D-核糖高产菌株的选育及其生产性试验

BacillussubtilisJSIM 10 18菌株是 1株产D 核糖的莽草酸营养缺陷型突变株 ,以此菌株为出发菌株 ,通过甲基磺酸乙酯和紫外线连续、间断诱变处理 ,获得了 1株次黄嘌呤、莽草酸双重缺陷型突变株No .2 71菌株。该突变株摇瓶发酵的D 核糖平均产量为 10 8 1g/L ,比亲株提高了 15 8g/L。在 30 0 0 0L发酵罐中 ,连续 5罐批 ,平均产D 核糖 96 4 g/L ,最高为 98g/L ,比亲株提高了 16 2 5 g/L。     

常压室温等离子体诱变与微生物液滴培养系统联用筛选L-组氨酸产生菌

利用常压室温等离子体（ARTP）诱变技术,对野生型谷氨酸棒杆菌（Corynebacterium glutamicum）ATCC 14067诱变处理。通过全自动高通量微生物液滴培养系统（MMC）和平板选育组氨酸结构类似物的抗性突变株。使用48孔板发酵初筛及摇瓶发酵复筛,最终从耐受3-氨基-1,2,4-三氮唑10 g/L和D-组氨酸8 g/L的抗性突变株中筛选得到菌株Cg-F4,其L-组氨酸产量为（0.561±0.016）g/L,并且经过7次连续传代实验验证了该菌株稳定性良好。     

2-酮基-D-葡萄糖酸产生菌球状节杆菌K1022抗噬菌体菌株的选育

从 2 酮基 D 葡萄糖酸产生菌球状节杆菌K10 2 2的异常发酵液中分离到 3种噬菌体 ,分别将其命名为KS2 11、KS2 12和KS2 13。采用紫外诱变或自然选育的方法 ,获得了 6株具有抗噬菌体能力的 2 酮基 D 葡萄糖酸高产菌株。研究了抗株C2 2 4和K2 32的遗传稳定性 ,并在 5 0kL的发酵罐上进行了发酵生产试验。结果表明 ,经多次传代 ,菌株C2 2 4和K2 32对噬菌体的抗性及其发酵产酸能力没有改变 ,可以应用于工业生产中     

L-组氨酸产生菌的选育

以谷氨酸棒杆菌 (Corynebacterium glutamicum )ATCC 1376 1为出发菌株 ,经硫酸二乙酯(DES)和亚硝基胍 (NTG)诱变处理 ,D 组氨酸 (D His)、6 氮鸟嘧啶 (6 AU)等结构类似物平板和以L 组氨酸 (L His)为惟一氮源平板定向筛选 ,获得一株L 组氨酸产生菌H 2 4 (D Hisr 6 AUrHisase- ) .在加有 15 0 g/L葡萄糖、35g/L硫酸铵以及 10mL/L玉米浆的发酵培养基中发酵 72h ,产L 组氨酸 1.6 g/L.     

纳他霉素高产菌株的选育及其发酵条件的研究

利用链霉素抗性突变选育纳他霉素高产菌株 ,即将经过紫外线诱变处理的纳他霉素(natamycin)产生菌褐黄孢链霉菌 (Streptomycesgilvosporeus)ATCC1 332 6菌株孢子涂布在含有链霉素最小抑制浓度 (0 6μg/mL)的培养基平板上 ,获得了 1 2 2株链霉素抗性突变株。其中纳他霉素产量高于出发菌株的有 1 3株。其中突变株SG 56的生产能力比出发菌株提高到 1 4 6 % ,研究了其发酵性能 ,优化了培养基组成和发酵工艺条件 ,使纳他霉素产量提高到 1 70 %。     

优良啤酒酵母嗜杀菌株Fu—1的选育

本文对具有优良生产性状的啤酒酵母2604菌株,经溴化乙锭(EB)诱变选育到呼吸缺陷型(P~o),嗜杀菌株y_(32)(K_2型)经单倍体分离、甲基磺酚乙酯(EMS)诱变选育出组氨酸缺陷型(his~-)突变株。然后经原生质体制备、融合、再生、检出、鉴定获得24株具有嗜杀特性的融合子。最后,经嗜杀特性测定、发酵度和双乙酰实验,选育出1株具有优良生产性状的嗜杀菌株,编号为Fu-1。     

L-谷氨酸生产菌的选育及其发酵条件的研究

采用黄色短杆菌（Brevibacterium flavum)T1为始发菌株，根据代谢控制发酵原理，利用紫外线、硫酸二乙酯进行诱变，定向选育出具有寡霉素抗性、谷氨酸氧肟酸盐抗性的温度敏感突变株TM106。然后，以温度敏感突变株TM106和产酸率高（10．5％以上）的天津短杆TG961为亲株。通过原生质体融合技术，成功地选育出了产酸率高的融合子CM021，并且该菌株系温度敏感型菌株，可用于谷氨酸强制发酵。在摇瓶条件下，产酸达13.6g/dI；在6m^3发酵罐上进行中试，产酸达14．6g/dI，转化率达62％。     

基于常压室温等离子体诱变技术选育高产色氨酸突变株的研究

本研究通过常压室温等离子体(ARTP)微生物育种技术,选育到一株高产L-色氨酸的突变株TRP-YP-3-2.与出发菌TRP-1201相比,该突变株生长及耗糖更快、产酸更高.TRP-YP-3-2色氨酸产量达到61.4 g/L,糖酸转化率达到19.25％,比出发菌分别提高了15.41％和22.77％.TRP-YP-3-2的产酸遗传稳定性研究发现经过30代培养后,仍具有很好的遗传稳定性.本研究不仅获得了一株高产色氨酸的突变株,具有很大的经济价值;同时确立的育种方法可为其他工业微生物的诱变育种提供有益的参考.     

乙酸胁迫下巴氏醋酸杆菌发酵过程中微环境水平的应答分析

以Acetobacter pasteurianus CICIM B7003及其高产突变株A. pasteurianus CICIM B7003-02为研究对象,通过2株巴氏醋酸杆菌发酵过程中胞内微环境分析来揭示其耐酸机制。在7. 5 L发酵罐中比较分批和半连续发酵过程中其发酵动力学以及和胞内微环境变化。结果显示,分批发酵中其平均产酸速率达0. 836 g/(L·h),相对亲本菌株提高29. 6%。半连续发酵中酸启动时间缩短了31 h,微环境分析表明,突变株中与产酸直接相关的乙醇脱氢酶(alcohol dehydrogenase,ADH)和乙醛脱氢酶(acetaldehyde dehydrogenase,ALDH)的最高酶活分别提高27. 0%和15. 2%,辅酶Q9含量提高69. 5%。突变株拥有更高比例的十八碳烯酸,比亲本株高71. 4%。突变株最高胞内ATP含量是亲本株2. 33倍,胞内ATP与比生长速率呈正相关,胞内谷氨酸和天冬氨酸含量分别比亲本株提高10. 7%和18. 3%。突变株主要依靠加强乙醇呼吸链,增强ATP合成和关键氨基酸代谢等机制的协同作用提高酸胁迫抗性     

产果糖基转移酶米曲霉菌株的选育

通过紫外线、紫外线和氯化锂复合、微波、微波和氯化锂复合4种诱变方法，对米曲霉GX-0010菌株进行了诱变，选育得到果糖基转移酶酶活高于GX-0010菌株的突变株8株，其中UVLi-3突变株的酶活最高，比GX-0010菌株高45．4％。     

灵芝固态发酵三七渣产漆酶的培养基配方优化

该试验通过单因素试验考察了酵母浸出粉添加量、硫酸铜添加量、培养基初始水含量、磷酸二氢钾添加量、硫酸镁添加量对灵芝固态发酵三七渣产漆酶的影响,并采用正交试验对培养基配方进行了优化。研究结果表明,优化的培养基配方为采用过60目筛的三七渣10 g,酵母浸出粉添加量3%,磷酸二氢钠添加量0.15%,硫酸铜添加量0.02%,硫酸镁添加量0.2%,培养基初始水含量60%,pH自然。在此优化条件下,灵芝固态发酵三七渣产生的漆酶活性可达8.69 U/g。     

利用响应面法优化丙酮酸发酵培养基

采用响应面法对丙酮酸产生菌光滑球拟酵母(Torulopsis glabrata)TP19的发酵培养基进行了优化。用Plackett-Burman方法对影响发酵各因素的效应进行评价,筛选出有显著效应的3个因素:硫酸铵、葡萄糖和烟酸;通过中心组合实验及响应面分析优化了这3个主要因素。采用优化后的条件进行摇瓶发酵,丙酮酸产量为42.4 g/L。进行5L自控发酵罐发酵,丙酮酸产量为44.8g/L,比优化前提高了16.2%。     

In vivo Mutational Analysis of Highly Conserved Amino Acid Residues of the Small Subunit Cpa1p of the Carbamylphosphate Synthetase of Saccharomyces cerevisiae

The role of selected amino acid residues located in the putative catalytic domain and of two conserved histidine residues within the small subunit of the carbamylphosphate synthetase (CPS) specific to the arginine biosynthesis pathway of the yeast Saccharomyces cerevisiae was studied using site-directed mutagenesis to change all residues to aspartic acid. Carbamylphosphate synthesis catalysed by modified CPS was tested in vivo. The C264D, H307D and H349D mutants were unable to grow on minimal medium, indicating the importance of these three residues for efficient CPS activity, whereas, four other mutated residues located in the catalytic site (including a proline residue) do not affect the growth rate. These results in comparison to those obtained with the CPS of Escherichia coli, implicate residues Cys 264 and His 349 in the glutaminase catalytic activity, and His 307 in the binding of glutamine to the active site. Using these three defective mutants, we investigated the in vivo utilization of ammonia by CPS. C264D and H307D mutants are able to use ammonia as a substrate when provided in sufficiently high concentrations (up to 200 mm). The H349D mutant, however, did not grow even at ammonium sulfate concentrations above 400 mm, suggesting that this substitution is critical to NH₃-dependent CPS activity although the ammonia binding site is presumably located within the large subunit of the enzyme. © 1997 by John Wiley & Sons, Ltd.     

L-赖氨酸高产菌选育的研究

以黄色短杆菌ATCC140 6 7为出发菌株 ,经亚硝基胍 (NTG)、硫酸二乙酯 (DES)逐级诱变处理 ,S 2 氨基乙基 L 半胱氨酸 (AEC)、磺胺胍 (SG)等氨基酸结构类似物及琥珀酸为唯一碳源平板定向育种 ,获得 1株L 赖氨酸高产菌XQ 8(AECrSucgThr- SGr)。在含有 16 0 g/L葡萄糖的培养基中 ,摇瓶发酵 72h ,L 赖氨酸积累可达 77～ 82 g/L。     

Evaluation of five selective media for isolation of catalase-negative gram-positive cocci from bulk tank milk

Five selective media including Edwards modified medium, Edwards modified medium supplemented with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L), Streptococcus selective medium, Streptosel agar, and thallium-crystal violet-toxin-ferric citrate medium were evaluated for the isolation of streptococci and streptococci-like organisms from raw milk. The sensitivity and specificity of these selective media for streptococci and streptococci-like organisms were determined by using American Type Culture Collection reference strains. Under experimental conditions Edwards modified medium with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L) showed the highest sensitivity (100%) and specificity (100%) for streptococci and streptococci-like organisms followed by thallium-crystal violettoxin-ferric citrate medium, Edwards modified medium, Streptococcus selective medium, and Streptosel agar. Edwards modified medium supplemented with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L) allowed growth of all streptococci and streptococci-like organisms, while inhibiting growth of the staphylococci and gram-negative reference strains. Bulk tank milk samples from 114 dairy herds were spiral plated onto Edwards modified medium with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L). A total of 344 isolates (at least three isolates from each sample) were randomly selected and identified to their species. This medium permitted growth of 328 streptococci and streptococci-like organisms belonging to genera Aerococcus, Enterococcus, Gemella, Lactococcus, Streptococcus, and Vagococcus. When Edwards modified medium supplemented with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L) was evaluated using bulk tank milk samples, the sensitivity and specificity of this medium for streptococci and streptococci-like organisms were observed to be 100 and 87.5%, respectively. The positive predictive value for streptococci and streptococci-like organisms was observed to be 99.4%. The results of the study indicate that Edwards modified medium supplemented with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L) can be used as a selective medium for the isolation of streptococci and streptococci-like organisms from bulk tank milk.     

碳氮源对Bacillus sp.B_(53)发酵产聚谷氨酸的影响

考察了 8种不同碳源和 7种不同氮源对Bacillussp B53 发酵产聚谷氨酸的影响。结果表明 ,柠檬酸、甘油和硫酸铵是合成聚γ 谷氨酸比较适宜的碳源和氮源 ,前体物质L 谷氨酸的存在是聚谷氨酸高产所必需的。经过正交试验和回归分析 ,确定最佳碳氮源配比为 :L Glu 2 0 g/L ,CTA 9 86 4g/L ,Glycerol 80 36 g/L ,(NH4) 2 SO47g/L ,其他培养基成分有MgSO4·7H2 O 0 5 g/L ,FeCl3 ·6H2 O 0 0 2 g/L ,K2 HPO41g/L ,CaCl2 ·2H2 O0 2 g/L ,MnSO4·H2 O 0 0 5 g/L。在既定发酵条件下 ,Bacillussp B53 在优化培养基上产生γ PGA 19 12 g/L比基础发酵培养上的 8 87g/L提高了 115 5 6 %。     

Strain Improvement Through UV and Chemical Mutagenesis for Enhanced Citric Acid Production in Molasses-Based Solid State Fermentation

Aspergillus niger was subjected to UV radiation and chemical mutagenesis to develop its hyper-producing mutants for enhanced citric acid production. Ethyl methane sulfonate (EMS) and Ethidium bromide (EB) were used for chemical mutagenesis of Aspergillus niger. UV, and chemically treated mutants of Aspergillus niger were identified by using 2-deoxy, D-glucose as selective marker. The selected mutants were cultured in solid state fermentation (SSF) of sugarcane molasses medium (10%) using corn cobs, banana stalk, sugarcane bagasse, wheat straw, and wheat bran as carrier substrates. After pH adjustment and sterilization, the triplicate flasks were inoculated with 5 mLof homogenous spore suspensions of selected mutants of A. niger and the flasks were subjected to SSF under still culture conditions. The mutant EB-3 (treated with 1 mg/mL ethidium bromide for 120 min) giving highest citric acid yield (64.2 mg/mL) in 72 h was selected as hyper-producing mutant. Citric acid production process using EB-3 mutant was then optimized to enhance citric acid production by the mutant in SSF. Aspergillus niger EB-3 mutant could produce 67.72 mg/mL citric acid in 72 h using banana stalks as support material under optimum conditions of pH (pH 6), incubation temperature (35°C) and inoculum size (5 mL) in SSF.     

Isolation and characterization of mutants of Propionibacterium strains

Procedures were developed to isolate and characterize mutants of strains of dairy propionibacteria. These procedures included the construction of minimal defined media to support growth of the strains, optimization of conditions of exposure of the strains to nitrosoguanidine, and identification of the phenotypes of the mutants that were generated. The minimal defined medium contained inorganic salts, adenosine, three vitamins, and sodium lactate as the carbon source, with cysteine, methionine, or cysteine plus methionine added as required by some strains. For mutagenesis, cells were exposed to either 100, 200, or 1000 micrograms/ml nitrosoguanidine, depending on the sensitivity of the strain, for 60 min at 35 degrees C. At least nine stable mutants were isolated and characterized for each of the five strains under study. The most frequent mutations generated were requirements for arginine, histidine, methionine, and uracil and alteration in pigment production.     

大肠杆菌发酵产L-色氨酸培养基及补料优化

为了进一步提高优化大肠杆菌发酵产L-色氨酸的产量,采用响应面法优化了原初始发酵培养基组合成分,建立了乙酸铵-玉米浆补料模式。结果表明优化后培养基组合:0.12%硫酸铵、0.7%磷酸二氢钾、0.15%一水柠檬酸、0.28%七水硫酸镁、0.05%酵母粉、0810 0.39%乙酸铵、0.3%玉米浆。5 L罐的培养验证表明,玉米浆和乙酸铵是影响L-色氨酸产量的主要成分因素,优化后L-色氨酸产量提高了35.4%,发酵结束达到24.53 g/L,单位菌体L-色氨酸产量提高了19.8%,达到0.272 g/OD,糖酸转化率提高了27%,为L-色氨酸发酵提供一定的参考。