Enhanced Production of a Novel Cyclic Hexapeptide Antibiotic (NW-G01) by Streptomyces alboflavus 313 Using Response Surface Methodology

NW-G01, produced by Streptomyces alboflavus 313, is a novel cyclic hexapeptide antibiotic with many potential applications, including antimicrobial activity and antitumor agents. This study developed a system for optimizing medium components in order to enhance NW-G01 production. In this study, Plackett-Burman design (PBD) was used to find the key ingredients of medium components, and then response surface methodology (RSM) was implemented to determine their optimal concentrations. The results of PBD revealed that the crucial ingredients related to the production of NW-G01 were (NH(4))(2)SO(4), peptone and CaCO(3). A prediction model has been built in the experiments of central composite design and response surface methodology, and its validation has been further verified. The optimal medium composition was determined (g/L): corn starch 15, glucose 15, peptone 3.80, (NH(4))(2)SO(4) 0.06, NaCl 1.5, CaCO(3) 1.30, MgSO(4)·7H(2)O 0.015, K(2)HPO(4)·3H(2)O 0.015, MnCl(2)·4H(2)O 0.015, FeSO(4)·7H(2)O 0.015, and ZnSO(4)·7H(2)O 0.015. Compared with NW-G01 production (5.707 mg/L) in non-optimized fermentation medium, the production of NW-G01 (15.564 mg/L) in optimized fermentation medium had a 2.73-fold increase.     

Optimization of Total Flavonoid Compound Extraction from Gynura medica Leaf Using Response Surface Methodology and Chemical Composition Analysis

Optimization of total flavonoid compound (TFC) extraction from Gynura medica leaf was investigated using response surface methodology (RSM) in this paper. The conditions investigated were 30-60% (v/v) ethanol concentration (X(1)), 85-95 °C extraction temperature (X(2)) and 30-50 (v/w) liquid-to-solid ratio (X(3)). Statistical analysis of the experiments indicated that temperature and liquid-to-solid ratio significantly affected TFC extraction (p < 0.01). The Box-Behnken experiment design showed that polynomial regression models were in good agreement with the experimental results, with the coefficients of determination of 0.9325 for TFC yield. The optimal conditions for maximum TFC yield were 55% ethanol, 92 °C and 50 (v/w) liquid-to-solid ratio with a 30 min extraction time. Extracts from these conditions showed a moderate antioxidant value of 54.78 μmol quercetin/g dry material (DM), 137.3 μmol trolox/g DM for 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 108.21 μmol quercetin/g DM, 242.31 μmol trolox/g DM for 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS(+)), respectively. HPLC-DAD-MS analysis showed that kaempferol-3-O-glucoside was the principal flavonoid compound in Gynura medica leaf.     

响应面法优化产类胡萝卜素红酵母液体发酵培养基的研究

采用响应面法对类胡萝卜素产生菌红酵母(Rhodotorula ap.D)的液体发酵培养基进行了优化.用PlackettBurman方法研究了酵母膏、硫酸铵和蔗糖等20个营养因素对类胡萝卜素产量的影响,结果表明,主要影响因素为酵母膏、硫酸铵和蔗糖(P＜0.05).通过中心组合实验及响应面分析优化了这三个因素,得到优化发酵培养基组成(g·L-1)为:酵母膏4.173,(NH4)2SO413.594,蔗糖60.533,MgSO10.3,K2HPO4 0.10,核黄素0.004.在此务件下红酵母产类胡萝卜素的最大产量为15.291mg·L-1,较原始培养基提高了58.65%.本实验得到了产类胡萝卜素红酵母液体发酵培养基的一个非常合适的模型.     

Extraction Optimization of Water-Extracted Mycelial Polysaccharide from Endophytic Fungus Fusarium oxysporum Dzf17 by Response Surface Methodology

Water-extracted mycelial polysaccharide (WPS) from the endophytic fungus Fusarium oxysporum Dzf17 isolated from Dioscorea zingiberensis was found to be an efficient elicitor to enhance diosgenin accumulation in D. zingigerensis cultures, and also demonstrated antioxidant activity. In this study, response surface methodology (RSM) was employed to optimize the extraction process of WPS from F. oxysporum Dzf17 using Box-Behnken design (BBD). The ranges of the factors investigated were 1-3 h for extraction time (X(1)), 80-100 °C for extraction temperature (X(2)), and 20-40 (v/w) for ratio of water volume (mL) to raw material weight (g) (X(3)). The experimental data obtained were fitted to a second-order polynomial equation using multiple regression analysis. Statistical analysis showed that the polynomial regression model was in good agreement with the experimental results with the determination coefficient (R(2)) of 0.9978. By solving the regression equation and analyzing the response surface contour plots, the extraction parameters were optimized as 1.7 h for extraction time, 95 °C for extraction temperature, 39 (v/w) for ratio of water volume (mL) to raw material weight (g), and with 2 extractions. The maximum value (10.862%) of WPS yield was obtained when the WPS extraction process was conducted under the optimal conditions.     

利用响应面法优化出芽短梗霉As3．933产普鲁兰多糖发酵培养基

采用响应面分析法对出芽短梗霉As3．933产普鲁兰多糖的发酵培养基进行优化。首先利用Plackett-Bur-man实验筛选出影响普鲁兰多糖产量的主要因素为酵母膏、（NH4）zSO4和K2HPO4,再利用最陡爬坡实验逼近最大响应区域,最后通过Box-Behnken实验并运用Design-Expert8．0软件优化发酵培养基。确定优化培养基组成为：蔗糖62．5g·L-1,（NH4）2S040．67g·L-1,酵母膏2．84g·L-1,K2HPO47．12g·L-1,NaCl1．25g·L-1,MgS04·7H200．25g·L-1,pH值6．5,优化后的普鲁兰多糖产量达到22．29g·L-1,较初始液体发酵培养基（17．32g·L-1）提高了28．70％。     

Ultrasound-Assisted Extraction of Syringin from the Bark of Ilex rotunda Thumb Using Response Surface Methodology

In this work, a rapid extraction method based on ultrasound-assisted extraction (UAE) of syringin from the bark of Ilex rotunda Thumb using response surface methodology (RSM) is described. The syringin was analyzed and quantified by high performance liquid chromatography coupled with UV detection (HPLC-UV). The extraction solvent, extraction temperature and extraction time, the three main factors for UAE, were optimized with Box-Behnken design (BBD) to obtain the highest extraction efficiency. The optimal conditions were the use of a sonication frequency of 40 kHz, 65% methanol as the solvent, an extraction time of 30 min and an extraction temperature of 40 °C. Using these optimal conditions, the experimental values agreed closely with the predicted values. The analysis of variance (ANOVA) indicated a high goodness of model fit and the success of the RSM method for optimizing syringin extraction from the bark of I. rotunda.     

Optimization of the Preparation of Fish Protein Anti-Obesity Hydrolysates Using Response Surface Methodology

The enzymatic condition for producing the anti-obesity hydrolysates from fish water-soluble protein was optimized with the aid of response surface methodology, which also derived a statistical model for experimental validation. Compared with neutral protease, papain and protamex, the porcine pancreas lipase inhibitory rate of hydrolysates from fish water-soluble protein was higher with alkaline protease. Results showed that the model terms were significant, the terms of lack of fit were not significant, and the optimal conditions for the hydrolysis by alkaline protease were initial pH 11, temperature 39 °C, enzyme dosage 122 U/mL and 10 h of hydrolysis time. Under these conditions, the porcine pancreas lipase and the α-amylase inhibitory rate could reach 53.04% ± 1.32% and 20.03 ± 0.89%, while predicted value were 54.63% ± 1.75%, 21.22% ± 0.70%, respectively. In addition, Lineweaver-Burk plots showed noncompetitive inhibition. The K_i value calculated was 84.13 mg/mL. These results demonstrated that fish water-soluble protein could be used for obtaining anti-obesity hydrolysates.     

响应面分析优化霉酚酸发酵培养基

借助Design Expert 7.0软件,对霉酚酸发酵培养基的主要成分进行优化研究。首先采用全因子设计找出玉米浆和蔗糖为影响霉酚酸产量的主要因素,通过最陡爬坡法逼近最大响应区域,再利用中心组合设计及响应面分析法进行回归分析,求得两因素的最优水平：玉米浆98 g/L,蔗糖53 g/L。经验证,发酵培养基优化后菌株产量提高了38.2%。     

Medium Optimization for Exopolysaccharide Production in Liquid Culture of Endophytic Fungus Berkleasmium sp. Dzf12

Berkleasmium sp. Dzf12, an endophytic fungus from Dioscorea zingiberensis, is a high producer of spirobisnaphthalenes with various bioactivities. The exopolysaccharide (EPS) produced by this fungus also shows excellent antioxidant activity. In this study, the experimental designs based on statistics were employed to evaluate and optimize the medium for EPS production in liquid culture of Berkleasmium sp. Dzf12. For increasing EPS yield, the concentrations of glucose, peptone, KH₂PO₄, MgSO₄·7H₂O and FeSO₄·7H₂O in medium were optimized using response surface methodology (RSM). Both the fractional factorial design (FFD) and central composite design (CCD) were applied to optimize the main factors which significantly affected EPS production. The concentrations of glucose, peptone and MgSO₄·7H₂O were found to be the main effective factors for EPS production by FFD experimental analysis. Based on the further CCD optimization and RSM analysis, a quadratic polynomial regression equation was derived from the EPS yield and three variables. Statistical analysis showed the polynomial regression model was in good agreement with the experimental results with the determination coefficient (adj-R²) as 0.9434. By solving the quadratic regression equation, the optimal concentrations of glucose, peptone and MgSO₄·7H₂O for EPS production were determined as 63.80, 20.76 and 2.74 g/L, respectively. Under the optimum conditions, the predicted EPS yield reached the maximum (13.22 g/L). Verification experiment confirmed the validity with the actual EPS yield as 13.97 g/L, which was 6.29-fold in comparison with that (2.22 g/L) in the original basal medium. The results provide the support data for EPS production in large scale and also speed up the application of Berkleasmium sp. Dzf12.     

响应面法优化杆菌肽发酵培养基

采用响应面分析法对杆菌肽产生菌的发酵培养基进行优化。采用Plackett-Burman实验筛选出影响杆菌肽产量的主要因素为豆粕、Na2S2O3、生物氮素,利用最陡爬坡路径逼近响应区域,应用Box-Behnken设计和响应面分析优化得到最佳发酵培养基,发酵单位较优化前提高了36.9%。     

Biochemical Properties of a New Cold-Active Mono- and Diacylglycerol Lipase from Marine Member Janibacter sp. Strain HTCC2649

Mono- and di-acylglycerol lipase has been applied to industrial usage in oil modification for its special substrate selectivity. Until now, the reported mono- and di-acylglycerol lipases from microorganism are limited, and there is no report on the mono- and di-acylglycerol lipase from bacteria. A predicted lipase (named MAJ1) from marine Janibacter sp. strain HTCC2649 was purified and biochemical characterized. MAJ1 was clustered in the family I.7 of esterase/lipase. The optimum activity of the purified MAJ1 occurred at pH 7.0 and 30 °C. The enzyme retained 50% of the optimum activity at 5 °C, indicating that MAJ1 is a cold-active lipase. The enzyme activity was stable in the presence of various metal ions, and inhibited in EDTA. MAJ1 was resistant to detergents. MAJ1 preferentially hydrolyzed mono- and di-acylglycerols, but did not show activity to triacylglycerols of camellia oil substrates. Further, MAJ1 is low homologous to that of the reported fungal diacylglycerol lipases, including Malassezia globosa lipase 1 (SMG1), Penicillium camembertii lipase U-150 (PCL), and Aspergillus oryzae lipase (AOL). Thus, we identified a novel cold-active bacterial lipase with a sn-1/3 preference towards mono- and di-acylglycerides for the first time. Moreover, it has the potential, in oil modification, for special substrate selectivity.     

响应曲面法优化小麦秸秆纤维素酶水解条件

利用响应曲面试验设计方法(RSM),选择底物质量分数、酶投加量、温度、pH值及水解时间为试验因子,还原糖(RS)产率为响应值,考察小麦秸秆纤维素酶水解过程中各影响因子对还原糖产率的影响,对小麦秸秆纤维素酶水解条件进行优化。结果表明,所考察的5个影响因子对还原糖产率均具有显著影响(p<0.05)。所得回归方程R² 值为 0.946 9,p<0.05,变异系数(C_V)值为4.37%,足够精度值为26.396,说明模型高度显著,可以在设计范围内对响应值进行预测。模型预测最佳水解条件为底物质量分数8.0%,酶投加量为35 FPU/g(以秸杆质量计),温度50 ℃,pH值5.4,水解时间96 h。利用最优水解条件进行验证试验,所得还原糖产率为60.73%,水解液中葡萄糖和木糖质量浓度分别为31.84和 16.74 g/L。     

响应面法优化微胶囊固定化粘红酵母生产L-苯丙氨酸的工艺条件

以微胶囊固定化粘红酵母为研究对象。考察了粘红酵母转化反式肉桂酸（t-CA）生成L-苯丙氨酸过程中转化体系温度、pH值、CNH＋4／C1-CA对L-苯丙氨酸积累量的影响,并通过响应面法对转化条件进行了优化,得到最佳转化条件为：温度30．75℃、pH值11．58、CNH＋4=4．77mol·L-1、C1-CA=10g·L-1,在该条件下,L-苯丙氨酸积累量为27．5g·L-1。     

Optimization of Ultrasound-Assisted Extraction of Artemisinin from Artemisia annua L. by Response Surface Methodology

Artemisinin is a compound extracted from Artemisia annua L. with a remarkable curative effect against malaria. It can be extracted using ultrasound-assisted extraction (UAE) and then detected via HPLC. In this study, response surface methodology (RSM) was used to optimize UAE conditions for obtaining the maximum yield of artemisinin. Three independent variables (ratio of solvent to material, extraction temperature, and ultrasonic power) were evaluated using the Box-Behnken experimental design, with the yield of artemisinin as a response variable. Experimental data were highly fitted to a mathematical-regression model using multiple linear regression (MLR). Based on response surface plots, the three independent variables exhibited interactive effects on the yield of artemisinin. The optimal extraction conditions were as follows: 42.71 mL/g ratio of solvent to material, 41.86°C extraction temperature and 120 W ultrasonic power. The predicted yield of artemisinin by model was 0.7848%, whereas the actual yield in the extracts was 0.7826% ± 0.0790% in adjusted optimal conditions, with a relative error of 0.28%. The results undoubtedly demonstrated that RSM could be used to explore the optimum conditions of artemisinin extraction.     

基于响应面法的注塑件残余应力优化

考虑模具温度、熔体温度、保压压力和保压时间等4个注塑工艺参数,利用响应面法进行实验设计,通过Moldflow注塑模拟软件对所设计的实验进行有限元分析,得到注塑工艺参数对制品残余应力值的影响.结果发现4个工艺参数中,熔体温度和保压压力对注塑制品残余应力值的影响极为显著,而模具温度和保压时间对制品残余应力值的影响则不显著.文章的研究结果为注塑工艺参数的调整、选取和优化,为获得残余应力值最小的优质塑料制品提供了方法和思路.     

Rapid Adsorption of Heavy Metals by Fe3O4/Talc Nanocomposite and Optimization Study Using Response Surface Methodology

Fe₃O₄/talc nanocomposite was used for removal of Cu(II), Ni(II), and Pb(II) ions from aqueous solutions. Experiments were designed by response surface methodology (RSM) and a quadratic model was used to predict the variables. The adsorption parameters such as adsorbent dosage, removal time, and initial ion concentration were used as the independent variables and their effects on heavy metal ion removal were investigated. Analysis of variance was incorporated to judge the adequacy of the models. Optimal conditions with initial heavy metal ion concentration of 100, 92 and 270 mg/L, 120 s of removal time and 0.12 g of adsorbent amount resulted in 72.15%, 50.23%, and 91.35% removal efficiency for Cu(II), Ni(II), and Pb(II), respectively. The predictions of the model were in good agreement with experimental results and the Fe₃O₄/talc nanocomposite was successfully used to remove heavy metals from aqueous solutions.     

响应曲面法优化槲皮素锌络合反应条件的研究

在单因素实验的基础上,利用响应曲面法对槲皮素与醋酸锌络合反应的工艺参数进行优化。响应曲面分析结果表明,反应温度,反应时间以及体系的pH值对槲皮素锌的产率存在显著的相关性,通过典型分析得到优化的络合反应条件为:反应温度为43℃,反应时间72min,pH9.3,在此条件下产率的理论值达86.7%。验证实验最大产率为82.93%。且槲皮素-锌在水中的溶解度为811.20μg/mL比原槲皮素的溶解度增大了169倍。     

A Newly Isolated Thermostable Lipase from Bacillus sp

A thermophilic lipolytic bacterium identified as Bacillus sp. L2 via 16S rDNA was previously isolated from a hot spring in Perak, Malaysia. Bacillus sp. L2 was confirmed to be in Group 5 of bacterial classification, a phylogenically and phenotypically coherent group of thermophilic bacilli displaying very high similarity among their 16S rRNA sequences (98.5–99.2%). Polymerase chain reaction (PCR) cloning of L2 lipase gene was conducted by using five different primers. Sequence analysis of the L2 lipase gene revealed an open reading frame (ORF) of 1251 bp that codes for 417 amino acids. The signal peptides consist of 28 amino acids. The mature protein is made of 388 amino acid residues. Recombinant lipase was successfully overexpressed with a 178-fold increase in activity compared to crude native L2 lipase. The recombinant L2 lipase (43.2 kDa) was purified to homogeneity in a single chromatography step. The purified lipase was found to be reactive at a temperature range of 55–80 °C and at a pH of 6–10. The L2 lipase had a melting temperature (Tm) of 59.04 °C when analyzed by circular dichroism (CD) spectroscopy studies. The optimum activity was found to be at 70 °C and pH 9. Lipase L2 was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) (100%), whereas phenylmethylsulfonyl fluoride (PMSF), pepstatin-A, 2-mercaptoethanol and dithiothreitol (DTT) inhibited the enzyme by over 40%. The CD spectra of secondary structure analysis showed that the L2 lipase structure contained 38.6% α-helices, 2.2% ß-strands, 23.6% turns and 35.6% random conformations.     

响应面法分析优化苯与乙烯的烷基化反应

对改性β-分子筛催化的苯与乙烯的烷基化反应的工艺条件进行了优化。通过单因素实验确定了反应温度、空速、乙烯与苯的摩尔比的取值范围,并用响应面法(Box-Behnken设计法)确定最佳工艺参数为:反应温度154℃,空速2h-1,乙烯与苯的摩尔比1∶3。在此条件下,苯的转化率达到56.274%、乙苯的选择性达到86.592%。     

Optimization of the Culture Medium Composition to Improve the Production of Hyoscyamine in Elicited Datura stramonium L. Hairy Roots Using the Response Surface Methodology (RSM)

Traditionally, optimization in biological analyses has been carried out by monitoring the influence of one factor at a time; this technique is called one-variable-at-a-time. The disadvantage of this technique is that it does not include any interactive effects among the variables studied and requires a large number of experiments. Therefore, in recent years, the Response Surface Methodology (RSM) has become the most popular optimization method. It is an effective mathematical and statistical technique which has been widely used in optimization studies with minimal experimental trials where interactive factors may be involved. This present study follows on from our previous work, where RSM was used to optimize the B5 medium composition in [NO(3-)], [Ca(2+)] and sucrose to attain the best production of hyoscyamine (HS) from the hairy roots (HRs) of Datura stramonium elicited by Jasmonic Acid (JA). The present paper focuses on the use of the RSM in biological studies, such as plant material, to establish a predictive model with the planning of experiments, analysis of the model, diagnostics and adjustment for the accuracy of the model. With the RSM, only 20 experiments were necessary to determine optimal concentrations. The model could be employed to carry out interpolations and predict the response to elicitation. Applying this model, the optimization of the HS level was 212.7% for the elicited HRs of Datura stramonium, cultured in B5-OP medium (optimized), in comparison with elicited HRs cultured in B5 medium (control). The optimal concentrations, under experimental conditions, were determined to be: 79.1 mM [NO(3-)], 11.4 mM [Ca(2+)] and 42.9 mg/L of sucrose.