大肠杆菌工程菌高密度发酵的初步研究

在5L发酵罐上以恒pH-补料发酵模式为基础,通过改变培养温度、时间以及补料浓度达到提高大肠杆菌产量的目的。实验结果表明,当恒pH值为6.5,补料类型甘油速度为35mL/h。连续补料6h,发酵12h结束,可使湿菌体量达到51.1g/L,初步达到了高密度发酵的要求。     

阿魏酸的制备

通过Perkin反应,以香兰素和乙酸酐为原料,在碳酸钾的催化下,合成乙酰阿魏酸,再经氨水催化水解、硫酸酸化处理,制备阿魏酸。采用正交实验研究了反应时间、催化剂用量、乙酸酐/香兰素物料比对反应的影响,结果表明乙酸酐/香兰素物料比对反应影响最大。该工艺与原有路线相比,具有成本低、收率高、污染少的优点。     

灵芝多糖的液态发酵研究

对泰山灵芝液态发酵灵芝多糖的培养基配方、发酵条件进行了优化选择，并通过分批发酵、补料分批发酵的方式获得了灵芝多糖液态发酵的一系列参数。通过60h的补料分批发酵，灵芝多糖的单位产量达到2．208g／L，为灵芝多糖的工业化发酵生产奠定了基础。     

Kitasatospora sp.MY 5-36菌株补料重复发酵生产ε-聚赖氨酸

以Kitasatospora sp. MY 5-36菌株为研究对象,首次采用带补料的重复发酵工艺发酵生产e-聚赖氨酸(e-PL),并对工艺参数进行初步研究. 在摇瓶上对重复发酵e-PL的可行性及稳定性进行了验证,并在5 L发酵罐上初步构建了带补料的重复发酵e-PL工艺. 研究结果表明,重复发酵过程中最佳留种量为10%(j). 通过对间歇补料、变速补料2种补料方式的考察,确定当残糖浓度下降至10 g/L时,采用变速补料并控制残糖浓度在10 g/L时能较好地进行带补料的重复发酵生产e-PL. 经5批次发酵后发酵体系仍具有较高的e-PL生产能力,e-PL批次平均终浓度为20 g/L,生产强度达到0.159 g/(L×h).     

固体碱催化Knoevenagel法合成阿魏酸的工艺研究

以香兰素和丙二酸为原料、苯为带水剂、固体碱为催化剂、吡啶为助催化剂,经Knoevenagel反应合成了阿魏酸,经重结晶提纯得到目标化合物,并对化合物进行了结构验证。结果表明,当n(香兰素)∶n(丙二酸)=1∶1.15、吡啶用量为5 wt%香兰素、固体碱催化剂用量为3 wt%香兰素时,反应效果最佳,阿魏酸的收率可达90%以上。     

四环素通氨补料工艺

四环素通氨补料是四环素发酵工艺的一种新方法。四环素发酵原工艺的缺点是发酵单位低。通氨补料是用通氨的方法创造菌种生长适应的 pH 值,强化菌种代谢,补充营养,以达到提高发酵单位的目的。东北制药总厂、沈阳市第二制药厂都采用了通氨补料     

阿魏酸异辛酯的一锅法合成

用香兰素、丙二酸二乙酯、异辛醇、冰醋酸为主要原料,以甘氨酸为催化剂,经Knoenenagel缩合反应,一锅法合成了阿魏酸异辛酯。研究了原料摩尔比、甘氨酸用量、反应时间等对阿魏酸异辛酯收率的影响。结果表明,最佳反应条件:香兰素6.08g(0.04mol),n(丙二酸二乙酯)/n(香兰素)=1.4,n(甘氨酸)/n(香兰素)=0.15,n(异辛醇)/n(丙二酸二乙酯)=12,反应时间8h,在该条件下目标产物阿魏酸异辛酯产率达75.2%。用核磁共振波谱及红外光谱对所得目标产物进行了表征。     

亚油酸对Lipstatin发酵生产的影响研究

亚油酸作为一种前体物质,对Lipstatin的生物合成有重要的影响。通过研究发酵培养基中亚油酸浓度对毒三素链霉菌发酵的影响,表明合适的亚油酸浓度能显著促进Lipstatin的生物合成。在此基础上,进一步研究了不同补料时间和补料速率对毒三素链霉菌发酵生产Lipstatin的影响,最终确定了最佳的补料时间和补料速率。在亚油酸浓度为20g·L^-1的发酵培养基中,接种后发酵36h时开始补料、补料速率为0．35g·L^-1·h^-1时,Lipstatin的发酵效价最高,为6．8g·L^-1。     

KF/K2CO3/γ-Al2O3催化合成反式阿魏酸

香兰素与丙二酸于乙酸正丁酯中,在KF/K2CO3/γ-Al2O3催化作用下,经Knoevenagel制得反式阿魏酸。收率为65.4%,纯度达到99.7%。     

补料发酵生产D-核糖工艺的研究

通过对补料时间、方式和补料液浓度变化(特别是补加氮源)对D-核糖发酵过程影响,确定了D-核糖发酵的补料工艺。按此工艺条件,发酵的时间为72 h,D-核糖的产量93.83 g/L,剩余葡萄糖浓度为4 g/L,转化率为0.284 3 g/g,生产强度为1.303 g/(L.h)。与不补料相比,D-核糖产量提高了95.9%。     

阿魏酸对蘑菇酪氨酸酶的抑制效应

The inhibitory effect of ferulic acid on the diphenolase activity of mushroom tyrosinase and the kinetic behavior were studied with L-3,4-dihydroxyphenylalanine （L-DOPA） as substrate. The inhibitor concentration leading to 50% relative activity lost （IC50） was estimated to be 0.15 mmol·L^-1. The inhibition mechanism obtained from Lineweaver-Burk plots shows that ferulic acid is a competitive inhibitor and the inhibition of tyrosinase by ferulic acid is a reversible reaction. The equilibrium constant for ferulic acid binding with the tyrosinase was determined to be 0.25 mmol·L^-1 for diphenolase. Keywords tyrosinase, ferulic acid, kinetics, inhibition, L-DOPA, diphenolase     

阿魏酸衍生物的设计、合成及除草活性

[目的]以天然产物阿魏酸为先导设计合成具有较强除草活性的衍生物。[方法]运用农药分子合理设计原理设计合成阿魏酸酰胺类似物,采用小杯法测定除草活性。[结果]合成了11个新型的阿魏酸衍生物;小杯法测定显示,在400 mg/L质量浓度下,化合物8f和8k的除草活性较好;半抑制浓度测定结果表明:8f对马唐根和茎的IC50值为分别为269.442、344.835 mg/L;8k对反枝苋根和茎的IC50值分别为187.168、224.912 mg/L。[结论]8f和8k具有较强的除草活性,值得进一步研究。     

Enhanced maize (Zea mays L.) pericarp browning: Associations with insect resistance and involvement of oxidizing enzymes

The kernel pericarp of a maize (Zea mays L.) inbred, Mp313E, that browns rapidly at milk stage when damaged and that is resistant toAspergillus flavus Link and the dusky sap beetleCarpophilus lugubris Murray compared to a susceptible inbred, SC212M, was examined for differing oxidizing enzymes (peroxidases) and their substrate specificity. Additional pericarp enzymes were constitutively produced by the Mp313E inbred compared to the SC212M inbred, as indicated by gel electrophoresis and isoelectric focusing. These enzymes oxidized relevent pericarp substrates such as ferulic acid. Similar results were seen with two varieties of maize containing theCh mutant gene, which brown upon senescence in that enhanced oxidation of ferulic acid was seen in homogenates of browned pericarp compared to that which was cold-shocked and did not brown. Corn powder that was browned by mixing oxidizing enzymes with corn pericarp components ferulic acid and coumaric acid were typically less preferred/more toxic to caterpillars such asHelicoverpa zea (Boddie) and sap beetles such asC. lugubris. Thus, enhanced browning of maize pericarp can promote resistance to insects and is at least partly influenced by the presence of oxidizing enzymes. This mechanism may also promote resistance to maize pathogens, including those that produce mycotoxins.     

Microbial decomposition of ferulic acid in soil

The suppression of plant growth by different phenolic acids is well known. This work was designed to determine if ferulic acid, a known phenolic inhibitor of plant growth, accumulates in the soil and if soil microorganisms could be isolated that metabolize it. Over 99% of the extractable ferulic acid was lost from decaying hackberry leaves in 300 days. During this time the amount in the top 15 cm of soil remained fairly constant at about 30 ppm, except for the March sample which was significantly higher than the rest. Addition of ferulic acid to soil caused an increase in CO₂ evolution and in numbers of a select group of microorganisms.Rhodotorula rubra andCepnalosporium curtipes, which actively metabolize ferulic acid, were isolated, but the metabolic pathways employed appear to be different from the reported one. The reported pathway for ferulic acid breakdown is ferulic acid to vanillic acid to protocatechuic acid to -keto-adipic acid.Rhodotorula Rubra was found to convert ferulic acid to vanillic acid, but no evidence was found for utilization of the rest of the pathway.Cephalosporium curtipes appears to use a different pathway or to metabolize intermediate compounds rapidly without accumulating them, because no phenolic compounds were found during the breakdown of ferulic acid. The presence in the soil of microorganisms that metabolize ferulic acid and other phenolic acids is ecologically significant because such organisms prevent long-term accumulations of these substances, which are toxic to many other microorganisms and higher plants.     

Production of vanillin from wheat bran hydrolyzates via microbial bioconversion

BACKGROUND: Wheat bran contains a large amount of ferulic acid, which can be released through enzymatic hydrolysis and bioconverted into vanillin. A previous study has shown that ferulic acid purification from bran carbohydrates with the Amberlite^® IRA 95 resin allowed an increased vanillin molar yield. In this work, alternative ferulic acid recovery methods were proposed and the possibility of exploiting the residual carbohydrate‐rich water phase was explored. RESULTS: Ferulic acid was recovered from crude wheat bran hydrolyzate by: (i) a hydrophobic sorbent cartridge (ISOLUTE ENV^+®): (ii) ethyl acetate extraction; and (iii) the resin previously employed. The highest recovery percentage (95%) was obtained with ISOLUTE ENV^+®, which also allowed an interesting vanillin molar yield from ferulic acid bioconversion (75% from 0.5 mmol L^?1 ferulic acid). The residual water phase was a good growth substrate for the microorganism operating the bioconversion. Cells grown on this matrix could efficiently bioconvert the recovered ferulic acid to vanillin CONCLUSION: The possibility of efficiently recovering ferulic acid from wheat bran hydrolyzates, bioconverting it into vanillin, and valorizing the sugar‐rich exhausted water fraction was demonstrated in this work. The approach allowed the production of a value‐added fine‐chemical from a food‐industry by‐product. Copyright © 2009 Society of Chemical Industry     

Allelopathic substances in ecosystems

Recovery studies were conducted with ferulic acid, a common allelopathic agent, using various soils and soil components. Ferulic acid was placed into sterile soil components (gibbsite, geothite, Georgia kaolin, and Utah bentonite), and different sterile soil materials (from different horizons in the same profile) varying in mineralogy and in organic matter content. The initial concentration of ferulic acid added to the soil materials was 1000 μg/g (5.149 mmol/g). The pH of the soil materials was adjusted and maintained at approximately 4.5 or 7.5. Samples were extracted with 0.03 M EDTA at days, 1, 4, 7, 10, and 13 after addition of ferulic acid. Concentrations of ferulic acid in the extracts were determined with a high performance liquid chromatograph. No breakdown products were detected. Models were developed to describe the recovery of ferulic acid from each soil material and soil component over time. Organic matter was the most active soil component involved in the irreversible retention of ferulic acid. The inorganic soil components were much less active than organic matter but appeared to be similar to each other in activity. Irreversible retention of ferulic acid by soil and soil components was greatest as pH 7.5.     

A Coenzyme‐Independent Decarboxylase/Oxygenase Cascade for the Efficient Synthesis of Vanillin

Vanillin is one of the most widely used flavor compounds in the world as well as a promising versatile building block. The biotechnological production of vanillin from plant‐derived ferulic acid has attracted much attention as a new alternative to chemical synthesis. One limitation of the known metabolic pathway to vanillin is its requirement for expensive coenzymes. Here, we developed a novel route to vanillin from ferulic acid that does not require any coenzymes. This artificial pathway consists of a coenzyme‐independent decarboxylase and a coenzyme‐independent oxygenase. When Escherichia coli cells harboring the decarboxylase/oxygenase cascade were incubated with ferulic acid, the cells efficiently synthesized vanillin (8.0 mM , 1.2 g L ^?1) via 4‐vinylguaiacol in one pot, without the generation of any detectable aromatic by‐products. The efficient method described here might be applicable to the synthesis of other high‐value chemicals from plant‐derived aromatics.     

Soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) Root Lignification Induced by Ferulic Acid. The Possible Mode of Action

Ferulic acid, in the form of feruloyl CoA, occupies a central position as an intermediate in the phenylpropanoid pathway. Due to the allelopathic function, its effects were tested on root growth, H(2)O(2) and lignin contents, and activities of cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) and peroxidase (POD, EC 1.11.1.7) from soybean (Glycine max (L.) Merr.) root seedlings. Three-day-old seedlings were cultivated in half-strength Hoagland's solution (pH 6.0), with or without 1.0 mM ferulic acid in a growth chamber (25 degrees C, 12/12 hr light/dark photoperiod, irradiance of 280 mumol m(-2) s(-1)) for 24 or 48 hr. Exogenously supplied ferulic acid induced premature cessation of root growth, with disintegration of the root cap, compression of cells in the quiescent center, increase of the vascular cylinder diameter, and earlier lignification of the metaxylem. Moreover, the allelochemical decreased CAD activity and H(2)O(2) level and increased the anionic isoform PODa5 activity and lignin content. The lignin monomer composition of ferulic acid-exposed roots revealed a significant increase of guaiacyl (G) units. When applied jointly with piperonylic acid (an inhibitor of the cinnamate 4-hydroxylase, C4H), ferulic acid increased lignin content. By contrast, the application of 3,4-(methylenedioxy) cinnamic acid (an inhibitor of the 4-coumarate:CoA ligase, 4CL) with ferulic acid did not. Taken together, these results suggest that ferulic acid may be channeled into the phenylpropanoid pathway (by the 4CL reaction) and, further, may increase the lignin monomer amount solidifying the cell wall and restricting the root growth.     

Influence of Pretreatment Stresses on Inhibitory Effects of Ferulic Acid, an Allelopathic Phenolic Acid

Experiments were conducted to determine the potential for acclimation (i.e., increased tolerance) to allelopathic phenolic acids resulting from pretreatment of seedlings with allelochemical (ferulic acid), drought, or nutrient stress. Cucumber seedlings were exposed to pretreatment stresses in a nutrient culture system for nine days, starting with day 3. Seedlings were subsequently treated for 5 hr with 0, 0.25, 0.5, or 0.75 mM ferulic acid. Acclimation (tolerance) was quantified by percentage inhibition of net phosphorus uptake. Seedlings grown with ferulic acid or drought pretreatment stresses were more tolerant to subsequent ferulic acid treatments (i.e., inhibition of net phosphorus uptake by ferulic acid was reduced). Nutrient pretreatment stress eliminated the inhibitory activity of ferulic acid on net phosphorus uptake. The results suggest that a general acclimation response to a variety of pretreatment stresses can confer an increased tolerance of plants to allelopathic phenolic acids such as ferulic acid.     

Radical Scavenging Activity of Lipophilized Products from Lipase-Catalyzed Transesterification of Triolein with Cinnamic and Ferulic Acids

Lipase-catalyzed transesterification of triolein with cinnamic and ferulic acids using an immobilized lipase from Candida antarctica (E.C. 3.1.1.3) was conducted to evaluate the antioxidant activity of the lipophilized products as model systems for enhanced protection of unsaturated oil. The lipophilized products were identified using ESI-MS. Free radical scavenging activity was determined using the DPPH radical method. The polarity of the solvents proved important in determining the radical scavenging activity of the substrates. Ferulic acid showed much higher radical scavenging activity than cinnamic acid, which has limited activity. The esterification of cinnamic acid and ferulic acid with triolein resulted in significant increase and decrease in the radical scavenging activity, respectively. These opposite effects were due to the effect of addition of electron-donating alkyl groups on the predominant mechanism of reaction (hydrogen atom transfer or electron transfer) of a species with DPPH. The effect of esterification of cinnamic acid was confirmed using ethyl cinnamate which greatly enhances the radical scavenging activity. Although, compared to the lipophilized cinnamic acid product, the activity was lower. The radical scavenging activity of the main component isolated from lipophilized cinnamic acid product using solid phase extraction, monocinnamoyl dioleoyl glycerol, was as good as the unseparated mixture of lipophilized product. Based on the ratio of a substrate to DPPH concentration, lipophilized ferulic acid was a much more efficient radical scavenger than lipophilized cinnamic acid.