Headspace ethylene accumulation on Stizolobium hassjoo hairy root culture producing L-3,4-dihydroxyphenylalanine

The addition of 1-aminocyclopropane-1-carboxylic acid (ethylene precursor), or 2-chloroethylphosphonic acid (ethephon, an ethylene-releasing compound) decreased root dry weight and l-DOPA (l-3,4-dihydroxyphenylalanine) accumulation in hairy root cultures of Stizolobium hassjoo. The inhibition caused by ethephon-mediated ethylene release was alleviated by 0.5 mg CoCl₂ l^–1 as an inhibitor of ethylene biosynthesis. The action of ethylene was inhibited by 1.5 mg AgNO₃ l^–1. Ethylene thus lowers hairy root formation and l-DOPA production; CoCl₂ decreases ethylene formation leading to a considerably improved root dry weight and l-DOPA production.     

Lateral root bridging as a strategy to enhance L-DOPA production in Stizolobium hassjoo hairy root cultures by using a mesh hindrance mist trickling bioreactor

Stizolobium hassjoo hairy roots exhibited a lateral root bridging behavior, enabling not only root dry weight but enhancement of intracellular L-DOPA content. When a single root tip was exerted a proper hindrance, the primary root growth was inhibited while lateral roots were profusely induced. The hindrance-induced lateral roots from individual primary root could bridge together under appropriate inoculation densities, leading to high density hairy root cultures producing secondary metabolites. In the present paper, a novel bioreactor was proposed based on a strategy of lateral root bridging by utilizing mesh as a hindrance, called "mesh hindrance mist trickling bioreactor (MHMTB)". Significant improvements of dry weight and L-DOPA production by using MHMTB were 1.8 and 2.2-folds, respectively, higher than those in the control run without the mesh hindrance within the root bed.     

应用响应面法优化发酵培养基提高达托霉素产量

[背景]达托霉素来自玫瑰孢链霉菌NRRL 11379的发酵产物,是重要的临床用抗生素.其原始产生菌发酵周期长,影响达托霉素的生产效率.本实验室前期在天蓝色链霉菌中重构了达托霉素的生物合成途径,有效地缩短了发酵周期,但重组菌株K10中达托霉素发酵产量很低,制约了后续的研究和开发.[目的]利用响应面法优化产达托霉素的重组菌...     

Establishment of transformed root cultures of Perezia cuernavacana producing the sesquiterpene quinone perezone

Summary The sesquiterpene quinone currently known as perezone is abundantly produced by the roots of Perezia cuernavacana. This compound is of biotechnological interest since it may be used as a pigment and has several pharmacological properties. In this work we demonstrate that perezone is also produced in transformed root cultures of P. cuernavacana. Hairy roots were induced by inoculation of internodal segments of sterile plants of P. cuernavacana with Agrobacterium rhizogenes AR12 strain. The axenic liquid MS medium cultures of the hairy roots isolated from the internodes showed active growth in the absence of growth regulators. The transformed nature of the tissue was confirmed by genomic integration (PCR and slot blot hybridization) and expression (enzyme activity) of the marker gus-gene. The production of perezone by a transformed root culture was evidenced by IR spectroscopy. Our results offer an alternative for enhanced production of perezone and represent an advantage over its extraction from natural plant populations which present problems in their agronomic culture.     

Application of response surface methodology for optimization of acidogenesis of cattail by rumen cultures

Acidogenesis of cattail using rumen cultures was carried out to produce volatile fatty acids (VFA) in this study. The influences of pH and substrate concentration on cattail degradation, VFA yield and microbial growth were investigated by using response surface methodology (RSM). Experimental results showed that a low substrate concentration and pH of 6.9 were optimal for acidogenesis of cattail. The highest cattail degradation efficiency, VFA yield and microbial yield were 75.9%, 0.41 g/g VS and 0.110 g/g VS, respectively. Further experiments confirmed that the main VFA in the acidogenesis of cattail were acetate, propionate and butyrate, while i-butyrate, valerate and i-valerate were also produced at low levels. The results suggested that acidogenesis using rumen cultures is a promising method for cattail disposal.     

Medium optimization for the production of recombinant nattokinase by Bacillus subtilis using response surface methodology

Nattokinase is a potent fibrinolytic enzyme with the potential for fighting cardiovascular diseases. Most recently, a new Bacillus subtilis/Escherichia coli (B. subtilis/E. coli) shuttle vector has been developed to achieve stable production of recombinant nattokinase in B. subtilis (Chen; et al. 2007, 23, 808-813). With this developed B. subtilis strain, the design of an optimum but cost-effective medium for high-level production of recombinant nattokinase was attempted by using response surface methodology. On the basis of the Plackett-Burman design, three critical medium components were selected. Subsequently, the optimum combination of selected factors was investigated by the Box-Behnken design. As a result, it gave the predicted maximum production of recombinant nattokinase with 71 500 CU/mL for shake-flask cultures when the concentrations of soybean hydrolysate, potassium phosphate, and calcium chloride in medium were at 6.100, 0.415, and 0.015%, respectively. This was further verified by a duplicated experiment. Moreover, the production scheme based on the optimum medium was scaled up in a fermenter. The batch fermentation of 3 L was carried out by controlling the condition at 37 degrees C and dissolved oxygen reaching 20% of air saturation level while the fermentation pH was initially set at 8.5. Without the need for controlling the broth pH, recombinant nattokinase production with a yield of 77 400 CU/mL (corresponding to 560 mg/L) could be obtained in the culture broth within 24 h. In particular, the recombinant B. subtilis strain was found fully stable at the end of fermentation when grown on the optimum medium. Overall, it indicates the success of this experimental design approach in formulating a simple and cost-effective medium, which provides the developed strain with sufficient nutrient supplements for stable and high-level production of recombinant nattokinase in a fermenter.     

响应面法优化獐牙菜苦苷生物转化条件

采用响应面法对獐牙菜苦苷的生物转化条件进行优化,首先应用Plackett-Burman方法筛选出对转化具有显著性影响的因素,再用中心响应面法的Response Optimizer工具对最优转化条件进行了分析和预测,最后对预测结果进行了验证。结果表明,獐牙菜新碱的最优化转化条件(mg/mL)为MgSO4.7H2O 5.14、MnSO4.4H2O 3.42、葡萄糖9.95、蛋白胨9.24、pH5.80。在此条件下獐牙菜新碱的最大转化产率为17.64%;红百金花内酯的最优转化条件(mg/mL)为MgSO4.7H2O 4.09、MnSO4.4H2O6.07、葡萄糖7.37、酵母膏7.37,pH5.73。在此条件下红百金花内酯的最大转化产率为8.81%。     

响应面法优化热带假丝酵母104菌株产羰基还原酶发酵培养基

通过菌种优选得到产高选择性羰基还原酶的热带假丝酵母(Candida tropicalis)104菌株,可不对称还原1-(3,5-二三氟甲基)苯基乙酮生成(S)-1-(3,5-二三氟甲基)苯基乙醇,对映体过量值>99.9%。采用部分因子实验设计考察发酵培养基中各组分对产酶的影响,结果表明,酵母粉、葡萄糖和NH4Cl浓度对产酶影响显著。继而采用最陡爬坡路径逼近最大响应区域,并结合中心组合实验和响应面对3个显著性因素进行分析,得到优化的发酵培养基组成:葡萄糖47.14g/L,酵母粉13.25g/L,NH4Cl2.71g/L,MgSO4·7H2O0.4g/L,KH2PO41g/L和K2HPO41g/L。采用该优化培养基,供试菌株的羰基还原酶活力达851.13U/L,较优化前提高了65.2%。     

Medium optimization by combination of response surface methodology and desirability function: an application in glutamine production

An optimization strategy based on desirability function approach (DFA) together with response surface methodology (RSM) has been used to optimize production medium in L-glutamine fermentation. Fermentation problems often force to reach a compromise between different experimental variables in order to achieve the most suitable strategy applying in industrial production. The importance of the use of multi-objective optimization methods lies in the ability to cope with this kind of problems. A sequential RSM with different combinations of glucose and (NH₄)₂SO₄ was performed to attain the optimal medium (OM-1) in glutamine production. Based on the result of RSM and the evaluation of production cost, a more economical optimal medium (OM-2) was obtained with the aid of DFA. In DFA study, glutamate, the main by-product in glutamine fermentation as another response was considered. Compared with OM-1 in validated experiment, similar amounts of glutamine were obtained in OM-2 while the concentration of glutamate and the production cost decreased by 53.6 and 7.1%, respectively.     

基于响应面法对香菇原生质体制备条件的优化

香菇Lentinula edodes是我国食用菌年产量最大的单品,优化香菇原生质体的制备条件,获得高质量、高活力的原生质体可以为香菇育种和遗传多样性分析提供技术保障。本研究利用单因素试验对稳渗剂浓度、酶解液类型、酶解时间、酶解温度4个因素的最优作用条件和相关性进行检验和筛选;依据单因素试验的结果,对酶解时间、酶解温度、酶解液种类进行响应面法3因素3水平试验优化分析。单因素试验结果表明,使用2%的溶壁酶+蜗牛酶+纤维素酶混合酶制剂为酶解液、0.6mol/L甘露醇为稳渗剂的效果显著高于其他处理(P<0.05)。经Box-Behnken设计得出香菇原生质体最佳制备条件为酶解时间3.25h,酶解液浓度1.7%,酶解温度30.49℃,获得原生质体产量为14.02×10⁶ CFU/mL,与理论产量(13.862×10⁶ CFU/mL)偏差小,可为后续原生质体融合育种和多样性分析提供重要基础。     

Medium optimization for antifungal active substances production from a newly isolated Paenibacillus sp. using response surface methodology

Statistics based experimental designs were used to optimize the medium for antifungal active substances production from a newly isolated Paenibacillus polymyxa Cp-S316 in shaker flask cultivation. The medium components having significant effect on the production were first identified using a fractional factorial design. Then steepest ascent method was employed to approach the experimental design space, followed by an application of response surface methodology for further optimization. A quadratic model was found to fit the antifungal active substances production. Response surface analysis revealed that the optimum values of the tested variables for the production of active substances were 12.3 (g/l) lactose, 17.5 (g/l) peptone, 0.4 (g/l) sodium nitrate, 4.5 (g/l) magnesium sulfate and 100 (g/l) potato. A production of 4687.71mug/ml, which was in agreement with the prediction, was observed in verification experiment. In comparison to the production of basal medium, 3.05-fold increase had been obtained.     

Process optimization of an auger pyrolyzer with heat carrier using response surface methodology

A 1 kg/h auger reactor utilizing mechanical mixing of steel shot heat carrier was used to pyrolyze red oak wood biomass. Response surface methodology was employed using a circumscribed central composite design of experiments to optimize the system. Factors investigated were: heat carrier inlet temperature and mass flow rate, rotational speed of screws in the reactor, and volumetric flow rate of sweep gas. Conditions for maximum bio-oil and minimum char yields were high flow rate of sweep gas (3.5 standard L/min), high heat carrier temperature (∼600 °C), high auger speeds (63 RPM) and high heat carrier mass flow rates (18 kg/h). Regression models for bio-oil and char yields are described including identification of a novel interaction effect between heat carrier mass flow rate and auger speed. Results suggest that auger reactors, which are rarely described in literature, are well suited for bio-oil production. The reactor achieved liquid yields greater than 73 wt.%.     

Medium optimization for the production of cold-active beta amylase by psychrotrophic Streptomyces MIUG 4 alga using response surface methodology

The aim of this work was to optimize the cultural and production parameters using statistical approach to the synthesis of cold-active beta amylase by Streptomyces MIUG 4 Alga in submerged fermentation (SmF). The process parameters influencing the enzyme production were identified using the Plackett-Burman design. Among the various parameters screened, the concentrations of glycerol, starch, and yeast extract were the most significant. The optimum levels of these significant parameters determined employing the response surface methodology (RSM) and central composite design (CCD) were as follows: glycerol (4.00%), starch (2.00%) and yeast extract (0.5%). By using the optimal fermentation medium! the cold-active beta amylase production was increased up to 246.62 AU, an approximate 2.6-fold improvement over the previous production (93.32 AU) with un-optimized medium. This is the first report on production of cold-active beta amylase by psychrotrophic Streptomyces sp. The present study indicates that the cold-adapted extracellular beta amylase of Streptomyces MIUG 4 Alga may have considerable potential for industrial application owing to its properties.     

Alkaloid production by transformed root cultures of Catharanthus roseus

Transformed roots of Catharanthus roseus were obtained following infection of detached leaves with Agrobacterium rhizogenes. Roots would not grow in full strength Gamborg's B5 medium but would grow satisfactorily if the medium was diluted to one half strength. Little alkaloid appeared in the growth medium but root tissue contained a high level and wide variety of alkaloids. Ajmalicine, serpentine, vindolinine and catharanthine were prominent components. Vinblastine could also be detected by a combination of HPLC and radioimmunoassay, though at a level of only 0.05g/g dry weight.Abbreviations B5 Gamborg's B5 nutrient salts - LC/MS combined liquid chromatography/mass spectrometry - FW fresh weight - Kb kilobase     

Formulation optimization of chelerythrine loaded O-carboxymethylchitosan microspheres using response surface methodology

The aims of this investigation were to develop a procedure to prepare chelerythrine (CHE) loaded O-carboxymethylchitosan (O-CMCS) microspheres by emulsion cross-linking method and optimize the process and formulation variables using response surface methodology (RSM) with a three-level, three-factor Box-Behnken design (BBD). The independent variables studied were O-CMCS/CHE ratio, O/W phase ratio, and O-CMCS concentration, dependent variables (responses) were drug loading content and encapsulation efficiency. Mathematical equations and response surface plots were used to relate the dependent and independent variables. The process and formulation variables were optimized to achieve maximum drug loading content and entrapment efficiency by the desirability function. The optimized microsphere formulation was characterized for particle size, shape, morphology and in vitro drug release. Results for mean particle size, drug loading content, entrapment efficiency, and in vitro drug release of CHE-loaded O-CMCS microspheres were found to be of 12.18 μm, 4.16 ± 3.36%, 57.40 ± 2.30%, and 54.5% at pH 7.4 after 70 h, respectively. The combination use of RSM, BBD and desirability function could provide a promising application for O-CMCS as controlled drug delivery carrier and help to develop procedures for a lab-scale microemulsion process.     

Medium optimization for the production of thermal stable beta-glucanase by Bacillus subtilis ZJF-1A5 using response surface methodology

Polysaccharides, such as barley flour, dextrin and soluble starch, were better carbon sources than monosaccharides and disaccharides, such as glucose and maltose, for cell growth of Bacillus subtilis ZJF-1A5 and beta-glucanase production. beta-Glucanase produced by B. subtilis ZJF-1A5 was associated partially with cell growth and increased significantly when cells entered stationary phase; yeast extract was the best nitrogen source, followed by soybean flour. All inorganic nitrogen sources chosen in the experiments were not favorable for cell growth and enzyme production. A fractional factorial design (2(6-2)) was applied to elucidate medium components that significantly affect beta-glucanase production. The concentration of barley flour, corn flour and soybean flour in medium were significant factors. The steepest ascent method was used to locate the optimal domain and a central composite design was used to estimate the quadratic response surface from which the factor levels for maximum production of beta-glucanase were determined. The composition of fermentation medium optimized with response surface methodology was (g/l): barley flour, 63.5; corn flour, 44.8; KH2PO4, 1.0; MgSO4 x 7H2O, 0.1; CaCl2, 0.1. beta-Glucanase activity was 251 U/ml at 48 h using optimized medium, 1.4 times higher than that in original medium.     

Statistical optimization of lipase catalyzed hydrolysis of methyloleate by response surface methodology

Hydrolysis of methyloleate was optimized using lipase from Chromobacterium viscosum immobilized on IRC-50. The optimization was studied by a statistical methodology using response surface methodology (RSM). The cumulative interactive effect of substrate, water concentration and time was studied in optimizing the hydrolysis of methyloleate. The interactive effect of substrate-time was found to be significant compared to substrate-water and time-water interactions. A well correlation was observed between the optimum values obtained from the response surface contour plots and from the quadratic regression model equation. The optimal values obtained for substrate, water and time were found to be in the experimental range chosen.     

Long-term stability of root cultures of tomato transformed with Agrobacterium rhizogenes R1601

Tomato explants were transformed with Agrobacter rhizogenesR1601 and root clones used to initiate longterm cultures inliquid and on solid media. The liquid cultures were maintainedfor 50 passages over 25 months in the presence and absence ofkanamycin. During this time the clones retained their high growthrates and antibiotic resistance phenotypes. The nptll gene wasdetected by PCR and dot blot hybridization in DNA from clonesat the 21st passage, and NPT-II enzyme activity was detectedin cell extracts prepared at the 45th passage. The solid cultureswere main tained for 12 passages over 12 months, either withcontinual selection, or with alternation during the last sixpassages between selective and non-selective media. The nptllgene was detected by PCR in DNA from cultures at the 5th passageand NPT-II enzyme activity was detected in cell extracts ofvigorously growing clones from the 12th passage. Passages inthe absence of selection had no discernible effect on the stabilityof the transformed state. The results indicate that the Ri-transformedstate can be maintained in tomato root clones during long periodsof culture. Key words: Agrobacterium rhizogenes, long-term cell culture, root clones, tomato, transformation     

Medium optimization of carbon and nitrogen sources for the production of spores from Bacillus amyloliquefaciens B128 using response surface methodology

The production of spores from Bacillus amyloliquefaciens B128 was investigated in shaker-flask cultivation. Optimization of the culture medium was carried out by using a two-step approach. A quick identification of a suitable source of carbon and nitrogen was obtained by a simple screening experiment, which was followed by an application of response surface methodology (RSM) for further optimization design. A five-level four-factor central composite design was employed to determine the maximum spore yield at optimum levels for lactose, tapioca, ammonium sulfate and peptone. Tapioca and peptone showed a significant linear main effect, while lactose and ammonium sulfate had no significant linear effect. The spore production was also significantly affected by lactose–ammonium sulfate and lactose–peptone. Optimum cultivation parameters were (g/L): 12.7 of lactose, 16.7 of tapioca, 1.8 of ammonium sulfate and 8.0 of peptone. The prediction spore yield was 5.93 × 10⁸ (no/mL). The actual experimental results were in agreement with the prediction.