基于响应面设计脂肪酶Novo435催化合成甘油二酯的工艺优化

以甘油、油酸为原料,优化在无溶剂体系中以固定化脂肪酶Novo435催化合成甘油二酯（diglyceride,DAG）的工艺。系统考察底物摩尔比（油酸／甘油）、反应温度、时间和加酶量等因素对油酸转化率和甘油二酯含量影响的基础上,利用响应面试验设计优化各主效因子,并经回归分析获得最优的工艺条件。所得最优条件：油酸与甘油底物摩尔比2．27、反应温度48．14℃、反应时间6．3h、加酶量1．68％。在此条件下,实验测得油酸转化率为45．42％,甘油二酯质量分数为70．01％,与响应面模型预测值吻合。     

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.     

Statistical optimization of the medium composition by response surface methodology to enhance schizophyllan production by Schizophyllum commune

The response surface methodology (RSM) involving central composite design (CCD) was employed to optimize the fermentation medium for the cell growth and schizophllan production by Schizophyllum commune CGMCC 5.113 in submerged culture at pH 6.5 and 26 degrees C. The four variables involved in this study were glucose, yeast extract, ammonium nitrate, and magnesium sulfate. The statistical analysis of the results showed that, in the range studied, glucose and yeast extract had a highly significant effect on schizophyllan production. The optimal medium for schizophyllan production calculated from the regression model of RSM was as follows: glucose, 18 g/l; yeast extract, 0.5 g/l; NH4NO3, 0.48 g/l; and MgSO4, 0.05 g/l, with a predicted maximum schizophyllan production of 11.74 g/l. These predicted values were experimentally validated. The excellent correlation between predicted and measured values justifies the validity of the response model. The results of bioreactor fermentation also show that the optimized medium enhanced schizophyllan production (12.80 g/l) by S. commune in a 5-1 fermenter.     

Purification of lipase from Cunninghamella verticillata and optimization of enzyme activity using response surface methodology

The fungus Cunninghamella verticillata was selected from isolates of oil-mill waste as a potent lipase producer as determined by the Rhodamine-B plate method. The lipase was purified from C. verticillata by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The purified enzyme was formed from a monomeric protein with molecular masses of 49 and 42 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 7.5 and the optimum temperature at pH 7.5 was 40 °C. The enzyme was stable between a pH range of 7.5 and 9.0 at 30 °C for 24 h. The enzyme activity was strongly inhibited by AgNO₃, NiCl₂, HgCl₂, CdCl₂ and EDTA. However, the presence of Ca²⁺, Mn²⁺ and Ba²⁺ ions enhanced the activity of the enzyme. The activity of purified lipase with respect to pH, temperature and salt concentration was optimized using a Box–Behnken design experiment. A polynomial regression model used in analysing this data, showed a significant lack of fitness. Therefore, quadratic terms were incorporated in the regression model through variables. Maximum lipase activity (100%) was observed with 2 mM CaCl₂, (pH 7.5) at a temperature of 40 °C. Regression co-efficient correlation was calculated as 0.9956.     

Optimization of enzymatic hydrolysis of mango kernel starch by response surface methodology

Amyloglucosidase (EC 3.2.1.3) from Aspergillus niger was employed for the saccharification of mango (Mangifera indica Linn) kernel starch. Response surface methodology based on a three-level three-factor Box-Behnken design was employed to optimize the important process variables such as substrate concentration (137.5-412.5 mg), enzyme concentration (4-12 mg) and temperature (35-55 °C). The sugar yield increased with both enzyme concentration and temperature, and decreased with substrate concentration. The response surface model indicated optimum conditions (substrate, 137.5 mg; enzyme, 12 mg; temperature, 55 °C) for obtaining 0.4851 mg sugar/mg substrate, which was also verified experimentally.     

A glycerol-inducible thermostable lipase from Bacillus sp.: medium optimization by a Plackett-Burman design and by response surface methodology

The production of a neutral lipase from a Bacillus sp. was improved tremendously (193-fold) following media optimization involving both the "one-at-a-time" and the statistical designing approaches. The present lipase was poorly induced by oils, instead its production was induced in the presence of sugars and sugar alcohols, mainly galactose, lactose, glycerol, and mannitol. A high inoculum density of 15% v/v (A550 = 0.8) led to maximum lipase production. Interestingly, the enzyme induction was growth independent, a property very different from most of the lipases investigated to date. The optimal composition of the growth medium to achieve maximum lipase production was determined to be as follows: NH4Cl, 35 g x L(-1); glycerol, 10 mL x L(-1); K2HPO4, 3 g x L(-1); KH2PO4, 1 g x L(-1); MgSO4.7H2O, 0.1 g x L(-1); glucose, 2 g x L(-1); MgCl2, 0.6 mmol x L(-1), with 15% inoculum density and an incubation period of 24 h. About 62 U x mL(-1) of enzyme production was achieved in the optimized medium.     

Activation of hepatic lipase catalyzed phosphatidylcholine hydrolysis by apolipoprotein E

The effect of apolipoproteins A-I, A-II, C-II, C-III and E on the hydrolysis of phosphatidylcholine and triacylglycerol by hepatic lipase was studied. Hepatic lipase catalyzed phospholipid hydrolysis was 1.8-fold activated by apolipoprotein E while the other apolipoproteins did not affect the hydrolysis by this enzyme. Triacylglycerol hydrolysis by hepatic lipase was 1.5-fold activated by apolipoprotein E while the other apolipoproteins inhibited hepatic lipase. These results suggest that lipoproteins containing apolipoprotein E may be preferred substrates for hepatic lipase.     

Statistical optimization of production conditions of alkaline pectin lyase from Bacillus cereus using response surface methodology

Alkaline pectin lyase finds applications in the degumming and retting of plant fibres, textile industry and pectic wastewater treatment where it degrades highly methylesterified pectin without prior action of any other pectinase. Response surface methodology (RSM) has been frequently utilized for the optimization of production process of industrially important enzymes from microbes. In the present work, fermentation conditions for the production of pectin lyase from Bacillus cereus were optimized using the factorial and central composite design of RSM. The cubic order polynomial regression model was found to be adequate and significant with a determination coefficient R² of 0.9505 (p?相似文献   

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.     

Preparation of stearoyl lactic acid ester catalyzed by lipases from Rhizomucor miehei and porcine pancreas optimization using response surface methodology

The esterification reaction between stearic acid and lactic acid using Rhizomucor miehei lipase and porcine pancreas lipase was optimized for maximum esterification using response surface methodology. The formation of the ester was found to depend on three parameters namely enzyme/substrate ratio, lactic acid (stearic acid) concentration and incubation period. The maximum esterification predicted by theoretical equations for both lipases matched well with the observed experimental values. In the case of R. miehei lipase, stearoyl lactic acid ester formation was found to increase with incubation period and lactic acid (stearic acid) concentrations with maximum esterification of 26.9% at an enzyme/substrate (E/S) ratio of 125 g mol⁻¹. In the case of porcine pancreas lipase, esterification showed a steady increase with increase in incubation period and lactic acid (stearic acid) concentration independent of the E/S ratios employed. In the case of PPL, a maximum esterification of 18.9% was observed at an E/S ratio of 25 g mol⁻¹ at a lactic acid (stearic acid) concentration of 0.09 M after an incubation period of 72 h. Received: 12 February 1999 / Received revision: 31 May 1999 / Accepted: 4 June 1999     

Statistical modelling and optimization of substrate composition for bacterial growth and cadmium removal using response surface methodology

In this study, substrate composition was optimized for the growth of Achromobacter xyloxidans and biosorption of Cd(II) from aqueous solution. Response surface methodology (RSM) was used to investigate the function of three independent operating variables, namely, peptone (2.5-10 g/L), beef extract (2.5-5.0 g/L) and incubation time (24-96 h), on dependent variables, i.e. sorption of Cd(II) ions, protein content and biomass growth of A. xyloxidans. The maximum Cd(II) removal efficiency of 69.2%, protein content 1.9 mg/L and growth 0.354 optical density was found at optimal conditions of peptone 10 g/L, incubation time 60 h and beef extract 2.5 g/L. The significance of independent variables and interactions between variables were tested by means of the analysis of variance (ANOVA) with 95% confidence limits and values of “Prob > F” less than 0.0500 indicate that model terms are significant. Fourier transfer infrared (FTIR) analysis was used to investigate sorption mechanism and involved functional groups in Cd(II) binding.     

Statistical optimization of alpha-amylase production by Streptomyces erumpens MTCC 7317 cells in calcium alginate beads using response surface methodology.

Alpha-amylase has a wide range of applications in starch industries, i.e. baking, brewing, distillery, etc. The alpha-amylase production from Streptomyces erumpens MTCC 7317 immobilized cells was compared with that of free cells. The immobilized cells of S. erumpens in calcium alginate beads were more effective for production of alpha-amylase (12.2% more yield) than free cells. Response surface methodology (RSM) was used to evaluate the effect of main variables, i.e. incubation period, pH and temperature on enzyme production with immobilized cells. A full factorial Central Composite Design (CCD) was applied to study these main factors that affected alpha-amylase production. The experimental results showed that the optimum incubation period, pH and temperature were 36 h, 6.0 and 50 degrees C, respectively for immobilized cells. Repeated batch fermentation of immobilized cells in shake flasks carried out in starch-beef extract medium showed that S. erumpens cells were physiologically active on the support even after four cycles of fermentation.     

Optimization of isoamyl acetate production by using immobilized lipase from Mucor miehei by response surface methodology

Immobilized lipase from Mucor miehei was employed for the esterification of isoamyl alcohol with acetic acid in n-heptane solvent. The important process variables studied were enzyme/substrate (E/S) ratio, alcohol (acid) concentration, and incubation period. Based on Box-Behnken design of experiments, a second order response function was developed. The percentage esterification increased with both E/S ratio and time and decreased with alcohol (acid) concentration. The model indicated optimum conditions for maximum esterification ranging from 20 to 99.6% in the alcohol (acid) concentration range of 0.031 to 0.3 M for a range of E/S ratios 8.33 to 50 g/mol, which were in good agreement with the experimental yields.     

响应面法快速优化曲霉Aspergillus sp.F044产脂肪酶培养条件

运用逐因子试验(Seriatim-Factorial Experiment)、Plackett-Burman、Response Surface Methodology(RSM)和单因子试验(Monofactorial Experiment)分析对产脂肪酶曲霉Aspergillus sp.F044产酶培养条件进行了快速优化。首先运用逐因子试验确定Aspergillus sp.F044产脂肪酶最适碳源和氮源,分别为麦芽糖和牛肉膏。在此基础上,通过Plackett-Burrman设计对影响其产酶相关因素进行评估并筛选出具有显著效应的橄榄油、牛肉膏、硫酸镁三个因素。然后通过实验拟合上述三因素与发酵液脂肪酶活力的一阶线性模型,沿着一阶模型给定的路径进行最速上升试验,在上升最高点处由中心组合试验和向应面分析确定其最优培养基组成;最后通过单因子试验确定最适发酵温度和摇床转速。试验优化的最优培养条件为:麦芽糖1.5%(w/v),硫酸铵7‰(w/v),磷酸氢二钾1‰(w/v),牛肉膏1.25%(w/v),硫酸镁2.11‰(w/v),橄榄油1.41%(v/v),自然pH,250r/min和30℃培养72h酶活达32.15U/mL。与初始11.18U相比,酶活提高2.88倍。     

Statistical optimization of glucose oxidase production from Aspergillus niger NRC9 under submerged fermentation using response surface methodology

Response surface methodology (RSM), employing the fractional factorial design (FFD) was used to optimize the fermentation medium for the production of glucose oxidase (GOD) from a marine isolate (NRC9) of Aspergillus niger under submerged fermentation. The design was employed by selecting glucose, CaCO_3, ammonium phosphate and MgSO₄ concentrations as model factors by ‘one variable at a time’ experiment. A second-order quadratic model and response surface method showed that the optimum concentrations (g/l) glucose, 100; CaCO₃, 25; (NH₄)₂HPO₄, 1.8 and 0.4 of MgSO₄, resulted in an improvement of GOD production (170?±?0.88 U/ml) as compared to the initial level (109.81?±?1.38 U/ml) after four days of incubation at 200 rpm and 30 °C, whereas its predicted value obtained by the quadratic model was 164.36 U/ml. Analysis of variance (ANOVA) showed a high coefficient of determination value (R ²) of 0.967, ensuring a satisfactory adjustment of the quadratic model with the experimental data. This is the first report on production of glucose oxidase from a marine fungal isolate, Aspergillus niger NRC9, using statistical experimental design and response surface methodology in optimization of its production under submerged fermentation.     

Statistical analysis and optimization of nitrogen,phosphorus, and inoculum concentrations for the biodegradation of petroleum hydrocarbons by response surface methodology

In order to optimize and evaluate the influence of nitrogen, phosphorus, and inoculum concentrations on the biodegradation of hydrocarbon contaminated effluents, experiments based on central composite design (CCD) method were carried out for 3 days, employing C1 mixed culture and intermittent aeration. The independent variables were nitrogen concentration (X ₁), phosphorus concentration (X ₂), and inoculum concentration (X ₃) and the removal of total petroleum hydrocarbons (TPH) was the dependent variable. The optimized nutrients ratio (C:N:P = 100:20:2.7) and inoculum concentration (1.32 g/l) provided TPH removal of 71.8% after processing for three days. Analysis using gas chromatography identified five hydrocarbons classes: paraffins, isoparaffins, olefins, naphthenics, and aromatics. The naphthenic compounds did not degrade as readily as the other hydrocarbons that were identified. The following degradation percentages were obtained: 87.1% for the paraffins, 77.7% for the isoparaffins, 78.6% for the olefins, 38.4% for the naphthenics, and 71.7% for the aromatics.     

Statistical optimization of l-leucine amino peptidase production from Streptomyces gedanensis IFO 13427 under submerged fermentation using response surface methodology

Response surface methodology (RSM) employing the central composite design (CCD) was used to optimize the fermentation medium for the production of l-leucine amino peptidase (LAP) from Streptomyces gedanensis IFO13427 under submerged fermentation. The design was employed by selecting substrate concentration, NaCl concentration and initial pH as model factors by ‘one variable at a time’ experiment. A second-order quadratic model and response surface method showed that the optimum conditions (soy bean 0.3%, NaCl, 0.03 M, and initial pH 7) resulted in the improvement of LAP production (25.69 IU/ml) as compared to the initial level (12.17 ± 0.23 IU/ml) after 72 h of fermentation, whereas its value predicted by the quadratic model was 24.56 IU/ml. Analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.9799, ensuring a satisfactory adjustment of the quadratic model with the experimental data. This is first report on LAP production by S. gedanensis using statistical experimental design and response surface methodology in submerged fermentation.     

Optimization of medium composition for lipase production by Candida rugosa NCIM 3462 using response surface methodology

A sequential optimization approach using statistical design of experiments was employed to enhance the lipase production by Candida rugosa in submerged batch fermentation. Twelve medium components were evaluated initially using the Plackett-Burman 2-level factorial design. The significant variables affecting lipase production were found to be glucose, olive oil, peptone, (NH4)2SO4, and FeCl3.6H2O. Various vegetable oils were tested in the second step, and among them, groundnut oil was found to be the best inducer for lipase production by C. rugosa. The third step was to identify the optimal values of the significant medium components with groundnut oil as the inducer using response surface methodology. The regression equation obtained from the experimental data designed using a central composite design was solved, and analyzing the response surface contour plots, the optimal concentrations of the significant variables were determined. A maximum lipase activity of 5.95 U.mL-1, which is 1.64 times the maximum activity obtained in the Plackett-Burman experimental trials, was observed. The optimum combination of medium constituents contained 19.604 g.L-1 glucose, 13.065 mL.L-1 groundnut oil, 7.473 g.L-1 peptone, 0.962 g.L-1 (NH4)2SO4, 0.0019 g.L-1 FeCl3.6H2O, and other insignificant components at the fixed level. A predictive model of the combined effects of the independent variables using response surface methodology and an artificial neural network was proposed. The unstructured kinetic models, logistic model, and Luedeking-Piret model were used to describe cell mass and lipase production. The parameters of the models were evaluated and the lipase production by C. rugosa was found to be growth associated.