Chia-Hung Kuo

Enhanced enzymatic hydrolysis of sugarcane bagasse by N-methylmorpholine-N-oxide pretreatment

The cellulose dissolution solvent used in Lyocell process for cellulose fiber preparation, N-methylmorpholine-N-oxide (NMMO) monohydrate, was demonstrated to be an effective agent for sugarcane bagasse pretreatment. Bagasse of 20wt% was readily dissolved in NMMO monohydrate at 130 degrees C within 1h. After dissolution, bagasse could be regenerated by rapid precipitation with water as a porous and amorphous mixture of its original components. The regenerated bagasse exhibited a significant enhancement on enzymatic hydrolysis kinetic. Not only the reducing sugars releasing rate but also hydrolysis yield was enhanced at least twofold as compared with that of untreated bagasse. The cellulose fraction of regenerated bagasse was nearly hydrolyzed to glucose after 72h hydrolysis with Cellulase AP3. The recycled NMMO demonstrated the same performance as the fresh one on bagasse pretreatment for hydrolysis enhancement. The regenerated bagasse was directly used in simultaneous saccharification and fermentation (SSF) for ethanol production by Zymomonas mobilis. No negative effect on ethanol fermentation was observed and ethanol yield approximately 0.15 g ethanol/g baggasse was achieved.

Lipase-immobilized biocatalytic membranes for biodiesel production.

Microbial lipase from Candida rugosa (Amano AY-30) has good transesterification activity and can be used for biodiesel production. In this study, polyvinylidene fluoride (PVDF) membrane was grafted with 1,4-diaminobutane and activated by glutaraldehyde for C. rugosa lipase immobilization. After immobilization, the biocatalytic membrane was used for producing biodiesel from soybean oil and methanol via transesterification. Response Surface Methodology (RSM) in combination with a 5-level-5-factor central composite rotatable design (CCRD) was employed to evaluate the effects of reaction time, reaction temperature, enzyme amount, substrate molar ratio and water content on the yield of soybean oil methyl ester. By ridge max analysis, the predicted and experimental yields under the optimum synthesis conditions were 97% and 95%, respectively. The lipase-immobilized PVDF membrane showed good reuse ability for biodiesel production, enabling operation for at least 165 h during five reuses of the batch, without significant loss of activity.

Ultrasonic-Assisted Extraction of the Botanical Dietary Supplement Resveratrol and Other Constituents of Polygonum cuspidatum

The roots of Polygonum cuspidatum produce several phenolic compounds, including trans-resveratrol (1), trans-piceid (2), and emodin (3), and are a commercial source of the botanical dietary supplement 1. Ultrasonic-assisted extraction technology and conventional shaking extraction procedures were compared for the extraction of 1-3 from P. cuspidatum roots, using 50% ethanol as a food grade solvent. These compounds were extracted successfully, and their mass transfer coefficients were calculated by fitting the experimental results to a model derived from Ficks second law. The results indicated that ultrasonic-assisted extraction had higher mass transfer efficacies and extraction yields for 1-3 as compared with conventional shaking extraction. Under the extraction conditions used (extraction temperature 50 °C; ultrasonic power 150 W), yields of 3.5, 9.2, and 7.8 mg/g were obtained for 1-3, respectively.

Effect of membranes with various hydrophobic/hydrophilic properties on lipase immobilized activity and stability

In this study, three membranes: regenerated cellulose (RC), glass fiber (GF) and polyvinylidene fluoride (PVDF), were grafted with 1,4-diaminobutane (DA) and activated with glutaraldehyde (GA) for lipase covalent immobilization. The efficiencies of lipases immobilized on these membranes with different hydrophobic/hydrophilic properties were compared. The lipase immobilized on hydrophobic PVDF-DA-GA membrane exhibited more than an 11-fold increase in activity compared to its immobilization on a hydrophilic RC-DA-GA membrane. The relationship between surface hydrophobicity and immobilized efficiencies was investigated using hydrophobic/hydrophilic GF membranes which were prepared by grafting a different ratio of n-butylamine/1,4-diaminobutane (BA/DA). The immobilized lipase activity on the GF membrane increased with the increased BA/DA ratio. This means that lipase activity was exhibited more on the hydrophobic surface. Moreover, the modified PVDF-DA membrane was grafted with GA, epichlorohydrin (EPI) and cyanuric chloride (CC), respectively. The lipase immobilized on the PVDF-DA-EPI membrane displayed the highest specific activity compared to other membranes. This immobilized lipase exhibited more significant stability on pH, thermal, reuse, and storage than did the free enzyme. The results exhibited that the EPI modified PVDF is a promising support for lipase immobilization.

High Yield of Wax Ester Synthesized from Cetyl Alcohol and Octanoic Acid by Lipozyme RMIM and Novozym 435

Wax esters are long-chain esters that have been widely applied in premium lubricants, parting agents, antifoaming agents and cosmetics. In this study, the biocatalytic preparation of a specific wax ester, cetyl octanoate, is performed in n-hexane using two commercial immobilized lipases, i.e., Lipozyme® RMIM (Rhizomucor miehei) and Novozym® 435 (Candida antarctica). Response surface methodology (RSM) and 5-level-4-factor central composite rotatable design (CCRD) are employed to evaluate the effects of reaction time (1–5 h), reaction temperature (45–65 °C), substrate molar ratio (1–3:1), and enzyme amount (10%–50%) on the yield of cetyl octanoate. Using RSM to optimize the reaction, the maximum yields reached 94% and 98% using Lipozyme® RMIM and Novozym® 435, respectively. The optimum conditions for synthesis of cetyl octanoate by both lipases are established and compared. Novozym® 435 proves to be a more efficient biocatalyst than Lipozyme® RMIM.

Optimized ultrasound-assisted extraction of phenolic compounds from Polygonum cuspidatum.

In this study the phenolic compounds piceid, resveratrol and emodin were extracted from P. cuspidatum roots using ultrasound-assisted extraction. Multiple response surface methodology was used to optimize the extraction conditions of these phenolic compounds. A three-factor and three-level Box-Behnken experimental design was employed to evaluate the effects of the operation parameters, including extraction temperature (30–70 °C), ethanol concentration (40%–80%), and ultrasonic power (90–150 W), on the extraction yields of piceid, resveratrol, and emodin. The statistical models built from multiple response surface methodology were developed for the estimation of the extraction yields of multi-phenolic components. Based on the model, the extraction yields of piceid, resveratrol, and emodin can be improved by controlling the extraction parameters. Under the optimum conditions, the extraction yields of piceid, resveratrol and emodin were 10.77 mg/g, 3.82 mg/g and 11.72 mg/g, respectively.

A novel enzyme-assisted ultrasonic approach for highly efficient extraction of resveratrol from Polygonum cuspidatum.

Resveratrol is a promising multi-biofunctional phytochemical, which is abundant in Polygonum cuspidatum. Several methods for resveratrol extraction have been reported, while they often take a long extraction time accompanying with poor extraction yield. In this study, a novel enzyme-assisted ultrasonic approach for highly efficient extraction of resveratrol from P. cuspidatum was developed. According to results, the resveratrol yield significantly increased after glycosidases (Pectinex® or Viscozyme®) were applied in the process of extraction, and better extraction efficacy was found in the Pectinex®-assisted extraction compared to Viscozyme®-assisted extraction. Following, a 5-level-4-factor central composite rotatable design with response surface methodology (RSM) and artificial neural network (ANN) was selected to model and optimize the Pectinex®-assisted ultrasonic extraction. Based on the coefficient of determination (R(2)) calculated from the design data, ANN model displayed much more accurate in data fitting as compared to RSM model. The optimum conditions for the extraction determined by ANN model were substrate concentration of 5%, acoustic power of 150W, pH of 5.4, temperature of 55°C, the ratio of enzyme to substrate of 3950 polygalacturonase units (PGNU)/g of P. cuspidatum, and reaction time of 5h, which can lead to a significantly high resveratrol yield of 11.88mg/g.

Enzymatic synthesis of rose aromatic ester (2-phenylethyl acetate) by lipase.

BACKGROUND 2-Phenylethyl acetate (2-PEAc) is a highly valued natural volatile ester with a rose-like odour that is widely used to add scent or flavour to cosmetics, soaps, foods and drinks. In this study, 2-PEAc was synthesised enzymatically by transesterification of vinyl acetate with 2-phenethyl alcohol catalysed by immobilised lipase (Novozym(®) 435) from Candida antarctic RESULTS Response surface methodology and a three-level/three-factor Box-Behnken design were used to evaluate the effects of time, temperature and enzyme amount on the molar conversion % of 2-PEAc. The results showed that temperature was the most important variable. Based on the ridge max analysis results, optimum enzymatic synthesis conditions were predicted as a reaction time of 79 min, a temperature of 57.8 °C and an enzyme amount of 122.5 mg. The predicted and experimental yields were 86.4 and 85.4% respectively. CONCLUSION Three immobilised lipases were screened and 15 reaction conditions were tested in order to find the combination for maximum yield. The optimisation of 2-PEAc synthesis catalysed by Novozym(®) 435 was successfully developed. The kinetic study of this transesterification reaction showed that it followed an ordered ping-pong bi-bi mechanism without any inhibition by reactants.

RSM and ANN modeling-based optimization approach for the development of ultrasound-assisted liposome encapsulation of piceid

Piceid, a naturally occurring derivative of resveratrol found in many plants, has recently been considered as a potential nutraceutical. However, its poorly water-soluble property could cause a coupled problem of biological activities concerning drug dispersion and absorption in human body, which is still unsolved now. Liposome, a well-known aqueous carrier for water-insoluble ingredients, is commonly applied in drug delivery systems. In this study, a feasible approach for solving the problem is that the targeted piceid was encapsulated into a liposomal formula as aqueous substrate to overcome its poor water-solubility. The encapsulation process was assisted by ultrasound, with investigation of lipid content, ultrasound power and ultrasound time, for controlling encapsulation efficiency (E.E%), absolute loading (A.L%) and particle size (PS). Moreover, both RSM and ANN methodologies were further applied to optimize the ultrasound-assisted encapsulation process. The data indicated that the most important effects on the encapsulation performance were found to be of lipid content followed by ultrasound time and ultrasound power. The maximum E.E% (75.82%) and A.L% (2.37%) were exhibited by ultrasound assistance with the parameters of 160mg lipid content, ultrasound time for 24min and ultrasound power of 90W. By methodological aspects of processing, the predicted E.E% and A.L% were respectively in good agreement with the experimental results for both RSM and ANN. Moreover, RMSE, R2 and AAD statistics were further used to compare the prediction abilities of RSM and ANN based on the validation data set. The results indicated that the prediction accuracy of ANN was better than that of RSM. In conclusion, ultrasound-assisted liposome encapsulation can be an efficient strategy for producing well-soluble/dispersed piceid, which could be further applied to promote human health by increased efficiency of biological absorption, and the process of ultrasound-mediated liposome encapsulation can be well established by a methodological approach using either RSM or ANN, but it is worth mentioning that the ANN model used here showed the superiority over RSM for predicting and optimizing encapsulation.

Knock-out of glucose dehydrogenase gene in Gluconacetobacter xylinus for bacterial cellulose production enhancement

Glucose dehydrogenase (GDH) locates in the cytoplasmic membrane of Gluconacetobacter xylinus oxidizes glucose to gluconic acid that decreases the conversion of glucose to bacterial cellulose (BC). In this study, a mutant of G. xylinus was generated by knocking-out the membrane bound GDH gene via homologous recombination of a defect GDH gene. The production of BC by G. xylinus mutant (GDH-KO strain) using glucose as a carbon source was investigated. Without the membrane bound GDH activity, the mutant strain still produces BC and increases glucose utilization efficiency for cellulose biosynthesis. In contrast, the wild-type strain oxidized a large fraction of glucose to gluconic acid that decreased the conversion yield of glucose to BC. Our results showed that the BC production from GDH-KO strain was about 40 and 230% higher than that of wild-type strain in static and shaken culture, respectively.