单因子-响应面法优化白地霉Y162产脂肪酶条件

对白地霉Y162液体发酵产脂肪酶的条件进行了优化。首先采用单因子实验筛选出最适碳源为橄榄油,氮源为黄豆粉和NH4Cl,无机盐为BaCl2和MgCl2。在此基础上,利用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出具有显著效应的橄榄油、BaCl2和NH4Cl三个最显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面中心组合设计对显著因素进行优化,得出橄榄油、BaCl2和NH4Cl最佳浓度分别为2.35％,0.36％,1.35％。优化后液体发酵液中脂肪酶活力提高到31.85 U/mL,比初始酶活力14.16 U/mL提高了2.25倍,表明单因子-响应面结合法可显著优化白地霉Y162液体发酵产脂肪酶条件。     

Optimized lipase-catalyzed synthesis of adipate ester in a solvent-free system

Immobilized Candida antarctica lipase-catalyzed esterification of adipic acid and oleyl alcohol was investigated in a solvent-free system (SFS). Optimum conditions for adipate ester synthesis in a stirred-tank reactor were determined by the response surface methodology (RSM) approach with respect to important reaction parameters including time, temperature, agitation speed, and amount of enzyme. A high conversion yield was achieved using low enzyme amounts of 2.5% w/w at 60°C, reaction time of 438 min, and agitation speed of 500 rpm. The good correlation between predicted value (96.0%) and actual value (95.5%) implies that the model derived from RSM allows better understanding of the effect of important reaction parameters on the lipase-catalyzed synthesis of adipate ester in an organic solvent-free system. Higher volumetric productivity compared to a solvent-based system was also offered by SFS. The results demonstrate that the solvent-free system is efficient for enzymatic synthesis of adipate ester.     

Statistical optimization of medium components and growth conditions by response surface methodology to enhance lipase production by Aspergillus carneus

A response surface approach has been used to study the production of an extracellular lipase from Aspergillus carneus, which has the property of immense industrial importance. Interactions were studied for five different variables (sunflower oil, glucose, peptone, agitation rate and incubation period), which were found influential for lipase production by one-at a time method. We report a 1.8-fold increase in production, with the final yield of 12.7 IU/ml in comparison to 7.2 U/ml obtained by one-at-a-time method. Using the statistical approach (response surface methodology (RSM)) the optimum values of these most influential parameters were as follows: sunflower oil (1%), glucose (0.8%), peptone (0.8%), agitation rate (200 rpm) and incubation period (96 h) at 37 °C. The subsequent verification experiment confirmed the validity of the model.     

Optimization of pancreatic lipase inhibition by Cudrania tricuspidata fruits using response surface methodology

The fruits of Cudrania tricuspidata (Carr.) Bur. (Moraceae) significantly inhibited pancreatic lipase, which plays a key role in fat absorption. Optimization of extraction conditions with minimum pancreatic lipase activity and maximum yield was determined using response surface methodology with three-level-three-factor Box–Behnken design (BBD). Regression analysis showed a good fit of the experimental data and the optimal condition was obtained as ethanol concentration, 74.5%; temperature 61.9 °C and extraction time, 13.5 h. The pancreatic lipase activity and extraction yield under optimal conditions were found to be 65.5% and 54.0%, respectively, which were well matched with the predicted value of 65.8% and 47.1%. Further fractionation of C. tricuspidata extract resulted in the isolation of compound 1, which was identified as 5,7,4′-trihydroxy-6,8-diprenylisoflavone. It inhibited pancreatic lipase activity with IC₅₀ value of 65.0 μM. HPLC analysis suggested positive correlation between pancreatic lipase inhibition and 5,7,4′-trihydroxy-6,8-diprenylisoflavone of C. tricuspidata fruits.     

Optimization of culture conditions for production of cis-epoxysuccinic acid hydrolase using response surface methodology

Response surface methodology, which allows for rapid identification of important factors and optimization of them to enhance enzyme production, was employed here to optimize culture conditions for the production of cis-epoxysuccinic acid hydrolase from Bordetella sp. strain 1–3. In the first step, a Plackett–Burman design was used to evaluate the effects of nine variables (yeast extract, cis-epoxysuccinic acid, KH₂PO₄, K₂HPO₄ · 3H₂O, MgSO₄ · 7H₂O, trace minerals solution, culture volume, initial pH and incubation time) on the enzyme production. Yeast extract, cis-epoxysuccinic acid and KH₂PO₄ had significant influences on cis-epoxysuccinic acid hydrolase production and their concentrations were further optimized using central composite design and response surface analysis. A combination of adjusting the concentration of yeast extract to 7.8 g/l, cis-epoxysuccinic acid to 9.8 g/l, and KH₂PO₄ to 1.12 g/l would favor maximum cis-epoxysuccinic acid hydrolase production. An enhancement of cis-epoxysuccinic acid hydrolase production from 5.6 U/ml to 9.27 U/ml was gained after optimization.     

Optimization of conditions for acid protease partitioning and purification in aqueous two-phase systems using response surface methodology

Aspergillopepsin I, an acid protease, was purified using an aqueous two-phase system that comprised various combinations of polyethylene glycol (PEG), NaH₂PO₄ and NaCl. Partition of the enzyme depended upon the molecular mass of the PEG and the presence of NaCl. With PEG 1500, 4000 and 6000, the partition coefficients were increased by 1,500-, 1,800- and 560-fold compared to values without NaCl. The presence of NaCl (8.75%, w/w) increased purification by 3.8, 9.5 and 2.8 times into these respective PEGs. The optimal aqueous two-phase system for acid protease purification was developed using response surface methodology. This system contained 17.3% of PEG 4000 (w/w), 15% NaH₂PO₄ (w/w) and 8.75% NaCl (w/w) and provided the best partition coefficient (Ke > 1,100) and yield over 99% in the same phase. The optimal ATPS purification factor of acid protease was over 5.     

响应面法优化粉被虫草中虫草素的提取工艺

【背景】粉被虫草是民间常用药用真菌,具有抗辐射等多种药理作用,虫草素是其重要的活性物质。【目的】探究粉被虫草中提取虫草素的影响因素,为制取虫草素提供参考。【方法】以虫草素得率为指标,用响应面法优化超声提取工艺。分别考察超声时间、液料比和超声功率对提取效率的影响,在单因素实验基础上进行3因素3水平的响应面分析实验。【结果】最优提取条件为超声时间606 s、液料比45.9:1、超声功率400 W,在此条件下虫草素得率为6.136 mg/g,与模型预测值接近,方程拟合良好。【结论】首次对粉被虫草中虫草素的提取条件进行了研究,将对制取虫草素提供参考,有利于粉被虫草的深度开发利用。     

Study on response surface methodology (RSM) of lipase-catalyzed synthesis of palm-based wax ester

The synthesis of wax ester using refined, bleached and deodorized (RBD) palm oil and oleyl alcohol catalyzed by lipozyme IM was carried out. Response surface methodology (RSM) based on a five-level, four-variable central composite rotatable design (CCRD) was used to evaluate the interactive effects of synthesis, of reaction time (2.5–10 h), temperature (30–70 °C), amount of enzyme (0.1–0.2 g) and substrate molar ratio (palm oil to oleyl alcohol, 1:1–1:5) on the percentage yield of wax esters. The optimum conditions derived via RSM were: reaction time 7.38 h, temperature 53.9 °C, amount of enzyme 0.149 g, and substrate molar ratio 1:3.41. The actual experimental yield was 84.6% under optimum condition, which compared well to the maximum predicted value of 85.4%.     

响应面分析法优化耐高温假黄色单胞菌硫氧化性能

【目的】采用响应面分析法对耐高温假黄色单胞菌的硫氧化性能进行优化。【方法】利用Plackett-Burman试验筛选影响菌株硫氧化性能的关键因子。通过最陡爬坡试验逼近最佳值区域,确定响应面试验中心轴,利用Box-Behnken设计和响应面分析法获得关键因素的最佳浓度。采用经典的改良硫酸钡比浊法测定硫酸根含量。【结果】牛肉膏、麦芽糖、镁离子(Mg~(2+))3个因素是影响菌株硫氧化性能的关键因素。响应面分析表明牛肉膏和镁离子的交互作用对硫酸根转化率的影响最大,优化后的结果为:麦芽糖(%)=0.07,牛肉膏(%)=0.11,Mg~(2+)(%)=0.04时,模型有最大值。模型的F值为52.60(P0.000 1),相关系数R2=0.980 2,说明该二次方程是显著的,该模型在整个回归区域内的拟合较好。经模拟堆肥试验验证该菌株可以有效增加堆肥中硫酸根含量。【结论】该模型可用于分析和预测耐高温假黄色单胞菌优化培养基配方,经优化后该菌株硫氧化性能大幅提升,硫酸根转化率由36.89%提高到80%以上,加入堆肥后与基础发酵条件下菌液相比更加有效增加了堆肥中SO_4~(2-)的含量,具有较好的应用前景。     

响应面法优化嗜热链球菌AR333电转化条件

【背景】嗜热链球菌AR333是本实验室从发酵乳中筛选出的一株高产活性胞外多糖乳酸菌。【目的】建立嗜热链球菌AR333高效电转化体系。【方法】通过单因素试验和Box-Behnken响应面法优化电转化条件。【结果】嗜热链球菌AR333最优电转化条件为甘氨酸浓度8.3g/L,OD_(600)为0.8,10%甘油(体积比)和0.5 mol/L蔗糖的电转缓冲液,pIB184质粒80 ng,电场强度14 kV/cm,0.4 mol/L山梨醇、2 mmol/L CaCl_2和20 mmol/L MgCl_2的LM17复苏培养基,复苏时间5 h。【结论】在最优电转化条件下,嗜热链球菌AR333电转化效率达到3.68×10~5 CFU/μg-DNA,比优化前提高了14倍,实现了嗜热链球菌AR333的高效遗传转化,为其功能解析和基因工程改造奠定基础。     

酶法合成2-氧-α-D-葡萄糖基-L-抗坏血酸(AA-2G)条件的研究

采用单因素优化法对环糊精葡萄糖苷转移酶(CGTase)合成糖基抗坏血酸(AA-2G)条件进行优化,AA-2G的产量为2.76 g/L,比未优化前0.46g/L提高了500%。再采用响应面法对AA-2G合成条件进行优化。由Plackett-Burman法筛选出三个主要因素为:pH、V_C和麦芽糊精浓度;由最陡爬坡实验得出最佳响应面区域;最后由Box-Behnken实验,得到最优条件为:pH 5.51,V_C36.16g/L,麦芽糊精28.54 g/L,转化时间24 h,温度37℃。在此条件下,AA-2G的理论产量为3.15 g/L,通过验证实验,得出AA-2G的产量为3.13 g/L,与预测的理论值接近,比单因素优化的结果(2.76g/L)提高了14%。     

Application of reverse micelle extraction process for amylase recovery using response surface methodology

The effect of different process variables of reverse micelle extraction process like pH, addition of surfactant (AOT) concentration and potassium chloride (KCl) concentration on amylase recovery has been studied and analysed. Solid-state fermentation was used for the production of amylase enzyme. Response surface methodology (RSM) using central composite rotatable design (CCRD) was employed to analyse and optimize the enzyme extraction process. The regression analysis indicates that the effect of AOT concentration, and KCl concentration were significant, whereas the effect of pH was non-significant on enzyme recovery. For the maximum recovery of enzyme, the optimum operating condition for pH, AOT concentration (M) and KCl concentration were 10.43, 0.05 and 1.00, respectively. Under these optimal conditions, the enzyme recovery was 83.16%.     

Use of response surface methodology to optimize culture medium for production of lipase with Candida sp. 99-125

Response surface methodology (RSM) was employed to optimize culture medium for production of lipase with Candida sp. 99-125. In the first step, a Plackett–Burmen design was used to evaluate the effects of different components in the culture medium. Soybean oil, soybean powder and K₂HPO₄ have significant influences on the lipase production. The concentrations of three factors were optimized subsequently using central composite designs and response surface analysis. The optimized condition allowed the production of lipase to be increased from 5000 to 6230 IU/ml in shake flask system. The lipase fermentation in 5 l fermenter reached 9600 IU/ml.     

Dual response surface-optimized process for feruloylated diacylglycerols by selective lipase-catalyzed transesterification in solvent free system

Feruloylated diacylglycerol (FDAG) was synthesized using a selective lipase-catalyzed the transesterification between ethyl ferulate and triolein. To optimize the reaction conversion and purity of FDAG, dual response surface was applied to determine the effects of five-level-five-factors and their reciprocal interactions on product synthesis. A total of 32 individual experiments were performed to study reaction temperature, reaction time, substrate molar ratio, enzyme loading, and water activity. The highest reaction conversion and selectivity towards FDAG were 73.9% and 92.3%, respectively, at 55 °C, reaction time 5.3 day, enzyme loading 30.4 mg/ml, water activity 0.08, and a substrate molar ratio of 3.7. Moreover, predicted values showed good validation with the experimental values when experiments corresponding to selected points on the contour plots were carried out.     

Optimization of extraction process of Glycyrrhiza glabra polysaccharides by response surface methodology

Experimental design was used to investigate the effect of three parameters (extraction time, extraction number and ratio of water to raw material) on polysaccharides yields. The ranges of the factors investigated were 3.5–4.5 h for extraction time (X₁), 4–6 for extraction number (X₂), and 25–35 for ratio of water to raw material (X₃). The statistical analysis of the experiment indicated that extraction time and ratio of water to raw material had significant effect on Glycyrrhiza glabra polysaccharides yields. The central composite design showed that polynomial regression models were in good agreement with the experimental results with the coefficients of determination of 0.924 for Glycyrrhiza glabra polysaccharides yield. The optimal condition for Glycyrrhiza glabra polysaccharides yield within the experimental range of the variables studied was at 4.3 h, 6, and 35. At this condition, the predicted yield of polysaccharides extracted was 3.6%.     

Kinetics and optimization of lipase-catalyzed synthesis of rose fragrance 2-phenylethyl acetate through transesterification

Lipase from Candida rugosa was immobilized on a polyvinylidene fluoride membrane for synthesis of rose flavor ester, 2-phenylethyl acetate. Response surface methodology (RSM) was employed for kinetic modeling of process and prediction the yield. The RSM was used in practice for determining the kinetic models by fitting the initial rate dates based on the equations of ping-pong bi–bi and order bi–bi model. The maximum reaction rate and kinetic constants were matched with the order bi–bi model. The specificity constant of the immobilized lipase was 10-folds higher than the free form indicated the enzyme–substrate affinity, and catalytic ability was enhanced after immobilization. Moreover, the effects of reaction parameters on the yield were evaluated by RSM using a Box–Behnken experimental design. Based on a ridge max analysis, the maximum conversion was 95.33 ± 2.57% at 38.78 h, 35.85 °C, and substrate mole ratio of 3.65:1. Furthermore, the order bi–bi kinetic model was simulated successfully in a batch reaction. A good prediction existed between the RSM results and integrated equation was found.     

响应面法优化细菌EHB01对铜绿微囊藻(Microcystis aeruginosa)的溶藻条件

【背景】水体富营养化导致的蓝藻水华对淡水资源造成了严重污染。利用环境友好型的溶藻菌可有效控制蓝藻的生长,是防治蓝藻水华形成的有效途径之一。【目的】优化溶藻细菌EHB01对铜绿微囊藻(Microcystis aeruginosa)的溶藻条件,以期为治理蓝藻水华污染提供高效的溶藻菌制剂。【方法】采用单因素试验对溶藻的发酵液浓度、温度、光照以及C:N和N:P进行分析,并对溶藻细菌EHB01发酵液的碳源、氮源和p H进行优化。基于单因素试验,选用中心组合试验设计(central composite design,CCD)确定关键因子的最佳数量水平,并以Desig-Expert 8.0.5进行回归分析,通过响应面分析获得溶藻效果最佳的参数。【结果】发酵液浓度对溶藻率的影响表现为持续上升;温度对溶藻率表现为先上升后下降;而光照、C:N和N:P均对细菌EHB01发酵液溶藻率的影响表现为先降低后上升的趋势。溶藻细菌EHB01发酵液所需的最佳碳源为蔗糖,氮源为硝酸钾,pH为7.5,优化条件下溶藻率达86.97%,与优化之前相比提高了21.72%。【结论】采用响应面法优化得出溶藻细菌EHB01发酵液最优的...     

响应面法优化枯草芽孢杆菌产脂肪酶的合成培养基

对枯草芽孢杆菌(Bacillus subtilis)CICC20034利用合成培养基液体发酵产脂肪酶的条件进行了优化。首先采用单因子实验筛选出最适诱导剂为三丁酸甘油酯,氮源为尿素,碳源为葡萄糖,无机盐为MgSO4。在此基础上,利用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出具有显著效应的三丁酸甘油酯、尿素、KH2PO4和培养基起始pH值4个最显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面中心组合设计对显著因素进行优化,获得最适合成培养基组分为:葡萄糖8g/L,尿素8.57g/L,三丁酸甘油酯2.62%,KH2PO42.59g/L,MgSO4.7H2O0.5g/L,TritonX-1000.5g/L,pH9.47。优化后的B.subtilis CICC 20034胞外脂肪酶活力达0.483U/ml,比初始酶活力0.072U/ml提高了6.7倍。     

Optimization of lipase-catalyzed biodiesel by response surface methodology

Biodiesel prepared by catalyzed mild transesterification has become of much current interest for bioenergy. The ability of a commercial immobilized lipase (Novo Industries--Bagsvaerd, Denmark) from Rhizomucor miehei (Lipozyme IM-77) to catalyze the transesterification of soybean oil and methanol was investigated in this study. Response surface methodology and 5-level-5-factor central composite rotatable design were employed to evaluate the effects on reaction time, temperature, enzyme amount, molar ratio of methanol to soybean oil, and added water content on percentage weight conversion to soybean oil methyl ester by transesterification. Based on ridge max analysis, the optimum synthesis conditions giving 92.2% weight conversion were: reaction time 6.3 h, temperature 36.5 degrees C, enzyme amount 0.9 BAUN (Batch Acidolysis Units NOVO), substrate molar ratio 3.4:1, and added water 5.8%.     

Optimization of ultrasonic-assisted extraction process of Poria cocos polysaccharides by response surface methodology

Polysaccharides production from Poria cocos was carried out using aqueous NaOH with the assistance of ultrasonic. Experimental design was used to investigate the effect of three parameters (extraction time, extraction concentration of NaOH, and ratio of aqueous NaOH to raw material) on polysaccharides yields. The ranges of the factors investigated were 1–3 min for extraction time (X₁), 0.5–1.0 mol/L for extraction concentration of NaOH (X₂), and 30–50 for ratio of aqueous NaOH to raw material (X₃). The statistical analysis of the experiment indicated that extraction concentration of NaOH had significant effect on P. cocos polysaccharides yields. The central composite design showed that polynomial regression models were in good agreement with the experimental results with the coefficients of determination of 0.9935 for P. cocos polysaccharides yield. The optimal condition for P. cocos polysaccharides yield within the experimental range of the variables studied was at 2.44 min, 0.789 mol/L, and 53.0. At this condition, the predicted yield of polysaccharides extracted was 82.3%.