运用试验设计方法提高胞外杂合b-葡聚糖酶在重组大肠杆菌中的表达

A genetically engineered Escherichia coli JM109 harboring pLF3 was used to produce a hybrid extracellular β-glucanase. Starting with enzyme production medium, glycerol and yeast extract combined with NaNO3 were screened to be the most suitable carbon and nitrogen source, respectively. Analysis of six components of the enzyme production medium by employing statistical optimization methods such as Plackett-Burman design and steepest ascent showed that yeast extract was the only significant variable and its best concentration for enzyme production was 12g·L^-1. After optimization of the medium, 297.71U·ml^-1 of β-glucanase activity in the medium and 352350U·g^-1 of β-glucanase selectivity could be obtained, which were 14 and 72 folds higher than those obtained from original medium, respectively. Even higher enzyme activities were achieved by batch cultivations in a conventional stirred bioreactor on the optimized medium.     

Production of Citric Acid from Apple Pomace Enzymolyzed by Cellulase

Cellulase can evidently increase the content of glucose and has a significant effect on the production of citric acid from apple pomace by Aspergillus niger. Based on experiments, a cellulolytic enzyme named cellulase A6 was found able to produce about 170 g glucose from 1 kg dried apple pomace after 12 h reaction, with cellulase concentration of 20 U/g in the medium at 50℃, natural pH without pretreatment of alkali. Using the treated apple pomace as a liquid state substrate, Aspergillus niger-C selected out was able to produce about 256 g citric acid from 1 kg dried apple pomace at 35℃ in 3 d or 30℃ in 5 d with flask rotation speed of 210 r/min, and the conversion of citric acid could reach 80% based on the amount of sugar consumed.     

原位氮饥饿发酵工艺中梯度补氮对谷氨酰胺合成酶的调控

The effects of uniform and gradient fed nitrogen on glutamine synthetase (GS), glutamate dehydrogenase(GDH) and glutamate synthase ((K)GAT)were investigated in glutamine production by fermentation of Corynebacterium glutamicum NS611 after 3 h of in-situ nitrogen starvation. It was shown that the strain in the later growth phase entered naturally into in-situ nitrogen starvation by controlling the initial concentration of urea and the biomass was slightly decreased. The pH value reached 6.5 again in the culture system, which confirmed the beginning of nitrogen starvation in the culture system. After 3 h nitrogen starvation the activity of GS was increased over two folds and the time of high activity of GS persisted three folds longer in the gradient fed nitrogen system than that in the normal fed batch. The higher activity of GDH was also maintained. The glutamine production increased by 72 % than the original technology of nitrogen starvation and the time of fermentation was shortened by above 12 h.     

运用试验读者设计方法提高胞外杂合β-葡聚糖酶在重组大肠杆菌中的表达

A genetically engineered Escherichia coli JM109 harboring pLF3 was used to produce a hybrid extracellular β-glucanase. Starting with enzyme production medium, glycerol and yeast extract combined with NaNO3 were screened to be the most suitable carbon and nitrogen source, respectively. Analysis of six components of the enzyme production medium by employing statistical optimization methods such as Plackett-Burman design and steepest ascent showed that yeast extract was the only significant variable and its best concentration for enzyme production was 12g·L-1. After optimization of the medium, 297.71U·ml-1 of β-glucanase activity in the medium and 352350U·g-1 of β-glucanase selectivity could be obtained, which were 14 and 72 folds higher than those obtained from original medium, respectively. Even higher enzyme activities were achieved by batch cultivations in a conventional stirred bioreactor on the optimized medium.     

Nitrogen removal characteristics of heterotrophic nitrification-aerobic denitrification by Alcaligenes faecalis C16

Alcaligenes faecalis C16 was found to have the ability to heterotrophically nitrify and aerobical y denitrify. In order to further understand its nitrogen removal ability and mechanism, the growth and ammonium removal response were investigated at different C/N ratios and ammonium concentrations in the medium with citrate and acetate as carbon source separately. Furthermore, experiments of nitrogen sources, production of nitrogen gas and enzyme assay were conducted. Results show that the bacterium converts NH4+-N and produces NH2OH during the growing phase and nitrite accumulation is its distinct metabolic feature. A. faecalis C16 is able to tolerate not only high ammonium concentration but also high C/N ratio, and the ammonium tolerance is associated with carbon source and C/N ratio. The nitrogen balance under different conditions shows that approximately 28%–45%of the initial ammonium is assimilated into the cells, 44%–60%is denitrified and several percent is converted to nitrification products. A. faecalis C16 cannot utilize hydroxylamine, nitrite or nitrate as the sole nitrogen source for growth. However, nitrate can be used when ammonium is simultaneously present in the medium. A possible pathway for nitrogen removal by C16 is suggested. The preliminary enzyme assay provides more evidence for this nitrogen removal pathway.     

Production of 2,3-Butanediol by Klebsiella Pneumoniae Using Glucose and Ammonium Phosphate

The production of 2,3-butanediol by Klebsiella pneumoniae from glucose supplemented with different salts was studied. A suitable medium composition was defined by response surface experiments. In a medium containing glucose and （NH4）2HPO4, the strain could convert 137.0g of glucose into 52.4g of 2,3-butanediol and 8.4g of acetoin in shaking flasks. The diol yield amounted to 90% of its theoretical value and the productivity was 1-1.5g.L^-1.h^-1. In fed-batch fermentation, the yield and productivity of diol were further enhanced by maintaining the pH at 6.0. Up to 92.4g of 2,3-butanediol and 13.1g of acetoin per liter were obtained from 215.0g of glucose per liter. The diol yield reached 98% of its theoretical value and the productivity was up to 2.1g.L^-1.h^-1.     

剩余活性污泥碱性发酵液用于厌氧交替好氧-缺氧序批式反应器生物除磷脱氮的碳源的研究(英文)

Activated sludge process has been widely used to remove phosphorus and nitrogen from wastewater. However, the nitrogen and phosphorus removal is sometimes unsatisfactory due to the low influent COD. Another problem with the activated sludge process is that large amount of waste activated sludge is produced, which needs further treatment. In this study, the waste activated sludge alkaline fermentation liquid was used as the main carbon source for phosphorus and nitrogen removal under anaerobic followed by alternating aerobic-anoxic conditions, and the results were compared with those using acetic acid as the carbon source. The use of alkaline fermentation liquid not only affected the transformations of phosphorus, nitrogen, intracellular polyhydroxyalkanoates and glycogen, but also led to higher removal efficiencies for phosphorus and nitrogen compared with acetic acid. It was observed that ammonium was completely removed with either alkaline fermentation liquid or acetic acid as the carbon source. However, the former resulted in higher removal efficiencies for phosphorus (95%) and nitrogen (82%), while the latter showed lower ones (87% and 74%, respectively). The presence of a large amount of propionic acid in the alkaline fermentation liquid was one possible reason for its higher phosphorus removal efficiency. Exogenous instead of endogenous denitrification was the main pathway for nitrogen removal with the alkaline fermentation liquid as the carbon source, which was responsible for its higher nitrogen removal efficiency. It seems that the alkaline fermentation liquid can replace acetic acid as the carbon source for phosphorus and nitrogen removal in anaerobic followed by alternating aerobic-anoxic sequencing batch reactor.     

氮源对利迪链菌素生产及相关次级代谢物分布的影响

The effects of nitrogen sources on streptolydigin production and distribution of secondary metabolites were investigated for flask cultured S.lydicus AS 4.2501.When peptone,asparamide,and glutamic acid were ex- amined as the nitrogen source,respectively,liquid chromatography-mass spectrometry（LC-MS）and photodiode array（PDA）analyses revealed the formation of two analogues of streptolydigin in the fermentation broth.When soybean meal was used as the source of nitrogen,three analogues of streptolydigin were detected.The use of am- monium sulfate as a source of nitrogen resulted in a lower pH value of the fermentation system,thus inhibiting streptolydigin biosynthesis and changing the metabolic profiling.Among the nitrogen sources that were made use of,glutamic acid was most favorable to the formation of streptolydigin.Simultaneously,this study also showed that the changing nitrogen sources resulted in altering the production and relative ratios of streptolydigin and its analogues.     

Biodegradation of Leather Waste by Enzymatic Treatment

The treatment of shavings, trimmings and splits of leather waste from tanneries has a potential to generate value-added products. In this study enzymatic treatment of leather waste was performed. This method utilizes alkaline protease produced by Bacillus subtilis in our laboratory by submerged fermentation. Optimum conditions of pH, time duration, temperature and concentration of enzyme were determined for maximum degradation of leather waste. The amount of degradation was measured by the release of amino acid hydroxyproline. Amino acid composition in the hydrolysate obtained by the enzyme hydrolysis was determined. This relative simple biotreatment of leather waste may provide a practical and economical solution.     

低高径比外循环气升式生物反应器带渣发酵生产有效霉素

Fermentation experiments to produce validamycins from crude substrates by Streptoniyces hygroscopi-cus were carried out in an external-loop airlift bioreactor (0.0115 m3 ) with a low ratio of height to diameter of the riser of 2.9 and a ratio of riser to downcomer diameter of 6.6. The influences of gas flow rate and liquid volume on fermentation of validamycins were investigated. Comparisons of validamycin fermentation were made among the external-loop airlift bioreactor, a mechanically stirred tank bioreactor (0.010m3 ) and shaking flasks. Under the same operation conditions including fermentation medium composition, inoculum ratio and culture temperature, the fermentation time in the external-loop airlift bioreactor (45 h) was shorter than that in the shaking flasks (100 h) and the same as that in the mechanically stirred tank bioreactor. After a total fermentation time of 45 h under optimized operation conditions, average validamycin concentration obtained in the external-loop airlift bioreactor was     

Enzymatische Hydrolyse und Fermentation von Apfeltrester

Apple pomace is a complex, carbohydrate-rich by-product of apple juice production and an interesting raw material for the production of platform chemicals such as ethanol and acetic acid. By simultaneous saccharification and fermentation, the pomace was degraded to 82 %. In the fermentation medium, the ethanol concentration was ∼5.7 vol %, which allows an economical further processing of the ethanol. Tank sediment (sludge) and accompanying substances (sorted out biomass) are also available for fermentative utilization, with acetic acid as the preferred product. The N-rich tank sediment can replace part of the required tap water and makes the addition of micronutrients unnecessary.     

A continuous alcohol fermentation byKluyveromyces fragilis using jerusalem artichoke

In this study, a continuous fermentation of inulin in the tubers of Jerusalem artichoke (JA) for alcohol production was investigated. Experiments were also conducted on the fermentation of mashed JA tubers, without extracting the juice. In a continuous fermentation of the juice of JA tubers, alcohol productivity was increased by 3.8 times as compared with that obtained in a batch fermentation. The liquefaction of mashed JA tubers by enzymes, pectinase and cellulase followed by fermentation of liquefied solution byK. fragilis was found as effective as direct fermentation of the juice. The results of this study are expected to provide valuable information in the utilization of Jarusalem artichoke for ethanol production.     

日本血吸虫核酸疫苗质粒适宜宿主菌的筛选及其发酵条件的优化

目的筛选二价日本血吸虫核酸疫苗质粒的适宜宿主菌,并对其发酵条件进行优化。方法通过综合考察细胞干重、质粒DNA产量及工程菌传代的质粒稳定性等参数,确定适宜宿主菌及其相适应的发酵培养基碳源、氮源和磷酸盐类型;进一步通过中心组合设计响应面试验,优化发酵培养基,并对优化的发酵培养基进行实验验证。结果确定适宜宿主菌为E.coliDH5α,优化的发酵培养基(m/v)为:NaCl1.0%,Yeast Extract1.0%,磷酸盐0.3%[其中m(K2HPO4)∶m(KH2PO4)=1∶4],甘油1.68%,牛肉汁2.28%,豆饼汁4.21%。在此发酵条件下,菌体干重由原来的1.57mg/ml增长至5.68mg/ml,质粒DNA产量由原来的3.94μg/ml增长至36.52μg/ml。结论已筛选出日本血吸虫核酸疫苗质粒适宜宿主菌,并优化了其发酵条件,明显提高了质粒DNA产量,具有大规模发酵生产应用价值。     

棉布支架固定化米根霉联产果胶酶及用于处理烟梗

利用果胶酶处理烟梗纤维生产乳酸等对环境友好,而应用新的发酵组合方式和优化发酵条件是提高产酶量,降低成本的有效途径。本文采用实验室研发的棉布支架固定化米根霉细胞的技术,优化了底物为果胶或烟梗的发酵产果胶酶的培养条件,还优化了处理烟梗果胶的条件。底物为果胶的最佳产酶条件为:转速190r/min,装液量50mL/250mL,发酵温度30℃,初始pH值5,初始孢子浓度0.75×10⁶个/mL。底物为烟梗的最佳产酶条件为:初始pH值4.6、初始孢子浓度0.5×10⁶个/mL等。在优化处理烟梗果胶的条件下,固定化米根霉产果胶酶连续法比游离的果胶酶法降解烟梗果胶的效果好,果胶降解率提高18.5%,达到74.1%。棉布支架固定化米根霉利用果胶发酵产果胶酶量较高,利用烟梗发酵脱胶效果较好。     

响应面法优化产低温脂肪酶工程菌Cl02的发酵条件

目的采用响应面法优化产低温脂肪酶工程菌Cl02的发酵条件。方法通过单因素试验考察培养基中酵母氮源含量、pH值、甘油含量和甲醇含量对酶活的影响,确定产酶的主要影响条件,在此基础上,根据Box-Benheken中心组合试验设计原理,采用三因素三水平的响应面分析法,综合考察各因子对低温脂肪酶酶活的影响,建立低温脂肪酶酶活的二次回归模型。结果培养基中酵母氮源含量、pH值和甲醇含量对酶活的影响显著。最适产低温脂肪酶条件为:酵母氮源含量7.3 g/L、pH 6.0、甲醇含量9.1 g/L,在此条件下,低温脂肪酶酶活可达42.25 IU/ml,比优化前(28.0 IU/ml)提高了50.9%。结论利用响应面法优化的发酵条件可显著提高工程菌的产酶能力,为规模化生产低温脂肪酶奠定了基础。     

新农抗2507发酵培养基优化的研究

农抗2507发酵培养基氮源多用黄豆饼粉。黄豆饼粉的优点是原材料质量稳定，染菌率低，但发酵效价相对较低。本研究对培养基配方进行改进，在黄豆饼粉配方中加人动物蛋白牛肉膏，提高了发酵水平。采用正交试验的方法，进行了农抗2507发酵培养基的筛选，最终得到最佳培养基配方：葡萄糖1．5％，玉米浆1％，黄豆饼粉2％，淀粉1．5％，牛肉膏O．35％，NaCl 0．5％，CaCO3 0．5％，(NH4)2SO4 0．5％。经摇瓶发酵试验，发酵水平比原始配方提高了696．9％。     

The fermentative production of gentamicins by Micromonospora purpurea

For fermentative production of gentamicins by Micromonospora purpurea it was necessary to adjust the initial pH value of the medium to 7.0–7.5. Glucose was the preferred carbon source. The production of gentamicins was performed in two steps. The first step was to grow the microbial cells and the second step was to inoculate the fermentation medium with the growing cell culture (6.0% v/v). The organism produced more antibiotic with organic nitrogen sources than with inorganic nitrogen source. Fodder yeast (50 and 40% total nitrogen) was a good nitrogen source both for microbial growth and the antibiotic production. The suitable concentrations of fodder yeast (50 and 40%) were 2 and 6 g/1 respectively.     

抗生素AGPM摇瓶发酵条件的正交实验

采用正交实验设计考察了培养基组成对新型抗生素AGPM发酵的影响,改进后在培养基组成为(g/L)：葡萄糖5, 玉米淀粉40, 黄豆饼粉16, 玉米浆2 ml, K2HPO4 1.0, MgSO4×7H2O 0.5, NaCl 0.5, 淀粉酶0.05及pH 5.5的条件下,单位发酵液的活性提高了18.9倍;同时表明较高的碳氮比对抗生素AGPM的合成有利.     

海洋微生物溶菌酶的发酵优化与中试生产

以海洋细菌S-12-86为试验菌株,采用摇瓶发酵优化的方式,研究培养基组分(碳源、氮源、碳源与氮源的比例、金属离子)与发酵条件(培养温度、接种体积分数、装液体积分数、起始pH值、产酶周期)对海洋微生物溶菌酶产量的影响,并进行中试放大试验。结果表明:该菌产酶最佳培养基组分为:葡萄糖10 g/L,蛋白胨5 g/L,MgSO45 g/L,CaCl22 g/L;最适发酵培养温度为30℃,接种体积分数为4.0%,装液体积分数为10.0%,起始pH值为8.0,发酵周期24 h。海洋细菌S-12-86发酵优化后的产酶量(25636.8 U/mL)较优化前的产酶量(14454.4 U/mL)提高了75.4%。海洋微生物溶菌酶中试发酵的产酶量达26697.87 U/mL。说明摇瓶发酵优化条件可以应用于海洋微生物溶菌酶中试生产上。