克雷伯氏菌发酵条件的响应面设计

以中心组合设计为基础利用响应面法,对克雷伯氏菌利用甘油生产1,3-丙二醇(1,3-PD)的培养条件进行优化,建立了以甘油、硫酸铵、pH值、发酵时间、和发酵温度对1,3-PD发酵生产的单个因素和交互影响的数学模型,得到发酵生产1,3-PD的最佳条件为:甘油49.9g/L;硫酸铵5.28g/L;pH值为7.19;培养时间84h;温度36.1℃。在此条件下,模型预测1,3-PD最大产量为19.03g/L。并在此条件下进行实际实验,1,3-PD的产量为18.33g/L,与模型预测接近。     

1,3-丙二醇氧化还原酶同功酶基因yqhD在肺炎克雷伯氏菌中的表达研究

以大肠杆菌(Escherichia coli)基因组为模板,PCR扩增出1,3-丙二醇氧化还原酶同功酶基因yqhD,经测序确认,将yqhD基因与四环素抗性基因TetR同时插入表达载体pUC18构建重组质粒pUC18-yqhD-TetR,重组质粒转化Klebsiella pneumoniae ME308;37℃,经1.0mmol/LIPTG诱导8h,重组肺炎克雷伯氏菌中1,3-丙二醇氧化还原酶同功酶的酶活力达到4.16U/mg,而对照菌株的酶活仅为0.62U/mg;将重组菌在摇瓶中进行微氧发酵培养,经IPTG诱导,可将60g/L甘油转化为33.8g/L1,3-丙二醇。     

发酵后期补加2种氮源对克雷伯氏菌合成1,3-丙二醇的影响

为探寻解决重要平台化合物1,3-丙二醇发酵后期菌体生长和1,3-丙二醇合成受限的方法,通过从菌体量、产物和关键酶活性及基因转录水平等方面,较全面地考察了发酵后期补加酵母膏和硫酸铵对克雷伯氏菌(Klebsiella pneumoniae)合成1,3-丙二醇的影响,结果为:添加两种氮源均有利于菌体生长;补加10 g/L酵母膏和硫酸铵,1,3-丙二醇产量由58.6 g/L分别提高到70.6 g/L和77.2 g/L;相对于酵母膏,硫酸铵对关键酶活性的增强更为明显,并使甘油脱氢酶(glycerol dehydrogenase,Dha D)在发酵后期始终维持较高水平,促进细胞生长和产物合成。此外,与补加酵母膏相比,补加硫酸铵后关键酶基因转录水平上调并不显著。表明硫酸铵主要通过直接激活关键酶活性促进细胞生长和产物合成。综上所述,发酵后期补加硫酸铵更利于1,3-丙二醇的生物合成,是提高发酵合成1,3-丙二醇水平的有效方式之一。     

克雷伯氏菌固定化技术发酵生产1,3-丙二醇

通过对4种常见的包埋材料固定化克雷伯氏菌发酵生产1,3-丙二醇的研究发现,海藻酸钙固定化无论在制备难易还是在发酵强度上都有很大优势,并进一步确定了海藻酸钙固定化的最优条件:海藻酸钠质量浓度6%,CaCl2质量浓度4%,交联时间4 h。相对于游离发酵,固定化发酵1,3-丙二醇终浓度提高了7.4%,发酵强度达到了1.04 g/(L.h)。此外,固定化克雷伯氏菌还能用于1,3-丙二醇半连续发酵。     

有氧条件下pH值调控方式对克雷伯氏肺炎杆菌发酵的影响

研究了有氧条件下,以粗甘油为底物,由克雷伯氏肺炎杆菌(Klebsiella pneumoniae)发酵生产1,3-丙二醇中不同通气量和3种不同的pH值调控方式(NaOH调控、NaOH和CaO联合调控、CaO调控)对菌体生长、1,3-丙二醇生成的影响.研究表明,空气通量为0.2L/rain最适合1,3-丙二醇的生产,1,3-丙二醇的最终浓度为17.96g/L;在有氧条件下,通过CaO调控pH值,可获得最高菌体密度,当发酵进行至48h时,1,3-丙二醇浓度可达62.4g/L,明显优于其他2种调控方式的1,3-丙二醇的发酵结果.     

强化乙醛酸和TCA循环对克雷伯氏菌合成1,3-丙二醇的影响

克雷伯氏菌(Klebsiella pneumoniae)代谢甘油合成1,3-丙二醇(1,3-propanediol,1,3-PDO)的过程中,存在乙酸溢流、TCA循环活性低的问题,影响1,3-PDO合成。该研究对K.pneumoniae中乙醛酸循环抑制因子iclR进行敲除,并过表达TCA循环中琥珀酸脱氢酶基因sdhC和苹果酸脱氢酶基因mdh,缓解乙酰辅酶A节点的碳流溢出。结果表明,敲除iclR后乙酸积累量降低了41%,1,3-PDO产量提高了8%。在K.pLric中单独过表达sdh C或mdh基因,2,3-丁二醇产量分别降低了47%和52%,1,3-PDO产量分别提高了7%和8%。共表达sdh C和mdh基因后,菌株的甘油利用能力增强,1,3-PDO产量比K.pLric提高了11%。5 L发酵罐分批补料发酵结果表明,K.pLric-sdh C-mdh的生物量明显提高,1,3-PDO产量达77.2 g/L,摩尔转化率为0.69 mol/mol。以上结果表明,敲除iclR激活乙醛酸循环、过表达sdh C和mdh强化TCA循环可以弱化乙酸溢流,增强菌株的甘油利用能力,促进合成1,3-PDO。     

不同醛脱氢酶对重组克雷伯氏菌生产3-羟基丙酸的影响

PCR扩增Cupriavidus necator Gab D4和Azospirillum brasilense KGSADH醛脱氢酶基因,分别与经改造获得卡那霉素抗性的p UC19质粒(p UC19Kan)连接,再转化至Klebsiella pneumoniae DSM2026,获得重组菌KpKan/KGSADH和KpKan/Gab D4。经摇瓶发酵,KpKan/KGSADH和KpKan/Gab D4的最高3-羟基丙酸(3-HP)产量分别为3.66与2.56 g/L,与未导入醛脱氢酶的K.pneumoniae对照菌相比,分别提高了352%与216%。对其他产物的研究表明,2株重组菌的1,3-丙二醇(1,3-PDO)的产量低于对照菌,而2,3-丁二醇(2,3-BD)和乙酸产量高于对照菌。该研究首次将C.necator Gab D4醛脱氢酶基因导入K.pneumoniae,并对比了KpKan/KGSADH与KpKan/Gab D4的3-HP产量,实验中确定的性能优良的发酵菌株为继续提高3-HP产量提供了帮助。     

1,3-丙二醇间歇发酵培养基的优化

从Na+、K+、NH4 +等 1价阳离子对甘油发酵生产 1,3-丙二醇过程中关键酶的活性影响分析出发 ,设计改进发酵培养基组成。通过间歇发酵 ,考察甘油初始质量百分比分别为 2 %、4 %和 6 %的发酵情况 ,结果表明 ,采用无铵培养基氨水调节 pH的发酵 ,甘油的转化率和 1,3 丙二醇的终浓度较高 ,而且与文献报道的培养基相比更为简单 ,有利于工业应用。     

用克雷伯氏菌批式流加发酵法生产1,3-丙二醇

通过对克雷伯氏菌在 7L发酵罐中厌氧间歇发酵甘油生产 1,3 丙二醇的实验研究 ,建立了一种与 pH调节相偶联的批式流加甘油发酵策略。考察了不同甘油维持浓度条件下的流加方式及不同培养方式对 1,3 丙二醇产率的影响。结果表明 ,甘油质量分数维持在 2 %的流加方式有利于 1,3 丙二醇的发酵生产 ,其在 30 5h内消耗甘油 2 80 g ,得到 1,3 丙二醇152 6 g ,摩尔转化率 6 5 5% ,生产强度 0 91g/L·h     

碳源对克氏菌合成PTT原料PDO的影响及对DhaB的表达调控

为提高1,3-丙二醇（PDO）产量,加快聚对苯二甲酸丙二醇酯（PTT）的产业化应用。考察了不同碳源对克雷伯氏菌合成PDO过程中碳流的影响和对关键酶DhaB的表达调控。结果表明,克雷伯氏菌不能直接利用葡萄糖合成PDO;以甘油为单一碳源时,PDO的产量仅为9 g/L,同时细胞生长受到一定抑制;而在甘油为底物的情况下,添加5 g/L的葡萄糖能够使菌体生物量提高66.7%、dhaB的转录上调20%、DhaB酶活提高64%,同时PDO产量提高1.5倍。上述结果表明混合碳源策略能够激活克雷伯氏菌PDO合成途径关键酶DhaB的表达,提高PDO的产量。     

Influence of heat shock on glycerol production in alcohol fermentation

The influence of single and double heat shocks induced during the exponential growth phase of the Saccharomyces cerevisiae fermentation of cultivar Sauvignon Blanc grape must was examined. Rapid temperature changes from 18 degrees C to 34 degrees C have been applied. The effect of the duration of exposure to a high temperature has been analyzed. By the applications of a single heat shock and a double heat shock, up to 8.2 g l(-1) and 11.0 g l(-1) glycerol have been produced, respectively. To prevent the evaporation of fine wine bouquet compounds during the temperature changes, reflux coolers on the top of bioreactors have been employed. By using this method, glycerol production was increased by up to 65%.     

Influence of equibiaxial extensional strain on stress relaxation of glycerol plasticised wheat gluten

BACKGROUND: Glycerol plasticised wheat gluten shows advantages for non‐food applications such as biodegradable package films and bioplastics because of their abundant resources, low cost, good biodegradability and suitable mechanical and oxygen barrier properties. The intention of this study was to evaluate to the equibiaxial stress relaxation of 400 g kg^?1 glycerol plasticised gluten at different biaxial strains at room temperature for a better understanding of the processibility. RESULTS: The plasticised gluten shows significant stress relaxation and the spectra span over six decades of time at biaxial strains from 0.03 to 1.51. Plotting the instantaneous modulus against strain reveals strain softening and strain hardening at strains below and above 0.25, respectively. The relaxation modulus as a function of time can be fitted to the Kohlrausch–Williams–Watts equation. CONCLUSION: These results suggest that stress relaxation of plasticised gluten is highly dependent on strain level and biaxial deformation accelerates the network relaxation by widening the distribution of relaxation times. Copyright © 2008 Society of Chemical Industry     

Influence of myosin heavy chain isoform expression and postmortem metabolism on the ATPase activity of muscle fibers

The objective of this study was to determine the effects of postmortem muscle pH and temperature declines on the actomyosin ATPase activity of muscle fibers expressing different MyHC isoforms. Using a quantitative histochemical procedure to determine ATPase activity, the maximum actomyosin ATPase activity was determined on individual fibers classified by MyHC expression. Samples were collected from the red (RST) and white (WST) semitendinosus muscles at 3 min and 24 h postmortem from electrically stimulated (ES) and control (NS) pork carcasses. In samples taken at 3 min postmortem, type I fibers had the lowest ATPase activity staining and type 2X and 2B had the highest activity staining, with type 2A fibers intermediate. Postmortem time and carcass treatment did not influence the ATPase activity staining of type I muscle fibers. ATPase activity staining of 2A fibers was lower (p<0.001) in 24 h samples than in 3 min samples from ES carcasses. In 3 min and NS-24 h samples, RST type 2A fibers had lower (p<0.05) activities than type 2A fibers from the WST. In type 2X fibers, ATPase activity staining decreased (p<0.01) from 3 min to 24 h postmortem in ES carcasses. This decrease was more severe in WST 2X fibers compared to RST 2X fibers. ATPase activity staining in type 2B fibers did not decrease from 3 min to 24 h postmortem in NS carcasses. In ES carcasses, activity staining of 2B fibers decreased (p<0.0001) with time postmortem. The results of the experiment indicate that fibers expressing fast MyHC isoforms have a higher ATPase activity early postmortem than slow muscle fibers but are more prone to inactivation by a rapid pH decline.     

Influence of glycerol and water activity on the properties of compressed egg white-based bioplastics

In this paper, the effects of glycerol (35%, 40% and 45%) and water activity (0.34 and 0.48) on the physical, mechanical and morphological properties of compressed egg white-based bioplastics are investigated. Lighter, more reddish and less yellowish sheets with a decreased thickness and second-order transition temperature, improved mechanical properties (increased flexibility and decreased rigidity and stiffness) and constant moisture content can be obtained by increasing water activity. Increasing the GLY content results in the same type of changes but to a lesser extent and increased moisture content. Increasing both, water activity and GLY, leads to a more pronounced effect for some properties. This study demonstrates that compressed egg white-based bioplastics with desired properties can be obtained by adjusting water activity and GLY content during their synthesis.     

Production of glycerol from glucose by coexpressing glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase in Klebsiella pneumoniae

As a valuable chemical, 1,3-propanediol (1,3-PD) could be biosynthesized by glycerol fermentation. However, no natural microorganisms that could directly convert glucose into 1,3-PD have been found so far. In this work, genes coding for two enzymes, glycerol-3-phosphate dehydrogenase (GPD, EC 1.1.1.8) and glycerol-3-phosphatase (GPP, EC 3.1.3.21), which were responsible for glycerol production, were organized into the plasmid pUC18K under control of the respective lac promoters. Two recombinant proteins were expressed successfully in wild-type Klebsiella pneumoniae. A glycerol concentration of 6.8 g l(-1) was obtained in flask culture. When glucose was exhausted, dihydroxyacetone was added and medium pH was adjusted to 7.0, and then a 1,3-PD concentration of 0.58 g l(-1) was achieved with engineered K. pneumoniae from glucose.     

叶面喷施丙三醇对烟叶碳氮代谢及硝酸盐积累的影响

  目的  探索丙三醇对白肋烟碳氮代谢及其硝酸盐积累的调控机理,建立新的白肋烟增效减害调控技术。  方法  以白肋烟品种TN90和TN86为材料,设置不同丙三醇浓度试验,并在确定丙三醇最佳适宜浓度的基础上,设置高氮和低氮两个处理,研究丙三醇对烟叶生物量积累、氮代谢关键酶活性、主要碳氮化合物含量及相关基因表达情况的影响。  结果  1）在高氮和低氮水平下,喷施丙三醇15 d后,烟叶生物量分别增加10.20%和13.36%,氮素积累量和氮素利用效率分别增加10.21%、3.15%、6.81%和3.11%,烟叶总糖和还原糖含量分别升高了25.15%、32.84%、47.76%和44.57%,但7 d后烟叶硝酸盐含量下降了53.22%和59.67%;2）低氮水平下喷施丙三醇烟叶色素、可溶性蛋白质和两糖含量与高氮条件下不喷施丙三醇结果相近;3）基因表达分析表明,喷施丙三醇显著促进了光反应途径、碳固定途径、蔗糖合成和氮代谢途径关键基因的表达,与对照相比,基因CP12-2、PPC16和NPF7.3的表达量增加0.5倍多。  结论  丙三醇能够提高烟叶碳氮代谢能力和氮素利用效率,提高烟叶碳水化合物含量和降低烟叶硝酸盐积累量,减氮配合喷施丙三醇是降低烟叶硝酸盐积累的有效途径。      

膜过滤对啤酒内源抗老化水平的影响

研究了实际生产中的微滤膜过滤对啤酒的老化与抗老化水平的影响,结果表明:在密闭环境,粗滤与精滤之后啤酒中的老化物质-醛类含量下降了4.11%,TBA值下降了2.13%,啤酒老化程度降低,口感更新鲜。与硅藻土过滤相比,微滤膜过滤过程中不会引入过渡态金属离子。抗老化物质SO2主要影响.OH的迟滞时间,R2为0.896 1,呈正相关,而膜过滤过程中SO2含量升高,使膜过滤后啤酒的迟滞时间延长。多酚物质可以减缓.OH的生成速率,膜过滤过程截留了啤酒中的抗老化物质多酚,使啤酒的DPPH清除率下降了5.70%,这使得啤酒中自由基生成速率升高,在老化后期抗老化水平下降。     

Effect of zinc source and dietary level on zinc metabolism in Holstein calves

Forty-eight Holstein male calves were stratified by origin and body weight and randomly assigned to one of 4 treatment groups. Dietary treatments were administered in 2 phases. In phase 1, treatment groups received the basal diet with no supplemental Zn (control), basal diet plus 20 mg of Zn/kg of DM as ZnSO4 or Zn proteinate (ZnProt), or basal diet plus 20 mg of Zn/kg of DM with 50% of the Zn supplied from each source (ZnM) for 98 d. In phase 2, calves continued to receive the same Zn source fed in phase 1; however, half of the calves in each treatment group were randomly selected to receive 500 mg of Zn/kg of DM (HiZnSO4, HiZnProt, HiZnM) for 14 d. Gain, feed intake, and feed efficiency of calves were not affected by treatment in either phase of the experiment. Treatment had no affect on plasma Zn concentration or alkaline phosphatase activity in phase 1, but liver Zn concentration was greater in calves fed ZnSO4 than those fed ZnProt. In phase 2, plasma Zn was greater in calves fed HiZnProt and HiZnM than in those fed HiZnSO4. Liver Zn was greater in calves fed HiZnProt than in those fed HiZnSO4. Duodenal Zn concentrations were greater in calves supplemented with HiZnProt and HiZnM than those supplemented with HiZnSO4. Liver metallothionein was greater in calves that received 500 mg of Zn/kg than in calves that received 20 mg of Zn/ kg, but was not affected by Zn source. Calves fed HiZnProt and HiZnM had greater kidney Zn concentrations than those fed HiZnSO4. Heart, spleen, testicular, and bone Zn concentrations were not affected by Zn source. Hoof wall samples contained nearly 3-fold greater Zn concentrations than hoof sole. Calves fed ZnSO4 had greater Zn concentration in hoof wall samples than those fed ZnM. Hoof sole Zn concentration was not affected by Zn source or concentration. Plasma and tissue Zn concentrations at harvest were generally similar in calves supplemented with 20 mg of Zn/kg from ZnSO4 or ZnProt. However, when supplemented at 500 mg of Zn/kg, ZnProt was absorbed to a greater extent than ZnSO4, based on higher plasma, liver, duodenal, and kidney Zn concentrations.