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1.
产1,3-丙二醇菌株的诱变和筛选 总被引:5,自引:0,他引:5
为提高克雷伯氏肺炎杆菌产1,3-丙二醇的能力,以离子束、紫外线和氯化锂为复合诱变法,建立了产酸圈和产物耐受相结合的平板筛选方法,获得可耐受高浓度1,3-丙二醇并且副产物中乙醇含量较少的优良突变菌株2株。与出发菌株相比,两株高产突变菌株Klebsiella pneumoniae LM 03和Klebsiella pneumoniae LM05的1,3-丙二醇产量分别提高了33% 和30% ,达到66.74 g/L和65.12 g/L;乙醇产量分别降低了38% 和24% ,降低为6.59 g/L和8.05 g/L。同时测定了诱变前后还原途径中甘油脱水酶(GDHt)和1,3-丙二醇氧化还原酶(PDOR)的酶活变化,研究表明诱变对GDHt有明显的促进作用,而对PDOR的影响不明显。该诱变和筛选方法目标明确、易操作、效率高,在1,3-PD工业规模的生物法生产中将具有良好的应用价值,而且对于其他具有工业应用价值的菌株筛选工作也具有一定的借鉴意义。 相似文献
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克雷伯氏菌(Klebsiella pneumonia)甘油歧化发酵生产1,3-丙二醇(1,3-PD)的过程中,乳酸是氧化途径最主要的副产物,乳酸的产生和积累,不仅限制了菌体本身的生长,而且严重影响了1,3-丙二醇的转化率。利用λRed重组技术对Klebsiella pneumonia中的酶乳酸脱氢酶基因(ldhA)进行改造。在λRed重组系统作用下,将带有300 bp的线性同源片段ldhA1-Cm-ldh A2与基因组DNA的同源重组,经过抗性筛选和PCR鉴定最终获得了ldhA基因缺失菌株K.pneumonia2-1ΔldhA。经过24 h发酵可知,乳酸最大产出浓度由原来的10.16 g/L降为0.49 g/L,1,3-PD由原来的78.83 g/L增长为85.76 g/L,甘油转化率由60.64%增长到65.97%,提高了5.33%。 相似文献
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利用原生质体诱变育种选育富硒能力强的酵母菌株* 总被引:1,自引:0,他引:1
利用原生质体诱变育种技术选育富硒能力强的酵母菌株,从13株啤酒酵母中筛选出一株富硒量高的诱变出发菌株,采用溶壁酶进行破壁,确定了原生质体制备的最适条件为酶浓度1g/100mL,酶解处理时间为120min,原生质体形成率为95.2%,再生率为21.8%,诱变后筛选出富硒量为821mg/kg,酵母干菌体收获量为0.88g/100mL的酵母菌A1。 相似文献
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紫杉醇产生菌Nodulisporium sylviforme原生质体诱变研究 总被引:5,自引:0,他引:5
对酶系组成、pH、酶解温度和酶解时间等影响树状多节孢原生质体制备和再生的因素和原生质体诱变进行了研究。结果表明 ,原生质体制备和再生的最佳条件为 :用pH5.5~ 6.0的0.7mol/LNaCl配制由 3%溶壁酶 + 3%蜗牛酶 + 1 %的溶菌酶 + 3%纤维素酶组成的复合酶系 ( 1ml酶液/2 5 0mg湿菌体 ) ,在 30℃恒温水浴条件下酶解 6h ;然后 ,将获得的原生质体过滤洗涤后 ,在含0.7mol/ LNaCl的PDA再生培养基上 ,采用双层平板培养法再生制备到的原生质体。树状多节孢紫杉醇产生菌原生质体诱变的最佳条件为 :30w紫外灯、距离 30cm、照射 5 0s;UV + 0.6%LiCl复合诱变、照射时间 40s,诱变菌株经初筛和复筛 ,选出了两株高产紫杉醇的原生质体诱变菌株———UV40-19和UL50-6,其产量从出发菌株紫杉醇的产量 ( 314.07μg /L)分别提高至 376.38μg/L和392.63μg/L。 相似文献
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以灰黄青霉菌(Penicillium griseofulvum HL)为出发菌株,试验得到灰黄青霉原生质体制备的优化条件为:菌体培养48h,用0.7mol/L的NaC1溶液作为渗透压稳定剂,用0.5%的蜗牛酶+0.5%的纤维素酶,在pH为6,30℃条件下酶解3h,所得原生质体数最多,达到3.14×107/ml.原生质体再生的最佳条件为采用双层平板培养法,在用0.7mol/L的蔗糖溶液配制的改进察氏培养基上其再生率最高,达到24.93%.灰黄青霉原生质体经过紫外线诱变,DES诱变,紫外线-DES诱变复合诱变,紫外线-氯化锂复合诱变选育异抗坏血酸高产菌株,通过对再生平板上长出的诱变菌株进行初筛和摇瓶复筛,最终获得一株异抗坏血酸产量较高的菌株ZD4,其产量为5.28mg/ml,提高到出发菌株产量(1.08 mg/ml)的488.9%,且连续传代6代遗传稳定. 相似文献
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High-level production of 1,3-propanediol from crude glycerol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> AKR102a 总被引:1,自引:0,他引:1
Wilkens E Ringel AK Hortig D Willke T Vorlop KD 《Applied microbiology and biotechnology》2012,93(3):1057-1063
The aim of this study was to optimize a biotechnological process for the production of 1,3-propanediol (1,3-PD) based on low-quality
crude glycerol derived from biodiesel production. Clostridium butyricum AKR102a was used in fed-batch fermentations in 1-L and 200-L scale. The newly discovered strain is characterized by rapid
growth, high product tolerance, and the ability to use crude glycerol at the lowest purity directly gained from a biodiesel
plant side stream. Using pure glycerol, the strain AKR102 reached 93.7 g/L 1,3-PD with an overall productivity of 3.3 g/(L*h).
With crude glycerol under the same conditions, 76.2 g/L 1,3-PD was produced with a productivity of 2.3 g/(L*h). These are
among the best results published so far for natural producers. The scale up to 200 L was possible. Due to the simpler process
design, only 61.5 g/L 1,3-PD could be reached with a productivity of 2.1 g/(L*h). 相似文献
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High production of 1,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain 总被引:11,自引:0,他引:11
Papanikolaou S Ruiz-Sanchez P Pariset B Blanchard F Fick M 《Journal of biotechnology》2000,77(2-3):191-208
Batch and continuous cultures of a newly isolated Clostridium butyricum strain were carried out on industrial glycerol, the major by-product of the bio-diesel production process. For both types of cultures, the conversion yield obtained was around 0.55 g of 1,3-propanediol formed per 1 g of glycerol consumed whereas the highest 1,3-propanediol concentration, achieved during the single-stage continuous cultures was 35-48 g l-1. Moreover, the strain presented a strong tolerance at the inhibitory effect of the 1,3-propanediol, even at high concentrations of this substance at the chemostat (e.g. 80 g l-1). 1,3-Propanediol was associated with cell growth whereas acetate and butyrate seemed non growth-associated products. At low and medium dilution rates (until 0.1 h-1), butyrate production was favoured, whereas at higher rates acetate production increased. The maximum 1,3-propanediol volumetric productivity obtained was 5.5 g l-1 h-1. A two-stage continuous fermentation was also carried out. The first stage presented high 1,3-propanediol volumetric productivity, whereas the second stage (with a lower dilution rate) served to further increase the final product concentration. High 1,3-propanediol concentrations were achieved (41-46 g l-1), with a maximum volumetric productivity of 3.4 g l-1 h-1. A cell concentration decrease was reported between the second and the first fermentor. 相似文献
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Hao J Lin R Zheng Z Sun Y Liu D 《Journal of industrial microbiology & biotechnology》2008,35(12):1615-1624
3-Hydroxypropionaldehyde (3-HPA) is a toxic intermediary metabolite in the biological route of 1,3-propanediol biosynthesis
from glycerol. 3-HPA accumulated in culture medium would arouse an irreversible cessation of the fermentation process. The
role of substrate (glycerol) on 3-HPA accumulation in aerobic fermentation was investigated in this paper. 1,3-Propanediol
oxidoreductase and glycerol dehydratase, two key enzyme catalyzing reactions of 3-HPA formation and consumption, were sensitive
to high concentration of 3-HPA. When the concentration of 3-HPA increased to a higher level in medium (ac 10 mmol/L), the
activity of 1,3-propanediol oxidoreductase in cell decreased correspondingly, which led to decrease of the 3-HPA conversion
rate, then the 3-HPA concentration increasing was accelerated furthermore. 3-HPA accumulation in culture medium was triggered
by this positive feedback mechanism. In the cell exponential growth phase, the reaction catalyzed by 1,3-propanediol oxidoreductase
was the rate limiting step in 1,3-propanediol production. The level of 3-HPA in culture medium could be controlled by the
substrate (glycerol) concentration, and lower level of glycerol could avoid 3-HPA accumulating to a high, lethal concentration.
In fed batch fermentation, under the condition of initial glycerol concentration 30 g/L, and keeping glycerol concentration
lower than 7–8 g/L in cell exponential growth phase, 3-HPA accumulation could not be incurred. Based on this result, a glycerol
feeding strategy was set up in fed batch fermentation. Under the optimized condition, 50.1 g/L of 1,3-propanediol was produced
in 24 h, and 73.1 g/L of final 1,3-propanediol concentration was obtained in 54 h. 相似文献
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Ringel AK Wilkens E Hortig D Willke T Vorlop KD 《Applied microbiology and biotechnology》2012,93(3):1049-1056
A new screening method was developed and established to find high-performance bacteria for the conversion of crude glycerol
to 1,3-propanediol. Three soil samples from palm oil-rich habitats were investigated using crude glycerol of a German biodiesel
plant. Nine promising 1,3-propanediol producers could be found. Because of a special pH buffer system, a fast evaluation on
microscale and high 1,3-propanediol concentrations up to 40 g L−1 could be achieved. Three strains demonstrated very high product tolerance and were identified as Clostridium butyricum. Two strains, AKR91b and AKR102a, grew and produced 1,3-propanediol in the presence of 60 g L−1 initial 1,3-propanediol, the strain AKR92a even in the presence of 77 g L−1 1,3-propanediol. The strains AKR91b and AKR102a tolerated up to 150 g L−1 crude glycerol and produced 80% of the 1,3-propanediol attained from pure glycerol of the same concentration. Further criteria
for the choice of a production strain were the pathogenicity (risk class), ability to grow on low-cost media, e.g., with less
yeast extract, and robustness, e.g., process stability after several bioconversions. Overall, the strain C. butyricum AKR102a was chosen for further process optimization and scale-up due to its high productivity and high final concentration
in a pH-regulated bioreactor. 相似文献
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Maria Metsoviti Kleopatra Paraskevaidi Apostolis Koutinas An-Ping Zeng Seraphim Papanikolaou 《Process Biochemistry》2012,47(12):1872-1882
The production of 1,3-propanediol, 2,3-butanediol and ethanol was studied, during cultivations of strain Klebsiella oxytoca FMCC-197 on biodiesel-derived glycerol based media. Different kinds of glycerol feedstocks and experimental conditions had an important impact upon the distribution of metabolic products; production of 1,3-propanediol was positively influenced by stable pH conditions and by the absence of N2 gas infusions throughout the fermentation. Thus, during batch bioreactor fermentations conducted at increasing glycerol concentrations, 1,3-propanediol at 41.3 g/L and yield ~47% (w/w) was achieved at initial glycerol concentration ~120 g/L. At even higher initial glycerol media (150 and 170 g/L), growth was not ceased, but 1,3-propanediol production declined. During fed-batch fermentation under optimal experimental conditions, 126 g/L of glycerol were converted into 50.1 g/L of 1,3-propanediol. In this experiment, also 25.2 g/L of ethanol (conversion yield ~20%, w/w) were formed. A batch-bioreactor culture was performed under non-sterilized conditions and the 1,3-propanediol production was almost equivalent to the sterilized process. Concerning 2,3-butanediol formation, the most detrimental parameter was the absence of N2 sparging and as a result, no 2,3-butanediol was produced. The presence of glucose as co-substrate seriously enhanced 2,3-butanediol production; when commercial glucose was employed as sole substrate, 32.1 g/L of 2,3-butanediol were formed. 相似文献
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1,3-丙二醇(1,3-PD)是一种重要的化工原料,发酵法生产1,3-PD是一条新颖且具有潜在竞争力的生产途径。本研究在前期工作的基础上,将分别来源于大肠杆菌和肺炎克雷伯氏菌的基因片段yqhD和dhaB串联表达,构建重组表达载体pYX212-zeocin-pGAP-yqhD-pGAP-dhaB;并得到重组酿酒酵母(Saccharomyces cerevisiae)W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB。该重组菌和对照S.cerevisiae分别以葡萄糖为底物摇瓶发酵72h后,重组酿酒酵母发酵液中1,3-PD含量约为1.5g/L;而对照菌株不产1,3-PD。以上结果表明本研究在国内首次成功构建了直接以葡萄糖为底物发酵生产1,3-PD的酿酒酵母基因工程菌。为进一步将dhaB、yqhD基因导入其他以葡萄糖为底物高产甘油的酵母宿主中表达,获得以葡萄糖为底物一步法发酵高产1,3-丙二醇工程菌打下了坚实的基础。 相似文献
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异源表达木糖异构酶基因对克雷伯氏菌合成1,3-丙二醇的影响 总被引:1,自引:1,他引:0
【目的】提高克雷伯氏菌胞内还原力以强化1,3-丙二醇合成。【方法】将来源于大肠杆菌的木糖异构酶基因在克雷伯氏菌中异源表达,构建重组菌。研究重组菌添加不同浓度木糖为辅底物与甘油共发酵过程中代谢产物和NADH的变化规律。【结果】与对照菌相比,重组菌细胞内还原力NADH提高了0.1?0.3倍,1,3-丙二醇产量达到23.31 g/L,提高20%,1,3-丙二醇转化率从0.60 mol/mol提高到0.73 mol/mol。【结论】木糖异构酶基因的表达强化了木糖代谢途径,经磷酸戊糖途径积累大量还原力,促进了1,3-丙二醇的生成。 相似文献
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Hao J Wang W Tian J Li J Liu D 《Journal of industrial microbiology & biotechnology》2008,35(7):735-741
Glycerol can be biologically converted to 1,3-propanediol, a key raw material required for the synthesis of polytrimethylene terephthalate and other polyester fibers. In 1,3-propanediol synthesis pathway, 3-hydroxypropionaldehyde (3-HPA) was an inhibitory intermediary metabolite. The accumulation of 3-HPA in broth would cause an irreversible cessation of the fermentation process. With the object of reducing 3-HPA level in the fermentation broth, dhaT gene which encodes 1,3-propanediol oxidoreductase (PDOR) was cloned and over expressed in 1,3-propanediol producing bacterium Klebsiella pneumoniae TUAC01. dhaT gene was linked downstream of the ptac promoter in an expressing vector pDK6 to form plasmid pDK-dhaT. The newly formed pDK-dhaT was transformed to K. pneumoniae TUAC01. Under the inducement of IPTG, PDOR was over-expressed when the constructed strain was cultured on an LB medium or a fermentation medium. A 5 L scale-up fermentation experiment was done to test the 3-HPA accumulation in broth, with the initial substrate glycerol 30 g/L; the peak levels of 3-HPA in broth were 7.55 and 1.49 mmol/L for control host strain and the constructed strain, respectively. In 50 g/L initial glycerol experiment, the peak level of 3-HPA in broth was 12.57 and 2.02 mmol/l for the control host strain and the constructed strain, respectively. Thus the fermentation cessation caused by the toxicity of 3-HPA was alleviated in the constructed strain. 相似文献