Determination of the maximum product yield from glucoamylase-producing Aspergillus niger grown in the recycling fermentor

Aspergillus niger has been grown in glucose- and maltose-limited recycling cultures to determine the maximum growth yield, the maximum product yield for glucoamylase production, and the maintenance requirements at very slow specific growth rates. Using the linear equation for substrate utilization, and using the experimental data from both recycling experiments, both the maximum growth yield, Y_xsm, and the maximum product yield, Y_psm, could be determined. The values estimated were 157 g biomass per mol maltose for Y_xsm and 100 g protein per mol maltose for Y_psm. Expressed on a C₁-basis these values are 0.52 and 0.36 C-mole per C-mol for respectively Y_xsm and Y_psm. The found value for Y_psm is half the value found for alkaline serine protease production in Bacillus lichoniformis, and it can be concluded that formation of extracellular protein is more energy consuming in filamentous fungi than in prokaryotic organisms. Maintenance requirements are no significant factor during growth of Aspergillus niger, and reported maintenance requirements are most probably due to differentiation.     

微生物转化浮萍资源生产蛋白酶的条件

利用微生物转化富含蛋白质的浮萍生产蛋白酶,为浮萍的高值化利用开辟一条新途径。在摇瓶转化的基础上,探索在5L发酵罐上沙雷氏菌(Serratiasp.)SYBC H转化浮萍生产蛋白酶的最佳转化条件。通过对温度、通风量、搅拌转速、pH等参数的研究,发现在5L发酵罐上,沙雷氏菌SYBC H转化浮萍生产蛋白酶的最佳条件为通风量6vvm,搅拌转速400r/min,发酵温度30℃。转化过程中,采用全自动控制pH值(9.0),蛋白酶的产量比不控制pH的对照组提高了23.3%。进一步研究发现,利用微生物沙雷氏菌SYBC H转化浮萍生产的蛋白酶具有耐有机溶剂的特性,在某些亲水性有机溶剂中保留90%以上的活性,是一种稀少珍贵的极端酶。     

Microbial synthesis of p-hydroxybenzoic acid from glucose.

A series of recombinant Escherichia coli strains have been constructed and evaluated for their ability to synthesize p-hydroxybenzoic acid from glucose under fed-batch fermentor conditions. The maximum concentration of p-hydroxybenzoic acid synthesized was 12 g/L and corresponded to a yield of 13% (mol/mol). Synthesis of p-hydroxybenzoic acid began with direction of increased carbon flow into the common pathway of aromatic amino acid biosynthesis. This was accomplished in all constructs with overexpression of a feedback-insensitive isozyme of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase. Expression levels of enzymes in the common pathway of aromatic amino acid biosynthesis were also increased in all constructs to deliver increased carbon flow from the beginning to the end of the common pathway. A previously unreported inhibition of 3-dehydroquinate synthase by L-tyrosine was discovered to be a significant impediment to the flow of carbon through the common pathway. Chorismic acid, the last metabolite of the common pathway, was converted into p-hydroxybenzoic acid by ubiC-encoded chorismate lyase. Constructs differed in the strategy used for overexpression of chorismate lyase and also differed as to whether mutations were present in the host E. coli to inactivate other chorismate-utilizing enzymes. Use of overexpressed chorismate lyase to increase the rate of chorismic acid aromatization was mitigated by attendant decreases in the specific activity of DAHP synthase and feedback inhibition caused by p-hydroxybenzoic acid. The toxicity of p-hydroxybenzoic acid towards E. coli metabolism and growth was also evaluated.     

Continuous cultivation of Clostridium thermobutyricum in a rotary fermentor system

The growth behavior of Clostridium thermobutyricum JW171K and its production of butyric acid were investigated under continuous cultivation in a recently developed rotary fermentor. Using low dilution rates (up to 40 times the shortest doubling time), the continuous culture conditions caused metabolic shifts from butyrate formation to the production of acetate. Using an 18-h volumetric retention time, no true steady state in butyrate formation was achieved after 22 days, although the optical density was stable. Acetate and butyrate were formed in an oscillatory mode with an alternating predominance between these two products, indicating an oscillation between the less exergonic acetate-forming but higher ATP (4ATP mol⁻¹ glucose) forming mode, and the more exergonic butyrate and 3ATP mol⁻¹ glucose forming mode. During the continuous culture drastic changes in cell morphology occurred and, at the lower dilution rates, long, granulose-containing, filamentous cells with rounded protuberances and swellings were observed. A maximal butyrate concentration of 18.4 g L⁻¹ and a productivity of about 2.4 g L⁻¹ per h (at 25–27 mM concentration in the broth) were obtained. Journal of Industrial Microbiology & Biotechnology (2000) 24, 7–13. Received 26 April 1999/ Accepted in revised form 16 August 1999     

球孢白僵菌固态罐发酵产孢培养的研究

建立了一套两升圆柱体玻璃发酵罐的固态通风培养装置及通风系统。以球孢白僵菌Bb371为生产菌,培养基220g(82％麸皮 4．5％黄豆粉 4．5％玉米粉 9％稻壳)作为生产原料,接种30ml,23℃培养温度下,分阶段调节通风量和空气湿度,培养5d,产孢量可达到116亿／g干培养基。     

重组低出血抗凝蛋白在毕赤酵母中的中试发酵工艺研究及其纯化与鉴定

目的:通过对毕赤酵母中试发酵工艺的改进,建立一种简便可行的重组低出血抗凝蛋白(EH)的中试发酵工艺,为EH蛋白的放大生产研究奠定基础。方法:首先通过摇瓶培养绘测毕赤酵母工程菌的生长曲线,然后根据生长曲线,将对数生长期的菌种经过两级摇瓶培养放大后,直接接种到500 L的发酵罐中放大培养,通过发酵液的D600nm值、溶氧值(DO2)及菌体湿重动态监测细菌的生长状态,并用流加甲醇的方法诱导表达目的蛋白;表达上清经超滤、两步离子交换层析纯化获得目的蛋白;用非还原型SDS-PAGE和HPLC检测目的蛋白的纯度;用SDS-PAGE和质谱方法分析目的蛋白的相对分子质量;用Western印迹验证目的蛋白;用凝块法检测目的蛋白的抗凝活性。结果:发酵结束时,上清中蛋白含量达1.41 g/L,经后期分离纯化,得到约21 g EH蛋白,SDS-PAGE分析可见EH蛋白在还原状态下表观相对分子质量约为13.2×103±0.2×103,质谱分析相对分子质量约为7.3×103±0.73×103;Western印迹表明检测条带为目的蛋白,能被抗水蛭素抗体特异性结合;非还原型SDS-PAGE和HPLC测得EH蛋白的纯度均高于95%;凝块法检测EH蛋白的抗凝比活性为512~1024 ATU/mg。结论:建立了一条简便可行的EH蛋白的中试放大发酵生产工艺。     

rhEPO-Fc融合蛋白的表达、纯化及质量研究

用无血清培养基培养分泌表达rhEPO-Fc融合蛋白的工程细胞株MY06（CHO细胞）,收集发酵培养上清,通过离心、过滤、亲和层析、离子交换层析、分子筛等方法对目的蛋白进行纯化,通过lowry法、SDS-PAGE、Western-blot、HPLC、ELISA及IEF等方法研究目的蛋白质量,以建立rhEPO-Fc融合蛋白的发酵、纯化工艺,并探寻该融合蛋白的质量检测方法。结果通过该工艺rhEPO-Fc蛋白表达量高达2g/L,蛋白纯化得率达45%以上,纯度可达98％以上,相对分子质量约为60KD,t1/2长达38h,免疫印迹证明具有天然EPO的免疫原性。研究结果表明该生产工艺可获得rhEPO-Fc的高效表达,纯化得率高,质量检验方法稳定可靠,适用于大规模生产。     

丙酮酸高产菌株的选育及中试研究

对T.glabrata WSH-IP12进行EMS诱变,挑选以NH4Cl为唯一氮源的平板上透明圈较大的菌株,经初筛和复筛后,发现T.glabrata WSH-IP303生产丙酮酸的能力强且稳定。以NH4Cl为唯一氮源摇瓶培养48h,其丙酮酸产量（35．1g/L）比出发菌株（21．4g/L)提高了64％。采用该菌株在300L罐上进行了4批发酵试验,丙酮酸产量最高可达58．4g/L,对葡萄糖产率0．562g/g。     

Effect of nutrient limitation and two-stage continuous fermentor design on productivities during "Clostridium ragsdalei" syngas fermentation

The effect of three limiting nutrients, calcium pantothenate, vitamin B₁₂ and cobalt chloride (CoCl₂), on syngas fermentation using “Clostridium ragsdalei” was determined using serum bottle fermentation studies. Significant results from the bottle studies were translated into single- and two-stage continuous fermentor designs. Studies indicated that three-way interactions between the three limiting nutrients, and two-way interactions between vitamin B₁₂ and CoCl₂ had a significant positive effect on ethanol and acetic acid formation. In general, ethanol and acetic acid production ceased at the end of 9 days corresponding to the production of 2.01 and 1.95 g L⁻¹ for the above interactions. Reactor studies indicated the three-way nutrient limitation in two-stage fermentor showed improved acetic acid (17.51 g g⁻¹ cells) and ethanol (14.74 g g⁻¹ cells) yield compared to treatments in single-stage fermentors. These results further support the hypothesis that it is possible to modulate the product formation by limiting key nutrients during C. ragsdalei syngas fermentation.     

The influence of vessel height and top-section size on the hydrodynamic characteristics of airlift fermentors

Fermentations of the yeast Saccharomyces cerevisiae were carried out in a 90 to 250-L working volume concentric tube airlift fermentor. Measurements of liquid circulation velocity, gas hold-up, and liquid mixing were made under varying conditions of gas flowrate, vessel height, and top-section size. Both liquid circulation velocity and mixing time increased with vessel height. Liquid velocity varied approximately in proportion to the square root of column height, supporting a theoretically based relationship. The effect of vessel height on gas hold-up was negligible. The height of the top-section had a significant effect on liquid mixing. Mixing time decreased with increasing size of the top-section up to a critical height. As the top-section was expanded beyond this height, little improvement in mixing was seen. This indicated the presence of a two-zone flow pattern in the top-section. Liquid velocity and gas hold-up were essentially independent of top-section height. (c) 1994 John Wiley & Sons, Inc.