产生物表面活性剂芽孢杆菌培养条件的优化

为了提高生物表面活性剂的表面活性,通过单因素及正交试验对已筛选的产生物表面活性剂芽孢杆菌的培养基及培养条件进行了优化,优化后的培养基成分为可溶性淀粉20 g/L,氯化铵2 g/L,KH2PO46 g/L,K2HPO42 g/L,MgSO4.7H2O 0.3 g/L,NaCl 2 g/L,CaCl20.08 g/L,EDTA 0.4 g/L。培养条件为4%接种量,种龄16 h,初始pH7,培养温度37℃,摇床转速160 r/min,发酵48 h。优化发酵条件后,发酵液表面张力由初始67.5 mN/m降低至24.8 mN/m,生物表面活性剂产量达到1.08 g/L。     

Immobilized Enzyme Three-Phase Reactors for Oxidation of Cephalosporin C

The enzymatic oxidation of Cephalosporin C (CEPHC) was catalyzed by D-aminoacid oxidase, from the red yeast Trigonopsis variabilis, immobilized on Duolite A365. The study was performed in two different three phase bioreactors, gas-liquid-solid (immobilized enzyme): the fluidized-bed batch reactor, fed continuously with oxygen and discontinuously with CEPHC, and the UF-membrane reactor continuously fed with both substrates. Only the first reactor allowed significant product yield (>70%) while the second was a very useful tool for laboratory investigation of both bioconversion kinetics and enzyme stability.

Optimum reaction temperature was 15d`C for the control of CEPHC spontaneous degradation (roughly 15% in 30 h), and enzyme deactivation (half-life greater than 30 h). Immobilization improved (one order of magnitude longer half-life) enzyme resistance to mechanical stresses induced by liquid stirring and gas bubbling. Roughly 0.04g of CEPHC was adsorbed per gram of enzyme carrier. The limiting step in oxygen transfer was the gas to liquid transport. In order to attain kinetic control of the bioconversion the mildest conditions were atmospheric gas pressure and oxygen flow rate equal to 2 × 10 ²NmL/s per mL of liquid phase.     

甘薯遗传作图策略研究与展望

甘薯分子遗传图谱的建立对甘薯分子育种技术体系的拓展和应用具有重要意义。当前,对甘薯分子遗传图谱的研究虽然取得一定的进展,但存在着很多技术瓶颈,如作图策略应用和优化等。总结了甘薯经典和分子遗传研究进展,剖析了甘薯分子遗传图谱作图的3种方法与策略;探讨和提出了提高甘薯作图效率和质量的途径主要是:优化作图群体质量、克服偏分离、整合多群体间遗传连锁图谱和选择合适的分子标记类型;并指出染色体关联在遗传作图中的重要性,提出甘薯分子育种领域亟待加强的方面,以期为今后甘薯精密分子图谱的建立及基于分子图谱的甘薯分子育种提供新的思路。     

High-level expression of soluble human β-defensin-2 in Escherichia coli

Human β-defensin-2 (hBD2) is a short cationic peptide with a broad antimicrobial spectrum. The coding sequence of hBD2 was cloned into pET-32a (+) to construct a fusion expression plasmid, pET32–hBD2, which was transformed into E. coli BL21 (DE3) for expression. The cultivation parameters of the expression vector harboring strain were optimized to produce the fusion protein in soluble form efficiently and to avoid the formation of insoluble inclusion bodies. The optimal conditions were determined as following: cultivation at 28 °C in MBL medium, induction at middle stage of exponential growth with 0.8 mM IPTG, and post-induction expression for 8 h. Under the above conditions, a high percentage of the target fusion protein (≥92.3%) was expressed in soluble form and the volumetric productivity of soluble fusion protein reached 1.3 g/l. The culture process was successfully scaled up in a 10 l bench-top fermentor.     

苦丁茶冬青的RAPD影响因素及实验条件的优化

以苦丁茶冬青为材料研究随机扩增多态DNA(RAPD)的影响因素及各种实验条件优化。研究结果表明：模板DNA的浓度适宜范围为20ng／反应-80ng／反应RAPD均可得到一致的结果;dNTVs的适宜浓度范围为200μmol／L-400μmol／L;Mg^2 适宜浓度范围为1．5mmol／L-2．0mmol／L;其合适的复性温度为35—37℃;2min的延伸时间,45次热循环。按照此优化的RAPD条件进行重复实验,实验结果重现性良好,因而确定了苦丁茶冬青RAPD反应体系之最佳的实验条件。     

南极交替单胞菌R11-5产卡拉胶酶的发酵条件优化

【背景】极地寒冷环境中发现了大量具有潜在应用前景的冷适应酶,同时也存在种类繁多的海藻多糖降解菌,因此极端环境微生物是筛选获得新颖、高效多糖降解酶的重要新源泉。由于筛选培养基通常并非野生菌发酵产酶的最优条件,为了使野生菌的产酶效率达到最高,需要对其培养条件进行优化,从而为其深入研究及开发利用提供依据。【目的】对一株产卡拉胶酶的南极菌株进行种属鉴定,并采用响应面法对该菌的发酵产酶条件进行优化。【方法】通过16SrRNA基因对产卡拉胶酶的南极菌株进行种属鉴定,采用响应面法优化南极菌株产酶发酵条件。【结果】该南极菌属于交替单胞菌属(Alteromonas),命名为交替单胞菌R11-5。发酵条件优化结果显示,7个环境因子影响交替单胞菌R11-5的产酶量。利用Design-Expert软件中的Plackett-Burman设计实验,筛选出影响交替单胞菌R11-5产酶量的4个主要因素分别为培养温度、牛肉膏浓度、卡拉胶浓度和Ca~(2+)浓度。通过Box-Behnken设计和响应面分析得到交替单胞菌R11-5最佳产酶发酵条件为:温度15.0°C,牛肉膏浓度11.0 g/L,卡拉胶浓度3.0 g/L,Ca~(2+)浓度5.0 mmol/L。优化后发酵上清液酶产量达到87.193 U/mL,与优化前相比提高了1.8倍。【结论】响应面法提高了南极交替单胞菌R11-5卡拉胶酶的产量,为其开发应用提供了科学依据。     

A群脑膜炎球菌荚膜多糖纯化工艺的优化

目的对A群脑膜炎球菌荚膜多糖纯化工艺的关键步骤进行分步研究,优化每一步工艺参数。方法优化十六烷基三甲基溴化铵的加入浓度、复合多糖的解离浓度和解离时间、不同厂家的苯酚、超滤和透析等工艺过程对荚膜多糖的影响。结果十六烷基三甲基溴化铵质量体积终浓度0.10%(w/v)沉淀效果更好,纯化获得的荚膜多糖产量更高相对分子质量更大。复合多糖解离浓度越高,纯化获得的荚膜多糖相对分子质量越小。延长复合多糖解离时间有利于提高荚膜多糖产量。不同厂家的苯酚、超滤和透析等工艺对荚膜多糖的产量和分子大小没有影响。结论现行A群脑膜炎球菌荚膜多糖纯化工艺复杂,优化后的工艺提高了荚膜多糖产量,缩短了工艺用时,增加了工艺稳定性。     

红树植物秋茄叶片双向电泳技术体系的建立及优化

对适用于秋茄(Kandelia candel)叶片蛋白质组学研究的双向电泳技术体系进行了优化.结果表明,采用酚抽提法提取的蛋白质浓度较低,约1.7 μg μL-1,SDS-PAGE电泳后几乎无条带;而采用改良TCA -丙酮沉淀法可显著提高提取液中蛋白质的含量,可达12.4 μgμL-1.秋茄叶片蛋白质主要分布在pH4～...     

响应面法优化枯草芽孢杆菌产脂肪酶的合成培养基

对枯草芽孢杆菌(Bacillus subtilis)CICC20034利用合成培养基液体发酵产脂肪酶的条件进行了优化。首先采用单因子实验筛选出最适诱导剂为三丁酸甘油酯,氮源为尿素,碳源为葡萄糖,无机盐为MgSO4。在此基础上,利用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出具有显著效应的三丁酸甘油酯、尿素、KH2PO4和培养基起始pH值4个最显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面中心组合设计对显著因素进行优化,获得最适合成培养基组分为:葡萄糖8g/L,尿素8.57g/L,三丁酸甘油酯2.62%,KH2PO42.59g/L,MgSO4.7H2O0.5g/L,TritonX-1000.5g/L,pH9.47。优化后的B.subtilis CICC 20034胞外脂肪酶活力达0.483U/ml,比初始酶活力0.072U/ml提高了6.7倍。     

毕赤酵母优化表达外源蛋白策略

毕赤酵母(Pichia pastoris)表达系统是一种异源蛋白表达的理想系统,但目前并非所有的外源蛋白都能在毕赤酵母中成功高效表达,不同的蛋白表现为不同表达水平、生物活性及稳定性。从遗传因素和表达条件综述了外源蛋白在毕赤酵母中的优化表达策略。