L-赖氨酸离子交换树脂的筛选

本文介绍一种从 L-赖氨酸发酵液中提取 L-赖氨酸的离子交换树脂——SR—1—3树脂。它对 L—赖氨酸的交换容量比国内普遍使用的732树脂提高25%左右。洗脱高峰集中,强度大,可望代替732树脂应用于 L-赖氨酸生产。     

枯草芽孢杆菌C1-B941的D-核糖发酵中间试验

使用转酮醇酶变异株—枯草芽孢杆菌C1－B941进行了D－核糖发酵中间试验。3000L发酵罐试验结果表明,发酵培养基中的葡萄糖浓度为18％时,发酵周期约为64h,发酵转化率达36．84％。发酵液经离子交换树脂纯化后,可以直接用于生产VBZ合成的中间体—N－D－核糖醇基－3,4－二甲苯胶。     

丙酮酸对肺炎克雷伯氏菌微氧发酵甘油产1,3-丙二醇的影响

微氧条件下,考察肺炎克雷伯氏菌发酵生产1,3-丙二醇过程中柠檬酸和丙酮酸对发酵过程的影响。摇瓶实验结果表明:添加柠檬酸能抑制菌体生长和1,3-丙二醇合成;丙酮酸对菌体生长和1,3-丙二醇合成有一定的促进作用。5 L发酵罐批式发酵表明:补料培养基中加入8 g/L丙酮酸,1,3-丙二醇的产量提高了约10.8%,转化率提高了约4.4%,比生长速率提高了约10.8%。上述结果初步表明,强化能量的产生能够有效促进1,3-丙二醇的合成,可以利用分子生物学手段强化丙酮酸的产生以促进1,3-丙二醇的合成。     

离子交换法在L-精氨酸发酵生产提取工艺中的应用研究

在发酵生产L-精氢酸的提取工艺中,对提取总收率影响较大的离子交换工艺进行了研究。结果表明,用国产强酸性001×7树脂对发酵液中的L-精氨酸进行动态交换吸附,当上柱流速控制在1/50vvm条件下,其交换量为1.135meq/ml湿树脂。用2.5mol/L氨水洗脱,流速控制在1/50vvm时,其洗脱效果最好。用国产201×4树脂进行脱色,每10ml树脂可脱色160ml以上的洗脱液,透光度大于90%,几乎不发生交换吸附L-精氨酸的现象。离子交换工序收率大于95%。     

枯草芽孢杆菌C1—B941的D—核糖发酵中间试验

使用转酮醇酶变异株－枯草芽孢杆菌Ｃ１－Ｂ９４１进行了Ｄ－核糖发酵中间试验。３０００Ｌ发酵罐试验结果表明,发酵培养基中的葡萄糖浓度为１８％时,发酵周期约为６４ｈ,发酵转化率达３６．８４％,发酵液经离子交换树脂纯化后,可以直接用于生产ＶＢ２合成的中间体－Ｎ－Ｄ－核糖醇基－３,４－二甲苯胺。     

固定化细胞利用离子交换树脂萃取发酵乳酸的研究

本文提出了利用海藻酸钙凝胶包埋固定化乳酸菌生产乳酸,用离子交换树脂从发酵液中分离出乳酸的新方法。该法成功地消除了产物乳酸对乳酸菌生长和产物乳酸形成的抑制作用,使发酵时间由１２０小时缩短到９６小时,乳酸的体积生产率由０．３２８ｇ／Ｌ·ｈ提高到０．４３２ｇ／Ｌ·ｈ。     

Eremomycin的发酵条件优化及发酵产物的分离纯化

研究确定Nocardia orientalis NRRL18098发酵生产eremomycin的最佳工艺条件,以及对发酵产物进行分离纯化并得到eremomycin的纯品。通过正交设计方法优化发酵培养基的组成。采用树脂吸附、中压液相色谱技术相结合的方法对发酵产物进行分离纯化。在优化条件下,eremomycin的摇瓶发酵单位达115 mg/L,提高了63.5%,并采用树脂吸附和中压液相色谱相结合的方法能有效地将eremomycin从发酵液中分离出来,制备获得eremomycin精制品。     

火龙果酵素在发酵过程中功能成分变化规律及其与抗氧化相关性

对火龙果酵素发酵487 d过程中总酸、有机酸、总多酚等功能成分的变化及其与抗氧化的相关性进行研究,其中抗氧化能力通过ABTS自由基清除能力和还原力来检测;此外,利用主成分分析法、因子分析法对不同发酵时间的火龙果酵素总酸含量、pH、总多酚含量、ABTS自由基清除能力和还原力5个评价指标进行分析.结果表明:随着发酵时间的延长,火龙果酵素发酵液中总酸、总多酚含量总体上呈逐步增加的趋势,发酵液的pH呈缓慢降低的趋势,从3.75降到3.40;利用高效液相色谱(HPLC)法检测有机酸,从火龙果原料和火龙果酵素发酵液中分别检测到9种和8种有机酸,原料中苹果酸的含量较多,发酵液中乳酸和醋酸的含量较多.与原料相比,酵素发酵液中新检测到了莽草酸;富马酸和抗坏血酸在原料中含量极少,酵素发酵液中则未检测到;火龙果经发酵后,检测到的8种有机酸的含量均有所增加.抗氧化分析结果表明:火龙果酵素发酵过程中ABTS自由基清除能力、还原力总体上呈逐步增加趋势;火龙果酵素发酵过程中总多酚含量、总酸含量、莽草酸和醋酸与ABTS自由基清除能力之间呈中度相关,总多酚含量还原力之间呈中度相关.主成分分析和因子分析结果显示:从检测指标中提取出2个因子,代表了抗氧化因子和环境因子;发酵至359d时,综合评价指标达到最高值且趋于稳定.     

pta基因敲除对L-色氨酸发酵的影响

[目的]探究pta基因缺失对大肠杆菌发酵生产L-色氨酸的影响.[方法]运用Red重组技术敲除pta基因,构建pta缺失株E.coli TRTH△pta.利用30 L发酵罐进行分批补料发酵试验,考察重组菌E.coliTRTHApta发酵0生产L-色氨酸过程中生物量、L-色氨酸产量、有机酸含量、发酵液中NH4+浓度及变化....     

从发酵液中高效提取L-缬氨酸的工艺研究

目的:利用有机膜过滤和离子交换法分离提取发酵液中的L-缬氨酸。方法:通过有机膜过滤,除去发酵液中的菌体及蛋白,滤液浓缩结晶获得L-缬氨酸产品,通过离子交换法从结晶母液中回收部分L-缬氨酸。结果:确定了有机微滤膜和超滤膜去除发酵液中菌体蛋白和色素的操作条件;确定了采用离子交换法提取L-缬氨酸的操作条件:选择732强酸性阳离子树脂,料液pH值为3.0左右,用0.4 mol/L的氨水以1.0 mL/min的速度洗脱,L-缬氨酸的收率为89.2%。结论:通过有机膜过滤和离子交换法分离提取发酵液中的L-缬氨酸,可以提高提取收率和产品质量。     

A novel downstream process for highly pure 1,3‐propanediol from an efficient fed‐batch fermentation of raw glycerol by Clostridium pasteurianum

An efficient downstream process without prior desalination was developed for recovering 1,3‐propanediol (1,3‐PDO) with high purity and yield from broth of a highly productive fed‐batch fermentation of raw glycerol by Clostridium pasteurianum. After removal of biomass and proteins by ultrafiltration, and concentration by water evaporation, 1,3‐PDO was directly recovered from the broth by vacuum distillation with continuous addition and regeneration of glycerol as a supporting agent. Inorganic salts in the fermentation broth were crystallized but well suspended by a continuous flow of glycerol during the distillation process, which prevented salt precipitation and decline of heat transfer. On the other hand, ammonium salt of organic acids were liberated as ammonia gas and free organic acids under vacuum heating. The latter ones formed four types of 1,3‐PDO esters of acetic acid and butyric acid, which resulted in yield losses and low purity of 1,3‐PDO (< 80%). In order to improve the efficiency of final 1,3‐PDO rectification, we examined alkaline hydrolysis to eliminate the ester impurities. By the use of 20% (w/w) water and 2% (w/w) sodium hydroxide, > 99% reduction of 1,3‐PDO esters was achieved. This step conveniently provided free 1,3‐PDO and the sodium salt of organic acids from the corresponding esters, which increased the 1,3‐PDO yield by 7% and prevented a renewed formation of esters. After a single stage distillation from the hydrolyzed broth and a followed active carbon treatment, 1,3‐PDO with a purity of 99.63% and an overall recovery yield of 76% was obtained. No wastewater with high‐salt content was produced during the whole downstream process. The results demonstrated that the monitoring and complete elimination of 1,3‐PDO esters are crucial for the efficient separation of highly pure 1,3‐PDO with acceptable yield from fermentation broth of raw glycerol.     

Enhanced hydrogen and 1,3‐propanediol production from glycerol by fermentation using mixed cultures

The conversion of glycerol into high value products, such as hydrogen gas and 1,3‐propanediol (PD), was examined using anaerobic fermentation with heat‐treated mixed cultures. Glycerol fermentation produced 0.28 mol‐H₂/mol‐glycerol (72 mL‐H₂/g‐COD) and 0.69 mol‐PD/mol‐glycerol. Glucose fermentation using the same mixed cultures produced more hydrogen gas (1.06 mol‐H₂/mol‐glucose) but no PD. Changing the source of inoculum affected gas production likely due to prior acclimation of bacteria to this type of substrate. Fermentation of the glycerol produced from biodiesel fuel production (70% glycerol content) produced 0.31 mol‐H₂/mol‐glycerol (43 mL H₂/g‐COD) and 0.59 mol‐PD/mol‐glycerol. These are the highest yields yet reported for both hydrogen and 1,3‐propanediol production from pure glycerol and the glycerol byproduct from biodiesel fuel production by fermentation using mixed cultures. These results demonstrate that production of biodiesel can be combined with production of hydrogen and 1,3‐propanediol for maximum utilization of resources and minimization of waste. Biotechnol. Bioeng. 2009; 104: 1098–1106. © 2009 Wiley Periodicals, Inc.     

腺苷钴胺素合成酶基因cobs的克隆及其在产1,3-丙二醇菌中的应用

甘油脱水酶是催化由甘油到1,3-丙二醇过程中的关键酶,它需要在辅酶B_(12)存在的情况下才能有效的进行催化;而在此催化过程中甘油脱水酶会出现失活现象,研究表明辅酶B_(12)可以有效的促使甘油脱水酶复活。因此,辅酶B_(12)在由甘油生物催化生产1,3-丙二醇过程中起到非常重要的作用。本研究利用PCR扩增技术,从Escherichia K-12菌株中扩增出产VB_(12)关键酶—腺苷钴胺素合成酶基因cobs,其序列与NCBI上已经公布的序列比对,同源性为99.6%,将基因cobs与产1,3-丙二醇关键酶基因dhaB、yqhD在Klebsiella pneumoniae中共表达,发酵结果显示重组菌所需额外添加的VB_(12)由原始菌株的0.01 g/L下降到0.004 g/L。     

Highly pure 1,3‐propanediol: Separation and purification from crude glycerol‐based fermentation

A new separation and purification process was developed for recovering 1,3‐propanediol (1,3‐PD) from crude glycerol‐based fermentation broth with high purity. The downstream process integrated chitosan flocculation, activated carbon decolorization, fixed bed cation exchange resin adsorption, and vacuum distillation. Breakthrough curves were measured considering the effect of sample concentration, flow rate, temperature, and resin stack height. Yoon–Nelson model was proposed to fit the fixed bed adsorption. The characteristic column parameters were calculated. Optimal condition for adsorption was 1,3‐PD, 30.0 g/L; flow rate, 1.00 mL/min; stacking height, 30.0 cm; and temperature, 298 K. Ethanol‐water (75%, 1 mL/min) was used as eluent to separate 1,3‐PD and glycerol with 95.3% 1,3‐PD elution rate. After vacuum distillation, the overall purity and yield of 1,3‐PD were 99.2% and 80.8% in the purification process, respectively. This is a simple and efficient downstream strategy for 1,3‐PD purification.     

Pathway and kinetic analysis of 1,3-propanediol production from glycerol fermentation by Clostridium butyricum

Stoichiometric analysis is applied to continuous glycerol fermentation by Clostridium butyricum to calculate theoretical maximum yields and to predict preferred pathways under different conditions. The upper limits of product concentration and productivity as a function of dilution rate in continuous culture is also predicted from product inhibition kinetic. The theoretical maximum propanediol yield (0.72 mol/mol glycerol) which is calculated for a culture without hydrogen and butyric acid formation agrees well with the experimental maximum value (around 0.71 mol/mol). Comparisons of experimental results (product concentration and productivity) with theoretical calculations and those of the glycerol fermentation by Klebsiella pneumoniae reveal that the production of 1,3-propanediol by C. butyricum is far below the optimum performance available with the present strain. One of the reasons is the relatively high formation of butyric acid under the culture conditions so far applied. The distribution of reducing equivalents to propanediol and hydrogen is also suboptimal. The utilization of the reducing power from pyruvate oxidation for propanediol production is about 60–70% of the theoretical maximum under the present experimental conditions.     

pH对克雷伯氏菌1,3-丙二醇合成关键酶酶活及发酵特性的影响

在5 L发酵罐进行甘油脉冲流加发酵,分析了不同pH值对克雷伯氏肺炎杆菌发酵特性的影响,pH 6.5为菌体最佳生长条件,克雷伯氏肺炎杆菌合成1,3-丙二醇的产量最高。在1,3-丙二醇合成速率较大的对数中前期,进行甘油脉冲流加发酵,提高甘油浓度促进甘油脱水酶、1,3-丙二醇氧化还原酶和甘油脱氢酶活性。不同pH值的脉冲试验表明,甘油脱水酶,2,3-丁二醇脱氢酶比酶活随着pH值的升高而升高,1,3-丙二醇氧化还原酶,乳酸脱氢酶比酶活在pH6.5最高,因此偏酸性的发酵条件和对数期维持一定的甘油浓度能够促进1,3-丙二醇的合成。     

Improved electrocompetence and metabolic engineering of Clostridium pasteurianum reveals a new regulation pattern of glycerol fermentation

Clostridium pasteurianum produces industrially valuable chemicals such as n‐butanol and 1,3‐propanediol from fermentations of glycerol and glucose. Metabolic engineering for increased yields of selective compounds is not well established in this microorganism. In order to study carbon fluxes and to selectively increase butanol yields, we integrated the latest advances in genome editing to obtain an electrocompetent Clostridium pasteurianum strain for further engineering. Deletion of the glycerol dehydratase large subunit (dhaB) using an adapted S. pyogenes Type II CRISPR/Cas9 nickase system resulted in a 1,3‐propanediol‐deficient mutant producing butanol as the main product. Surprisingly, the mutant was able to grow on glycerol as the sole carbon source. In spite of reduced growth, butanol yields were highly increased. Metabolic flux analysis revealed an important role of the newly identified electron bifurcation pathway for crotonyl‐CoA to butyryl‐CoA conversion in the regulation of redox balance. Compared to the parental strain, the electron bifurcation pathway flux of the dhaB mutant increased from 8 to 46% of the overall flux from crotonyl‐CoA to butyryl‐CoA and butanol, indicating a new, 1,3‐propanediol‐independent pattern of glycerol fermentation in Clostridium pasteurianum.     

Engineered Saccharomyces cerevisiae that produces 1,3‐propanediol from d‐glucose

Aims: Saccharomyces cerevisiae is a safe micro‐organism used in fermentation industry. 1,3‐Propanediol is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensively chemical synthesis. The aim of this study is to engineer a S. cerevisiae strain that can produce 1,3‐propanediol at low cost. Methods and Results: By using d ‐glucose as a feedstock, S. cerevisiae could produce glycerol, but not 1,3‐propanediol. In this study, we have cloned two genes yqhD and dhaB required for the production of 1,3‐propanediol from glycerol, and integrated them into the chromosome of S. cerevisiae W303‐1A by Agrobacterium tumefaciens‐mediated transformation. Both genes yqhD and dhaB functioned in the engineered S. cerevisiae and led to the production of 1,3‐propanediol from d ‐glucose. Conclusion: Saccharomyces cerevisiae can be engineered to produce 1,3‐propanediol from low‐cost feedstock d ‐glucose. Significance and Impact of the Study: To our knowledge, this is the first report on developing S. cerevisiae to produce 1,3‐propanediol by using A. tumefaciens‐mediated transformation. This study might lead to a safe and cost‐efficient method for industrial production of 1,3‐propanediol.     

Efficient production of 1,3-propanediol from fermentation of crude glycerol with mixed cultures in a simple medium

The objective of this study was to examine the applicability of mixed cultures for 1,3-propanediol (1,3-PDO) production from crude glycerol. Three different sources of mixed cultures were tested, where the mixed culture from a municipal wastewater treatment plant showed the best results. 1,3-PDO can be produced as the main product in this mixed culture with typical organic acids like acetic and butyric acids as by-products. The yield was in the range of 0.56–0.76 mol 1,3-PDO per mol glycerol consumed depending on the glycerol concentration. A final product concentration as high as 70 g/L was obtained in fed-batch cultivation with a productivity of 2.6 g/L h. 1,3-PDO can be kept in the culture several days after termination of the fermentation without being degraded. Degradation tests showed that 1,3-PDO is degraded much slower than other compounds in the fermentation broth. In comparison to 1,3-PDO production in typical pure cultures, the process developed in this work with a mixed culture achieved the same levels of product titer, yield and productivity, but has the decisive advantage of operation under complete non-sterile conditions. Moreover, a defined fermentation medium without yeast extract can be used and nitrogen gassing can be omitted during cultivation, leading to a strong reduction of investment and production costs.     

Influence of iron,phosphate and methyl viologen on glycerol fermentation of Clostridium butyricum

 The effect of methyl viologen addition, and iron and phosphate limitation on product distribution during glycerol fermentation of Clostridium butyricum DSM 5431 was investigated in continuous culture. Special attention was paid to the gaseous products H₂ and CO₂, which were measured on-line. In all three cases, an increased yield of 1,3-propanediol linked to a decreased hydrogen release was observed, indicating that a higher proportion of electrons was channelled from reduced ferredoxin towards NADH₂ production. The specific substrate consumption rates and the specific production rates revealed that this increase in propanediol yield was not obtained at the expense of glycolysis products but by an increased substrate conversion (overflow metabolism). The acetate/ butyrate ratio during glycerol fermentation was essentially influenced by the availability of iron. It was substantially increased when the culture turned from iron excess to iron-limited conditions. Therefore iron limitation proved to be a suitable means to achieve high 1,3-propanediol yields and to reduce butyrate formation. Received: 29 August 1995 / Accepted: 20 September 1995