酶法提取薏苡仁油的优化

本文应用响应面分析的方法研究了酶用量、酶解温度和pH对薏苡仁油提取率的影响,并得到了最佳的提取条件。研究结果表明,酶用量为1.569%、酶解温度在47.7℃、酶解pH为4.75。响应的薏苡仁油提取率为10.948%。油脂质量分析结果表明,酶法处理并没有显著改变薏苡仁油的性质。     

Optimizing the medium compositions for accumulation of the novel FR-008/Candicidin derivatives CS101 by a mutant of Streptomyces sp. using statistical experimental methods

Medium compositions for the production of the novel derivatives of FR-008/Candicidin which was produced by a mutant of Streptomyces sp. FR-008 were optimized using two statistical methods including Plackett–Burman design (P–B), which was applied to find the key ingredients for the best medium composition, and response surface methodology (RSM), which was used to determine the optimal concentrations of these components. Results indicated that peptone, copper sulfate and glycerol had significant effects on the production. Under the proposed optimized conditions, the CS101 experimental yield (191.259 mg/L) closely matched the yield (203.536 mg/L) predicted by the statistical model. The optimization of the medium contributed to 10-fold higher antibiotic production than that of the control. It was first revealed that copper could stimulate FR-008/Candicidin and their derivatives synthesis at an optimal concentration in this paper, moreover, the basis of this phenomenon was also explained by investigating the enhancement of the enzymatic pathways.     

Optimized synthesis of lipase-catalyzed l-ascorbyl laurate by Novozym® 435

l-Ascorbyl laurate is a fatty acid derivative of l-ascorbic acid which can be widely used as a natural antioxidant in both lipid containing food and cosmetic applications. To avoid any possible harmful effects from chemically synthesized product, the enzymatic synthesis appears to be the best way to satisfy the consumer demand for natural antioxidants. The ability of immobilized lipase from Candida antarctica (Novozym^® 435) to catalyze the direct esterification between l-ascorbic acid and lauric acid was investigated. Response surface methodology (RSM) and 5-level-4-factor central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction time (2–10 h), temperature (25–65 °C), enzyme amount (10–50% w/w of l-ascorbic acid), and substrate molar ratio of l-ascorbic acid to lauric acid (1:1–1:5) on percentage molar conversion to l-ascorbyl laurate. Based on the analysis result of ridge max, the optimal enzymatic synthesis conditions were predicted as follows: reaction time 6.7 h, temperature 30.6 °C, enzyme amount 34.5%, substrate molar ratio 1:4.3; and the optimal actual yield was 93.2%.     

Optimization of chemoenzymatic Baeyer–Villiger oxidation of cyclohexanone to ε-caprolactone using response surface methodology

ε-Caprolactone (ε-CL) has attracted a great deal of attention and a high product concentration is of great significance for reducing production cost. The optimization of ε-CL synthesis through chemoenzymatic Baeyer–Villiger oxidation mediated by immobilized Trichosporon laibacchii lipase was studied using response surface methodology (RSM). The yield of ε-CL was 98.06% with about 1.2 M ε-CL concentration that has a substantial increase mainly due to both better stability of the cross-linked immobilized lipase used and the optimum reaction conditions in which the concentration of cyclohexanone was 1.22 M, the molar ratio of cyclohexanone:urea hydrogen peroxide (UHP) was 1:1.3, and the reaction temperature was 56.5°C. Based on our experimental results, it can be safely concluded that there are three reactions in this reaction system, not just two reactions, in which the third reaction is that the acetic acid formed reacts with UHP to form peracetic acid in situ catalyzed by the immobilized lipase. A quadratic polynomial model based on RSM experimental results was developed and the R² value of the equation is 0.9988, indicating that model can predict the experimental results with high precision. The experimental results also show that the molar ratio of cyclohexanone to UHP has very significant impact on the yield of ε-CL (p < .0006).     

Combination of classical and statistical approaches to enhance the fermentation conditions and increase the yield of Lipopeptide(s) by Pseudomonas sp. OXDC12: its partial purification and determining antifungal property

Around 200 different lipopeptides (LPs) have been identified to date, most of which are produced via Bacillus and Pseudomonas species. The clinical nature of the lipopeptide (LP) has led to a big surge in its research. They show antimicrobial and antitumor activities due to which mass-scale production and purification of LPs are beneficial. Response surface methodology (RSM) approach has emerged as an alternative in the field of computational biology for optimizing the reaction parameters using statistical models. In the present study, Pseudomonas sp. strain OXDC12 was used for production and partial purification of LPs using Thin Layer Chromatography (TLC). The main goal of the study was to increase the overall yield of LPs by optimizing the different variables in the fermentation broth. This was achieved using a combination of one factor at a time (OFAT) and response surface methodology (RSM) approaches. OFAT technique was used to optimize the necessary parameters and was followed by the creation of statistical models (RSM) to optimize the remaining variables. Maximum mycelial growth inhibition (%) against the fungus Mucor sp. was 61.3% for LP. Overall, the combination of both OFAT and RSM helped in increasing the LPs yield by 3 folds from 367mg/L to 1169mg/L.     

Optimisation and Characterisation of Lipase-Catalysed Synthesis of a Kojic Monooleate Ester in a Solvent-Free System by Response Surface Methodology

Kojic acid is widely used to inhibit the browning effect of tyrosinase in cosmetic and food industries. In this work, synthesis of kojic monooleate ester (KMO) was carried out using lipase-catalysed esterification of kojic acid and oleic acid in a solvent-free system. Response Surface Methodology (RSM) based on central composite rotatable design (CCRD) was used to optimise the main important reaction variables, such as enzyme amount, reaction temperature, substrate molar ratio, and reaction time along with immobilised lipase from Candida Antarctica (Novozym 435) as a biocatalyst. The RSM data indicated that the reaction temperature was less significant in comparison to other factors for the production of a KMO ester. By using this statistical analysis, a quadratic model was developed in order to correlate the preparation variable to the response (reaction yield). The optimum conditions for the enzymatic synthesis of KMO were as follows: an enzyme amount of 2.0 wt%, reaction temperature of 83.69°C, substrate molar ratio of 1:2.37 (mmole kojic acid:oleic acid) and a reaction time of 300.0 min. Under these conditions, the actual yield percentage obtained was 42.09%, which is comparably well with the maximum predicted value of 44.46%. Under the optimal conditions, Novozym 435 could be reused for 5 cycles for KMO production percentage yield of at least 40%. The results demonstrated that statistical analysis using RSM can be used efficiently to optimise the production of a KMO ester. Moreover, the optimum conditions obtained can be applied to scale-up the process and minimise the cost.     

Culture conditions optimization of hyaluronic acid production by Streptococcus zooepidemicus based on radial basis function neural network and quantum-behaved particle swarm optimization algorithm

This study aimed to optimize the culture conditions (agitation speed, aeration rate and stirrer number) of hyaluronic acid production by Streptococcus zooepidemicus. Two optimization algorithms were used for comparison: response surface methodology (RSM) and radial basis function neural network coupling quantum-behaved particle swarm optimization algorithm (RBF-QPSO). In RBF-QPSO approach, RBF is employed to model the microbial HA production and QPSO algorithm is used to find the optimal culture conditions with the established RBF estimator as the objective function. The predicted maximum HA yield by RSM and RBF-QPSO was 5.27 and 5.62 g/l, respectively, while a maximum HA yield of 5.21 and 5.58 g/l was achieved in the validation experiments under the optimal culture conditions obtained by RSM and RBF-QPSO, respectively. It was indicated that both models provided similar quality predictions for the above three independent variables in terms of HA yield, but RBF model gives a slightly better fit to the measured data compared to RSM model. This work shows that the combination of RBF neural network with QPSO algorithm has good predictability and accuracy for bioprocess optimization and may be helpful to the other industrial bioprocesses optimization to improve productivity.     

Artificial Intelligence versus Statistical Modeling and Optimization of Cholesterol Oxidase Production by using Streptomyces Sp.

Cholesterol oxidase (COD) is a bi-functional FAD-containing oxidoreductase which catalyzes the oxidation of cholesterol into 4-cholesten-3-one. The wider biological functions and clinical applications of COD have urged the screening, isolation and characterization of newer microbes from diverse habitats as a source of COD and optimization and over-production of COD for various uses. The practicability of statistical/ artificial intelligence techniques, such as response surface methodology (RSM), artificial neural network (ANN) and genetic algorithm (GA) have been tested to optimize the medium composition for the production of COD from novel strain Streptomyces sp. NCIM 5500. All experiments were performed according to the five factor central composite design (CCD) and the generated data was analysed using RSM and ANN. GA was employed to optimize the models generated by RSM and ANN. Based upon the predicted COD concentration, the model developed with ANN was found to be superior to the model developed with RSM. The RSM-GA approach predicted maximum of 6.283 U/mL COD production, whereas the ANN-GA approach predicted a maximum of 9.93 U/mL COD concentration. The optimum concentrations of the medium variables predicted through ANN-GA approach were: 1.431 g/50 mL soybean, 1.389 g/50 mL maltose, 0.029 g/50 mL MgSO₄, 0.45 g/50 mL NaCl and 2.235 ml/50 mL glycerol. The experimental COD concentration was concurrent with the GA predicted yield and led to 9.75 U/mL COD production, which was nearly two times higher than the yield (4.2 U/mL) obtained with the un-optimized medium. This is the very first time we are reporting the statistical versus artificial intelligence based modeling and optimization of COD production by Streptomyces sp. NCIM 5500.     

Extraction,development and validation of HPLC-UV method for rapid and sensitive determination of colchicine from Colchicum autumnale L. Bulbs

Colchicum autumnale L. also known as the autumn crocus, contains colchicine alkaloid having antifungal properties. The tuber of this plant is rich in terms of colchicine. In this research an ultrasound-assisted extraction (UAE) method was optimized for the extraction of colchicine from Colchicum autumnale L. bulbs before high-performance liquid chromatography with UV detection (HPLC-UV). Optimization of various extraction parameters was performed using response surface methodology (RSM) to evaluate the maximum colchicine yield from Colchicum autumnale L. bulbs. The Box-Behnken design (BBD) and RSM were used to investigate the effect of three key parameters (extraction time (20–60?min), extraction temperature (40–80?°C) and ultrasound power (500–700?W) on extraction efficiency. The variance analysis suggested that the dependent response variable of yield of colchicine may be expressed by a quadratic polynomial model. The optimal theoretical extraction conditions were found to be an ultrasonication power of 602.4?W, an extraction time of 42?min and a temperature of 64?°C. Under these conditions, the optimum foreseen yield was 0.237%. The experimental colchicine yield obtained by following the optimized conditions was found to be 0.238%. These values are very well compatible with each other.     

New insights on nucleoside 2′-deoxyribosyltransferases: a versatile Biocatalyst for one-pot one-step synthesis of nucleoside analogs

In recent years, glycosiltransferases have arisen as standard biocatalysts for the enzymatic synthesis of a wide variety of natural and non-natural nucleosides. Such enzymatic synthesis of nucleoside analogs catalyzed by nucleoside phosphorylases and 2′-deoxyribosyltransferases (NDTs) has demonstrated to be an efficient alternative to the traditional multistep chemical methods, since chemical glycosylation reactions include several protection–deprotection steps. This minireview exhaustively covers literature reports on this topic with the final aim of presenting NDTs as an efficient option to nucleoside phosphorylases for the synthesis of natural and non-natural nucleosides. Detailed comments about structure and catalytic mechanism of described NDTs, as well as their possible biological role, substrate specificity, and advances in detection of new enzyme specificities towards different non-natural nucleoside synthesis are included. In addition, optimization of enzymatic transglycosylation reactions and their application in the synthesis of natural and non-natural nucleosides have been described. Finally, immobilization of NDTs is shown as a practical procedure which leads to the preparation of very interesting biocatalysts applicable to industrial nucleoside synthesis.     

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.     

Optimized butyl butyrate synthesis catalyzed by Thermomyces lanuginosus lipase

Butyl butyrate is an ester present in pineapple flavor, which is very important for the food and beverages industries. In this work, the optimization of the reaction of butyl butyrate synthesis catalyzed by the immobilized lipase Lipozyme TL‐IM was performed. n‐Hexane was selected as the most appropriate solvent. Other reaction parameters such as temperature, substrate molar ratio, biocatalyst content and added water, and their responses measured as yield, were evaluated using a fractional factorial design, followed by a central composite design (CCD) and response surface methodology. In the fractional design 2^4–1, the four variables were tested and temperature and biocatalyst content were statistically significant and then used for optimization on CCD. The optimal conditions for butyl butyrate synthesis were found to be 48°C; substrate molar ratio 3:1 (butanol:butyric acid); biocatalyst content of 40% of acid mass. Under these conditions, over 90% of yield was obtained in 2 h. Enzyme reuse was tested by washing the biocatalyst with n‐hexane or by direct reuse. The direct reuse produced a rapid decrease on enzyme activity, while washing with n‐hexane allowed reusing the enzyme for five reactions cycles keeping approximately 85% of its activity. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1416–1421, 2013     

Magnetic chitosan beads for covalent immobilization of nucleoside 2′-deoxyribosyltransferase: application in nucleoside analogues synthesis

Cross-linked magnetic chitosan beads were prepared in presence of epichlorohydrin under alkaline conditions, and subsequently incubated with glutaraldehyde in order to obtain an activated support for covalent attachment of nucleoside 2′-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT). Changing the amount of magnetite (Fe₃O₄) and epichlorohydrin (EPI) led to different macroscopic beads to be used as supports for enzyme immobilization, whose morphology and properties were characterized by scanning electron microscopy, spin electron resonance (ESR), and vibrating sample magnetometry (VSM). Once activated with glutaraldehyde, the best support was chosen after evaluation of immobilization yield and product yield in the synthesis of thymidine from 2′-deoxyuridine and thymine. In addition, optimal conditions for highest activity of immobilized LrNDT on magnetic chitosan were determined by response surface methodology (RSM). Immobilized biocatalyst retained 50 % of its maximal activity after 56.3 h at 60 °C, whereas 100 % activity was observed after storage at 40 °C for 144 h. This novel immobilized biocatalyst has been successfully employed in the enzymatic synthesis of 2′-deoxyribonucleoside analogues as well as arabinosyl-nucleosides such as vidarabine (ara-A) and cytarabine (ara-C). Furthermore, this is the first report which describes the enzymatic synthesis of these arabinosyl-nucleosides catalyzed by an immobilized nucleoside 2′-deoxyribosyltransferase. Finally, the attached enzyme to magnetic chitosan beads could be easily recovered and recycled for 30 consecutive batch reactions with negligible loss of catalytic activity in the synthesis of 2,6-diaminopurine-2′-deoxyriboside and 5-trifluorothymidine.     

Comparison of estimation capabilities of response surface methodology (RSM) with artificial neural network (ANN) in lipase-catalyzed synthesis of palm-based wax ester

Background  

Wax esters are important ingredients in cosmetics, pharmaceuticals, lubricants and other chemical industries due to their excellent wetting property. Since the naturally occurring wax esters are expensive and scarce, these esters can be produced by enzymatic alcoholysis of vegetable oils. In an enzymatic reaction, study on modeling and optimization of the reaction system to increase the efficiency of the process is very important. The classical method of optimization involves varying one parameter at a time that ignores the combined interactions between physicochemical parameters. RSM is one of the most popular techniques used for optimization of chemical and biochemical processes and ANNs are powerful and flexible tools that are well suited to modeling biochemical processes.     

Integrated reactor concepts for the enzymatic kinetic synthesis of cephalexin

Integrated process concepts for enzymatic cephalexin synthesis were investigated by our group, and this article focuses on the integration of reactions and product removal during the reactions. The last step in cephalexin production is the enzymatic kinetic coupling of activated phenylglycine (phenylglycine amide or phenylglycine methyl ester) and 7-aminodeacetoxycephalosporanic acid (7-ADCA). The traditional production of 7-ADCA takes place via a chemical ring expansion step and an enzymatic hydrolysis step starting from penicillin G. However, 7-ADCA can also be produced by the enzymatic hydrolysis of adipyl-7-ADCA. In this work, this reaction was combined with the enzymatic synthesis reaction and performed simultaneously (i.e., one-pot synthesis). Furthermore, in situ product removal by adsorption and complexation were investigated as means of preventing enzymatic hydrolysis of cephalexin. We found that adipyl-7-ADCA hydrolysis and cephalexin synthesis could be performed simultaneously. The maximum yield on conversion (reaction) of the combined process was very similar to the yield of the separate processes performed under the same reaction conditions with the enzyme concentrations adjusted correctly. This implied that the number of reaction steps in the cephalexin process could be reduced significantly. The removal of cephalexin by adsorption was not specific enough to be applied in situ. The adsorbents also bound the substrates and therewith caused lower yields. Complexation with beta-naphthol proved to be an effective removal technique; however, it also showed a drawback in that the activity of the cephalexin-synthesizing enzyme was influenced negatively. Complexation with beta-naphthol rendered a 50% higher cephalexin yield and considerably less byproduct formation (reduction of 40%) as compared to cephalexin synthesis only. If adipyl-7-ADCA hydrolysis and cephalexin synthesis were performed simultaneously and in combination with complexation with beta-naphthol, higher cephalexin concentrations also were found. In conclusion, a highly integrated process (two reactions simultaneously combined with in situ product removal) was shown possible, although further optimization is necessary.     

Solvent selection and optimization of α‐chymotrypsin‐catalyzed synthesis of N‐Ac‐Phe‐Tyr‐NH2 using mixture design and response surface methodology

A peptide, N‐Ac‐Phe‐Tyr‐NH₂, with angiotensin I‐converting enzyme (ACE) inhibitor activity was synthesized by an α‐chymotrypsin‐catalyzed condensation reaction of N‐acetyl phenylalanine ethyl ester (N‐Ac‐Phe‐OEt) and tyrosinamide (Tyr‐NH₂). Three kinds of solvents: a Tris–HCl buffer (80 mM, pH 9.0), dimethylsulfoxide (DMSO), and acetonitrile were employed in this study. The optimum reaction solvent component was determined by simplex centroid mixture design. The synthesis efficiency was enhanced in an organic‐aqueous solvent (Tris‐HCl buffer: DMSO: acetonitrile = 2:1:1) in which 73.55% of the yield of N‐Ac‐Phe‐Tyr‐NH₂ could be achieved. Furthermore, the effect of reaction parameters on the yield was evaluated by response surface methodology (RSM) using a central composite rotatable design (CCRD). Based on a ridge max analysis, the optimum condition for this peptide synthesis included a reaction time of 7.4 min, a reaction temperature of 28.1°C, an enzyme activity of 98.9 U, and a substrate molar ratio (Phe:Tyr) of 1:2.8. The predicted and the actual (experimental) yields were 87.6 and 85.5%, respectively. The experimental design and RSM performed well in the optimization of synthesis of N‐Ac‐Phe‐Tyr‐NH₂, so it is expected to be an effective method for obtaining a good yield of enzymatic peptide. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

The chlorination of barbituric acid and some of its derivatives by chloroperoxidase

Barbituric acid and some of its derivatives are presented as new substrates for the chloroperoxidase from Caldariomyces fumago. These compounds are rapidly converted to the 5-chloro or 5,5-dichloro derivatives, in very high yield. The reaction path is discussed and the kinetics of the reactions are investigated. It is shown that neither the concentration nor the structure of the organic substrate has any influence on the rate of halogenation. The enzymatic chlorination of 1-methyl-5-phenylbarbituric acid does not proceed in a stereoselective manner. The results are compared with the present theories concerning the enzymatic reaction mechanism, and the current research on this topic is evaluated. The available data do not as yet permit a definitive choice of reaction mechanism.     

β-galactosidase-catalyzed synthesis of 3-O-β-D-galactopyranosyl-sn-glycerol: Optimization by response surface methodology

In this study, the synthesis of 3-O-β-D-galactopyranosyl-sn-glycerol (GG) was performed by the reverse hydrolysis of D-galactose and glycerol using β-galactosidase from Kluyveromyces lactis. Four process variables, reaction temperature (30.0–45.0?°C), reaction time (24–48?h), enzyme concentration (150.00–350.00?U/mL), and substrate molar ratio (glycerol:D-galactose, 7.5:12.5?mmol/mmol) were investigated and optimized via response surface methodology (RSM) for optimal GG synthesis. Both quadratic equations and the optimal reaction conditions were established. Results showed that the four variables, i.e., reaction temperature, reaction time, enzyme concentration, and substrate molar ratio had significant (p?β-galactosidase concentration and 8.65:1.00 of substrate molar concentration ratio (glycerol: D-galactose) at 39.8?°C and 48?h of reaction. Under these conditions, the GG concentration was 140.03?g/L and GG yield was 55.71%, which both were close to the predicted values (143.26?g/L and 56.73%). This finding proves the RSM to be a useful tool in optimizing process conditions for GG synthesis.     

Response surface methodology as a decision-making tool for optimization of culture conditions of green microalgae Chlorella spp. for biodiesel production

We have evaluated process optimization and the interactive effects of a number of variables using a Box–Behnken design of response surface methodology (RSM). The process variables nitrate, phosphate, glucose and pH were optimized to enhance the cell growth rate, lipid accumulation and other biochemical parameters of Chlorella spp. The most significant increase in lipid production (dry cell weight basis) occurred at limited concentrations of nitrate and phosphate, 1 % glucose and pH 7.5. The addition of nitrates during the mid-lag and mid-exponential phases produced the maximum inhibitory effect on lipid accumulation and the presence of yeast extract led to a further enhancement of lipid accumulation. Of all the media tested, BG-11 was the best suited medium for algal biomass production and chlorophyll content. A significant increase in algal biomass was observed in BG-11 supplemented with bicarbonate and glucose (1 %). The maximum specific growth rate observed was on 9th day of culturing. Results of optimization of process variables through response surface methodology and optimization of various other conditions reflect cutting edge research directed towards increasing algal biomass and lipid content for biodiesel production using an efficient economical technological approach.     

Optimization of lipase-catalyzed synthesis of acetylated EGCG by response surface methodology

(−)-Epigallocatechin-3-O-gallate (EGCG) acetylated derivatives, which can be widely used as a natural antioxidant in both lipid containing food and cosmetic applications, were prepared by lipase catalyzed acylation of EGCG with vinyl acetate. Response surface methodology (RSM) and 5-level-4-factor central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction time (6–10 h), temperature (30–50 °C), enzyme amount (1.5–2.5% (w/w) of substrate), and substrate molar ratio of EGCG to vinyl acetate (0.5–1.5) on conversion of EGCG. By using multiple regression analysis, the experimental data were fitted to a second order polynomial model. The most suitable combination of variables was 40 °C, 2.12%, 10 h and 1.13 for the reaction temperature, the enzyme amount, the reaction time, and EGCG/vinyl acetate mole ratio, respectively. At these optimal conditions, the conversion yield reached 87.37%. The presence of mono-, di- and tri-acetylated derivatives in acetylated EGCG was confirmed by LC–MS-MS and identified as 5″-O-acetyl-EGCG, 3″, 5″-2-O-acetyl-EGCG and 5′, 3″, 5″-3-O-acetyl-EGCG by NMR.