percentage of reduced ³⁵S recovered as acid-volatile sulfide (% AVS_red) ranged from 15.8 to 44.9% in spring, and was significantly reduced at each station in summer (range 14.5 to 31.4%). An analysis of variance indicated that seasonality accounted for 31.5% of the total variance in total rates of sulfate reduction, with only 7.5% of the variance accounted for by depth into the sediment; there were no station differences. In both seasons, there were no clear patterns in the proportion of radiolabel incorporated into the AVS and chromium-reducible sulfur (CRS) pools with depth into the sediment. These results are contrary to the seasonal patterns and pathways of sulfate reduction in temperate coastal sediments.     

DHA高产菌Schizochytrium sp.FJU-512的分离及其18S rRNA基因序列比较分析

采用松花粉垂钓法分离到一株Docosahexaenoicacid(DHA)高产菌FJU 512.该菌株DHA含量高(占总脂肪酸的56. 24 % ),其它长链杂酸含量少(仅有docosapentaenoicacid, DPA),极具开发应用价值.高密度培养可获得33gL-1生物量.该菌株行二分裂生长,没有分生胞子.对其18SrRNA基因进行了克隆测序并登录GenBank(AY758384).依据18SrRNA基因建立的系统进化树表明:该菌与Schizochytriumlimacinum具有紧密的亲源关系. 图7表2参29     

Reduction of wastewater toxicity and change of microbial community in a hydrolysis acidification reactor pre-treating trimethylolpropane wastewater

Trimethylolpropane (TMP) wastewater is one of the most toxic petrochemical wastewater. Toxicants with high concentrations in TMP wastewater often inhibit the activity of microorganisms associated with biological treatment processes. The hydrolysis acidification process (HAP) is widely used to pretreat petrochemical wastewater. However, the effects of HAP on the reduction of wastewater toxicity and the relevant underlying mechanisms have rarely been reported. In this study, an HAP reactor was operated for 240 days, fed with actual TMP wastewater diluted by tap water in varying ratios. The toxicity of TMP wastewater was assessed with the inhibition ratio of oxygen uptake rate. When the organic loading rates were lower than 7.5 kg COD/m³/d, the toxicity of TMP wastewater was completely eliminated. When the actual TMP wastewater was directly fed into the reactor, the toxicity of TMP wastewater decreased from 100% to 34.9%. According to the results of gas chromatographymass spectrometry analysis, four main toxicants contained in TMP wastewater, namely, formaldehyde, 2-ethylacrolein, TMP and 2-ethylhexanol, were all significantly removed, with removal efficiencies of 93.42%, 95.42%, 72.85% and 98.94%, respectively. Compared with the removal efficiency of CODCr, the reduction rate of toxicity is markedly higher by HAP. In addition, the change of microbial community in the HAP reactor, along the operation period, was studied. The results revealed that, compared with the seed sludge, Firmicutes became the dominant phylum (abundance increased from 0.51% to 57.08%), followed by Proteobacteria and Bacteroidetes (abundance increased from 59.75% to 25.99% and from 4.70% to 8.39%, respectively).

Open image in new window

     

Effect of different irrigation methods on yield of red hot pepper and plant mortality caused by Phytophthora capsici Leon

This experiment was conducted in 1999 and 2000 in a field naturally infected by Phytophthora capsici to determine the effects of different irrigation methods, namely, basin (B), closed-end furrow (F), drip (D) and sprinkler (S) irrigation on dry yield of chili pepper (Capsium annuum L. K. Maras Type). Water use efficiency (WUE) and plant mortality were also measured. The averages of total applied water to the B, F, D and S plots for the two years were 937.7 mm, 920.6 mm, 886.5 and 913.4 mm, respectively. Thus, the mean seasonal water use by pepper varied from 1020.7 to 1109.7 mm. The highest water use was measured in B, followed by use for S, D and F methods. The highest mean potential dry yield with 1.58 t ha(-1) was obtained using D method. This was followed by S (1.36 t ha(-1)), B (1.13 t ha(-1)), and F (0.81 t ha(-1)) methods. In terms of plant mortality, crops having B irrigation had the highest (93.9%), followed by lessen amount to the S (3.2%), F (3.1%), and D methods (1.7%). Only the B irrigation method stimulated the development of P. capsici disease. On the other hand, mean WUEs varied between 0.7 and 1.7 kg ha(-1)mnm(-1) for Irrigation methods. WUE for D method was slightly higher than S method, but considerably higher than B and F methods.     

浮游动物DNA宏条形码标志基因比较研究

DNA宏条形码技术作为一种新型生物监测方法,在未来生态环境监测中有巨大的应用潜力。目前,浮游动物DNA宏条形码监测仍在发展阶段,需要首先对其(采样方法、引物选择和数据分析等)进行标准化和调整,然后才能用于常规流域生态监测。其中,如何选择合适的PCR扩增引物是DNA宏条形码生物监测标准化的关键问题之一。本研究比较了COI、18SV9和16S通用引物在浮游动物DNA宏条形码监测中的差异,为初步建立规范化的浮游动物DNA宏条形码监测方法提供技术支撑。结果表明,16S引物对浮游动物具有更好的特异性,其产生的操作分类单元(operational taxonomic unit, OTU)有88.1%属于浮游动物。虽然18SV9引物具有更高的物种覆盖度,不仅能扩增出浮游动物,还能扩增出大量藻类和真菌,但其物种识别敏感性较差,不适合浮游动物物种水平多样性监测。COI引物的浮游动物物种特异性、物种覆盖度和物种识别敏感性都适中,检出的浮游动物物种数量高于18SV9引物和16S引物,更加适合浮游动物DNA宏条形码监测。