Applied Biochemistry and Biotechnology (v.164, #2)

Resolution of racemic glycidyl (o, m, p)-methylphenyl ethers by using a newly isolated Botryosphaeria dothidea ZJUZQ007 with epoxide hydrolase activity affords enantiopure epoxides with enantiomeric excesses (e.e.s) of 91–99% and enantiomeric ratios (E) of 18.1 to 48.6. The (R)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ethers whereas the (S)-epoxides was obtained from glycidyl p-methylphenyl ether. Substitution pattern of the methyl group exerted effects both on configurations of the remaining epoxides and enantioselectivities of epoxide hydrolase. The observations were explained by enzyme-substrate docking studies. It is the first example showing that for kinetic resolution of glycidyl methylphenyl ethers, fungal species of B. dothidea was applied.
Keywords: Botryosphaeria dothidea ; Docking study; Enantioselectivity; Epoxide hydrolase; Glycidyl methylphenyl ethers

Imprinted polymers are now being increasingly considered for active biomedical uses such as drug delivery. In this work, the use of molecularly imprinted polymers (MIPs) in designing new drug delivery devices was studied. Imprinted polymers were prepared from methacrylic acid (functional monomer), ethylene glycol dimethacrylate (cross-linker), and bromhexine (as a drug template) using bulk polymerization method. The influence of the template/functional monomer proportion and pH on the achievement of MIPs with pore cavities with a high enough affinity for the drug was investigated. The polymeric devices were further characterized by FT-IR, thermogravimetric analysis, scanning electron microscopy, and binding experiments. The imprinted polymers showed a higher affinity for bromhexine and a slower release rate than the non-imprinted polymers. The controlled release of bromhexine from the prepared imprinted polymers was investigated through in vitro dissolution tests by measuring absorbance at λ max of 310 nm by HPLC-UV. The dissolution media employed were hydrochloric acid at the pH level of 3.0 and phosphate buffers, at pH levels of 6.0 and 8.0, maintained at 37.0 and 25.0 ± 0.5 °C. Results from the analyses showed the ability of MIP polymers to control the release of bromhexine In all cases The imprinted polymers showed a higher affinity for bromhexine and a slower release rate than the non-imprinted polymers. At the pH level of 3.0 and at the temperature of 25 °C, slower release of bromhexine imprinted polymer occurred.
Keywords: Molecularly imprinted polymer; Bromhexine; Bulk polymerization; Drug release; Biological systems

Specific Poly-phenolic Compounds in Cell Culture of Vitis vinifera L. cv. Gamay Fréaux by Inga Mewis; Iryna M. Smetanska; Carsten T. Müller; Christian Ulrichs (148-161).
Cell cultures established from plants represent an attractive alternative to whole plants for effective production of bioactive secondary metabolites. Cell culture from Vitis vinifera L. cv. Gamay Fréaux accumulated high amounts of hydroxycinnamic acid derivatives and anthocyanins. Two new compounds were identified: 3-O-glucosylresveratrol, a stilbene derivative, abundant in cell suspension culture, and a hydroxyphenol, 4-(3,5-dihydroxyphenyl)-phenol, abundant in callus culture. The major anthocyanin monoglucosides present in cell suspension culture were cyanidin 3-O-glucoside and peonidin 3-O-glucoside, and the major cinnamoyl derivatives were cyanidin 3-O-p-coumaryl glucoside and peonidin 3-O-p-coumaryl glucoside. Three minor anthocyanin compounds were found in V. vinifera cell culture: delphinidin 3-O-glucoside, petunidin 3-O-glucoside, and delphinidin 3-O-p-coumaryl glucoside. Anthocyanin levels of cell suspension cultures increased significantly—about eight fold—after 4-day cultivation in new medium. Salicylic acid at a concentration of 50 μM did not enhance anthocyanin accumulation in cell suspension culture, and similar levels of jasmonic acid significantly reduced the anthocyanin content.
Keywords: Resveratrol derivative; Anthocyanin; Cell culture; Vitis vinifera ; Elicitation; Wine

Production of Lactate in Escherichia coli by Redox Regulation Genetically and Physiologically by Huimin Liu; Junhua Kang; Qingsheng Qi; Guanjun Chen (162-169).
Escherichia coli grows fermentatively in glucose-containing medium under anaerobic condition with formation of a mixture of organic acids (lactate, acetate, formate, and succinate) and ethanol to accommodate reducing equivalents generated during glycolysis. In this paper, we tried to improve the lactate accumulation in E. coli by redox regulation genetically and physiologically. Our results indicated that genetic regulation of the host by reducing the reductive by-product may improve the lactate production. In addition, lactate accumulation was also improved under reduced and anaerobic cultivation conditions. Engineered E. coli SDU4 was able to accumulate lactate under strictly anaerobic conditions to 100 g/L with a yield of 1.97 mol/mol glucose.
Keywords: Escherichia coli ; Metabolic engineering; Redox; Lactate

Solid-State Fermentation of Palm Kernel Cake with Aspergillus flavus in Laterally Aerated Moving Bed Bioreactor by Yoke Phooi Wong; Horng Yuan Saw; Jidon Janaun; Kamatam Krishnaiah; Auti Prabhakar (170-182).
Solid-state fermentation (SSF) was employed to enhance the nutritive values of palm kernel cake (PKC) for poultry feeding. Aspergillus flavus was isolated from local PKC and utilized to increase the mannose content of PKC via the degradation of β-mannan in PKC; evaluation was done for batch SSF in Erlenmeyer flasks and in a novel laterally aerated moving bed (LAMB) bioreactor. The optimum condition for batch SSF in flasks was 110% initial moisture content, initial pH 6.0, 30 °C, 855 μm particle size, and 120 h of fermentation, yielding 90.91 mg mannose g−1 dry PKC (5.9-fold increase). Batch SSF in the LAMB at the optimum condition yielded 79.61 mg mannose g−1 dry PKC (5.5-fold increase) within just 96 h due to better heat and mass transfer when humidified air flowed radially across the PKC bed. In spite of a compromise of 12% reduction in mannose content when compared with the flasks, the LAMB facilitated good heat and mass transfer, and improved the mannose content of PKC in a shorter fermentation period. These attributes are useful for batch production of fermented PKC feed in an industrial scale.
Keywords: Aspergillus flavus ; Bioreactor; Laterally aerated moving bed; Palm kernel cake; Poultry feed; Solid-state fermentation

A Subtractively Optimized DNA Microarray Using Non-sequenced Genomic Probes for the Detection of Food-Borne Pathogens by Jin Yong Lee; Byoung Chan Kim; Kwan Jong Chang; Joo-Myung Ahn; Jee-Hoon Ryu; Hyo-Ihl Chang; Man Bock Gu (183-193).
In this study, we present the successful detection of food-borne pathogens using randomly selected non-sequenced genomic DNA probes-based DNA microarray chips. Three food-borne pathogens, Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), and Bacillus cereus, were subjected for the preparation of the DNA microarray probes. Initially, about 50 DNA probes selected randomly from non-sequenced genomic DNA of each pathogen were prepared by using a set of restriction enzyme pairs. The proto-type of DNA microarray chip for detecting three different pathogens simultaneously was fabricated by using those DNA probes prepared for each pathogen. This proto-type DNA microarray has been tested with three target pathogens and additional seven bacteria, and successfully verified with a few cross-hybridized probes. After this primary verification of the DNA microarray hybridization, this proto-type DNA microarray chip was redesigned and successfully optimized by eliminating a few cross-hybridized probes. The specificity of this redesigned DNA microarray chip to each pathogen was confirmed without any serious cross-hybridizations, and its multiplexing capability in its pathogen detection was found to be possible. This randomly selected non-sequenced genomic DNA probes-based DNA microarray was successfully proved to be the high-throughput simultaneous detection chip for the detection of food-borne pathogens, without knowing the exact sequence information of the target bacteria. This could be the first fabrication of DNA microarray chip for the simultaneous detection of different kinds of food-borne pathogens.
Keywords: Food-borne pathogen; DNA microarray chip; Non-sequenced genomic DNA probes; Cross-hybridization; Multiplexed detection

Near-Infrared Chemometric Approach to Exhaustive Analysis of Rice Straw Pretreated for Bioethanol Conversion by Yoshiki Horikawa; Tomoya Imai; Rie Takada; Takashi Watanabe; Keiji Takabe; Yoshinori Kobayashi; Junji Sugiyama (194-203).
We report a simple analytical procedure combining near-infrared (NIR) spectroscopy with multivariate analysis to detect the saccharification efficiency of pretreated rice straw. Three types of sample preparation methods were tested to develop a powerful calibration model, with the disk sample used as the standard protocol. From the spectra dataset of NaOH-treated biomass, we obtained a good calibration for the saccharification ratio and some major structural components by partial least-squares regression. Adding dataset from hot water and dilute sulfuric acid pretreatments to NaOH sample dataset, an acceptable calibration model to predict the saccharification ratio as well as the glucose, xylose, and lignin contents was generated. NIR has a great potential for rapid screening of saccharification efficiency of pretreated biomass, which would allows us to control the quality of processing toward better bioethanol production.
Keywords: NIR spectroscopy; Multivariate analysis; Calibration model; Saccharification ratio

Cellulase-Producing Bacteria from Thai Higher Termites, Microcerotermes sp.: Enzymatic Activities and Ionic Liquid Tolerance by Kitipong Taechapoempol; Thammanoon Sreethawong; Pramoch Rangsunvigit; Weerachart Namprohm; Bandhit Thamprajamchit; Sirirat Rengpipat; Sumaeth Chavadej (204-219).
The three highest hydrolysis-capacity-value isolates of Bacillus subtilis (A 002, M 015, and F 018) obtained from Thai higher termites, Microcerotermes sp., under different isolation conditions (aerobic, anaerobic, and anaerobic/aerobic) were tested for cellulase activities—FPase, endoglucanase, and β-glucosidase—at 37 °C and pH 7.2 for 24 h. Their tolerance to an ionic liquid, 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), was also investigated. The results showed that the isolate M 015 provided the highest endoglucanase activity whereas the highest FPase and β-glucosidase activities were observed for the isolate F 018. The isolate F 018 also showed the highest tolerance to [BMIM]Cl in the range of 0.1–1.0 vol.%. In contrast, the isolate A 002 exhibited growth retardation in the presence of 0.5–1.0 vol.% [BMIM]Cl.
Keywords: Cellulose; Cellulase-producing bacteria; Microcerotermes sp.; Bacillus subtilis ; Ionic liquid; 1-butyl-3-methylimidazolium chloride

Saponins are secondary metabolites that are widely distributed in the plant kingdom and are often the active components in medicinal herbs. Hence, saponins have a potential for the pharmaceutical industry as antibacterial, virucidal, anti-inflammatory, and anti-leishmanial drugs. However, their commercial application is often hindered because of practical problems, such as low and variable yields and limited availability of natural resources. In vitro cultures provide an alternative to avoid problems associated with field production; they offer a system in which plants are clonally propagated and yield is not affected by environmental changes. Additionally, treatment of in vitro cultures with elicitors such as methyl jasmonate may increase the production of saponins up to six times. In vitro cultures are amenable to metabolic engineering by targeting specific genes to enhance saponin production or drive production towards one specific class of saponins. Hitherto, this approach is not yet fully explored because only a limited number of saponin biosynthesis genes are identified. In this paper, we review recent studies on in vitro cultures of saponin-producing plants. The effect of elicitation on saponin production and saponin biosynthesis genes is discussed. Finally, recent research efforts on metabolic engineering of saponins will also be presented.
Keywords: Triterpene saponins; Saponin biosynthesis; In vitro culture; Elicitation; Metabolic engineering

Suppression of Pulmonary Tumor Promotion and Induction of Apoptosis by Crocus sativus L. Extraction by Saeed Samarghandian; Jalil Tavakkol Afshari; Saeideh Davoodi (238-247).
Crocus sativus L., commonly known as saffron, is the raw material for one of the most expensive spice in the world, and it has been used in folk medicine for centuries. We investigated the potential of the ethanolic extract of saffron to induce cytotoxic and apoptosis effects in carcinomic human alveolar basal epithelial cells (A549), a commonly used cell culture system for in vitro studies on lung cancer. The cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum treated with different concentrations of the ethanolic extract of saffron for two consecutive days. Cell viability was quantitated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Apoptotic cells were determined using annexin V–fluorescein isothiocyanate by flow cytometry. Saffron could decrease the cell viability in the malignant cells as a concentration- and time-dependent manner. The IC50 values against the A549 cell lines were determined as 1,200 and 650 μg/ml after 24 and 48 h, respectively. Saffron-induced apoptosis of the A549 cells in a concentration-dependent manner, as determined by flow cytometry histogram of treated cells that induced apoptotic cell death, is involved in the toxicity of saffron. It might be concluded that saffron could cause cell death in the A549 cells, in which apoptosis plays an important role. Saffron could also be considered as a promising chemotherapeutic agent in lung cancer treatment in future.
Keywords: A549; Annexin V; Apoptosis; Crocus sativus L. (Iridaceae); MTT