L-鸟氨酸高产菌的原生质体融合育种

以谷氨酸高产菌S9114和谷氨酸棒杆菌GS538为出发菌株,应用原生质体融合技术,定向选育出一株L-鸟氨酸高产菌株RH169,该菌株能在发酵液中积累L-鸟氨酸,质量浓度可达19.3 g.L-1。     

胶胨状芽孢杆菌拮抗菌株活性物质形成条件及其性质初步研究

探索了培养基组分和培养条件对胶胨状芽孢杆菌拮抗菌株抗菌活性形成的影响。确定了菌株的最适发酵条件 :即以 1 %麦芽糖为碳源 ,以 0 1 % (NH4 ) 2 SO4 为氮源 ,32℃～ 37℃摇瓶培养 ,在培养过程中保持pH近中性有利于菌株生长和积累活性产物。从发酵液中获得抗生物质粗品 ,该物质对G+ 的蜡样芽孢杆菌 ,金黄色葡萄球菌和G- 的大肠杆菌等均有较强抗菌活性。经热处理、酶处理和生化定性分析 ,表明此活性产物为肽类化合物     

一株可积累鸟苷菌株的纯培养条件研究

菌株Bacillus.subtilis.S3 68是以鸟苷生产菌株B .subtilis.A0 66为出发菌经诱变所得。对该菌株进行培养条件研究的过程中 ,发现该菌株可以在摇瓶纯培养条件下积累鸟苷。试验结果表明 :发酵过程中 ,腺嘌呤的用量 0 .3 5mg/ml时 ,发酵液中鸟苷积累量最大 ,培养基中腺嘌呤的用量高于或低于 0 .3 5mg/ml均不利于鸟苷产物的积累 ;培养基中味精、硫酸铵、硫酸镁、磷酸二氢钾及Mn2 +用量显著影响发酵液中鸟苷积累水平 ;培养基中生物素、蛋氨酸、精氨酸、组氨酸、氯化钙及Fe2 +、Zn2 +用量与鸟苷积累的相关性不显著     

管碟法测定苏云金杆菌上清液中增效物质含量的研究

以草生欧文氏杆菌LS0 0 5为指示菌 ,用管碟法测定苏云金杆菌KN-11发酵液中增效物质的相对浓度。结果表明管碟法和生物测定法在测定增效物质的代谢曲线有较好的一致性 ,均表明 ,菌株KN-11的增效物质在营养体时 (约 9h)开始产生 ,到芽孢初步形成时(约 2 0h)已达到最高值的 80 %以上 ,之后是一个缓慢的积累过程。     

管蝶法测定苏云金杆菌上清液中增效物质含量的研究

以草生欧文氏杆菌LS005为指示菌,用管蝶法测定苏云金杆菌KN－11发酵液中增效物质的相对浓度。结果表明管蝶法和生物测定法在测定增效物质的代谢曲线有较好的一致性,均表明,菌株KN－11的增效物质城营养体时（约9h）开始产生,到芽孢初步形成时（约20h）已达到最高值的80％以上,之后是一个缓慢的积累过程。     

一株蜂粮源拮抗细菌的分离鉴定及其抑菌物质特性

【目的】为发掘和利用蜂粮中拮抗菌资源,对分离获得的拮抗细菌菌株PC2进行分类鉴定,并测定其发酵液抑菌物质基本特性。【方法】采用改良牛津杯双层平板法测定菌株发酵液抑菌谱及温度、p H、紫外线和蛋白酶对其抑菌活性稳定性的影响,菌株鉴定结合形态学、生理生化特征和16S r RNA基因序列分析,硫酸铵沉淀法和盐酸沉淀有机溶剂提取法进行抑菌活性物质的初步分离。【结果】从3种蜂粮中分离筛选得到17株拮抗菌株,其中1株细菌PC2以马铃薯葡萄糖液体培养基发酵制备的无菌发酵液对7种供试菌株具有较强抑制作用,经形态、生理生化特征及16S r RNA基因序列分析,将其初步鉴定为解淀粉芽胞杆菌(Bacillus amyloliquefaciens)。菌株发酵液抑菌活性对温度、酸和紫外线具有较强的稳定性,对蛋白酶K、胃蛋白酶、碱性蛋白酶处理敏感。菌株发酵液存在抑菌蛋白和脂肽类物质。【结论】菌株PC2在食品保鲜和农业生防中具有潜在的开发应用价值。     

具有杀线虫活性的郭霍氏芽孢杆菌发酵条件优化及稳定性评价

【背景】芽孢杆菌是一类常见的生防菌,在植物病害防治中展现出巨大潜力。【目的】对一株具有杀线虫活性的郭霍氏芽孢杆菌(Bacillus kochii) DDWB进行发酵条件优化和稳定性评价,为菌株的开发提供理论支持。【方法】以发酵液上清杀线虫率及细菌发酵液OD600值为指标,通过单因素法与正交试验设计,对菌株的培养基与发酵参数进行优化;同时对菌株发酵液酸碱、温度、紫外、遗传及储存稳定性进行评价。【结果】DDWB菌株培养基优化后为:蔗糖2%,酵母提取物1%,氯化钾2%;优化后初始pH值为8.0,装液量为150 mL/250 mL锥形瓶,发酵时间为48h,接种量为8%,转速为160r/min,发酵温度为31℃;稳定性测定结果显示发酵液对酸碱敏感,紫外光照射4 h后活性物质发生降解,但发酵液对温度不敏感且杀线虫活性相关基因可稳定遗传。【结论】本研究优化了DDWB菌株的发酵条件,并对发酵液中活性物质的稳定性进行了多因素评价,使得菌株可以快速扩繁并长期稳定保持对根结线虫的抑制活性,为进一步评价菌株田间防效和研究生防机制奠定了基础。     

甲基营养型L-丝氨酸产生菌的选育

以甲基营养型假单胞菌J-12为出发菌株,经硫酸二乙酯（DES）和亚硝基胍（NTG）诱变处理,D-丝氨酸结构类似物平板和高浓度甘氨酸平板定向筛选,获得1株三-丝氨酸高产菌N-13,其发酵液中L-丝氨酸产量较出发菌株提高97．9％。在含有20g/L甘氨酸和7mL／L甲醇的培养基中,L-丝氨酸积累可达4．81g／L。     

低双乙酰啤酒酵母菌株BEZ112的选育

以啤酒酿造生产菌株啤酒酵母(Saccharomyces cerevisiae)FB作为出发菌株,用甲基磺酸乙酯(EMS)诱变,经分离筛选得到一株优良的啤酒酵母菌株BEZ112。该菌株的絮凝性、发酵度、酒精度、发酵液的总酯和总高级醇的含量等特性保持了亲株的优良性状。但以12°Bx麦芽汁为培养基用500mL三角瓶在12℃下发酵,该菌株发酵至第4d,发酵液中的双乙酰含量达到峰值(0.291mg/L),比出发菌株FB发酵4d的峰值降低了30％,发酵至第8d,BEZ112发酵液中的双乙酰含量比出发菌株FB的降低了23％。以12°Bx麦芽汁为培养基用500L罐在12℃下发酵8d,BEZ112发酵液中的双乙酰含量(0.091mg/L)比出发菌株FB的(0.124mg/L)降低了27％。发酵得到的啤酒口感纯正清爽。     

深黄被孢霉利用不同碳源产油脂比较

本研究主要探讨深黄被孢霉M2菌株对生物质全糖的利用,考察其碳源同化能力、不同碳源下产脂情况以及对玉米皮渣的利用能力。研究结果表明,M2菌株能够利用葡萄糖、木糖、阿拉伯糖和甘露糖进行生长和油脂积累。M2菌株以6%糖浓度的玉米皮渣水解液为底物发酵培养,油脂微生物生物量达18.2g/L,干菌体油脂含量45.7%,单位体积发酵液油脂产量为8.3g/L。     

Studies on the Accumulation of Orotic Acid by Escherichia coli K12

More than 300 mg/liter of orotic acid was found to accumulate in the supernatants of the cultures of wild type strains of E. coli K12. The pyrimidine precursor was accumulated in a synthetic medium such as glucose-ammonium sulfate medium. The substance was isolated from the culture, crystallized, and identified as orotic acid. Orotic acid was excreted mainly during logarithmic phase of the bacterial growth. Yeast extract or nutrient broth stimulated bacterial growth, but suppressed orotic acid accumulation. E. coli strains other than K12 failed to accumulate orotic acid.

The results suggest that the accumulation of orotic acid is specific to E. coli K12.     

酿酒酵母中过表达URA5及URA3基因催化合成UMP的初步研究

为提高乳清酸到尿嘧啶核苷酸(UMP)的转化效率,利用PCR方法扩增酿酒酵母乳清酸磷酸核糖转移酶基因URA5, 并将其连接到携带乳清苷酸脱羧酶基因URA3的表达载体pYX212中,构建了重组质粒pYX212-URA5,然后转化到酿酒酵母BJX12中进行表达,并进行转化乳清酸到UMP的初步研究。试验结果表明: pYX212-URA5/ BJX12发酵培养40h后以32 mM乳清酸为底物催化产生UMP的量约为7 mM。明显高于同等条件下pYX212/ BJX12的UMP产量2.7 mM和对照组野生型BJX12的UMP产量2.4 mM。     

Studies on the Accumulation of Orotic Acid by Escherichia coli K12

The mechanism whereby Escherichia coli K12 accumulates orotic acid in culture fluid was studied. Pyrimidine compounds were incorporated effectively into cells of E. coli K12, stimulated the growth, and depressed the accumulation; while purine compounds were not so much consumed by the microorganism for its growth, and affected the accumulation to a lesser extent. On the other hand, E. coli B unable to accumulate orotic acid utilized less effectively pyrimidine compounds for its growth than strain K12.

It is supposed, therefore, that in the de novo pathway for pyrimidine synthesis in E. coli K12 the step from orotic acid to 5′-UMP is genetically depressed so that orotic acid is accumulated when pyrimidine compounds, that would cause a feedback inhibition of orotic acid synthesis upon incorporation, are not supplemented.     

直接利用糖质原料生产L-苹果酸菌种的选育

从土壤中筛选到一株产L－苹果酸的黄曲霉菌,经紫外线、亚硝基胍（NTG）、硫酸二乙酯（DES）和^60Co辐射等诱变处理,并经多次分离得到一株性能稳定的高产L－苹果酸突变株黄曲霉HA5800,35℃,200r/min摇瓶发酵120h,产L－苹果酸72g/L、糖酸转化率为74．22％,经山东省卫生防疫站检定,该菌株不产黄曲霉素素B1。     

对黄豆苷原具转化作用的耐氧突变株Aeroto-AUH-JLC140未知代谢产物分离、鉴定及抗氧化活性测定

【目的】与原出发菌株AUH-JLC140相比,耐氧突变株Aeroto-AUH-JLC140在其生长过程中产生一种未知物质,且该未知物质的产生与添加底物黄豆苷原无关。对该未知物质进行分离纯化和结构鉴定,并测定其产生动态及抗氧化活性。【方法】利用高效液相色谱对未知物质进行分离,经紫外吸收图谱、质谱、核磁共振氢谱和碳谱等分析,对未知物质进行结构鉴定;通过1,1-二苯基-2-苦味酰基自由基(DPPH)清除试验测定其抗氧化活性。【结果】Aeroto-AUH-JLC140产生的未知物质被鉴定为吲哚,接种后15 h菌株产吲哚最高,所产吲哚量为19.89 mg/L。浓度为0.2 mmol/L(即23.40 mg/L)的吲哚除对DPPH自由基具有明显清除作用外,还能有效降低脑心浸液(BHI)液体培养基的氧化-还原电位。【结论】耐氧突变株Aeroto-AUH-JLC140产生的未知代谢产物为吲哚,菌株通过产生吲哚降低培养基氧化-还原电位,进而为该菌株的生长提供适宜的低氧微环境。     

Naphthalene oxidation by a Pseudomonas putida strain carrying a mutant plasmid

Naphthalene oxidation by a parent and a mutant strain of Pseudomonas putida was studied. The parent strain contained a plasmid NPL-1 which controlled oxidation of naphthalene to salicylic acid and was capable of oxidizing salicylate. The mutant strain did not oxidize salicylate because of a mutation in salicylate hydroxylase; it contained also a mutant plasmid NPL-41 which determined constitutive synthesis of naphthalene oxygenase. Salicylic acid which accumulated as a product of naphthalene catabolism in the cultural broth of the wild strain was found to undergo further oxidation by the population of growing cells. The content of salicylic acid in the cultural broth of the mutant strain reached maximum and then remained constant. An anion-exchange resin was tested in order to prevent the inhibition of naphthalene oxygenase by salicylate and to increase the yield of salicylic acid. The transmissible character of the mutant plasmid NPL-41 makes it possible, with the aid of conjugation, to construct Pseudomonas strains which would oxidize naphthalene to salicylic acid without further degradation of this compound.     

中长链聚羟基脂肪酸酯(mcl PHA)在嗜水气单胞菌Ⅰ型PHA合酶缺失突变株中的合成

拥有Ⅰ型聚羟基脂肪酸酯(PHA)舍酶基因的嗜水气单胞菌CGMCC 0911株可利用月桂酸而不能利用葡萄糖作为碳源积累PHBHHx。将氯霉素抗性基因(cm)插入到该基因中,获得带有Ⅰ型PHA合酶断裂基因(phaC：：Cm)的自杀质粒pFH10。自杀质粒DFH10通过接合作用转入嗜水气单胞菌CGMCC 0911株中并发生体内同源重组,Cm被整合到基因组上,获得Ⅰ型PHA合酶缺失突变株。DNA序列测定证明了这一结果。GC分析表明,突变株不再产生PHBHHx,但却可利用月桂酸或葡萄糖积累中长链PHA,明显表明野生型嗜水气单胞菌基因组中存在另一个编码Ⅱ型PHA合酶的基因,且只有Ⅰ型PHA合酶被钝化后,这个功能被隐藏的Ⅱ型PHA合酶才可在细胞中发挥作用。     

Pyrimidine biosynthetic pathway of Pseudomonas fluorescens

Pyrimidine biosynthesis in Pseudomonas fluorescens strain A126 was investigated. In this study, de novo pyrimidine biosynthetic pathway mutant strains were isolated using both conventional mutagenesis and transposon mutagenesis. The resulting mutant strains were deficient for either aspartate transcarbamoylase, dihydroorotase or orotate phosphoribosyltransferase activity. Uracil, uridine or cytosine could support the growth of every mutant strain selected. In addition, the aspartate transcarbamoylase mutant strains could utilize orotic acid to sustain their growth while the orotidine-5'-monophosphate decarboxylase mutant strains grew slowly upon uridine 5'-monophosphate. The wild-type strain and the mutant strains were used to study possible regulation of de novo pyrimidine biosynthesis in P. fluorescens. Dihydroorotase specific activity more than doubled after the wild-type cells were grown in orotic acid relative to unsupplemented minimal-medium-grown cells. Starving the mutant strains of pyrimidines also influenced the levels of several de novo pyrimidine biosynthetic pathway enzyme activities.     

Studies on the Utilization of Hydrocarbon by Microorganisms

An uracil-requiring mutant (KY7122) of Arthrobacter paraffineus KY4303 (ATCC15591) was found to accumulate orotic acid and orotidine on n-paraffine as a sole carbon source.

Both substances were definitely indentified as orotic acid and orotidine, from the results on column and paper chromatography, UV and IR absorption spectra, elementary analysis and analyses of hydrolysate.

Cultural conditions for orotic acid and orotidine fermentation were then investigated. As the carbon source n-paraffines from C₁₄ to C₁₆ were the most suitable for the fermentation, and sorbitol, fructose and mannitol were best utilized for the growth, and orotidine produced from them were twice as much as those from hydrocarbon. The addition of 200 mg of uracil and 2 g of C. S. L to 1 liter of medium was most optimal for orotic acid and orotidine fermentation.

Orotic acid and orotidine accumulations were enhanced by the addition of either l-tyrosine, l-leusine, l-threonine, gluconate or meat extract.     

Preliminary report on the biological effects of space flight on the producing strain of a new immunosuppressant, Kanglemycin C

Kanglemycin C (K-C) is a new immunosuppressant isolated from the culture broth of Nocardia mediterranei var. kanglensis 1747-64. To improve the productivity of K-C and to study the biological effects of space flight on its producing strain, spores from five K-C producing strains (U-10, U-15, U-7, M-13, γ-33) mutated from the wild strain N. mediterranei var. kanglensis 1747-64 were carried into space by an unmanned spaceship, “Shenzhou III” (Divine Vessel III) on March 25, 2002. Comparatively, the strain U-7 was the highest K-C producing strain among the above five starting strains when cultivated in 500-ml Erlenmeyer flasks. After a 6 day and 18 h flight, the treated spores went through serial screening processes to screen for high-yield K-C mutant strains, using thin layer chromatography and high performance liquid chromatography (HPLC). The K-C yield produced by one mutant strain, designated as F-16, derived from the starting strain U-7 was increased by up to 200% when compared to that produced by the starting strain U-7 in 500-ml Erlenmeyer flasks after careful postflight HPLC analysis. Another mutant strain, designated as F-210, derived from the starting strain M-13 showed reduced productivity of K-C as well as exhibited changes in some morphological and physiological characteristics. For example, the broth color of the strain F-210 changed from yellow to purple after 96 h of culture, but that of the ground control strain M-13 remained yellow. Similarly, the mycelium morphological change from filamentous to coccoid of F-210 occurred later than that of ground control M-13. Examination of the survivability of postflight spores indicated that exposure to radiation, during the 162 h of space flight, plays a critical role in the survival rates of spores such that spores exposed to strong radiation exhibited lower survival rates than spores exposed to weak radiation.Jianqin Zhou and Chenghang Sun have contributed equally to this work.