Applying the Taguchi robust design to the optimization of the asymmetric reduction of ethyl 4-chloro acetoacetate by bakers' yeast

This study examined the characteristics and operational parameters of the asymmetric reduction of ethyl 4-chloro acetoacetate by bakers’ yeast in order to produce S-4-chloro-3-hydroxybutyric acid ethyl ester. Eight operational variables were also optimized using the Taguchi method with consideration of the freshness of yeast cells as a noise factor. An L₁₈ orthogonal array was used to design the experiments. The reaction yield and the product's optical purity were considered as two product quality variables. A desirability function was applied to combine these two qualities as a single objective function. Additionally, the signal-to-noise (SN) ratio was used to estimate the variability in product quality. Optimization was undertaken not only to yield the best performance, but also to minimize the variation in quality. The confirmation experiments indicated that the reaction performance and the robustness of the product quality under the optimized conditions were higher than those obtained in other experiments in this study. Our results further demonstrate that the product's optical purity could be increased to >95% by adjusting the operational level of the main factors.     

Protein structure prediction in a 210-type lattice model: parameter optimization in the genetic algorithm using orthogonal array

We have applied the orthogonal array method to optimize the parameters in the genetic algorithm of the protein folding problem. Our study employed a 210-type lattice model to describe proteins, where the orientation of a residue relative to its neighboring residue is described by two angles. The statistical analysis and graphic representation show that the two angles characterize protein conformations effectively. Our energy function includes a repulsive energy, an energy for the secondary structure preference, and a pairwise contact potential. We used orthogonal array to optimize the parameters of the population, mating factor, mutation factor, and selection factor in the genetic algorithm. By designing an orthogonal set of trials with representative combinations of these parameters, we efficiently determined the optimal set of parameters through a hierarchical search. The optimal parameters were obtained from the protein crambin and applied to the structure prediction of cytochrome B562. The results indicate that the genetic algorithm with the optimal parameters reduces the computing time to reach a converged energy compared to nonoptimal parameters. It also has less chance to be trapped in a local energy minimum, and predicts a protein structure which is closer to the experimental one. Our method may also be applicable to many other optimization problems in computational biology.     

Bacillus megaterium WZ009催化合成(R)-4-氯-3-羟基丁酸乙酯和(S)-3-羟基-γ-丁内酯

以外消旋4-氯-3-羟基丁酸乙酯为唯一C源的富集培养筛选得到一株菌株WZ009,经16S rDNA测序鉴定为巨大芽胞杆菌(Bacillus megaterium)。B.megaterium WZ009静息细胞可以立体选择性催化(S)-4-氯-3-羟基丁酸乙酯水解和脱氯反应得到光学纯的(R)-4-氯-3-羟基丁酸乙酯(e.e.≥99%)和(S)-3-羟基-γ-丁内酯(e.e.≥95%)。笔者对B.megaterium WZ009不对称催化反应影响因素(温度、pH、中和剂、底物浓度、时间进程以及细胞重复利用)进行优化研究,确定了该反应体系最优条件:底物浓度200 mmol/L,中和剂氨水,pH 7.2,40℃反应12 h,转化率达到50.6%,底物对映体过量值为99.6%。该生物催化合成(R)-4-氯-3-羟基丁酸乙酯和(S)-3-羟基-γ-丁内酯过程具有良好的工业化应用前景。     

桑木层孔菌液体培养条件的研究

对桑木层孔菌(Phellinus mori)液体发酵条件进行了研究,以生物量和胞外多糖为指标,通过L16(45)和L9(34)正交表进行了两次正交试验,筛选出桑木层孔菌最适液体培养条件为:麦芽糖30 g/L,酵母浸粉和蛋白胨15 g/L(质量比2 1),KH2PO4和CaCl25.5 g/L(质量比1 1),初始pH6.0;通过单因素试验筛选出最适装液量为120 mL/250 mL,最适接种量为10%。在此条件下液体发酵培养7 d后,桑木层孔菌生物量达到23.375 g/L,胞外多糖产量达到3.993 g/L。     

BP神经网络优化微生物浸矿工艺

以低品位黄铜矿溶液为原料,浸矿制备Cu2＋能有效提高低品位黄铜矿的利用价值。基于浸矿过程中存在多因素影响的现象,通过正交试验与神经网络分析方法,对浸矿条件（接种量、矿石品位、Fe2＋添加量及浸矿溶液pH）实行优化。结果表明：在正交试验组中最佳试验结果为浸矿产128．753mg／LCu^2＋;BP神经网络优化后的最佳实验组合为微生物接种量12％、矿石品位0．3％、添加Fe^2＋24g/L及浸矿溶液pH1．7,该条件下验证试验产Cu^2＋ 141．352mg／L,通过正交试验及神经网络优化提高了微生物浸出低品位黄铜矿酸性溶液Cu^2＋的产量。     

Enhanced Specificity of TPMT*2 Genotyping Using Unidirectional Wild-Type and Mutant Allele-Specific Scorpion Primers in a Single Tube

Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques.     

荧光假单胞菌短链脱氢酶的克隆、表达及酶学性质分析

为了研究荧光假单胞菌中短链脱氢酶的生理角色和催化特性,从荧光假单胞菌Pseudomonas fluorescens GIM1.49基因组DNA克隆表达了一个短链脱氢酶的编码基因pfd,并分析了该基因产物的酶学性质。基因pfd全长684 bp,编码227个氨基酸,推算分子量为24.2 kDa。将携带短链脱氢酶基因的重组质粒pET28b-pfd转入大肠杆菌BL21(DE3) 进行表达,得到了28 kDa的表达产物。重组荧光假单胞菌短链脱氢酶 (PFD) 能氧化4-氯-3-羟基丁酸乙酯、1-苯乙醇、苯甲醇、仲丁醇和还原4-氯-乙酰乙酸乙酯、2-溴-苯乙酮、4-溴-苯乙酮等底物。以4-氯-3-羟基丁酸乙酯为底物时活力最高,Km值为186.90 mmol/L,Vmax为89.56 U/mg。氧化4-氯-3-羟基丁酸乙酯时,最适反应温度和pH分别为12 ℃和10.5,倾向于利用NAD+作辅酶;而还原4-氯-乙酰乙酸乙酯时,最适温度和pH为24 ℃和8.8,倾向于利用NADPH作辅酶。重组PFD能耐受50％ (V/V) 的甲醇等有机助溶剂,Ca2+ (1 mmol/L) 和EDTA (5 mmol/L) 对其酶活有一定的促进作用。上述结果表明,重组PFD是一个新型的短链脱氢酶,其代谢角色推测与卤代次级醇的氧化降解有关。     

Optimization of gibberellic acid production by immobilized Gibberella fujikuroi mycelium in fluidized bioreactors

An orthogonal experimental design L9 (3(4)) was used to investigate effects of temperature, pH, C:N ratio (glucose-C, NH4Cl-N) and concentrations of rice flour on production of gibberellic acid by Gibberella fujikuroi in 3.5 l fluidized bioreactors. The gibberellic acid production in a fluidized bioreactor could reach 3.90 g l(-1), more than 3-times greater than previously reported for submerged and solid fermentations. pH, rice flour concentration and C:N ratio were the factors that most influenced the production of gibberellic acid; pH being the most important. The response surface of gibberellic acid production to changes in pH and C:N ratio or rice flour concentration indicated that greatest production was found with a C:N ratio of 36.8 and pH 5 while the optimum concentration for rice flour was 2 g l(-1) and production increased with increased pH. The effect of temperature on the production of gibberellic acid was also significant and greatest production was at 30 degrees C.     

Optimizing the spectrofluorimetric determination of cefdinir through a Taguchi experimental design approach

The aim of this work is to optimize a spectrofluorimetric method for the determination of cefdinir (CFN) using the Taguchi method. The proposed method is based on the oxidative coupling reaction of CFN and cerium(IV) sulfate. The quenching effect of CFN on the fluorescence of the produced cerous ions is measured at an emission wavelength (λ_em) of 358 nm after excitation (λ_ex) at 301 nm. The Taguchi orthogonal array L₉ (3⁴) was designed to determine the optimum reaction conditions. The results were analyzed using the signal‐to‐noise (S/N) ratio and analysis of variance (ANOVA). The optimal experimental conditions obtained from this study were 1 mL of 0.2% MBTH, 0.4 mL of 0.25% Ce(IV), a reaction time of 10 min and methanol as the diluting solvent. The calibration plot displayed a good linear relationship over a range of 0.5–10.0 µg/mL. The proposed method was successfully applied to the determination of CFN in bulk powder and pharmaceutical dosage forms. The results are in good agreement with those obtained using the comparison method. Finally, the Taguchi method provided a systematic and efficient methodology for this optimization, with considerably less effort than would be required for other optimizations techniques. Copyright © 2015 John Wiley & Sons, Ltd.     

一株联苯降解菌的筛选及其降解条件研究

通过选择性富集培养,从济南市南部山区所取的土样中分离得到1株联苯降解菌,实验证明该菌株能以联苯作为惟一的碳源和能源生长,初步命名为LB07,经形态学、生理生化鉴定和16SrRNA基因序列对比分析,确定该菌属于巨大芽胞杆菌。分别研究pH、温度、底物浓度、装液量和培养时间各个单因素对联苯降解率的影响。然后设计正交实验确定菌株降解联苯的最佳优化条件,最佳培养条件为联苯初始质量浓度150mg／L,温度35℃,pH=6．5,装液量为250mL三角瓶装50mL,培养时间10d,菌株LB07降解联苯的性能最好,降解率达到94％。     

吉林长白山刺五加总皂苷超声提取工艺研究

以吉林长白山野生刺五加果实为原料,采用正交试验设计通过香草醛—高氯酸显色法对不同超声处理条件下刺五加果实粗提物中总皂甙得率进行测定,优化野生刺五加果实总皂甙超声提取工艺.结果表明:超声提取吉林长白山野生刺五加果实总皂甙的最佳工艺条件为料液比1∶20、乙醇体积分数70％、超声波功率450W、超声提取时间50 min,测得刺五加粗提物中总皂甙含量为3.57 mg/g,该提取方法简便、快捷,可用于长白山野生刺五加果实总皂甙提取和测定.     

Optimization of lactic acid production in SSF by Lactobacillus amylovorus NRRL B-4542 using Taguchi methodology

Lactic acid production parameter optimization using Lactobacillus amylovorus NRRL B-4542 was performed using the design of experiments (DOE) available in the form of an orthogonal array and a software for automatic design and analysis of the experiments, both based on Taguchi protocol. Optimal levels of physical parameters and key media components namely temperature, pH, inoculum size, moisture, yeast extract, MgSO4 . 7H20, Tween 80, and corn steep liquor (CSL) were determined. Among the physical parameters, temperature contributed higher influence, and among media components, yeast extract, MgSO4 . 7H20, and Tween 80 played important roles in the conversion of starch to lactic acid. The expected yield of lactic acid under these optimal conditions was 95.80% and the actual yield at optimum conditions was 93.50%.     

A novel spectrofluorimetric method for the assay of pseudoephedrine hydrochloride in pharmaceutical formulations via derivatization with 4-chloro-7-nitrobenzofurazan

A new highly sensitive and specific spectrofluorimetric method has been developed to determine a sympathomimetic drug pseudoephedrine hydrochloride. The present method was based on derivatization with 4-chloro-7-nitrobenzofurazan in phosphate buffer at pH 7.8 to produce a highly fluorescent product which was measured at 532 nm (excitation at 475 nm). Under the optimized conditions a linear relationship and good correlation was found between the fluorescence intensity and pseudoephedrine hydrochloride concentration in the range of 0.5-5 μg mL(-1). The proposed method was successfully applied to the assay of pseudoephedrine hydrochloride in commercial pharmaceutical formulations with good accuracy and precision and without interferences from common additives. Statistical comparison of the results with a well-established method showed excellent agreement and proved that there was no significant difference in the accuracy and precision. The stoichiometry of the reaction was determined and the reaction pathway was postulated.     

黑木耳ISSR-PCR反应体系的正交优化

采用正交试验设计的方法,对黑木耳ISSR-PCR(简单重复序列区间-多聚酶链反应)反应体系中的5种主要因素(Taq聚合酶,Mg2+,模板DNA,dNTP及引物)4个水平进行优化筛选,确立了适合黑木耳ISSR分析的优化反应体系(20μL),通过梯度PCR试验筛选得到相应引物的最佳退火温度。     

脂肪酶抑制剂产生菌Bacillus sp.LF深层发酵条件研究

随着生活水平的不断提高 ,肥胖在中国也开始成为影响人们健康的主要危险之一。目前市场上常见的减肥产品主要是作用于大脑 ,通过抑制食欲减少食物的摄入 ,由于这类药可能产生大脑、心血管及神经系统的副作用 ,不宜长期使用。瘦素 (Leptin)是一种肥胖基因表达的多肽激素 ,也是通过食欲中枢发挥作用 ,可以抑制食欲 ,提高代谢率 ,达到减肥的目的。因而另一类减肥药品脂肪酶抑制剂近年来受到国内外重视。脂肪酶抑制剂可直接阻断人体对脂肪的吸收 ,它不需要通过影响中枢神经系统来抑制人体的食欲 ,而是阻止脂肪在胃肠道的吸收。文献报道脂肪酶抑…     

Application of scale-down experiments in the study of citric acid biosynthesis

Problems associated with the development of submersed citric acid biosynthesis on sugar beet molasses for process improvement were solved by scale-down experiments: the determination of optimal temperature profile, optimal pH value, influence of OTR and inoculum quality. The optimal pH value at the start of the process was 6.4 with 3% of inoculum volume and temperature profile between 28–30°C. In the case of OTR which is scale-dependent, we prefer to use DOC pilot plant data for the transfer of technology.     

趋磁细菌W M-1 的培养条件优化研究

采用正交设计实验法研究了趋磁细菌WM-1产磁性细胞的培养条件。并利用L16(44)方案,用16个实验完成了4种培养条件、4个水平的优化工作。研究结果表明,培养基的pH值是影响趋磁细菌WM-1产磁性细胞的重要因素,正交实验结果在90％的置信区间可信。在最优的培养条件下,即pH为7．0,氧气的浓度为4％,m酒石酸:m琥珀酸为1:1,NaNO3 100 mg l-1条件下,磁性细胞的浓度提高到6．5×107 cells ml-1,比优化前提高了约8．3％．趋磁细菌WM-1磁滞回线的测量表明,Hc=230 Oe,Ms=0．9 emu／g dry wt．Cells,及Mr／Ms=0．50。     

Two spectroscopic methods for estimation of anti-migraine medications: Sumatriptan and Zolmitriptan based on nucleophilic substitution reaction

Sensitive and selective spectrophotometric and spectrofluorimetric methods have been developed for the estimation of two anti-migraine drugs, namely sumatriptan succinate (SUM) and zolmitriptan (ZOL). These methods depend on producing a yellow-coloured product after the reaction of the two drugs with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). The reaction products exhibited maximum absorbance at 481 nm in borate buffer of pH 9 and fluorescence emission peak at 540 nm after excitation at 470 nm for the two drugs. The linear ranges were 5–60 μg/ml for SUM and 5–50 μg/ml for ZOL in the spectrophotometric method (Method I), whereas this was 0.4–4 μg/ml for SUM and 0.5–5 μg/ml for ZOL in the spectrofluorimetric method (Method II). The method validity was assessed according to International Council for Harmonisation (ICH) guidelines. Statistical analysis of the results obtained from the proposed and comparison methods confirmed that the proposed methods were highly accurate and precise. The suggested methods could be used for the determination of the mentioned drugs in both pure form and in tablets.     

瑞拉菌素产生菌GAO-1-GR-2发酵工艺条件优化研究

对瑞拉菌GAO-1-GR-2的发酵条件进行了单因素研究,通过正交试验设计对该菌株的培养基进行了筛选。并对其发酵条件进行了研究,结果表明：有机氮源以酵母粉和黄豆饼粉为宜;碳源以葡萄糖和饴糖最好,但考虑到发酵成苓,应选用葡萄糖2％。发酵培养基应呈中性,以采用自然pH为宜,pH超过8,或小于7,则会抑制发酵产物活性;通气量为1：0．5最好,能降低20％耗氧量;摇床转速140r／min,培养温度30℃,发酵周期72h。抗生素发酵原粉效价能达到4000μg／mL。     

拟茎点霉ISSR-PCR反应体系的优化

旨在探讨TaqDNA聚合酶、dNTP、Mg2+、引物和模板DNA等因素对ISSR-PCR扩增的影响,采用单因子试验和正交设计方法相结合建立并优化拟茎点霉反应体系。在此基础上进行引物筛选,同时通过梯度PCR试验,确定引物的最佳退火温度。通过对19个拟茎点霉菌株的检验,结果表明已确立的体系稳定可靠,对不同模板均有较好的适用性和通用性,为利用ISSR技术对拟茎点霉进行遗传分析奠定基础。